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Paul J. Verma and Huseyin Sumer (eds.), Cell Reprogramming: Methods and Protocols, Methods in Molecular Biology,
vol. 1330, DOI 10.1007/978-1-4939-2848-4_1, © Springer Science+Business Media New York 2015
Chapter 1
Cellular Reprogramming in Basic and Applied Biomedicine:
The Dawn of Regenerative Medicine
Wendy Dean
Abstract
Fertilization triggers a cascade of cellular and molecular events restoring the totipotent state and the
potential for all cell types. However, the program quickly directs differentiation and cellular commitment.
Under the genetic and epigenetic control of this process, Waddington likened this to a three-dimensional
landscape where cells could not ascend the slope or traverse once canalized thus leading to cell fate deci-
sions and the progressive restriction of cellular potency. But this is not the only possible outcome at least
experimentally. Somatic cell nuclear transfer and overexpression of key transcription factors to generate
induced pluripotent cells have challenged this notion. The return to pluripotency and the reinstatement of
plasticity and heterogeneity once thought to be the exclusive remit of the developing embryo can now be
replicated in vitro. The following chapter introduces some of these ideas and suggests that the fundamental
principles learned may constitute the first step toward the opportunity for specific tissue renewal and
replacement in healthy aging and the treatment of chronic diseases—the age of regenerative medicine.
Key words Cellular reprogramming, Regenerative medicine, Induced pluripotent cells, Healthy aging
1 Introduction—Filling in Waddington’s Canal
Cellular reprogramming entered the realm of our imagination in
2006 when Shinya Yamamaka announced that a “four-factor cock-
tail” could transform differentiated fibroblasts into induced, plu-
ripotent stem cells [1]. This inspired a more prescriptive and
defined way of achieving the alchemic transmogrification of defined
cellular states. The significance of the breakthrough discovery of
reprogramming fully differentiated cells back to a pluripotent state
in what developmentally constitutes a retrograde cellular transition
was quickly acknowledged with the joint awarding of a Nobel Prize
in Physiology or Medicine in 2012 to Profs Shinya Yamanaka and
Sir John Gurdon for their complementary work in nuclear repro-
gramming. Their discoveries had laid much of the groundwork for
the concept of experimentally induced retrograde progression to
induced pluripotent stem (iPS) cells.](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-20-2048.jpg)
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But exactly how does the forced overexpression of a handful of
transcription factors and chromatin-binding molecules transform
the defined cellular state of a differentiated cell and progress it back
up Waddington’s ascending landscape to assume a pluripotent
phenotype—in essence, a stem cell?
The simple answer is, at present, that we do not know.
However, the full impact of modern genome-wide investigation
and the sheer force of numbers of researchers worldwide leading
this investigation make the prospect of significant mechanistic
understanding only a matter of time and the translation to patient-
specific regenerative medicine a reality in our lifetime. In the course
of these studies there is a real prospect of collateral benefit; much
will be learned about the potential to identify and manipulate
endogenous stem cell populations that function in tissue repair and
replacement throughout life. Indeed the intense study of the pro-
cesses of cellular regression may well hold the key to understanding
healthy aging and offer an explanation for the growing number of
centenarians in our societies, which has seen a fivefold rise over the
last 30 years (Office for National Statistics UK; BBC news, 27th
Sept 2013).
Cellular reprogramming is the conversion of one specific cell
type to another. Arguably, we could well consider that develop-
ment in its usual forward-only direction could constitute a form of
cellular reprogramming. Here, the highly specialized and fully dif-
ferentiated oocyte is reprogrammed on fertilization to restore an
ephemeral totipotent state that is quickly followed by a series of
progressively more differentiated cellular decisions passing through
ever more restricted multipotent junctures to give rise to the fully
formed neonatal animal. In the 1940s Conrad Waddington
described this process in his classical model of the epigenetic land-
scape where one genotype allowed for the generation of multiple
cellular phenotypes [2]. Waddington illustrated the hierarchical
progression of the undifferentiated state by a series of channels
which were progressively more restricted and increasingly sepa-
rated; thus once the cellular state was “fated” thereafter the lineage
was restricted and incapable of either returning to a more undif-
ferentiated state or a different germ cell layer [3]. In the postgen-
omic era, these ideas together with classical developmental and
cellular biology have formed the basis of our understanding of the
field of epigenetics.
However, today cellular reprogramming more often refers to
those landmark methods which included transdifferentiation or
direct cell conversion, somatic cell nuclear transfer (SCNT) and
experimental reprogramming, the basis of the generation of iPS
cells [4]. The chapters which follow outline the details of how to
establish these various models of developmental and cellular pro-
cesses that set the experimental scene for understanding the mech-
anisms underpinning these transitions and serve to allow us
Wendy Dean](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-21-2048.jpg)
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unprecedented opportunities in basic, agricultural and biomedical
science to improve health and wellbeing, to enhance food security,
and offer therapeutic solutions to the treatment of chronic disor-
ders in humans.
By way of an introduction to these methods I will outline some
of the origins and common themes which these methods share and
contrast points where they differ. These experimental approaches
have certainly been instrumental in driving a deeper and more
comprehensive understanding of mammalian development and
stem cell biology in general and will undoubtedly continue to drive
fundamental and applied questions in these areas. Perhaps most
exciting, as a result of these experimental systems, fundamentally
held beliefs about the prescriptive nature of developmental pro-
cesses and tissue regeneration upon damage are now being chal-
lenged. The prospect of significant improvement of health span, on
a patient specific basis, is now within sight.
While the focus of this book is the experimental details that
facilitate cellular reprogramming, before embarking on an outline
of these techniques it may be worth touching, if only briefly, on
some processes that occur naturally which are capable of achieving
the same end. Transdifferentiation and cell fusion, much like that
of experimental heterokaryons, do occur naturally [5, 6].
Transdifferentiation constitutes a change in cellular fate, which can
facilitate the transit between lineages in the most extreme case and
between a differentiated cell type and its less differentiated fore-
runner within a given lineage. Here, one distinction that is often
applied is that both of these processes take place by a direct cell
conversion and not via a pluripotent intermediate. In mammals
transdifferentiation can be achieved experimentally by both gain of
function through overexpression and loss of function mechanisms
of one or a few factors and in this way bears some resemblance to
iPS production. Interestingly, these induced transitions can be
studied in vitro using stem cell models such as an ES cell, a proxy
for the inner cell mass of the blastocyst stage in mammals. In what
seems a reversion of the very first cellular decision in development,
ES cells can be driven to acquire trophoblast stem (TS) cell-like
fates [7–9] which implies that the experimental manipulation
endows the cell with permission, and capacity, for the lowering of
the epigenetic barrier that ordinarily separates and defines these
first two cell lineages.
Cell fusion and transdifferentiation have shared a common
past. In 2002 two significant papers identified the potential of ES
cells co-cultured with either neural stem cells or bone marrow cells
to subsequently undergo differentiation to a variety of cell types.
However, this occurred not by dedifferentiation, which was the
first explanation, but by transdifferentiation via spontaneous cell
fusion [10, 11]. At the time this caused a significant rethink in the
field but supplied positive benefit in the greater degrees of vigor
Cellular Reprogramming for Biomedicine](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-22-2048.jpg)
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that were thereafter required of these types of experiments [12].
Perhaps more importantly, this did highlight the fact that these
processes could occur, albeit at a low frequency, establishing the
proof of principle that similar cell–cell fusion events that allow cell
fate transitions may take place in vivo. Thinking along these experi-
mental lines may well be of benefit in particular to the adult stem
cell field.
While SCNT and iPS cell reprogramming are seemingly dia-
metrically opposed they share interesting common origins in the
ferment of mammalian experimental embryology and cell biology
in the 1980s. The premise of SCNT had been based on classical
developmental experiments carried out by Spemann in the 1920s
answering the question of totipotency of nuclei at least early in
development [13]. This was extended by the seminal work of
Briggs and King in the 1950s [14] followed closely by John
Gurdon [15] illustrating that in amphibian models differentiated
nuclei could be transplanted to the enucleated oocyte and give rise
to an adult organism. While this confirmed nuclear conservation,
they also showed that the regenerative potency with nuclear donors
isolated from more advanced, and hence more differentiated tis-
sues, was progressively restricted [16]. As a whole this progressive
restriction, i.e., the very idea that Waddington described as canali-
zation, seemed to be holding up.
In 1983, McGrath and Solter published a method of nuclear
transplantation in mammals using a fusogenic virus [17]. This laid
the ground work for the flurry of reports of “cloning” in mammals
from embryonic cells in the sheep by Steen Willadsen [18] to the
landmark achievement of Campbell and Wilmut in 1996 of the
generation of a live cloned sheep, Dolly, from an adult, fully dif-
ferentiated, mammary cell nucleus [19]. To date cloning has been
successful in more than 15 mammalian species including the extinct
Pyrenean ibex and a handful of other endangered species [20].
While cloning in most species has been a success, among endan-
gered species cloning has been more difficult. Of these only the
mouflon sheep survived for more than a few days after birth [21].
Clearly the oocyte, in conjunction with modulation of widespread
chromatin remodeling, can reinstruct a terminal program to relive
its developmental past; something once thought to be unachiev-
able under any circumstance [22].
The induction of stem cells starting from differentiated fibro-
blasts is an extreme form of cell fate conversion and hence may
constitute an extreme form of transdifferentiation. Here, the con-
trast to the reprogramming in SCNT is stark. The cellular as well as
the nuclear status of the fibroblast must be dedifferentiated and
ultimately progressed to the pinnacle of the canalized landscape in
order to form pluripotent stem cells. In this form of reprogram-
ming the cell is a most unsuitable environment with little of its own
capacity to direct retrograde dedifferentiation unto pluripotency.
Wendy Dean](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-23-2048.jpg)
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The earliest incarnations of this process were first described in
Lasser et al. [23] where overexpression of a defined transcription
factor (TF), MyoD, was able to drive fibroblasts toward a muscle
cell fate. While this worked best in mesodermally derived cells,
similar results were also obtained in ectodermal and endodermal
derivatives hinting at the now familiar concept that forced over-
expression of TFs, defining for a given cell type, greatly assists in
the transdifferentiation toward that cell type [24]. In practice this
is but a short step away in taking this idea forward toward a des-
tination in stem cell populations—in essence the seed of the
“four-factor cocktail” had been planted. Over the intervening
years intra-germ layer conversion was demonstrated for a vast
number of TF combinations. Interestingly, the dynamics of the
transition were highly variable with both the starting cell type
and the order of expression of the TF cocktail able to influence
the cellular outcome. In fact, only relatively recently has this
approach succeeded in “long distance” direct conversion; start-
ing with fibroblasts a “three-factor” cocktail was able to generate
functional neurons [25].
Induced pluripotent stems cells have changed the way we think
about cellular differentiation, cell fate commitment, and the unidi-
rectional nature of development [26]. Beyond that, the very nature
of the stably differentiated cell has been challenged along with the
ideas of the epigenome that serve to reinforce and fix that state.
While remarkable in the insights that derived from conversion of
cell types both within and across germ layer boundaries, direct cell
conversion has significant limitations. Ideally, and in keeping with
the need to be able to supply adequate numbers of any cell type in
any lineage, stem cells seem like the best option and those equiva-
lent to embryonic stem cells would allow unrestricted and ethically
uncomplicated extension to therapeutic applications in the treat-
ment of disease.
Applying the lessons of intra-lineage conversion, Takahashi
and Yamanaka focused their attention on transcription factor net-
works associated with pluripotency and self-renewal, both hall-
marks of pluripotent embryonic stem (ES) cells. Distilling the list
to the now well known “four-factor cocktail,” of Oct3/4, Sox2,
Klf4 and c-Myc (OSKM), and transfecting them into either fetal or
adult mouse, and later human, fibroblasts lead eventually to the
generation of the first iPS cells [1]. Remarkably, in mouse and
human, expression from the delivery systems is eventually taken
over by the endogenous loci thereby supplying a continuous source
of the essential factors characteristic of the target ES cells. Although
highly inefficient, these cells fulfilled their potential being able to
differentiate into all three germ layers and in the generation of
both chimeric animals and entirely iPS-derived mice by tetraploid
complementation, the gold standard for demonstrating pluripo-
tency. Interestingly, a large proportion of the domestic animal iPS
Cellular Reprogramming for Biomedicine](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-24-2048.jpg)
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systems fail to either activate the endogenous loci or silence the
transgenes in the course of iPS reprogramming.
Better and more efficacious delivery systems that did not
involve viral vectors, requisite for use in humans, have now been
achieved. Many iterations and reiterations of the “essential factors”
have also taken place with replacements now in common use. In
this respect it is remarkable that the “four factors” have been found
to be so broadly able to direct iPS cell generation across such a
wide cross section of mammalian species. In a few cases, in bovine
[27] and the endangered class of Felids [28] is an additional factor,
namely Nanog, required for iPS cell reprogramming. In the goat
and sheep, eight factors have been reported to be required to
reprogram primary ear fibroblasts [29, 30].
Second- and third-generation reprogramming approaches to
iPS cells now exist which employ either small molecule inhibitors
or transfection of families of microRNAs alone or in combination
with the Yamanaka factors [31, 32]. MicroRNAs are particularly
abundant in pluripotent ES cells; among the most abundant, the
miR301/367 in humans and the miR290 cluster in the mouse, are
themselves up-regulated by the OSKM quartet and mutually rein-
force the pluripotent state thereby driving cells toward this termi-
nus. Coupled to their ability to down-regulate de novo methylation
the up-regulation of the miR290 cluster also enhances, among
other functions, the kinetics of the mesenchymal to epithelial tran-
sition (MET) requisite for reprogramming to iPS status [33–35].
Incidentally, alteration of the culture environment has also proven
to enhance iPS cell reprogramming.
The ability to generate ES cells in mouse and human has been
a breakthrough in pioneering the idea of replacement therapies for
faulty genes together with functional and mechanistic studies in all
biological disciplines, which ultimately underpin applied research.
In domestic species of agricultural and veterinary importance,
while some species have been amenable to the generation of
embryonic-like stem cells especially in light of improvements trans-
lated from the mouse, many have yet to achieve the same unre-
stricted claims to pluripotency. Here, iPS cell generation may prove
to be the solution as is the case in the equine system. Equine
ES-like cells possess only some of the full repertoire of the pluripo-
tent spectrum while equine iPS cells seem to be fully functional
and able to contribute to teratomas in engraftment experiments
[36]. Targeting of iPS cells once established may not prove univer-
sally simple. For example, human ES cells are refractory to conven-
tional genome editing via homologous recombination achieving
only very low efficiencies compared to the mouse and hence other
targeted methodologies such as zinc finger proteins, TALENs and
CRISPR are required [37].
The development of SNCT has long been regarded as a means
by which rare and endangered species might be rescued from
Wendy Dean](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-25-2048.jpg)
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impending extinction. Indeed, even some now extinct species have
been reanimated by NT where appropriate recipient species
hybrids still survive. It would now seem possible that iPS genera-
tion may provide additional avenues to help in supporting efforts
to save endangered species offering prospects of generation of
gametes in vitro from iPS cells as has been achieved with ES cells
[38–40]. Despite the relative ease in which the iPS generation has
been successful across a very wide swath of mammalian species, the
generation of gametes may not prove as simple; nonetheless, there
is reason for great optimism that the species variation among germ
cell maturation can be overcome and functional gametes gener-
ated across the diverse class of Mammalia. Failing the ability to
generate full maturation of gametes, iPS cells may well allow for
unprecedented mechanistic studies into germ cell development
across a wide selection of species many of whom may offer better
and closer physiological comparisons to humans without serious
ethical limitations [38, 41].
2 The Epigenome and Life in Culture
With the unparalleled promise of personalized medicine and gen-
eration of patient-specific tissue by stem cell therapies, replacement
and renewal no longer seems like a distant prospect. Less ambi-
tious but potentially more beneficial is the ability to test patient-
specific matching of drug treatment by using iPS cells either
directly or on tissue-specific differentiation. Veterinary drug test-
ing and biopharmaceutical companies may well screen and develop
treatments tailored by genetically typing patient groups to offer
the best fit for regulation of metabolic disorders using iPS cells
derived from specifically defined allelic profiling.
However, the question remains about the role of the epig-
enome and the influence of culture-based rearing of cells and tis-
sues especially where tissue engraftment is required. Here, lessons
from ES cells as a proxy for iPS cells will be highly informative. It
has long been recognized that cells in culture, including embry-
onic stem cells acquire increasing levels of DNA methylation, as a
function of the duration of life in culture, a significant barrier to
both dedifferentiation via SCNT and iPS reprogramming.
Recent evaluation of the DNA methylation profile of primed
vs naïve ES cells has shed light on this question. Small molecule
inhibitors (aka 2i) that both enhance ES cell derivation and reduce
their heterogeneity in culture have focused attention on the role of
the composition of the culture media and the DNA methylome in
mouse [42–45] and in human ES cells [46]. Thus the presence of
conventional serum can affect the pluripotential capacity of ES
cells by significant modulation of DNA methylation, notably by
increasing methylation and decreasing naïve pluripotency. In as
Cellular Reprogramming for Biomedicine](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-26-2048.jpg)
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much as microRNA families that are associated with iPS cell repro-
gramming negatively regulate DNA methyltransferases and hence
DNA methylation, these two common components (i.e., serum
and microRNAs) seem to be at odds with one another for the
reprogramming process. Loss of DNA methylation, especially tied
to natural reprogramming, has been a dominant interest in the
field of epigenetics. The discovery of another significant pathway
able to down-regulate DNA methylation by methylcytosine
oxidation-coupled to repair pathways may be able to offer some
answers [47, 48]. A family of three enzymes, the ten-eleven-
translocation or TETS, iteratively oxidizing the methyl group on
cytosine to hydroxymethyl cytosine (5hmC) eventually leads to
this loss of DNA methylation via the return to the cytosine group.
Enzymatically, this reaction requires reduced Fe2+
and
α-ketoglutarate as cofactors and is hence very sensitive to the media
conditions and gaseous environment during culture.
Ascorbic acid, Vitamin C (VitC), has been known to enhance
iPS generation in mouse and humans for some time. Here acting
not via the 2i pathway but rather by alleviating the senescence road-
block, in the presence of VitC the histone demethylases Jhdm1a/1b
are stimulated [49]. Interestingly, TET1 is involved via its func-
tional domain in the formation of 5hmc at loci critical for MET in
a VitC-dependent manner [50]. In a systematic screen, the absence
of all H3K9me2 and me3 histone methylases, which include
Suv39H1 and 2, G9A and SetDB1, were found to work synergisti-
cally with VitC to enhance iPS cell reprogramming [50]. The mod-
ulation of H3K9me2/me3 is mechanistically linked to loss of DNA
methylation [51]. As such the presence of VitC in somatic cell
reprogramming is tied to loss of DNA methylation likely via repli-
cation-dependent passive mechanisms that involve loss of H3K9
methylation as well.
Whether or not the acquisition of DNA methylation during
culture of iPS cells will constitute a barrier to their widespread
application is not yet clear. In mouse ES cells maintained in stan-
dard serum-based culture conditions CpG methylation is high.
However, what happens to this hypermethylation once it is intro-
duced into a cellular context in vivo or upon tissue derivation has
not been systematically explored. In a simple but elegant test of
this question the results of a recent experiment gives us cause for
optimism. ES cells carrying a GFP reporter were used to make
chimeric animals by the classical blastocyst injection method. These
chimeric embryos were collected at E17.5 and the GFP-positive
cells isolated by flow cytometry and subsequently evaluated for lev-
els of DNA methylation. While the original ES cells were heavily
methylated, those GFP-positive cells isolated from tissues of these
embryos showed reduced levels of DNA methylation that were not
significantly different from the GFP-negative host cells. In essence,
in dividing cells within an in vivo environment, the DNA
Wendy Dean](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-27-2048.jpg)
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methylation levels had been returned to normal [52]. Whether this
is universally true in other species needs to be proven.
Collectively, we are closing in on solutions to overcome many
of the barriers that currently limit unbridled enthusiasm and realis-
tic optimism for the promise of iPS cell-based application to regen-
erative medicine. The regulation of the epigenome is amongst one
of the most complicated barriers which unify the challenges of both
SCNT and iPS cell reprogramming irrespective of the application
[53]. At present the incredible rate of research output in this area is
rivaled only by that of the stem cell biology (which is overlapping
with iPS cells). Lessons learned in driving the program back to the
top of the Waddington landscape have revealed that pathways at
intermediate heights may well provide equally good or better van-
tage points for obtaining multipotent stem cell populations both
in vitro and that are resident in vivo, that might offer solutions to
contemporary obstacles. Indeed, direct cell conversion has chal-
lenged our belief about the distance between differentiated lineages
and the depth of the canalization. Late in 2014, the direct conver-
sion of fibroblasts into thymic epithelial-like cells giving rise to a
functional thymus-like organ on transplantation of aggregates
together with T-cell precursors and support cells was reported [54].
The chapters that follow offer practical solutions and guide-
lines on how to overcome the obstacles that currently impede our
progress in experimental reprogramming. Innovation will come
when we challenge the dogma and invite fresh eyes to use our
methods and supply their own new questions. The 2012 Nobel
Prize for Medicine and Physiology to Dr. Shinya Yamananka and
Sir John Gurdon acknowledged the start of exciting and indeed
remarkable discoveries in reprogramming. No doubt the first of
very many!
References
1. Takahashi K, Yamanaka S (2006) Induction of
pluripotent stem cells from mouse embryonic
and adult fibroblast cultures by defined factors.
Cell 126:663–676
2. Waddington CH (1942) Canalization of devel-
opment and the inheritance of acquired charac-
ters. Nature 150:563–565
3. Waddington CH (1957) The strategy of the
genes. George Allen & Unwin, London, UK
4. Jopling C, Boue S, Izpisua Belmonte JC
(2011) Dedifferentiation, transdifferentiation
and reprogramming: three routes to regenera-
tion. Nat Rev Mol Cell Biol 12:79–89
5. Nygren JM, Jovinge S, Breitbach M et al
(2004) Bone marrow-derived hematopoietic
cells generate cardiomyocytes at a low fre-
quency through cell fusion, but not transdif-
ferentiation. Nat Med 10:494–501
6. Orlic D, Kajstura J, Chimenti S et al (2001)
Bone marrow cells regenerate infarcted myo-
cardium. Nature 410:701–705
7. Niwa H, Miyazaki J, Smith AG (2000)
Quantitative expression of Oct-3/4 defines
differentiation, dedifferentiation or self-
renewal of ES cells. Nat Genet 24:372–376
8. Niwa H, Toyooka Y, Shimosato D et al (2005)
Interaction between Oct3/4 and Cdx2 deter-
mines trophectoderm differentiation. Cell
123:917–929
9. Lu CW, Yabuuchi A, Chen L et al (2008) Ras-
MAPK signaling promotes trophectoderm for-
mation from embryonic stem cells and mouse
embryos. Nat Genet 40:921–926
10. Ying QL, Nichols J, Evans EP et al (2002)
Changing potency by spontaneous fusion.
Nature 416:545–548
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Paul J. Verma and Huseyin Sumer (eds.), Cell Reprogramming: Methods and Protocols, Methods in Molecular Biology,
vol. 1330, DOI 10.1007/978-1-4939-2848-4_2, © Springer Science+Business Media New York 2015
Chapter 2
Synthetic mRNA Reprogramming of Human Fibroblast Cells
Jun Liu and Paul J. Verma
Abstract
Reprogramming of somatic cells, such as skin fibroblasts, to pluripotency was first achieved by forced
expression of four transcription factors using integrating retroviral or lentiviral vectors, which result in
integration of exogenous DNA into cellular genome and present a formidable barrier to therapeutic appli-
cation of induced pluripotent stem cells (iPSCs). To facilitate the translation of iPSC technology to clinical
practice, mRNA reprogramming method that generates transgene-free iPSCs is a safe and efficient method,
eliminating bio-containment concerns associated with viral vectors, as well as the need for weeks of screen-
ing of cells to confirm that viral material has been completely eliminated during cell passaging.
Key words Reprogramming, Transgene-free, Induced pluripotent stem cells, Modified mRNA,
Transfection
1 Introduction
The discovery that induced pluripotent stem cells (iPSCs) can be
generated from differentiated cell types, e.g., skin fibroblasts,
through the overexpression of a set of defined transcription fac-
tors holds the promise for regenerative medicine and cell-based
autologous therapies [1, 2]. The initial approach utilized retrovi-
ral vectors to deliver OCT4, KLF4, SOX2, and c-MYC to repro-
gram mouse and human fibroblasts to iPSCs. However, this
approach carries the risk associated with integration of exotic
transgene sequences into the genome and therefore is precluded
for cell-based therapeutic applications in patients. A variety of
technologies have been developed for transgene integration-free
pluripotency reprogramming, such as using adenoviral vectors [3,
4], non-integrating DNA plasmid-based vectors [5–9], protein
transduction [10, 11], Sendai viral vectors [12, 13], microRNA-
based reprogramming [14, 15], and modified mRNA-based
reprogramming approach [16, 17]. The modified mRNA technol-
ogy is a non-viral, non-integrating, clinically relevant reprogram-
ming method, and completely eliminates the risk of genomic](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-34-2048.jpg)
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Paul J. Verma and Huseyin Sumer (eds.), Cell Reprogramming: Methods and Protocols, Methods in Molecular Biology,
vol. 1330, DOI 10.1007/978-1-4939-2848-4_3, © Springer Science+Business Media New York 2015
Chapter 3
MicroRNA-Mediated Reprogramming of Somatic Cells
into Induced Pluripotent Stem Cells
Shelley E.S. Sandmaier and Bhanu Prakash V.L. Telugu
Abstract
MicroRNAs or miRNAs belong to a class of small noncoding RNAs that play a crucial role in posttran-
scriptional regulation of gene expression. Nascent miRNAs are expressed as a longer transcript, which are
then processed into a smaller 18–23-nucleotide mature miRNAs that bind to the target transcripts and
induce cleavage or inhibit translation. MiRNAs therefore represent another key regulator of gene expres-
sion in establishing and maintaining unique cellular fate. Several classes of miRNAs have been identified to
be uniquely expressed in embryonic stem cells (ESC) and regulated by the core transcription factors Oct4,
Sox2, and Klf4. One such class of miRNAs is the mir-302/367 cluster that is enriched in pluripotent cells
in vivo and in vitro. Using the mir-302/367 either by themselves or in combination with the Yamanaka
reprogramming factors (Oct4, Sox2, c-Myc, and Klf4) has resulted in the establishment of induced plu-
ripotent stem cells (iPSC) with high efficiencies. In this chapter, we outline the methodologies for estab-
lishing and utilizing the miRNA-based tools for reprogramming somatic cells into iPSC.
Key words ESC, IPSC, miRNA, Pluripotency, Reprogramming
Abbreviations
iPSC Induced pluripotent stem cells
ESC Embryonic stem cells
TALENS Transcription activator-like effector nucleases
CRISPR Clustered regularly interspaced short palindromic repeat
ZFN Zinc finger nucleases
1 Introduction
MicroRNAs (miRNAs) are short noncoding RNAs that bind target
mRNAs via complete or incomplete sequence complementarity and
regulate stability and translatability of the message [1–3]. Nascent
miRNAs are transcribed from endogenous loci via Pol II RNA
polymerase as 85–100 base pair nascent transcripts, which are then](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-46-2048.jpg)
![30
processed by Drosha and Dicer into mature miRNAs of 18–23
nucleotides in length [3, 4]. The mature miRNAs are characterized
by a “seed sequence” at the 5′-end between nucleotides 2–8, exhib-
iting perfect complementarity with the target gene [2]. After a
miRNA recognizes and binds to the target mRNA, it inhibits trans-
lation in either of the two ways: (1) targeting the mRNA for cleav-
age if the miRNA shares perfect complementarity with the sequence
or (2) in the case of partial complementarity prevents assembly of a
ribosome initiation complex and initiation of translation [3]. Due
to the ability of miRNAs to bind to target sequences, albeit with
poor complementarity, one miRNA is often capable of binding to a
cohort of mRNAs and inhibiting translation. Accordingly, many
genes can often be regulated by a candidate miRNA [1, 2].
In embryonic stem cells (ESC), several classes of miRNAs have
been identified to be specifically enriched, indicating a possible role
in maintaining pluripotency [5]. Potentially several more exist
based on the putative ability of certain transcripts to form hairpin
miRNA precursors [5]. A much stronger evidence for the role of
miRNAs in maintaining pluripotency comes from the discovery
that several of the miRNA genes have binding sites for core pluri-
potency genes Oct4, Sox2, and Nanog in their promoters [6]. In
ESC, miRNAs specifically target genes which affect varying prop-
erties of pluripotency such as transcription factors, cell cycle genes,
and genes involved in epigenetic regulation. Regulation of pluripo-
tency by such diverse cellular mechanisms is necessary to ensure
greater stability of ESC [2].
Considering the abundance of miRNAs in ESC, and their
putative role in regulating pluripotency, the ability of miRNAs to
aid in the production and maintenance of induced pluripotent
stem cells (iPSCs) has been increasingly studied. iPSCs have tradi-
tionally been generated from somatic cells via retroviral delivery
of Oct4, Sox2, Klf4, and c-myc (OSKM) reprogramming factors,
as was first reported by Takahashi and Yamanaka [7]. However,
the induction of pluripotency by OSKM is rather inefficient
(0.001–0.01 %), yielding very few colonies per million cells
infected with retrovirus [7, 8]. Recently, iPSCs have been
produced with greater efficiency by incorporating specific miRNA
clusters shown to be involved in regulating the pluripotent state
[9, 10]. Specifically, a well-studied mir-302/367 cluster, which has
been shown to play a role in regulating cell cycle, and is regulated
by the core pluripotency factors Oct4, Sox2, Nanog, and Tcf3, has
been utilized in the reprogramming efforts [6, 11]. Mir-302/367
cluster comprises of five miRNAs, mir-302a, -b, -c, -d, and
mir-367, expressed as a polycistronic construct and located within
intron 8 of the LARP7 gene in humans, with homologs in several
species including cattle, pigs, and mice [6, 9]. Interestingly, the
seed sequences of the four miRNAs (302a/b/c/d) are
identical, and share high degree of conservation across species.
When used in combination with the traditional OSKM factors in
Shelley E.S. Sandmaier and Bhanu Prakash V.L. Telugu](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-47-2048.jpg)
![31
reprogramming experiments, the number of iPSC colonies has
been shown to be enhanced by at least two orders of magnitude
(0.1–0.8 %) [9, 10]. In fact, cells can be reprogrammed to pluri-
potency with mir-302/367 and a histone deacetylase (HDAC)
inhibitor, valproic acid, alone, and show ESC-like morphology
sooner than cells reprogrammed with OSKM. Moreover, these
iPSCs are capable of contributing to all three germ layers as well
as giving rise to germ-line chimeras in mice [9]. Human iPSCs
have also been generated using miRNAs with or without the addi-
tion of OSKM into the genome [9, 12–14]. Therefore, our and
several other laboratories have adopted miRNAs as a standard fac-
tor in reprogramming iPSC (Manuscript in preparation). The use
of miRNA is especially important in reprogramming somatic cells
from livestock species, where the efficiencies of reprogramming
are even lower, and the conditions for optimal culture not com-
pletely understood. In this manuscript, the procedures for making
and using miRNA-based vectors for reprogramming somatic cells
from the domestic animal species, pig, are discussed. However,
the methods discussed below can easily be adopted for other
model organisms.
2 Materials
Store all reagents and media at 4 °C unless otherwise noted.
1. Complete media: 440 mL HyClone High Glucose DMEM
(ThermoScientific), 50 mL 10 % fetal calf serum (FCS), 2.5 mL
100× GlutaMAX (Gibco), 5 mL 100× nonessential amino
acids, and 5 mL 100× sodium pyruvate (see Note 1).
2. iPSC media: 382.5 mL HyClone DMEM F12
(ThermoScientific), 100 mL knockout serum replacer (KSR)
(Gibco), 2.5 mL GlutaMAX, 5 mL nonessential amino acids,
10 mL sodium bicarbonate solution 7.5 %, and 8 ng/mL
FGF2 (R&D Systems).
3. 0.25 % trypsin–EDTA for dissociation and harvesting of cells.
4. Dimethyl sulfoxide (DMSO) for cryopreservation.
5. CF-1 mice OR irradiated mouse embryonic fibroblasts (MEFs).
6. Phosphate-buffered saline (PBS).
7. T-75 flasks.
8. Cryovial freezing container filled with 2-propanol.
1. 293-FT cells (Life Technologies) viral packaging cells.
2. Geneticin (G418).
3. Gelatin.
4. Polyjet (Signagen).
2.1 Cell Culture
2.2 Lentivirus
Production
miRNA Mediated Reprogramming of Somatic Cells](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-48-2048.jpg)
![37
Paul J. Verma and Huseyin Sumer (eds.), Cell Reprogramming: Methods and Protocols, Methods in Molecular Biology,
vol. 1330, DOI 10.1007/978-1-4939-2848-4_4, © Springer Science+Business Media New York 2015
Chapter 4
Generation of Footprint-Free Induced Pluripotent Stem
Cells from Human Fibroblasts Using Episomal Plasmid
Vectors
Dmitry A. Ovchinnikov, Jane Sun, and Ernst J. Wolvetang
Abstract
Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease
and are set to transform regenerative medicine and drug screening over the next decade. The generation
of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of
these potential uses. Here we describe in detail the generation of human iPSC from control and disease-
carrying individuals’ fibroblasts using episomal plasmids.
Key words Human induced pluripotent stem cells, Reprogramming, Episomal plasmid vectors,
Fibroblasts, Transfection, Genomic integration
1 Introduction
Lentiviral or retroviral delivery of reprogramming factors has been
a powerful tool in pioneering the field of cell reprogramming.
However, the concerns associated with the disruption of the
genome at the viral integration sites, number and position and the
unpredictable nature of transgene silencing, as well as their
potential reactivation following differentiation have made
integration-dependent methods unsuitable for clinical applica-
tions. Indeed, with the wealth of currently available alternative
technologies there is really no need to modify the genomic DNA
of the target cell when generating induced pluripotent stem cells.
Researchers have a number of options ranging from piggybac or
sleeping beauty transposon-based or Cre recombinase-aided meth-
ods to excise integrated reprogramming cassettes, or to avoid
DNA-integrating methods altogether and use mRNA, helper-
dependent adenoviral, Sendai virus-derived or episomal vector-
based methods [1–6]. Here we describe a protocol for the
generation of human iPSCs from fibroblasts using episomal plasmid](https://image.slidesharecdn.com/2621055-250607150208-b98c9c02/75/Cell-Reprogramming-Methods-And-Protocols-Paul-J-Verma-Huseyin-Sumer-54-2048.jpg)
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- 1. Cell Reprogramming MethodsAnd Protocols Paul J Verma Huseyin Sumer download https://ebookbell.com/product/cell-reprogramming-methods-and- protocols-paul-j-verma-huseyin-sumer-5242110 Explore and download more ebooks at ebookbell.com
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- 5. CellRepro- gramming Paul J.Verma Huseyin SumerEditors Methods and Protocols Methods in Molecular Biology 1330
- 6. ME T HO D S I N MO L E C U L A R BI O L O G Y Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651
- 8. Cell Reprogramming Methods andProtocols Editedby Paul J.Verma StemCellandGeneticEngineeringGroup,DepartmentofMaterialsEngineering, FacultyofEngineering,MonashUniversity,Clayton,VIC,Australia;SouthAustralianResearch& DevelopmentInstitute(SARDI),TurretfieldResearchCentre,Rosedale,SA,Australia Huseyin Sumer SwinburneUniversityofTechnology,Hawthorn,VIC,Australia
- 9. ISSN 1064-3745 ISSN1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-2847-7 ISBN 978-1-4939-2848-4 (eBook) DOI 10.1007/978-1-4939-2848-4 Library of Congress Control Number: 2015955417 Springer New York Heidelberg Dordrecht London © Springer Science+Business Media New York 2015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper Humana Press is a brand of Springer Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com) Editors Paul J. Verma Stem Cell and Genetic Engineering Group Department of Materials Engineering Faculty of Engineering Monash University Clayton, VIC, Australia South Australian Research & Development Institute (SARDI) Turretfield Research Centre Rosedale, SA, Australia Huseyin Sumer Swinburne University of Technology Hawthorn, VIC, Australia
- 10. v Cell Reprogramming: Methodsand Protocols is a comprehensive review of cellular repro- gramming technology in vertebrates, aimed at reprogramming differentiated cells and germ line transmission of pluripotent stem cells. The emphasis here is on providing readily repro- ducible techniques for inducing pluripotency in somatic cells for disease modeling and the generation of cloned embryos and animals in a number of key research and commercially important species. Additional chapters dealing with such reprogramming-related issues such as analysis of mitochondrial DNA in reprogrammed cells and the isolation of repro- gramming intermediates are also included. A section providing alternative cutting-edge methods for nuclear transfer, as well as techniques for the production of germ line chimeras from embryonic stem cells and induced pluripotent stem cells is also incorporated. This is complimented with the neonatal care and management of somatic cell nuclear transfer derived offspring. Cell Reprogramming also provides an understanding of the factors involved in nuclear reprogramming, which is imperative for the success of reprogramming. This volume will prove beneficial to molecular biologists, stem cell biologists, clinicians, biotechnologists, students, veterinarians, and animal care technicians involved with reprogramming, nuclear transfer, and transgenesis. Clayton, VIC, Australia Paul J. Verma Hawthorn, VIC, Australia Huseyin Sumer Preface
- 12. vii Preface. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I BACKGROUND 1 Cellular Reprogramming in Basic and Applied Biomedicine: The Dawn of Regenerative Medicine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Wendy Dean PART II DE NOVO REPROGRAMMING 2 Synthetic mRNA Reprogramming of Human Fibroblast Cells . . . . . . . . . . . . . 17 Jun Liu and Paul J. Verma 3 MicroRNA-Mediated Reprogramming of Somatic Cells into Induced Pluripotent Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Shelley E.S. Sandmaier and Bhanu Prakash V.L. Telugu 4 Generation of Footprint-Free Induced Pluripotent Stem Cells from Human Fibroblasts Using Episomal Plasmid Vectors. . . . . . . . . . . . . . . . 37 Dmitry A. Ovchinnikov, Jane Sun, and Ernst J. Wolvetang 5 Reprogramming of Human Fibroblasts with Non-integrating RNA Virus on Feeder-Free or Xeno-Free Conditions . . . . . . . . . . . . . . . . . . . . . . . . 47 Pauline T. Lieu PART III LIVESTOCK, DOMESTIC AND ENDANGERED SPECIES 6 Inducing Pluripotency in Cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Luis F. Malaver-Ortega, Amir Taheri-Ghahfarokhi, and Huseyin Sumer 7 Generation of Induced Pluripotent Stem Cells (iPSCs) from Adult Canine Fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Sehwon Koh and Jorge A. Piedrahita 8 Derivation of Equine-Induced Pluripotent Stem Cell Lines Using a piggyBac Transposon Delivery System and Temporal Control of Transgene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Kristina Nagy and Andras Nagy 9 Generation of Avian Induced Pluripotent Stem Cells. . . . . . . . . . . . . . . . . . . . 89 Yangqing Lu, Franklin D. West, Brian J. Jordan, Robert B. Beckstead, Erin T. Jordan, and Steven L. Stice Contents
- 13. viii 10 Generation ofInduced Pluripotent Stem Cells from Mammalian Endangered Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Inbar Friedrich Ben-Nun, Susanne C. Montague, Marlys L. Houck, Oliver Ryder, and Jeanne F. Loring PART IV GERM-LINE TRANSMISSION OF PLURIPOTENT STEM CELLS 11 Generation of Efficient Germ-Line Chimeras Using Embryonic Stem Cell Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 William A. Ritchie 12 Generation of Viable Mice from Induced Pluripotent Stem Cells (iPSCs) Through Tetraploid Complementation . . . . . . . . . . . . . . . 125 Lan Kang and Shaorong Gao 13 Cloning Endangered Felids by Interspecies Somatic Cell Nuclear Transfer. . . . 133 Martha C. Gómez and C. Earle Pope 14 Generation of Chimeras from Porcine Induced Pluripotent Stem Cells . . . . . . 153 Franklin D. West, Steve L. Terlouw, John R. Dobrinsky, Yangqing Lu, Erin T. Jordan, and Steven L. Stice 15 A Novel Method of Somatic Cell Nuclear Transfer with Minimum Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 S.M. Hosseini, F. Moulavi, and M.H. Nasr-Esfahani 16 Neonatal Care and Management of Foals Derived by Somatic Cell Nuclear Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Aime K. Johnson and Katrin Hinrichs PART V INFLUENCING REPROGRAMMING AND GENOME EDITING 17 Isolation of Reprogramming Intermediates During Generation of Induced Pluripotent Stem Cells from Mouse Embryonic Fibroblasts . . . . . . 205 Christian M. Nefzger, Sara Alaei, and Jose M. Polo 18 Analysis of Mitochondrial DNA in Induced Pluripotent and Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 William Lee, Richard D.W. Kelly, Ka Yu Yeung, Gael Cagnone, Matthew McKenzie, and Justin C. St. John 19 Genome Modification of Pluripotent Cells by Using Transcription Activator-Like Effector Nucleases (TALENs). . . . . . . . . . . . . . . . . . . . . . . . . . 253 Amir Taheri-Ghahfarokhi, Luis F. Malaver-Ortega, and Huseyin Sumer Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Contents
- 14. ix SARA ALAEI •Department of Anatomy and Developmental Biology, Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia ROBERT B. BECKSTEAD • Department of Animal and Dairy Science, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA INBAR FRIEDRICH BEN-NUN • Department of Chemical Physiology, Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA, USA GAEL CAGNONE • The Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, Clayton, VIC, Australia WENDY DEAN • Epigenetics Programme, The Babraham Institute, Cambridgeshire, UK JOHN R. DOBRINSKY • JRD Biotechnology, Oregon, WI, USA SHAORONG GAO • National Institute of Biological Sciences, NIBS, Beijing, People’s Republic of China; School of Life Sciences and Technology, Tongji University, Shanghai, People’s Republic of China MARTHA C. GÓMEZ • Audubon Nature Center for Research of Endangered Species, New Orleans, LA, USA KATRIN HINRICHS • Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA S.M. HOSSEINI • Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran MARLYS L. HOUCK • San Diego Zoo Institute for Conservation Research, Escondido, CA, USA JUSTIN C. ST. JOHN • The Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, Clayton, VIC, Australia AIME K. JOHNSON • JT Vaughn Large Animal Teaching Hospital, College of Veterinary Medicine, Auburn University, Auburn, AL, USA BRIAN J. JORDAN • Department of Animal and Dairy Science, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA ERIN T. JORDAN • Department of Animal and Dairy Science, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA LAN KANG • Institute of Cancer Stem Cell, Dalian Medical University, Dalian, People’s Republic of China; National Institute of Biological Sciences, NIBS, Beijing, People’s Republic of China RICHARD D.W. KELLY • The Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, Clayton, VIC, Australia SEHWON KOH • Department of Cell Biology, Duke University, Durham, NC, USA; Duke University Medical Center, Duke University, Durham, NC, USA WILLIAM LEE • The Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, Clayton, VIC, Australia Contributors
- 15. x PAULINE T. LIEU• Global R&D, Life Technologies Corporation, Carlsbad, CA, USA JUN LIU • Stem Cell and Genetic Engineering Group, Department of Materials Engineering, Faculty of Engineering, Monash University—Clayton Campus, Clayton, VIC, Australia JEANNE F. LORING • Department of Chemical Physiology, Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA, USA; Department of Reproductive Medicine, University of California, San Diego, La Jolla, CA, USA YANGQING LU • Department of Animal and Dairy Science, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA; JRD Biotechnology, Oregon, WI, USA; State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, China LUIS F. MALAVER-ORTEGA • Monash Institute for Medical Research, Monash University, Clayton, VIC, Australia; Australian Animal Health Laboratories, CSIRO Biosecurity Flagship, East Geelong, VIC, Australia MATTHEW MCKENZIE • The Molecular Basis of Mitochondrial Disease Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, Clayton, VIC, Australia SUSANNE C. MONTAGUE • Department of Chemical Physiology, Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA, USA F. MOULAVI • Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran ANDRAS NAGY • Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada; Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada KRISTINA NAGY • Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M.H. NASR-ESFAHANI • Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran CHRISTIAN M. NEFZGER • Department of Anatomy and Developmental Biology, Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia DMITRY A. OVCHINNIKOV • Stem Cell Engineering Group, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, Australia JORGE A. PIEDRAHITA • Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; Genomics Program, North Carolina State University, Raleigh, NC, USA; Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC, USA JOSE M. POLO • Department of Anatomy and Developmental Biology, Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia C. EARLE POPE • Audubon Nature Center for Research of Endangered Species, New Orleans, LA, USA WILLIAM A. RITCHIE • Roslin Embryology Ltd., Macmerry, Tranent, Scotland, UK; Monash Biomed Private Limited, Delhi, India OLIVER RYDER • San Diego Zoo Institute for Conservation Research, Escondido, CA, USA STEVEN L. STICE • Department of Animal and Dairy Science, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA Contributors
- 16. xi HUSEYIN SUMER •Department of Chemistry and Biotechnology, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC, Australia JANE SUN • Stem Cell Engineering Group, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, Australia SHELLEY E.S. SANDMAIER • Department of Animal and Avian Sciences, University of Maryland, College Park, MD, USA; Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD, USA AMIR TAHERI-GHAHFAROKHI • Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran BHANU PRAKASH V.L. TELUGU • Department of Animal and Avian Sciences, University of Maryland, College Park, MD, USA; Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD, USA STEVE L. TERLOUW • Minitube of America, Mt. Horeb, WI, USA PAUL J. VERMA • Stem Cell and Genetic Engineering Group, Department of Materials Engineering, Monash University, Clayton, VIC, Australia; South Australian Research and Development Institute, Turretfield Research Centre, Rosedale, SA, Australia FRANKLIN D. WEST • Department of Animal and Dairy Science, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA ERNST J. WOLVETANG • Stem Cell Engineering Group, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, Australia KA YU YEUNG • The Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, Clayton, VIC, Australia; Molecular Basis of Metabolic Disease, Division of Metabolic and Vascular Health, Warwick Medical School, The University of Warwick, Coventry, UK Contributors
- 18.
- 20. 3 Paul J. Vermaand Huseyin Sumer (eds.), Cell Reprogramming: Methods and Protocols, Methods in Molecular Biology, vol. 1330, DOI 10.1007/978-1-4939-2848-4_1, © Springer Science+Business Media New York 2015 Chapter 1 Cellular Reprogramming in Basic and Applied Biomedicine: The Dawn of Regenerative Medicine Wendy Dean Abstract Fertilization triggers a cascade of cellular and molecular events restoring the totipotent state and the potential for all cell types. However, the program quickly directs differentiation and cellular commitment. Under the genetic and epigenetic control of this process, Waddington likened this to a three-dimensional landscape where cells could not ascend the slope or traverse once canalized thus leading to cell fate deci- sions and the progressive restriction of cellular potency. But this is not the only possible outcome at least experimentally. Somatic cell nuclear transfer and overexpression of key transcription factors to generate induced pluripotent cells have challenged this notion. The return to pluripotency and the reinstatement of plasticity and heterogeneity once thought to be the exclusive remit of the developing embryo can now be replicated in vitro. The following chapter introduces some of these ideas and suggests that the fundamental principles learned may constitute the first step toward the opportunity for specific tissue renewal and replacement in healthy aging and the treatment of chronic diseases—the age of regenerative medicine. Key words Cellular reprogramming, Regenerative medicine, Induced pluripotent cells, Healthy aging 1 Introduction—Filling in Waddington’s Canal Cellular reprogramming entered the realm of our imagination in 2006 when Shinya Yamamaka announced that a “four-factor cock- tail” could transform differentiated fibroblasts into induced, plu- ripotent stem cells [1]. This inspired a more prescriptive and defined way of achieving the alchemic transmogrification of defined cellular states. The significance of the breakthrough discovery of reprogramming fully differentiated cells back to a pluripotent state in what developmentally constitutes a retrograde cellular transition was quickly acknowledged with the joint awarding of a Nobel Prize in Physiology or Medicine in 2012 to Profs Shinya Yamanaka and Sir John Gurdon for their complementary work in nuclear repro- gramming. Their discoveries had laid much of the groundwork for the concept of experimentally induced retrograde progression to induced pluripotent stem (iPS) cells.
- 21. 4 But exactly howdoes the forced overexpression of a handful of transcription factors and chromatin-binding molecules transform the defined cellular state of a differentiated cell and progress it back up Waddington’s ascending landscape to assume a pluripotent phenotype—in essence, a stem cell? The simple answer is, at present, that we do not know. However, the full impact of modern genome-wide investigation and the sheer force of numbers of researchers worldwide leading this investigation make the prospect of significant mechanistic understanding only a matter of time and the translation to patient- specific regenerative medicine a reality in our lifetime. In the course of these studies there is a real prospect of collateral benefit; much will be learned about the potential to identify and manipulate endogenous stem cell populations that function in tissue repair and replacement throughout life. Indeed the intense study of the pro- cesses of cellular regression may well hold the key to understanding healthy aging and offer an explanation for the growing number of centenarians in our societies, which has seen a fivefold rise over the last 30 years (Office for National Statistics UK; BBC news, 27th Sept 2013). Cellular reprogramming is the conversion of one specific cell type to another. Arguably, we could well consider that develop- ment in its usual forward-only direction could constitute a form of cellular reprogramming. Here, the highly specialized and fully dif- ferentiated oocyte is reprogrammed on fertilization to restore an ephemeral totipotent state that is quickly followed by a series of progressively more differentiated cellular decisions passing through ever more restricted multipotent junctures to give rise to the fully formed neonatal animal. In the 1940s Conrad Waddington described this process in his classical model of the epigenetic land- scape where one genotype allowed for the generation of multiple cellular phenotypes [2]. Waddington illustrated the hierarchical progression of the undifferentiated state by a series of channels which were progressively more restricted and increasingly sepa- rated; thus once the cellular state was “fated” thereafter the lineage was restricted and incapable of either returning to a more undif- ferentiated state or a different germ cell layer [3]. In the postgen- omic era, these ideas together with classical developmental and cellular biology have formed the basis of our understanding of the field of epigenetics. However, today cellular reprogramming more often refers to those landmark methods which included transdifferentiation or direct cell conversion, somatic cell nuclear transfer (SCNT) and experimental reprogramming, the basis of the generation of iPS cells [4]. The chapters which follow outline the details of how to establish these various models of developmental and cellular pro- cesses that set the experimental scene for understanding the mech- anisms underpinning these transitions and serve to allow us Wendy Dean
- 22. 5 unprecedented opportunities inbasic, agricultural and biomedical science to improve health and wellbeing, to enhance food security, and offer therapeutic solutions to the treatment of chronic disor- ders in humans. By way of an introduction to these methods I will outline some of the origins and common themes which these methods share and contrast points where they differ. These experimental approaches have certainly been instrumental in driving a deeper and more comprehensive understanding of mammalian development and stem cell biology in general and will undoubtedly continue to drive fundamental and applied questions in these areas. Perhaps most exciting, as a result of these experimental systems, fundamentally held beliefs about the prescriptive nature of developmental pro- cesses and tissue regeneration upon damage are now being chal- lenged. The prospect of significant improvement of health span, on a patient specific basis, is now within sight. While the focus of this book is the experimental details that facilitate cellular reprogramming, before embarking on an outline of these techniques it may be worth touching, if only briefly, on some processes that occur naturally which are capable of achieving the same end. Transdifferentiation and cell fusion, much like that of experimental heterokaryons, do occur naturally [5, 6]. Transdifferentiation constitutes a change in cellular fate, which can facilitate the transit between lineages in the most extreme case and between a differentiated cell type and its less differentiated fore- runner within a given lineage. Here, one distinction that is often applied is that both of these processes take place by a direct cell conversion and not via a pluripotent intermediate. In mammals transdifferentiation can be achieved experimentally by both gain of function through overexpression and loss of function mechanisms of one or a few factors and in this way bears some resemblance to iPS production. Interestingly, these induced transitions can be studied in vitro using stem cell models such as an ES cell, a proxy for the inner cell mass of the blastocyst stage in mammals. In what seems a reversion of the very first cellular decision in development, ES cells can be driven to acquire trophoblast stem (TS) cell-like fates [7–9] which implies that the experimental manipulation endows the cell with permission, and capacity, for the lowering of the epigenetic barrier that ordinarily separates and defines these first two cell lineages. Cell fusion and transdifferentiation have shared a common past. In 2002 two significant papers identified the potential of ES cells co-cultured with either neural stem cells or bone marrow cells to subsequently undergo differentiation to a variety of cell types. However, this occurred not by dedifferentiation, which was the first explanation, but by transdifferentiation via spontaneous cell fusion [10, 11]. At the time this caused a significant rethink in the field but supplied positive benefit in the greater degrees of vigor Cellular Reprogramming for Biomedicine
- 23. 6 that were thereafterrequired of these types of experiments [12]. Perhaps more importantly, this did highlight the fact that these processes could occur, albeit at a low frequency, establishing the proof of principle that similar cell–cell fusion events that allow cell fate transitions may take place in vivo. Thinking along these experi- mental lines may well be of benefit in particular to the adult stem cell field. While SCNT and iPS cell reprogramming are seemingly dia- metrically opposed they share interesting common origins in the ferment of mammalian experimental embryology and cell biology in the 1980s. The premise of SCNT had been based on classical developmental experiments carried out by Spemann in the 1920s answering the question of totipotency of nuclei at least early in development [13]. This was extended by the seminal work of Briggs and King in the 1950s [14] followed closely by John Gurdon [15] illustrating that in amphibian models differentiated nuclei could be transplanted to the enucleated oocyte and give rise to an adult organism. While this confirmed nuclear conservation, they also showed that the regenerative potency with nuclear donors isolated from more advanced, and hence more differentiated tis- sues, was progressively restricted [16]. As a whole this progressive restriction, i.e., the very idea that Waddington described as canali- zation, seemed to be holding up. In 1983, McGrath and Solter published a method of nuclear transplantation in mammals using a fusogenic virus [17]. This laid the ground work for the flurry of reports of “cloning” in mammals from embryonic cells in the sheep by Steen Willadsen [18] to the landmark achievement of Campbell and Wilmut in 1996 of the generation of a live cloned sheep, Dolly, from an adult, fully dif- ferentiated, mammary cell nucleus [19]. To date cloning has been successful in more than 15 mammalian species including the extinct Pyrenean ibex and a handful of other endangered species [20]. While cloning in most species has been a success, among endan- gered species cloning has been more difficult. Of these only the mouflon sheep survived for more than a few days after birth [21]. Clearly the oocyte, in conjunction with modulation of widespread chromatin remodeling, can reinstruct a terminal program to relive its developmental past; something once thought to be unachiev- able under any circumstance [22]. The induction of stem cells starting from differentiated fibro- blasts is an extreme form of cell fate conversion and hence may constitute an extreme form of transdifferentiation. Here, the con- trast to the reprogramming in SCNT is stark. The cellular as well as the nuclear status of the fibroblast must be dedifferentiated and ultimately progressed to the pinnacle of the canalized landscape in order to form pluripotent stem cells. In this form of reprogram- ming the cell is a most unsuitable environment with little of its own capacity to direct retrograde dedifferentiation unto pluripotency. Wendy Dean
- 24. 7 The earliest incarnationsof this process were first described in Lasser et al. [23] where overexpression of a defined transcription factor (TF), MyoD, was able to drive fibroblasts toward a muscle cell fate. While this worked best in mesodermally derived cells, similar results were also obtained in ectodermal and endodermal derivatives hinting at the now familiar concept that forced over- expression of TFs, defining for a given cell type, greatly assists in the transdifferentiation toward that cell type [24]. In practice this is but a short step away in taking this idea forward toward a des- tination in stem cell populations—in essence the seed of the “four-factor cocktail” had been planted. Over the intervening years intra-germ layer conversion was demonstrated for a vast number of TF combinations. Interestingly, the dynamics of the transition were highly variable with both the starting cell type and the order of expression of the TF cocktail able to influence the cellular outcome. In fact, only relatively recently has this approach succeeded in “long distance” direct conversion; start- ing with fibroblasts a “three-factor” cocktail was able to generate functional neurons [25]. Induced pluripotent stems cells have changed the way we think about cellular differentiation, cell fate commitment, and the unidi- rectional nature of development [26]. Beyond that, the very nature of the stably differentiated cell has been challenged along with the ideas of the epigenome that serve to reinforce and fix that state. While remarkable in the insights that derived from conversion of cell types both within and across germ layer boundaries, direct cell conversion has significant limitations. Ideally, and in keeping with the need to be able to supply adequate numbers of any cell type in any lineage, stem cells seem like the best option and those equiva- lent to embryonic stem cells would allow unrestricted and ethically uncomplicated extension to therapeutic applications in the treat- ment of disease. Applying the lessons of intra-lineage conversion, Takahashi and Yamanaka focused their attention on transcription factor net- works associated with pluripotency and self-renewal, both hall- marks of pluripotent embryonic stem (ES) cells. Distilling the list to the now well known “four-factor cocktail,” of Oct3/4, Sox2, Klf4 and c-Myc (OSKM), and transfecting them into either fetal or adult mouse, and later human, fibroblasts lead eventually to the generation of the first iPS cells [1]. Remarkably, in mouse and human, expression from the delivery systems is eventually taken over by the endogenous loci thereby supplying a continuous source of the essential factors characteristic of the target ES cells. Although highly inefficient, these cells fulfilled their potential being able to differentiate into all three germ layers and in the generation of both chimeric animals and entirely iPS-derived mice by tetraploid complementation, the gold standard for demonstrating pluripo- tency. Interestingly, a large proportion of the domestic animal iPS Cellular Reprogramming for Biomedicine
- 25. 8 systems fail toeither activate the endogenous loci or silence the transgenes in the course of iPS reprogramming. Better and more efficacious delivery systems that did not involve viral vectors, requisite for use in humans, have now been achieved. Many iterations and reiterations of the “essential factors” have also taken place with replacements now in common use. In this respect it is remarkable that the “four factors” have been found to be so broadly able to direct iPS cell generation across such a wide cross section of mammalian species. In a few cases, in bovine [27] and the endangered class of Felids [28] is an additional factor, namely Nanog, required for iPS cell reprogramming. In the goat and sheep, eight factors have been reported to be required to reprogram primary ear fibroblasts [29, 30]. Second- and third-generation reprogramming approaches to iPS cells now exist which employ either small molecule inhibitors or transfection of families of microRNAs alone or in combination with the Yamanaka factors [31, 32]. MicroRNAs are particularly abundant in pluripotent ES cells; among the most abundant, the miR301/367 in humans and the miR290 cluster in the mouse, are themselves up-regulated by the OSKM quartet and mutually rein- force the pluripotent state thereby driving cells toward this termi- nus. Coupled to their ability to down-regulate de novo methylation the up-regulation of the miR290 cluster also enhances, among other functions, the kinetics of the mesenchymal to epithelial tran- sition (MET) requisite for reprogramming to iPS status [33–35]. Incidentally, alteration of the culture environment has also proven to enhance iPS cell reprogramming. The ability to generate ES cells in mouse and human has been a breakthrough in pioneering the idea of replacement therapies for faulty genes together with functional and mechanistic studies in all biological disciplines, which ultimately underpin applied research. In domestic species of agricultural and veterinary importance, while some species have been amenable to the generation of embryonic-like stem cells especially in light of improvements trans- lated from the mouse, many have yet to achieve the same unre- stricted claims to pluripotency. Here, iPS cell generation may prove to be the solution as is the case in the equine system. Equine ES-like cells possess only some of the full repertoire of the pluripo- tent spectrum while equine iPS cells seem to be fully functional and able to contribute to teratomas in engraftment experiments [36]. Targeting of iPS cells once established may not prove univer- sally simple. For example, human ES cells are refractory to conven- tional genome editing via homologous recombination achieving only very low efficiencies compared to the mouse and hence other targeted methodologies such as zinc finger proteins, TALENs and CRISPR are required [37]. The development of SNCT has long been regarded as a means by which rare and endangered species might be rescued from Wendy Dean
- 26. 9 impending extinction. Indeed,even some now extinct species have been reanimated by NT where appropriate recipient species hybrids still survive. It would now seem possible that iPS genera- tion may provide additional avenues to help in supporting efforts to save endangered species offering prospects of generation of gametes in vitro from iPS cells as has been achieved with ES cells [38–40]. Despite the relative ease in which the iPS generation has been successful across a very wide swath of mammalian species, the generation of gametes may not prove as simple; nonetheless, there is reason for great optimism that the species variation among germ cell maturation can be overcome and functional gametes gener- ated across the diverse class of Mammalia. Failing the ability to generate full maturation of gametes, iPS cells may well allow for unprecedented mechanistic studies into germ cell development across a wide selection of species many of whom may offer better and closer physiological comparisons to humans without serious ethical limitations [38, 41]. 2 The Epigenome and Life in Culture With the unparalleled promise of personalized medicine and gen- eration of patient-specific tissue by stem cell therapies, replacement and renewal no longer seems like a distant prospect. Less ambi- tious but potentially more beneficial is the ability to test patient- specific matching of drug treatment by using iPS cells either directly or on tissue-specific differentiation. Veterinary drug test- ing and biopharmaceutical companies may well screen and develop treatments tailored by genetically typing patient groups to offer the best fit for regulation of metabolic disorders using iPS cells derived from specifically defined allelic profiling. However, the question remains about the role of the epig- enome and the influence of culture-based rearing of cells and tis- sues especially where tissue engraftment is required. Here, lessons from ES cells as a proxy for iPS cells will be highly informative. It has long been recognized that cells in culture, including embry- onic stem cells acquire increasing levels of DNA methylation, as a function of the duration of life in culture, a significant barrier to both dedifferentiation via SCNT and iPS reprogramming. Recent evaluation of the DNA methylation profile of primed vs naïve ES cells has shed light on this question. Small molecule inhibitors (aka 2i) that both enhance ES cell derivation and reduce their heterogeneity in culture have focused attention on the role of the composition of the culture media and the DNA methylome in mouse [42–45] and in human ES cells [46]. Thus the presence of conventional serum can affect the pluripotential capacity of ES cells by significant modulation of DNA methylation, notably by increasing methylation and decreasing naïve pluripotency. In as Cellular Reprogramming for Biomedicine
- 27. 10 much as microRNAfamilies that are associated with iPS cell repro- gramming negatively regulate DNA methyltransferases and hence DNA methylation, these two common components (i.e., serum and microRNAs) seem to be at odds with one another for the reprogramming process. Loss of DNA methylation, especially tied to natural reprogramming, has been a dominant interest in the field of epigenetics. The discovery of another significant pathway able to down-regulate DNA methylation by methylcytosine oxidation-coupled to repair pathways may be able to offer some answers [47, 48]. A family of three enzymes, the ten-eleven- translocation or TETS, iteratively oxidizing the methyl group on cytosine to hydroxymethyl cytosine (5hmC) eventually leads to this loss of DNA methylation via the return to the cytosine group. Enzymatically, this reaction requires reduced Fe2+ and α-ketoglutarate as cofactors and is hence very sensitive to the media conditions and gaseous environment during culture. Ascorbic acid, Vitamin C (VitC), has been known to enhance iPS generation in mouse and humans for some time. Here acting not via the 2i pathway but rather by alleviating the senescence road- block, in the presence of VitC the histone demethylases Jhdm1a/1b are stimulated [49]. Interestingly, TET1 is involved via its func- tional domain in the formation of 5hmc at loci critical for MET in a VitC-dependent manner [50]. In a systematic screen, the absence of all H3K9me2 and me3 histone methylases, which include Suv39H1 and 2, G9A and SetDB1, were found to work synergisti- cally with VitC to enhance iPS cell reprogramming [50]. The mod- ulation of H3K9me2/me3 is mechanistically linked to loss of DNA methylation [51]. As such the presence of VitC in somatic cell reprogramming is tied to loss of DNA methylation likely via repli- cation-dependent passive mechanisms that involve loss of H3K9 methylation as well. Whether or not the acquisition of DNA methylation during culture of iPS cells will constitute a barrier to their widespread application is not yet clear. In mouse ES cells maintained in stan- dard serum-based culture conditions CpG methylation is high. However, what happens to this hypermethylation once it is intro- duced into a cellular context in vivo or upon tissue derivation has not been systematically explored. In a simple but elegant test of this question the results of a recent experiment gives us cause for optimism. ES cells carrying a GFP reporter were used to make chimeric animals by the classical blastocyst injection method. These chimeric embryos were collected at E17.5 and the GFP-positive cells isolated by flow cytometry and subsequently evaluated for lev- els of DNA methylation. While the original ES cells were heavily methylated, those GFP-positive cells isolated from tissues of these embryos showed reduced levels of DNA methylation that were not significantly different from the GFP-negative host cells. In essence, in dividing cells within an in vivo environment, the DNA Wendy Dean
- 28. 11 methylation levels hadbeen returned to normal [52]. Whether this is universally true in other species needs to be proven. Collectively, we are closing in on solutions to overcome many of the barriers that currently limit unbridled enthusiasm and realis- tic optimism for the promise of iPS cell-based application to regen- erative medicine. The regulation of the epigenome is amongst one of the most complicated barriers which unify the challenges of both SCNT and iPS cell reprogramming irrespective of the application [53]. At present the incredible rate of research output in this area is rivaled only by that of the stem cell biology (which is overlapping with iPS cells). Lessons learned in driving the program back to the top of the Waddington landscape have revealed that pathways at intermediate heights may well provide equally good or better van- tage points for obtaining multipotent stem cell populations both in vitro and that are resident in vivo, that might offer solutions to contemporary obstacles. Indeed, direct cell conversion has chal- lenged our belief about the distance between differentiated lineages and the depth of the canalization. Late in 2014, the direct conver- sion of fibroblasts into thymic epithelial-like cells giving rise to a functional thymus-like organ on transplantation of aggregates together with T-cell precursors and support cells was reported [54]. The chapters that follow offer practical solutions and guide- lines on how to overcome the obstacles that currently impede our progress in experimental reprogramming. Innovation will come when we challenge the dogma and invite fresh eyes to use our methods and supply their own new questions. The 2012 Nobel Prize for Medicine and Physiology to Dr. Shinya Yamananka and Sir John Gurdon acknowledged the start of exciting and indeed remarkable discoveries in reprogramming. No doubt the first of very many! References 1. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676 2. Waddington CH (1942) Canalization of devel- opment and the inheritance of acquired charac- ters. Nature 150:563–565 3. Waddington CH (1957) The strategy of the genes. George Allen & Unwin, London, UK 4. Jopling C, Boue S, Izpisua Belmonte JC (2011) Dedifferentiation, transdifferentiation and reprogramming: three routes to regenera- tion. Nat Rev Mol Cell Biol 12:79–89 5. Nygren JM, Jovinge S, Breitbach M et al (2004) Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low fre- quency through cell fusion, but not transdif- ferentiation. Nat Med 10:494–501 6. Orlic D, Kajstura J, Chimenti S et al (2001) Bone marrow cells regenerate infarcted myo- cardium. Nature 410:701–705 7. Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self- renewal of ES cells. Nat Genet 24:372–376 8. Niwa H, Toyooka Y, Shimosato D et al (2005) Interaction between Oct3/4 and Cdx2 deter- mines trophectoderm differentiation. Cell 123:917–929 9. Lu CW, Yabuuchi A, Chen L et al (2008) Ras- MAPK signaling promotes trophectoderm for- mation from embryonic stem cells and mouse embryos. Nat Genet 40:921–926 10. Ying QL, Nichols J, Evans EP et al (2002) Changing potency by spontaneous fusion. Nature 416:545–548 Cellular Reprogramming for Biomedicine
- 29. 12 11. Terada N,Hamazaki T, Oka M et al (2002) Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 416:542–545 12. Wells WA (2002) Is transdifferentiation in trouble? J Cell Biol 157:15–18 13. Spemann H (1938) Embryonic development and induction. Hafner, New York, NY 14. Briggs R, King TJ (1952) Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs. Proc Natl Acad Sci U S A 38:455–463 15. Gurdon JB (1962) Adult frogs derived from the nuclei of single somatic cells. Dev Biol 4:256–273 16. Gurdon JB (1962) The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J Embryol Exp Morphol 10:622–640 17. McGrath J, Solter D (1983) Nuclear transplan- tation in the mouse embryo by microsurgery and cell fusion. Science 220:1300–1302 18. Willadsen SM (1986) Nuclear transplantation in sheep embryos. Nature 320:63–65 19. Wilmut I, Schnieke AE, McWhir J et al (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385:810–813 20. Lanza RP, Cibelli JB, Diaz F et al (2000) Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning 2:79–90 21. Loi P, Ptak G, Barboni B et al (2001) Genetic rescue of an endangered mammal by cross- species nuclear transfer using post-mortem somatic cells. Nat Biotechnol 19:962–964 22. McGrath J, Solter D (1984) Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro. Science 226:1317–1319 23. Lassar AB, Paterson BM, Weintraub H (1986) Transfection of a DNA locus that mediates the conversion of 10 T1/2 fibroblasts to myo- blasts. Cell 47:649–656 24. Weintraub H, Tapscott SJ, Davis RL et al (1989) Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci U S A 86:5434–5438 25. Lujan E, Chanda S, Ahlenius H et al (2012) Direct conversion of mouse fibroblasts to self- renewing, tripotent neural precursor cells. Proc Natl Acad Sci U S A 109:2527–2532 26. Ladewig J, Koch P, Brustle O (2013) Leveling Waddington: the emergence of direct pro- gramming and the loss of cell fate hierarchies. Nat Rev Mol Cell Biol 14:225–236 27. Sumer H, Liu J, Malaver-Ortega LF et al (2011) NANOG is a key factor for induction of pluripotency in bovine adult fibroblasts. J Anim Sci 89:2708–2716 28. Verma R, Liu J, Holland MK et al (2013) Nanog is an essential factor for induction of pluripotency in somatic cells from endangered felids. Biores Open Access 2:72–76 29. Ren J, Pak Y, He L et al (2011) Generation of hircine-induced pluripotent stem cells by somatic cell reprogramming. Cell Res 21:849–853 30. Sartori C, DiDomenico AI, Thomson AJ et al (2012) Ovine-induced pluripotent stem cells can contribute to chimeric lambs. Cell Reprogram 14:8–19 31. Judson RL, Babiarz JE, Venere M et al (2009) Embryonic stem cell-specific microRNAs pro- mote induced pluripotency. Nat Biotechnol 27:459–461 32. Mikkelsen TS, Hanna J, Zhang X et al (2008) Dissecting direct reprogramming through inte- grative genomic analysis. Nature 454:49–55 33. Benetti R, Gonzalo S, Jaco I et al (2008) A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyl- transferases. Nat Struct Mol Biol 15:268–279 34. Sinkkonen L, Hugenschmidt T, Berninger P et al (2008) MicroRNAs control de novo DNA methylation through regulation of transcrip- tional repressors in mouse embryonic stem cells. Nat Struct Mol Biol 15:259–267 35. Subramanyam D, Lamouille S, Judson RL et al (2011) Multiple targets of miR-302 and miR- 372 promote reprogramming of human fibro- blasts to induced pluripotent stem cells. Nat Biotechnol 29:443–448 36. Nagy K, Sung HK, Zhang P et al (2011) Induced pluripotent stem cell lines derived from equine fibroblasts. Stem Cell Rev 7:693–702 37. Liu Y, Rao M (2011) Gene targeting in human pluripotent stem cells. Methods Mol Biol 767:355–367 38. Hayashi K, Ohta H, Kurimoto K et al (2011) Reconstitution of the mouse germ cell specifi- cation pathway in culture by pluripotent stem cells. Cell 146:519–532 39. Hayashi K, Saitou M (2013) Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. Nat Protoc 8:1513–1524 40. Nayernia K, Nolte J, Michelmann HW et al (2006) In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Dev Cell 11:125–132 Wendy Dean
- 30. 13 41. Imamura M,Hikabe O, Lin ZY et al (2014) Generation of germ cells in vitro in the era of induced pluripotent stem cells. Mol Reprod Dev 81:2–19 42. Ficz G, Hore TA, Santos F et al (2013) FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency. Cell Stem Cell 13:351–359 43. Habibi E, Brinkman AB, Arand J et al (2013) Whole-genome bisulfite sequencing of two dis- tinct interconvertible DNA methylomes of mouse embryonic stem cells. Cell Stem Cell 13:360–369 44. Leitch HG, McEwen KR, Turp A et al (2013) Naive pluripotency is associated with global DNA hypomethylation. Nat Struct Mol Biol 20:311–316 45. Yamaji M, Ueda J, Hayashi K et al (2013) PRDM14 ensures naive pluripotency through dual regulation of signaling and epigenetic pathways in mouse embryonic stem cells. Cell Stem Cell 12:368–382 46. Takashima Y, Guo G, Loos R et al (2014) Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158:1254–1269 47. Kriaucionis S, Heintz N (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324:929–930 48. Tahiliani M, Koh KP, Shen Y et al (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324:930–935 49. Wang T, Chen K, Zeng X et al (2011) The his- tone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C- dependent manner. Cell Stem Cell 9:575–587 50. Chen J, Guo L, Zhang L et al (2013) Vitamin C modulates TET1 function during somatic cell reprogramming. Nat Genet 45:1504–1509 51. Lehnertz B, Ueda Y, Derijck AA et al (2003) Suv39h-mediated histone H3 lysine 9 methyla- tion directs DNA methylation to major satellite repeats at pericentric heterochromatin. Curr Biol 13:1192–1200 52. Ludwig G, Nejman D, Hecht M et al (2014) Aberrant DNA methylation in ES cells. PLoS One 9:e96090 53. Hemberger M, Dean W, Reik W (2009) Epigenetic dynamics of stem cells and cell lin- eage commitment: digging Waddington’s canal. Nat Rev Mol Cell Biol 10:526–537 54. Bredenkamp N, Ulyanchenko S, O’Neill KE et al (2014) An organized and functional thy- mus generated from FOXN1-reprogrammed fibroblasts. Nat Cell Biol 16:902–908 Cellular Reprogramming for Biomedicine
- 32. Part II De NovoReprogramming
- 34. 17 Paul J. Vermaand Huseyin Sumer (eds.), Cell Reprogramming: Methods and Protocols, Methods in Molecular Biology, vol. 1330, DOI 10.1007/978-1-4939-2848-4_2, © Springer Science+Business Media New York 2015 Chapter 2 Synthetic mRNA Reprogramming of Human Fibroblast Cells Jun Liu and Paul J. Verma Abstract Reprogramming of somatic cells, such as skin fibroblasts, to pluripotency was first achieved by forced expression of four transcription factors using integrating retroviral or lentiviral vectors, which result in integration of exogenous DNA into cellular genome and present a formidable barrier to therapeutic appli- cation of induced pluripotent stem cells (iPSCs). To facilitate the translation of iPSC technology to clinical practice, mRNA reprogramming method that generates transgene-free iPSCs is a safe and efficient method, eliminating bio-containment concerns associated with viral vectors, as well as the need for weeks of screen- ing of cells to confirm that viral material has been completely eliminated during cell passaging. Key words Reprogramming, Transgene-free, Induced pluripotent stem cells, Modified mRNA, Transfection 1 Introduction The discovery that induced pluripotent stem cells (iPSCs) can be generated from differentiated cell types, e.g., skin fibroblasts, through the overexpression of a set of defined transcription fac- tors holds the promise for regenerative medicine and cell-based autologous therapies [1, 2]. The initial approach utilized retrovi- ral vectors to deliver OCT4, KLF4, SOX2, and c-MYC to repro- gram mouse and human fibroblasts to iPSCs. However, this approach carries the risk associated with integration of exotic transgene sequences into the genome and therefore is precluded for cell-based therapeutic applications in patients. A variety of technologies have been developed for transgene integration-free pluripotency reprogramming, such as using adenoviral vectors [3, 4], non-integrating DNA plasmid-based vectors [5–9], protein transduction [10, 11], Sendai viral vectors [12, 13], microRNA- based reprogramming [14, 15], and modified mRNA-based reprogramming approach [16, 17]. The modified mRNA technol- ogy is a non-viral, non-integrating, clinically relevant reprogram- ming method, and completely eliminates the risk of genomic
- 35. 18 integration and mutagenesisinherent to DNA and viral-based technologies. Moreover, the mRNA reprogramming approach offers a robust and dose-titratable of multiple different mRNA expression, which allows for stoichiometry control of individual factors required during reprogramming. We have efficiently generated iPSCs from the skin fibroblasts of a type 1 diabetes patient using a Stemgent® mRNA reprogram- ming system. Here, we describe a stepwise protocol for the genera- tion of mRNA-derived iPSCs from primary human fibroblasts using a Stemgent® synthetic modified mRNA, focusing on material preparation (including primary human fibroblasts, feeder cells, inducing medium, and conditioned medium), plating cells, trans- fecting cells, identifying iPSC colonies, picking and passaging iPSC colonies. The protocol described here is for reprogramming of human fibroblasts to pluripotency, however, which has broad applicability in other species. 2 Materials This protocol describes the use of the Stemgent® mRNA repro- gramming system to reprogram four wells of human skin dermal fibroblasts at one time in a 6-well plate format. Material prepara- tion should begin 1 week prior to starting the experiment. All materials should be prepared under sterile conditions in a biologi- cal safety cabinet. 1. Pluriton medium. Thaw the 500 mL bottle of Pluriton medium completely at 4 °C (see Note 1). Once the medium bottle has thawed completely, add 5 mL of penicillin/streptomycin (100×) to the bottle. Pipet thoroughly to mix. Pipet 40 mL aliquots of the medium into seven 50 mL conical tubes (280 mL total). Freeze the seven medium aliquots at −20 °C. Store the remaining 220 mL of medium at 4 °C for use during the first week for generating NuFF-conditioned Pluriton medium. 2. Pluriton supplement. Thaw the 200 μL vial of supplement on ice (see Note 2). Pipet 4 μL of supplement directly into the bottom of 50 sterile, low protein-binding microcentrifuge tubes. Freeze and store the supplement aliquots at −70 °C for up to 3 months. 3. B18R Recombinant protein. Thaw the 40 μg vial of B18R pro- tein (eBioscience, #34-8185-85; 0.5 mg/mL stock concentra- tion, 80 μL total volume) on ice (see Note 3). Pipet 4 μL of the B18R protein directly into the bottom of 20 sterile, low protein-binding microcentrifuge tubes. Freeze and store the protein aliquots at −70 °C for up to 3 months. 2.1 Tissue and Cell Culture Reagents Jun Liu and Paul J. Verma
- 36. 19 4. mRNA cocktail.Thaw the individual vials containing each mRNA reprogramming factor on ice. Keep mRNA vials on ice at all times (see Note 4). Using RNase-free aerosol-barrier tips, combine the mRNA factors according to the table below in a sterile, 1.5 mL RNase-free microcentrifuge tube on ice. Oct4 mRNA 385.1 μl Sox2 mRNA 119.2 μl Klf4 mRNA 155.9 μl c-Myc mRNA 147.7 μl Lin28 mRNA 82.5 μl nGFP mRNA 110.6 μl mRNA cocktail mix 1000 μl Pipet the contents of the tube to mix thoroughly. Aliquot 50 μL of the mRNA cocktail into 20 individual sterile, 1.5 mL RNase-free microcentrifuge tubes. Freeze and store the ali- quots at −70 °C. 5. Human fibroblast medium: 10 % serum (fetal bovine/calf serum), DMEM—high glucose with sodium pyruvate and L-glutamine added and 1 % penicillin–streptomycin. Filter- sterilize medium using a 0.22 μm pore size, low protein- binding filter. Store at 4 °C for up to 2 weeks. 6. Human iPSC culture medium: 20 % Knockout serum replace- ment, DMEM/F-12, 1 % Non-essential amino acids, 1 % L-glutamine, 0.1 % β-mercaptoethanol, 8 ng/mL basic fibro- blast growth factor, and 1 % penicillin-streptomycin. Filter- sterilize medium using a 0.22 μm pore size, low protein-binding filter. Store at 4 °C for up to 2 weeks. 7. MEF culture medium: 10 % serum (fetal bovine/calf serum), DMEM—high glucose with sodium pyruvate and L-glutamine added and 1 % penicillin–streptomycin. Filter-sterilize medium using a 0.22 μm pore size, low protein-binding filter. Store at 4 °C for up to 2 weeks. 3 Methods 1. Thaw one vial of inactivated NuFF cells containing approxi- mately 4×106 cells. 2. Incubate the cells in the T75 flask using human fibroblast medium at 37 °C and 5 % CO2 for overnight. 3. Aspirate the NuFF culture medium from the T75 tissue cul- ture flask. 3.1 Generating NuFF-Conditioned Pluriton Medium Synthetic mRNA Reprogramming of Human Fibroblast Cells
- 37. 20 4. Add 10mL of PBS to the cells to wash. 5. Add 25 mL of Pluriton medium supplemented with 25 μL of bFGF (to a final bFGF concentration of 4 ng/mL) to the T75 flask (see Note 5). 6. Incubate the cells overnight at 37 °C and 5 % CO2. 7. After 24 h incubation, the medium in the T75 flask can be col- lected as NuFF-conditioned Pluriton medium and be frozen at −20 °C, and replaced with 25 mL fresh Pluriton medium sup- plemented with bFGF to a final concentration of 4 ng/mL. 8. Repeat the collection and exchange of medium daily through day 6. 9. Thaw all aliquots of previously collected NuFF-conditioned Plurito medium at 4 °C. 10. Collect final 25 mL of NuFF-conditioned Pluriton medium from the NuFF cells in the T75 flask. 11. Pool all thawed NuFF-conditioned Pluriton medium and filter using a 0.22 μm pore size, low protein-binding filter. 12. Dispense filtered NuFF-conditioned Pluriton medium into 40 mL aliquots and re-freeze at −20 °C until use. 1. Punch biopsies are obtained from volunteer’s non-sun exposed buttock skin with ethics approval and patient consent (see Note 6). Punch biopsy size is about 6–8 mm in diameter. 2. In sterile hood transfer the skin sample to a 100-mm sterile dish containing 10 mL of PBS. 3. Dissect the dermis from the rest of the skin (epidermis and subcutaneous tissue) using scalpel and forceps. 4. Mince the dermis into small pieces (~1 mm3 ) and place about three or four fragments on the bottom of a well of 6-well plates, separated from one another. 5. Allow explants to air-dry for 15 min. 6. Gently add 2 mL of fibroblast medium to cover each tissue piece. Place the plates in the 5 % CO2 incubator at 37 °C. 7. Incubate for 7 days without touching the flask to allow cells to migrate out of tissue fragments. 8. Change the medium once per week, until substantial number of fibroblasts is observed. 9. When 80 % confluent, passage 1:3 using 0.25 % trypsin/ EDTA. A small aliquot should be taken for mycoplasma testing by PCR. 10. Begin reprogramming at passage 3 and freeze down backup vials in liquid nitrogen for storage. 3.2 Human Dermal Fibroblast Isolation Jun Liu and Paul J. Verma
- 38. 21 1. Add 1mL of sterile 0.2 % gelatin (in ddH2O) in each of 4 wells of a 6-well tissue culture plate. Incubate the plate for a mini- mum of 30 min at 37 °C and 5 % CO2. 2. Thaw 1×106 inactivated NuFF cells in a 37 °C waterbath until only a small ice crystal remains (see Note 7). 3. Transfer the NuFF cells to a 15 mL conical tube and add 5 mL of human fibroblast medium to the cells while gently agitating the contents of the tube. 4. Centrifuge the cells for 4 min at 200×g. 5. Aspirate the supernatant and resuspend the cell pellet in 8 mL of human fibroblast medium. 6. Aspirate the gelatin solution from the four wells of the pre- pared 6-well plate and add 2 mL of NuFF cell suspension to each of the four wells. 7. Incubate the cells overnight at 37 °C and 5 % CO2. The procedure is appropriate for dermal fibroblasts in culture in a T75 flask and may not be applicable to all target cell types. For target cells other than fibroblasts, harvest the cells according to an appropriate protocol and plate in the format described below. 1. Remove the culture medium from the T75 flask of cells to be harvested. 2. Wash the cells with 10 mL of PBS in the flask. 3. Add 3 mL of 0.05 % Trypsin/EDTA to the flask and incubate for 5 min at 37 °C and 5 % CO2. 4. Add 6 mL of human fibroblast medium (or appropriate target cell medium containing serum) to the flask to neutralize the Trypsin/EDTA. 5. Transfer the cell suspension to a 15 mL conical tube and cen- trifuge for 5 min at 200×g. 6. Remove the supernatant and resuspend the pellet in 5 mL of human fibroblast medium. 7. Count the cells in solution and calculate the live cell density. 8. Aspirate the culture medium from NuFF feeder cells and plate the target cells in three independent wells of the NuFF feeder plate at densities of 5×103 , 1×104 , 2.5×104 cells per well in 2 mL total volume per well. Plate human BJ fibroblasts in a well with NuFF feeder cells at density of 1×104 as control. 9. Incubate the cells at 37 °C and 5 % CO2. At day 1 of transfection, the cells must be cultured in the medium with 200 ng/mL B18R for 2 h before the first transfection with mRNA. 3.3 NuFF Feeder Cells Plating 3.4 Target Cell Plating 3.5 Transfection 3.5.1 Day 1 Transfection Synthetic mRNA Reprogramming of Human Fibroblast Cells
- 39. 22 1. Add 10mL of Pluriton medium to a sterile 100 mm dish. 2. Incubate the medium for 2 h at 37 °C and 5 % CO2 to equili- brate the medium (see Note 8). 3. Thaw one vial of Pluriton supplement and one vial of B18R protein on ice. 4. Add 4 μl of the supplement and 4 μl of the B18R protein to the medium to generate Pluriton reprogramming medium (with B18R protein). 5. Aspirate the target cell medium from each of the 4 wells to be transfected. 6. Add 2 mL of Pluriton reprogramming medium (with B18R protein) to each of the four wells. 7. Incubate the cells for 2 h at 37 °C and 5 % CO2 prior to transfecting. 8. Thaw one 50 μL aliquot of the mRNA cocktail on ice (Tube 1). 9. Using RNase-free, aerosol-barrier pipette tips, add 200 μL of Opti-MEM to the tube containing the mRNA cocktail and pipet gently to mix (Tube 1). 10. In a second sterile, RNase-free 1.5 mL microcentrifuge tube, add 225 μl of Opti-MEM and 25 μL of RNAiMAX, mix gently (Tube 2). 11. Transfer the entire contents of Tube 2 to the mRNA cocktail solution in Tube 1 to generate the mRNA transfection com- plex and pipet gently 3–5 times. 12. Incubate the mRNA transfection complex at room tempera- ture for 15 min to allow the mRNA to properly complex with the transfection reagent. 13. In a dropwise manner, add 120 μL of the mRNA transfection complex to each of the four wells to be transfected. 14. Gently rock the 6-well plate from side to side and front back to distribute the mRNA transfection complex evenly across the wells. 15. Incubate the cells for 4 h at 37 °C and 5 % CO2. 16. Add 10 mL of medium to a sterile 100 mm dish and incubate the medium for at least 2 h at 37 °C and 5 % CO2 to equilibrate the medium. 17. Just prior to use, add 4 μL of supplement and 4 μL of the B18R protein to the equilibrated medium to generate Pluriton reprogramming medium (with B18R protein). 18. After the target cells have been transfected for 4 h, aspirate the medium containing the mRNA transfection complex from each well (see Note 9). Jun Liu and Paul J. Verma
- 40. 23 19. Add 2mL of the equilibrated Pluriton reprogramming medium (with B18R protein) to each well. 20. Incubate the cells overnight at 37 °C and 5 % CO2. The transfection procedure must be repeated each day from Day 2 to Day 6 exactly as done on Day 1. Monitor the cell cultures daily, observing cell proliferation rates, morphology changes, and nGFP expression in each well (Fig. 1). 1. Prepare the mRNA transfection complex as described for Day 1 (see Note 10). 2. Transfect cells as described for Day 1. 3. Equilibrate Pluriton medium and prepare Pluriton reprogram- ming medium (with B18R protein) as described for Day 1. 4. Change medium after 4 h of transfection and incubate the cells overnight at 37 °C and 5 % CO2. Starting at Day 7, NuFF-conditioned Pluriton reprogramming medium must be used in place of Pluriton reprogramming medium. Transfection of the target cells must be continued as done previ- ously from Day 1 to Day 6. The protocol for generating and pre- paring NuFF-conditioned Pluriton reprogramming medium is detailed in Subheading 3.1. Continue to monitor the cell cultures 3.5.2 Day 2–6 Transfection 3.5.3 Day 7–18 Transfection Fig. 1 Observation of target cells during day 1 to day 5. Transfected cells will begin to appear in small clusters with a more compacted morphology compared with the fibroblasts at day 5. The nGFP expression should appear in the transfected cells Synthetic mRNA Reprogramming of Human Fibroblast Cells
- 41. 24 daily, as morphologicalchanges become more pronounced between Day 7 and Day 18 (Fig. 2). 1. Prepare the mRNA transfection complex as described for Day 1 (see Note 10). 2. Transfect cells as described for Day 1. 3. Equilibrate NuFF-conditioned Pluriton medium and prepare NuFF-conditioned Pluriton reprogramming medium (with B18R protein) as described for Day 1. 4. After 4 h of transfection, remove the medium containing the transfection reagent and add 2 mL of equilibrated NuFF- conditioned Pluriton reprogramming medium (with B18R protein) to each well, as described for Day 1. 5. Incubate the cells overnight at 37 °C and 5 % CO2. 1. Prepare MEF feeder cells in 12-well plates 1 day before iPSC colony pickup. 2. Thaw one aliquot of Pluriton supplement on ice and add 4 μL of the supplement to 10 mL of Pluriton medium to generate Pluriton reprogramming medium. 3. Aspirate the MEF culture medium from 12-well MEF feeder plates. 4. Add 1 mL of PBS to each well to rinse and aspirate the PBS. 3.6 Pickup and Culture of iPSC Colonies Fig. 2 Morphological changes of an emerging colony and colony pickup. (a, b, c) Morphological changes char- acteristic of an iPSC cluster marked with a yellow dashed circles. (c) The iPSC colony was manually cut into eight pieces, which should be transferred to an individual well of a 12-well plate with a newly pated feeder layer. (d, e, f) Health human iPSC colonies with defined colony edges and the uniform and compact iPSC within the colonies Jun Liu and Paul J. Verma
- 42. 25 5. Add 1mL of human iPSC culture medium to each of the rinsed wells. 6. Aspirate the medium from the well of the 6-well plate that the primary iPSCs will be picked from. 7. Add 2 mL of Pluriton reprogramming medium to the well of iPSCs to be picked. 8. Using a stereo microscope, locate iPSC colonies based on morphology. Using a glass picking tool or 1 mL insulin syringe, gently divide the colony into approximately 4–9 pieces (see Note 11). 9. Using a pipettor with a sterile 10 μL pipet tip, transfer the detached colony pieces out of the reprogramming well and into an individual well of the prepared 12-well plate (see Note 12). 10. Repeat the picking and replating process for the next iPSC colonies. Pick one colony at a time and transfer the cell aggre- gates of each to a new well of the prepared 12-well inactivated MEF feeder plate. 11. After six iPSC colonies have been picked and replated, return both the 12-well plate and the primary reprogrammed colo- nies on the 6-well plate to the incubator at 37 °C and 5 % CO2. After allowing the cells to incubate for at least 30 min, an addi- tional six primary iPSC colonies can be picked and replated on a new prepared 12-well MEF feeder plate. Repeat this process (steps 10–12) in increments of six iPSC colonies at a time until a sufficient number of colonies have been picked. 12. The iPSCs are cultured in human iPSC culture medium, or adapted to other proven ESC culture conditions. The cell cul- ture medium must be changed every day to provide necessary nutrients and growth factors for the maintenance of human iPSCs (see Note 13). 4 Notes 1. The 500 mL bottle of Pluriton medium may take up to 2 days to thaw completely at 4 °C. Approximately 220 mL of Pluriton medium will be used during the first week of the protocol and for generating NuFF-conditioned Pluriton medium. The remaining medium should be aliquoted and stored at −20 °C until use. After thawing, the shelf-life of Pluriton medium is 2 weeks when stored at 4 °C. 2. The 200 μL vial of Pluriton supplement must be aliquoted in single-use vials and frozen at −70 °C until use in order to mini- mize degradation of components in the supplement. One 4 μL aliquot will be used for each daily 10 mL medium preparation. Synthetic mRNA Reprogramming of Human Fibroblast Cells
- 43. 26 Once the single-usealiquots have been thawed they must be used immediately and cannot be re-frozen. 3. The B18R protein must be aliquoted into single-use vials and frozen at −70 °C until use. All vials of the B18R protein must be kept on ice at all times in order to minimize degradation of the protein. One aliquot will be used for each day of transfec- tion. Once the single-use aliquots have been thawed they must be used immediately and cannot be re-frozen. 4. Create a master mRNA cocktail and aliquot the mix into single-use volumes. This can be done up prior to beginning the reprogramming experiment. Combine all mRNA factors according to the volumes in the table below. When reprogram- ming 4 wells at a time, aliquot the mRNA cocktail into 20 single-use vials, one of which will be used for each day of trans- fection. The mRNA cocktail, as prepared below, has a molar stoichiometry of 3:1:1:1:1:1 for the Oct4, Sox2, Klf4, c-Myc, Lin28 and nGFP mRNAs, respectively. Each mRNA factor is supplied at a concentration of 100 ng/L. Once the single-use aliquots have been thawed they cannot be re-frozen. 5. The total number of cells plated in the flask will determine the volume of Pluriton medium that can be effectively conditioned each day. If 3×106 to 4×106 NuFF cells have been plated in the T75 flask, 25 mL of Pluriton medium can be conditioned each day. If less than 3×106 cells were plated in the flask, add 2 mL of Pluriton medium per 2.5×105 cells plated. A mini- mum of 2.25×106 NuFF cells (18 mL medium) should be used in one T75 flask. 6. Before any material can be collected from a human volunteer, ethical approval for the research must be obtained form the local institutional ethics committee. Only trained and autho- rized personnel should perform skin biopsies, and every sub- ject for whom skin is taken must give written informed consent. It is essential that the designation of the cell strain is unam- biguous. It should be unique and maintain donor anonymity. 7. Inactivated NuFF cells should be evenly plated at a density of 2.5×105 cells per well of a 6-well plate in a total volume of 2 mL of human fibroblast medium per well. If one vial of NuFF cells contains more than 1×106 cells, the remainder of the NuFF cells should be plated in a separate T75 flask to be used to generate NuFF-conditioned Pluriton medium (see Subheading 3.1 “Generating conditioned Pluriton medium”). 8. If reprogramming under low oxygen conditions, the medium should be equilibrated at low O2 tensions. 9. Do not leave the mRNA transfection complex in the culture medium for longer than 4 h, as prolonged exposure to the Jun Liu and Paul J. Verma
- 44. 27 RNAiMAX transfection reagentwill result in increased cellular toxicity. 10. Cells undergoing reprogramming must be transfected with the mRNA reprogramming factor cocktail every day. It is impor- tant to transfect the cells at the same time each day in order to maintain sufficient levels of mRNA transcripts to allow for con- tinual expression of the mRNA factors. 11. It is important to break up the colony into smaller cell aggre- gates, but not into single cells. 12. Transfer all of the pieces from one colony into a single well of the 12-well plate. 13. For the first few passages after a picking from the reprogrammed cultures, the cells should be passaged manually (without enzymes or centrifugation) at low split ratios to build robust, dense cultures. The cells can be split using an enzymatic proto- col for routine culture once there are a large number of human iPSC colonies in the well(s). Human iPSCs are generally pas- saged every 4–7 days in culture, but the actual passaging sched- ule and split ratio for each passage will vary depending on the cell culture’s quality and growth. It is important to passage the cells before the culture becomes overgrown. Acknowledgement This work was supported by the Victorian Government’s Infrastructure Operational Program and collaboration with Stemgent, Inc. References 1. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676 2. Takahashi K, Tanabe K, Ohnuki M et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872 3. Stadtfeld M, Nagaya M, Utikal J et al (2008) Induced pluripotent stem cells generated with- out viral integration. Science 322:945–949 4. Yu J, Hu K, Smuga-Otto K et al (2009) Human induced pluripotent stem cells free of vector and transgene sequences. Science 324:797–801 5. Jia F, Wilson KD, Sun N et al (2010) A nonvi- ral minicircle vector for deriving human iPS cells. Nat Methods 7:197–199 6. Okita K, Matsumura Y, Sato Y et al (2011) A more efficient method to generate integration- free human iPS cells. Nat Methods 8:409–412 7. Okita K, Nakagawa M, Hyenjong H et al (2008) Generation of mouse induced pluripo- tent stem cells without viral vectors. Science 322:949–953 8. Woltjen K, Michael IP, Mohseni P et al (2009) piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458:766–770 9. Yusa K, Rad R, Takeda J et al (2009) Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat Methods 6:363–369 10. Kim D, Kim CH, Moon JI et al (2009) Generation of human induced pluripotent Synthetic mRNA Reprogramming of Human Fibroblast Cells
- 45. 28 stem cells bydirect delivery of reprogramming proteins. Cell Stem Cell 4:472–476 11. Zhou H, Wu S, Joo JY et al (2009) Generation of induced pluripotent stem cells using recom- binant proteins. Cell Stem Cell 4:381–384 12. Fusaki N, Ban H, Nishiyama A et al (2009) Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not inte- grate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 85:348–362 13. Ban H, Nishishita N, Fusaki N et al (2011) Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc Natl Acad Sci U S A 108:14234–14239 14. Judson RL, Babiarz JE, Venere M et al (2009) Embryonic stem cell-specific microRNAs pro- mote induced pluripotency. Nat Biotechnol 27:459–461 15. Miyoshi N, Ishii H, Nagano H et al (2011) Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell 8:633–638 16. Warren L, Manos PD, Ahfeldt T et al (2010) Highly efficient reprogramming to pluripo- tency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7:618–630 17. Mandal PK, Rossi DJ (2013) Reprogramming human fibroblasts to pluripotency using modi- fied mRNA. Nat Protoc 8:568–582 Jun Liu and Paul J. Verma
- 46. 29 Paul J. Vermaand Huseyin Sumer (eds.), Cell Reprogramming: Methods and Protocols, Methods in Molecular Biology, vol. 1330, DOI 10.1007/978-1-4939-2848-4_3, © Springer Science+Business Media New York 2015 Chapter 3 MicroRNA-Mediated Reprogramming of Somatic Cells into Induced Pluripotent Stem Cells Shelley E.S. Sandmaier and Bhanu Prakash V.L. Telugu Abstract MicroRNAs or miRNAs belong to a class of small noncoding RNAs that play a crucial role in posttran- scriptional regulation of gene expression. Nascent miRNAs are expressed as a longer transcript, which are then processed into a smaller 18–23-nucleotide mature miRNAs that bind to the target transcripts and induce cleavage or inhibit translation. MiRNAs therefore represent another key regulator of gene expres- sion in establishing and maintaining unique cellular fate. Several classes of miRNAs have been identified to be uniquely expressed in embryonic stem cells (ESC) and regulated by the core transcription factors Oct4, Sox2, and Klf4. One such class of miRNAs is the mir-302/367 cluster that is enriched in pluripotent cells in vivo and in vitro. Using the mir-302/367 either by themselves or in combination with the Yamanaka reprogramming factors (Oct4, Sox2, c-Myc, and Klf4) has resulted in the establishment of induced plu- ripotent stem cells (iPSC) with high efficiencies. In this chapter, we outline the methodologies for estab- lishing and utilizing the miRNA-based tools for reprogramming somatic cells into iPSC. Key words ESC, IPSC, miRNA, Pluripotency, Reprogramming Abbreviations iPSC Induced pluripotent stem cells ESC Embryonic stem cells TALENS Transcription activator-like effector nucleases CRISPR Clustered regularly interspaced short palindromic repeat ZFN Zinc finger nucleases 1 Introduction MicroRNAs (miRNAs) are short noncoding RNAs that bind target mRNAs via complete or incomplete sequence complementarity and regulate stability and translatability of the message [1–3]. Nascent miRNAs are transcribed from endogenous loci via Pol II RNA polymerase as 85–100 base pair nascent transcripts, which are then
- 47. 30 processed by Droshaand Dicer into mature miRNAs of 18–23 nucleotides in length [3, 4]. The mature miRNAs are characterized by a “seed sequence” at the 5′-end between nucleotides 2–8, exhib- iting perfect complementarity with the target gene [2]. After a miRNA recognizes and binds to the target mRNA, it inhibits trans- lation in either of the two ways: (1) targeting the mRNA for cleav- age if the miRNA shares perfect complementarity with the sequence or (2) in the case of partial complementarity prevents assembly of a ribosome initiation complex and initiation of translation [3]. Due to the ability of miRNAs to bind to target sequences, albeit with poor complementarity, one miRNA is often capable of binding to a cohort of mRNAs and inhibiting translation. Accordingly, many genes can often be regulated by a candidate miRNA [1, 2]. In embryonic stem cells (ESC), several classes of miRNAs have been identified to be specifically enriched, indicating a possible role in maintaining pluripotency [5]. Potentially several more exist based on the putative ability of certain transcripts to form hairpin miRNA precursors [5]. A much stronger evidence for the role of miRNAs in maintaining pluripotency comes from the discovery that several of the miRNA genes have binding sites for core pluri- potency genes Oct4, Sox2, and Nanog in their promoters [6]. In ESC, miRNAs specifically target genes which affect varying prop- erties of pluripotency such as transcription factors, cell cycle genes, and genes involved in epigenetic regulation. Regulation of pluripo- tency by such diverse cellular mechanisms is necessary to ensure greater stability of ESC [2]. Considering the abundance of miRNAs in ESC, and their putative role in regulating pluripotency, the ability of miRNAs to aid in the production and maintenance of induced pluripotent stem cells (iPSCs) has been increasingly studied. iPSCs have tradi- tionally been generated from somatic cells via retroviral delivery of Oct4, Sox2, Klf4, and c-myc (OSKM) reprogramming factors, as was first reported by Takahashi and Yamanaka [7]. However, the induction of pluripotency by OSKM is rather inefficient (0.001–0.01 %), yielding very few colonies per million cells infected with retrovirus [7, 8]. Recently, iPSCs have been produced with greater efficiency by incorporating specific miRNA clusters shown to be involved in regulating the pluripotent state [9, 10]. Specifically, a well-studied mir-302/367 cluster, which has been shown to play a role in regulating cell cycle, and is regulated by the core pluripotency factors Oct4, Sox2, Nanog, and Tcf3, has been utilized in the reprogramming efforts [6, 11]. Mir-302/367 cluster comprises of five miRNAs, mir-302a, -b, -c, -d, and mir-367, expressed as a polycistronic construct and located within intron 8 of the LARP7 gene in humans, with homologs in several species including cattle, pigs, and mice [6, 9]. Interestingly, the seed sequences of the four miRNAs (302a/b/c/d) are identical, and share high degree of conservation across species. When used in combination with the traditional OSKM factors in Shelley E.S. Sandmaier and Bhanu Prakash V.L. Telugu
- 48. 31 reprogramming experiments, thenumber of iPSC colonies has been shown to be enhanced by at least two orders of magnitude (0.1–0.8 %) [9, 10]. In fact, cells can be reprogrammed to pluri- potency with mir-302/367 and a histone deacetylase (HDAC) inhibitor, valproic acid, alone, and show ESC-like morphology sooner than cells reprogrammed with OSKM. Moreover, these iPSCs are capable of contributing to all three germ layers as well as giving rise to germ-line chimeras in mice [9]. Human iPSCs have also been generated using miRNAs with or without the addi- tion of OSKM into the genome [9, 12–14]. Therefore, our and several other laboratories have adopted miRNAs as a standard fac- tor in reprogramming iPSC (Manuscript in preparation). The use of miRNA is especially important in reprogramming somatic cells from livestock species, where the efficiencies of reprogramming are even lower, and the conditions for optimal culture not com- pletely understood. In this manuscript, the procedures for making and using miRNA-based vectors for reprogramming somatic cells from the domestic animal species, pig, are discussed. However, the methods discussed below can easily be adopted for other model organisms. 2 Materials Store all reagents and media at 4 °C unless otherwise noted. 1. Complete media: 440 mL HyClone High Glucose DMEM (ThermoScientific), 50 mL 10 % fetal calf serum (FCS), 2.5 mL 100× GlutaMAX (Gibco), 5 mL 100× nonessential amino acids, and 5 mL 100× sodium pyruvate (see Note 1). 2. iPSC media: 382.5 mL HyClone DMEM F12 (ThermoScientific), 100 mL knockout serum replacer (KSR) (Gibco), 2.5 mL GlutaMAX, 5 mL nonessential amino acids, 10 mL sodium bicarbonate solution 7.5 %, and 8 ng/mL FGF2 (R&D Systems). 3. 0.25 % trypsin–EDTA for dissociation and harvesting of cells. 4. Dimethyl sulfoxide (DMSO) for cryopreservation. 5. CF-1 mice OR irradiated mouse embryonic fibroblasts (MEFs). 6. Phosphate-buffered saline (PBS). 7. T-75 flasks. 8. Cryovial freezing container filled with 2-propanol. 1. 293-FT cells (Life Technologies) viral packaging cells. 2. Geneticin (G418). 3. Gelatin. 4. Polyjet (Signagen). 2.1 Cell Culture 2.2 Lentivirus Production miRNA Mediated Reprogramming of Somatic Cells
- 49. 32 5. Polybrene. 6. Packagingplasmids and vectors with genes of interest, maxiprepped. 7. Valproic acid (Stemgent), store at −20 °C. 3 Methods Cells should always be incubated at 37 °C in 5 % CO2 unless otherwise noted. Passaging of cells is always done with 0.25 % trypsin–EDTA unless otherwise noted. As an alternative to generating your own MEFs for use as feeder cells, irradiated MEFs from this mouse strain are available for purchase. 1. Set up a mating by placing one 8-week-old CF-1 female in a cage with one CF-1 male. Check daily in the morning to determine the presence of a copulatory plug. The first sighting of a plug will be considered day 0.5 of gestation. 2. On day 13.5 of gestation, sacrifice pregnant females by cervical dislocation. Remove the uterus, isolate the embryos, and per- form the following steps in a laminar flow hood. Remove limbs and internal organs of the fetuses, and mince the remainder of the fetuses in 3 mL of 0.25 % trypsin–EDTA using a sterile scalpel blade (see Note 2). Allow cells to digest for 30–60 min in a 37 °C incubator with 5 % CO2. Halt the reaction with 6 mL of complete media. 3. Centrifuge cells at 800×g for 10 min and aspirate supernatant. Wash cells twice more with 6 mL complete media and centrifu- gation. Plate cells in 150 mm dishes. 4. After 1–2 days of culture, trypsinize cells and freeze in 92 % complete media+8 % DMSO in liquid nitrogen (see Notes 3 and 4). In order to irradiate, thaw the frozen vials and grow in T-75 flasks (see Note 5). Passage cells 2–5 times at a ratio of 1:5. Remove media and add PBS to irradiate. After irradiation, count cells and freeze as before with a density of 5–10×106 cells per vial. 1. Thaw one vial of 293-FT cells and put into a T-75 flask with 17 mL complete media. On day 2, feed cells with complete media containing 500 μg/mL of G418. 2. On day 3, passage cells using 3 mL of 0.25 % trypsin–EDTA at 1:4 using complete media+G418. Keep the passaged cells in a T-75 flask. 3. On day 4, passage cells as before and count using a hemocy- tometer. Seed 4×106 cells per 100 mm dish (see Note 6) in complete media which does not contain G418. 3.1 Production of Mouse Embryonic Fibroblasts 3.2 Production of Lentivirus for Transduction Shelley E.S. Sandmaier and Bhanu Prakash V.L. Telugu
- 50. 33 4. The nextday (day 0), transfect cells. Two hours before transfection, refresh the media with 5 mL of complete media per 100 mm dish. Because the lentivirus is divided into multi- ple parts to ensure safety, each plasmid will be infected into an individual 100 mm dish to produce lentivirus of one type only. Mix the following amounts of DNA in 250 μL of plain DMEM per single reaction (see Note 7): (a) pMD2.G (VSV-G): 3.15 μg per dish. (b) psPAX2: 5.85 μg per dish. (c) Plasmid containing OSK: 6 μg per dish. (d) Plasmid containing MLN: 6 μg per dish. (e) Plasmid containing miR-302/367: 6 μg per dish. 5. In a separate mixture, add 30 μL of Polyjet to 220 μL of plain DMEM per single reaction. Mix gently. Add 250 μL Polyjet mixture to each DNA solution dropwise and gently finger flick to mix. Incubate for 15 min at room temperature (see Note 8). 6. Add mixture dropwise to prepared 293-FT cells. Gently shake dish left and right, and then forward and backward to mix, and incubate. 1. On day 0 (the same day you transfect 293-FT cells), thaw the frozen porcine fibroblasts and seed one vial per T-75 flask (see Note 9). One day later, trypsinize the cells and count; each well of a 6-well plate will need 1×105 cells. 1. 18 h post-transfection (day 1), aspirate and discard the supernatant and feed 293-FT cells with 9 mL per 100 mm dish of complete media containing only 2 % FCS (see Note 1). 2. On day 2 (48 h post-transfection) collect the supernatant and centrifuge at 200×g for 10 min to separate any cells collected. Add 1.2 μL Polybrene per 0.5 mL of complete media per well of a 6-well plate containing porcine fibroblasts. Then add 1.5 mL per well of combined transfected supernate. Incubate cells with lentivirus for 6 h only, then aspirate, and feed with 2 % FCS media. Repeat transduction of target cells as above 24 h later, on day 3. 1. Feed each well of the 6-well plate with 1.5 mL of complete media. When cells become confluent, passage with .05 % trypsin at a ratio of 1:6 onto two 100 mm dishes containing MEF feeder cells. Feed each dish with 7.5 mL of complete media for 1 more day. 2. The next day, switch to feeding with iPSC media which con- tains 0.5 μM valproic acid. Feed daily with 7.5 mL for 7 days. 3. After the 7 days on valproic acid, continue to feed daily with iPSC media alone. When fibroblasts begin to overgrow, split 3.3 Preparation of Porcine Fibroblasts 3.4 Transduction of Porcine Fibroblasts 3.5 Reprogramming of Fibroblasts to iPSCs miRNA Mediated Reprogramming of Somatic Cells
- 51. 34 plates with trypsinand plate on fresh MEFs at a ratio of 1:4 or greater once or twice during the initial reprogramming period (see Note 10). Continue feeding cells with 7.5 mL iPSC media and check for colonies daily. 4. After colonies begin to appear, they are manually picked with pulled Pasteur pipettes and moved individually to a single well of a 24-well plate, with a layer of MEFs (see Note 11). 5. As necessary, colonies can gradually be moved up to a 12-well and then a 6-well plate. 6. Once colonies have appeared, there are several things to do right away to establish good (or eliminate poor) cell lines. These include AP staining (see Note 12), PCR amplification for pluripotency genes (Oct4, Nanog), and analysis of morpho- logical characteristics of iPSCs. 4 Notes 1. FCS should be stored at −20 °C. Avoid multiple freeze-thaw cycles by aliquoting serum and thawing single aliquots for storage at 4 °C. GlutaMAX can be stored at room temperature or 4 °C. Even though several of these reagents are shipped sterile, filter sterilize the complete mixture to ensure sterility. For complete media containing only 2 % FCS, add 10 mL FCS to 480 mL DMEM. Keep all other additive amounts the same. 2. Use each fetus as a separate replicate. That is, each fetus should require 3 mL trypsin and should be performed in a separate tube. 3. We use a dry trypsinization throughout. To do this, add the appropriate amount of trypsin solution to the flask or well and immediately remove the excess. Allow cells to incubate for 5 min, and then add complete media back to the well for pas- saging. This minimizes the amount of stress on cells by provid- ing a bare minimum of trypsin while also allowing for single-cell passage. 4. Freezing should be done slowly (−1 °C/min); we use Mr. Frosty freezing containers filled with 2-propanol that allow for slow freezing. Put vials of cells into freezing container, and place into −80 °C freezer. Keep in freezer overnight and trans- fer to liquid nitrogen the next day for long-term storage. 5. When thawing cells, do so quickly to avoid damage. Thaw at 37 °C, and immediately add 1 mL complete media drop by drop. Pipette gently up and down a few times and transfer to 15 mL tube containing 3 mL media. Centrifuge cells at 200×g for 5 min. Remove supernatant, and add complete media to resuspend cells. Shelley E.S. Sandmaier and Bhanu Prakash V.L. Telugu
- 52. 35 6. 100 mmdishes should be pretreated with gelatin. Add 5 mL gelatin and incubate for 3–5 h. After this time, aspirate gelatin and seed cells as indicated. 7. Because you will most likely be performing multiple replicates of transfections, make a master mix of DNA and DMEM. Remember to keep different plasmids in separate master mixes. 8. Again, make a master mix of Polyjet and DMEM to account for the multiple dishes you will be transfecting. Do not add DNA to Polyjet solution—always add Polyjet to DNA. 9. If cells are sparsely populated, thaw the cells a few days earlier and passage them once using trypsin and complete media. 10. Usually, passaging during this period is done around day 10 and again around day 20. Be vigilant about noticing any changes in cell population as soon as they occur, as overgrowth of feeders can inhibit generation of iPSCs. 11. Pulled Pasteur pipettes are made by heating the glass over a flame near the end of the pipette and, once warm, bending the glass to create an L-shape at the end. When manually pas- saging, use the bent end of the pipette to scrape colonies off the plate, then collect the media containing cells, and transfer or split onto the new plate. 12. When performing an AP stain, always move cells to a different plate from those you want to continue culturing. The fixative used in AP staining can kill cells in adjacent wells if you decide to stain in the same plate you are keeping colonies you want to maintain. Acknowledgements This work was supported in part by funds from Maryland Agriculture Experimental Station (MAES) Seed Grant, Maryland Stem Cell Research Fund (MSCRF) Exploratory Grant, and the Department of Animal and Avian Sciences, University of Maryland. References 1. Ying SY, Chang DC, Lin SL (2008) The microRNA (miRNA): overview of the RNA genes that modulate gene function. Mol Biotechnol 38:257–268 2. Huang XA, Lin H (2012) The miRNA regula- tion of stem cells. Wiley Interdiscip Rev Membr Transp Signal 1:83–95 3. Cullen BR (2013) MicroRNAs as mediators of viral evasion of the immune system. Nat Immunol 14:205–210 4. Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, Kim VN (2004) MicroRNA genes are transcribedbyRNApolymeraseII.EMBOJ23:4051– 4060 5. Houbaviy HB, Murray MF, Sharp PA (2003) Embryonic stem cell-specific MicroRNAs. Dev Cell 5:351–358 6. Card DA, Hebbar PB, Li L, Trotter KW, Komatsu Y, Mishina Y, Archer TK (2008) Oct4/Sox2-regulated miR-302 targets cyclin miRNA Mediated Reprogramming of Somatic Cells
- 53. 36 D1 in humanembryonic stem cells. Mol Cell Biol 28:6426–6438 7. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676 8. Di Stefano B, Maffioletti SM, Gentner B, Ungaro F, Schira G, Naldini L, Broccoli V (2011) A microRNA-based system for selecting and main- taining the pluripotent state in human induced pluripotent stem cells. Stem Cells 29:1684–1695 9. Anokye-Danso F, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y, Zhang Y, Yang W, Gruber PJ, Epstein JA, Morrisey EE (2011) Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8:376–388 10. Judson RL, Babiarz JE, Venere M, Blelloch R (2009) Embryonic stem cell-specific microR- NAs promote induced pluripotency. Nat Biotechnol 27:459–461 11. Marson A, Levine SS, Cole MF, Frampton GM, Brambrink T, Johnstone S, Guenther MG, Johnston WK, Wernig M, Newman J, Calabrese JM, Dennis LM, Volkert TL, Gupta S, Love J, Hannett N, Sharp PA, Bartel DP, Jaenisch R, Young RA (2008) Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell 134:521–533 12. Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi DL, Kano Y, Nishikawa S, Tanemura M, Mimori K, Tanaka F, Saito T, Nishimura J, Takemasa I, Mizushima T, Ikeda M, Yamamoto H, Sekimoto M, Doki Y, Mori M (2011) Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell 8:633–638 13. Zhang Z, Wu WS (2013) Sodium butyrate promotes generation of human iPS cells through induction of the miR302/367 cluster. Stem Cells Dev 22(16):2268–2277 14. Lin SL, Chang DC, Chang-Lin S, Lin CH, Wu DT, Chen DT, Ying SY (2008) Mir-302 repro- grams human skin cancer cells into a pluripo- tent ES-cell-like state. RNA 14:2115–2124 Shelley E.S. Sandmaier and Bhanu Prakash V.L. Telugu
- 54. 37 Paul J. Vermaand Huseyin Sumer (eds.), Cell Reprogramming: Methods and Protocols, Methods in Molecular Biology, vol. 1330, DOI 10.1007/978-1-4939-2848-4_4, © Springer Science+Business Media New York 2015 Chapter 4 Generation of Footprint-Free Induced Pluripotent Stem Cells from Human Fibroblasts Using Episomal Plasmid Vectors Dmitry A. Ovchinnikov, Jane Sun, and Ernst J. Wolvetang Abstract Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease- carrying individuals’ fibroblasts using episomal plasmids. Key words Human induced pluripotent stem cells, Reprogramming, Episomal plasmid vectors, Fibroblasts, Transfection, Genomic integration 1 Introduction Lentiviral or retroviral delivery of reprogramming factors has been a powerful tool in pioneering the field of cell reprogramming. However, the concerns associated with the disruption of the genome at the viral integration sites, number and position and the unpredictable nature of transgene silencing, as well as their potential reactivation following differentiation have made integration-dependent methods unsuitable for clinical applica- tions. Indeed, with the wealth of currently available alternative technologies there is really no need to modify the genomic DNA of the target cell when generating induced pluripotent stem cells. Researchers have a number of options ranging from piggybac or sleeping beauty transposon-based or Cre recombinase-aided meth- ods to excise integrated reprogramming cassettes, or to avoid DNA-integrating methods altogether and use mRNA, helper- dependent adenoviral, Sendai virus-derived or episomal vector- based methods [1–6]. Here we describe a protocol for the generation of human iPSCs from fibroblasts using episomal plasmid
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- 56. Tammany, a Republicanmay be pardoned for suggesting that the wisdom of Tammany is due to the wisdom of the Old Serpent. Certainly, many innocent persons have been accused of dalliance with the foul fiend on much worse primâ facie evidence than that which is furnished by the universal admission that Tammany, out of the most uncompromising materials, has succeeded in achieving exploits which antecedently would have been absolutely impossible. For Tammany, although preserving and maintaining from first to last a discipline which is the despair of all the other political machines in the country, has never been without fierce internecine fights. It has cast out leader after leader, and the ferocity of the feuds within Tammany has exceeded that of any of the combats which have been waged against the common enemy. Nevertheless, notwithstanding all schisms, all reverses, all exposures, Tammany remains to this day the strongest, the best disciplined, and the most feared political organisation in the world.
- 57. TAMMANY HALL, OPENED1860. Mr. Croker, in the series of interviews which I reported in the October number of the Review of Reviews, argued with much force and plausibility that it was contrary to the law of human nature that an organisation could live and last so long if it were composed of Thugs and desperados, and that witness no doubt is true. Even so stout and stalwart an opponent of Tammany as Dr. Albert Shaw has frequently felt himself constrained to admit that the insane fashion in which New York has been governed rendered even the rule of Tammany preferable to the constitutional and legal chaos which was the only substitute. Dr. Shaw, speaking of the system under which New York has hitherto been governed, said:—
- 58. To know itsins and outs is not so much like knowing the parts and the workings of a finely adjusted machine as it is like knowing the obscure topography of the great Dismal Swamp considered as a place of refuge for criminals. Again he wrote:— In New York, the absurdly disjointed and hopelessly complex array of separate boards, functions, and administrative powers, first makes it impossible for the community to focalise responsibility anywhere in the formal mechanism of municipal government, and then makes it possible for an irresponsible self-centred political and mercenary society like Tammany to gain for itself the real control, and thus to assume a domination that ought to be centred in some body or functionary directly accountable to the people. Government by a secret society like Tammany is better than the chaos of a disjointed government for which there can be no possible location of central responsibility. It is not for me to dogmatise where experts, native to New York, hopelessly disagree. But viewed from the outside the secret of Tammany’s success seems to lie chiefly in the fact that Tammany has from the first been really a democratic organisation. No one was too poor, too wicked, or too ignorant to be treated by Tammany as a man and a brother if he would stand in with the machine and join the brotherhood. This secret of Tammany—the open secret—was explained to me in Chicago by a saloon-keeper of more than dubious morals who had been a Tammany captain in New York. I saw him the night after Dr. Parkhurst had scored his first great success over the politicians of New York. The ex-Tammany Captain shook his head when I asked him what he thought of Dr. Parkhurst’s campaign. He had no use for Dr. Parkhurst. For a time, he thought, he might advertise himself, which was no doubt his object, but after that everything would go on as before. The one permanent institution in New York was Tammany.
- 59. I asked himto explain his secret. “Suppose,” said I, “that I am a newly arrived citizen in your precinct, and come to you and wish to join Tammany, what would be required of me?” “Sir,” said he, “before anything would be required of you we would find out all about you. I would size you up myself, and then after I had formed my own judgment I would send two or three trusty men to find out all about you. Find out, for instance, whether you really meant to work and serve Tammany, or whether you were only getting in to find out all about it. If the inquiries were satisfactory then you would be admitted to the ranks of Tammany, and you would stand in with the rest.” “What should I have to do?” “Your first duty,” said he, “would be to vote the Tammany ticket whenever an election was on, and then to hustle around and make every other person whom you could get hold of vote the same ticket.” “And what would I get for my trouble?” I asked. “Nothing,” said he, “unless you needed it. I was twenty years captain and I never got anything for myself, but if you needed anything you would get whatever was going. It might be a job that would give you employment under the city, it might be a pull that you might have with the alderman in case you got into trouble, whatever it was you would be entitled to your share. If you get into trouble, Tammany will help you out. If you are out of a job Tammany will see that you have the first chance of whatever is going. It is a great power, is Tammany. Whether it is with the police, or in the court, or in the City Hall, you will find Tammany men everywhere, and they all stick together. There is nothing sticks so tight as Tammany.” Therein, no doubt, this worthy ex-captain revealed the great secret, of Tammany’s success. Tammany is a brotherhood. Tammany men stick together, and help each other.
- 60. The record ofTammany, however, hardly bears out the claim made for it by Mr. Croker as to the honesty and purity of its administration. From its very early days Tammany has had a bad record for dishonesty and utter lack of scruple. As early as 1837, two Tammany leaders, who had held the federal offices of Collector of the Port of New York, and of United States District Attorney for the Southern district of New York, skipped to Europe after embezzling, the one £250,000, the other £15,000. About twenty years later, another Tammany leader, who was appointed Postmaster for New York, advanced £50,000 of post-office money in order to carry Pennsylvania for Buchanan. These, however, were but bagatelles compared with the carnival of plunder which was established when Tweed was Tammany Boss. It was not until about the middle of the century that Tammany laid the hand upon the agency which for nearly fifty years has been the sceptre of its power. A certain Southerner, rejoicing in the name of Rynders, who was a leading man in Tammany in the Forties, organised as a kind of affiliated institution the Empire Club, whose members were too disreputable even for Tammany. These men, largely composed of roughs and rowdies, who rejoiced in the expressive title of the Bowery Plug Uglies, were the first to lay their hand upon the immigrant and utilise him for the purpose of carrying elections. Mr. Edwards, writing in McClure’s Magazine, says:— It was the Empire Club, indeed, which taught the political value of the newly-arrived foreigner. Its members approached the immigrants at the piers on the arrival of every steamship or packet; conducted them into congenial districts; found them employment in the city works, or perhaps helped them to set up in business as keepers of grog-shops. “Politics in Louisiana,” General Grant is reported to have said on one occasion, “are Hell.” They seem to have been very much like hell in the days when the Plug Uglies with Rynders at their head ruled the roast at Tammany. Mr. Edwards tells a story which sheds a lurid ray of light on the man and manners of that time. Mr. Godwin, who
- 61. preceded Mr. Godkinin the incessant warfare which the Evening Post has waged against Tammany, had given more than usual offence to Rynders. That worthy, therefore, decided to assassinate the editor as he was taking his lunch at the hotel. Mike Walsh, however, a plucky Irishman, interfered, and enabled Godwin to make his escape. When the intended victim had gone out— Rynders stepped up to Walsh and said: “What do you mean by interfering in this matter? It is none of your affair.” “Well, Godwin did me a good turn once, and I don’t propose to see him stabbed in the back. You were going to do a sneaking thing; you were going to assassinate him, and any man who will do that is a coward.” “No man ever called me a coward, Mike Walsh, and you can’t.” “But I do, and I will prove that you are a coward. If you are not one, come upstairs with me now. We will lock ourselves into a room; I will take a knife and you take one; and the man who is alive after we have got through, will unlock the door and go out.” Rynders accepted the challenge. They went to an upper room. Walsh locked the door, gave Rynders a large bowie-knife, took one himself, and said: “You stand in that corner, and I’ll stand in this. Then we will walk towards the centre of the room, and we won’t stop until one or the other of us is finished.” Each took his corner. Then Walsh turned and approached the centre of the room. But Rynders did not stir. “Why don’t you come out?” said Walsh. Rynders, turning in his corner, faced his antagonist, and said: “Mike, you and I have always been friends; what is the use of our fighting now? If we get at it, we shall both be killed, and there is no good in that.” Walsh for a moment said not a word; but his lip curled, and he looked upon Rynders with an expression of utter contempt. Then he said: “I told you you were a coward, and now I prove it. Never speak to me again.”
- 62. Mike Walsh, thehero of this episode of the bowie-knife, is notable as having been the first man to publicly accuse Tammany of tampering with the ballot-box. He was not the last by any means; but Tammany seems to have begun well, for, says Mr. Edwards:— Roscoe Conkling once said, chatting with a group of friends, that Governor Seward had told him that the Tammany frauds committed by the Empire Club in New York City in 1844 unquestionably gave Polk the meagre majority of five thousand which he obtained in New York State, and by which he was brought to the Presidency. FERNANDO WOOD. It is not surprising that with this beginning things went on from bad to worse until Mike Walsh, a few years before the War, publicly declared in a great Democratic meeting in the city:—
- 63. “I tell younow, and I say it boldly, that in this body politic of New York there is not political or personal honesty enough left to drive a nail into to hang a hat upon.” There is a fine picturesqueness about this phrase which enables it to stick like a burr to the memory. It was not, however, until the Irish emigration began in good earnest that Tammany found its vocation. Fernando Wood was first elected to the Mayoralty in 1854. Fernando Wood was a ward politician who first became known to the public by a prosecution in which it was proved that he had cheated his partner by altering the figures in accounts. He did not deny the charge, but pleaded statutory limitation. Having thus succeeded in avoiding gaol, he promptly ran for the Mayoralty, and was duly elected. With him came what Mr. Godkin calls “the organisation of New York politics on a criminal basis.” The exploits of Fernando Wood, however, were thrown entirely into the shade by the lurid splendour of his successor. This was William M. Tweed, the famous “Boss” Tweed, who began his life as a journeyman, and ended it in Ludley Street Gaol, after having ruled New York for years, as if he were a Turkish Pasha. After serving apprenticeship as a Member of the New York Senate, Deputy Street Commissioner, and President of the Board of Supervisors, he gradually made his way upwards until he was recognised as Boss of Tammany. It was not, however, until the year 1868 that he succeeded in giving the public a true taste of his quality. Even hardened Tammany politicians were aghast at the colossal frauds which he practised at the polls—frauds not only unique in their dimensions, but in the exceeding variety and multiplicity of their methods. On January 1st, 1869, Tweed and his allies began to plunder the city in a fashion which might have made the mouth of a Roman proconsul water. His ally, Connolly, was made Comptroller, while Tweed himself found ample scope for his fraudulent genius in the posts of Deputy Street Commissioner and Supervisor. In the first year he issued fraudulent warrants for £750,000. The money was spent fast and furiously. Tweed was a fellow of infinite variety, and
- 64. he seemed almostto revel in the diversity of methods by which he could plunder the public. One very ingenious and simple fraud was his securing an Act of the Legislature, making a little paper which he owned the official organ of the City Government. In that capacity he drew £200,000 a year from the rates and taxes, as compensation for printing the report of the proceedings of the Common Council. Mr. Edwards says:— He established a printing company, whose main business was the printing of blank forms and vouchers, for which in one year two million eight hundred thousand dollars was charged. Another item was a stationer’s company, which furnished all the stationery used in the public institutions and departments, and this company alone received some three millions a year. On an order for six reams of cap paper, the same amount of letter paper, two reams of notepaper, two dozen pen-holders, four small ink-bottles, and a few other articles, all worth not more than fifty dollars, a bill of ten thousand dollars was rendered and paid. The frauds upon which the conviction of Tweed was obtained consisted in the payment of enormously increased bills to mechanics, architects, furniture-makers, and, in some instances, to unknown persons, for supplies and services. It was the expectation that an honest bill would be raised all the way from sixty to ninety per cent. In the first months of the ring’s stealing the increase was about sixty per cent. Some of the bills were increased by as much as ninety per cent., but the average increase was such as to make it possible to give sixty-seven per cent. to the ring, the confederates being allowed to keep thirty-three per cent.; and of that thirty-three per cent. probably at least one-half was a fraudulent increase. After a time the outrageous nature of his stealings provoked a revolt in Tammany itself. It is to this which Mr. Croker looks back with such proud complacency as marking the advent of reformed Tammany. Tweed was beaten at the elections, and his opponents secured a majority on the Board of Aldermen. Thereupon the resourceful rascal promptly went down to Albany, bought up a sufficient number of
- 65. Congressmen and senatorsto give him control of the Legislature, and so secured a new Charter for New York, which legislated his opponents out of office. By this Charter a board of audit was created which consisted of Tweed, Connolly and Mayor Hall. What followed is thus described by the Nation:— The “Board” met once for but ten minutes, and turned the whole “auditing” business over to Tweed. This sounds like a joke, but is true. Tweed then went to work, and “audited” as hard as he could, Garvey and other scamps bringing in the raw material in the shape of “claims,” and he never stopped till he had “audited” about 6,000,000 dols. worth. Connolly’s part in the little game then came in, and that worthy citizen drew his warrants for the money, which that simple-minded “scholar and gentleman” the Mayor endorsed, without having the least idea what was going on. Tweed’s share of the plunder amounted to about 1,000,000 dols. in all. The Joint Committee, reporting on the condition of the city’s finances, declared that the discoverable stealings of three years are 19,000,000 dols., which is probably only half the real total. Never was a more unblushing rascal, as Mr. Tilden said in his account of Tweed’s sovereignty. The Tammany Ring controlled the State Legislature, the police, and every department or functionary of the law; several of the judges on the bench were its servile instruments, and issued decrees at its command; it secured the management of the election “machine,” and “ran” it at its own free will and pleasure; a large part of the press was absolutely at its disposal. In the course of three years it had paid to eleven newspapers the sum of 2,329,482 dols. (about £466,000) nominally for advertisements, most of which were never even published, or never seen. Not only the City government, but the lion’s share of the State government also had fallen into the hands of “Boss” Tweed and his confederates. Millions of dollars were stolen by the conspirators by means of “street openings,” “improvements,” new pavements, and other frauds. The Ring took from the public treasury a sum amounting to over £1,500,000 for furnishing and “repairing” a
- 66. new Court-house. Thecharges for plastering alone came to about £366,000. For carpets, warrants were drawn for £120,000, although there were scarcely any carpets in the building. The floors were either bare, or covered with oil-cloth. Nearly £100,000 was alleged to have been paid for iron safes, and over £8,200 for “articles” not defined and never found. The total sum stolen was over £4,000,000. WILLIAM M. TWEED. Tweed’s brief but dazzling career—for he was indeed a hero clad in Hell-fire—is said by President Andrews to have cost the City of New York 160,000,000 dols. The fine levied by Germany on the City of Paris after the War of 1870-1 was only one-fourth that amount. Fraud may be more costly than War. The total direct property loss occasioned by the great fire at Chicago in 1871, when three square miles of buildings were burned down, and 98,500 persons rendered
- 67. homeless, was only30,000,000 dols. above the plunder of Tweed and his gang. Thus Fraud can be almost as ruinous as Fire. MR. TILDEN. Tweed was a fellow, if not of infinite jest like poor Yorick, at least of infinite insolent humour. In 1871 he boasted that he had amassed a fortune of 20,000,000 dols. Nor did he in the least scruple to avow the means by which he acquired it. President Andrews, of Brown University, in telling the history of the last quarter century, says, “He used gleefully to show his friends the safe where he kept money for bribing legislators, finding those of the Tammany-Republican stripe easiest game. Of the contractor who was decorating his country place at Greenwich he inquired, pointing to a statue, ‘Who the hell is that?’ ‘That is Mercury, the god of merchants and thieves,’ was the reply. ‘That’s bully,’ said Tweed; ‘put him over the front door.’”
- 68. Tweed was tothe last popular with the masses of the people. Even when the whole town was ringing with proofs of his guilt, he stood as candidate for the Senate of New York State, and was elected. He had distributed in the poorer districts some £10,000 worth of coal and flour, and one of his champions brought down the house by declaring that “Tweed’s heart has always been in the right place, and, even if he is a thief, there is more blood in his little finger and more marrow in his big toe than the men who are abusing him have in their whole bodies.” This man, with this excessive development of marrow in his big toe, was ultimately run down by Mr. Tilden and the Committee of Seventy. Connolly, the Comptroller, weakened and made terms with his opponents by appointing Mr. Green as Deputy-Comptroller. Mr. Green had little difficulty in laying hands upon all that was necessary in order to secure the prosecution and conviction of Tweed. Tweed’s two infamous judges were driven from the bench, and he himself was clapped into gaol. He made his escape, and sought refuge in Spain. He was, however, delivered up to the American authorities, and reconducted to prison, where he died. To the last Tweed retained possession of much of his ill-gotten wealth. An offer which was made to surrender the residue of his millions in return for his liberty was rejected. Tweed thought himself on the whole, an ill-used man. The judge who tried Tweed declared that he had perverted the “power with which he was clothed in a manner more infamous, more outrageous, than any instance of a like character which the history of the civilised world afforded.” But Tweed himself declared that he believed he had done right, and was willing to “submit himself to the just criticism of any and all honest men.” From this it would seem that Mr. Croker is not alone in his imperturbable consciousness of public rectitude. Tweed on one occasion admitted that he had perhaps erred, but he explained he was not to blame. The fault lay with human nature in the first place, and with the system under which New York was governed in the second. Therein, no doubt, he was right. “Human
- 69. nature,” he said,“could not resist such temptations as were offered to men who were in power in New York, so long as the disposition of the offices of the city was at their command.” The most outrageous thing that Tweed ever did was to pass a bill through the State Legislature at Albany, giving the judges unlimited power to punish summarily whatever they chose to consider to be contempt. By this law, which was fortunately vetoed by the Governor, every newspaper in New York would have been gagged as effectually as the press of Constantinople. After Tweed fell, Tammany was reorganised under Honest John Kelly and Richard Croker. Mr. Godkin declares that Honest John Kelly was only honest in name. He says:— John Kelly practised the great Greek maxim “not too much of anything,” simply made every candidate pay handsomely for his nomination, pocketed the money himself, and, whether he rendered any account of it or not, died in possession of a handsome fortune. His policy was the very safe one of making the city money go as far as possible among the workers by compelling every office-holder to divide his salary and perquisites with a number of other persons. The same system had prevailed down to the year 1894, when Tammany, for the first time in many years, was driven from power. Just before the upset, the New York Evening Post published the records of the twenty-eight men who now or recently composed the Executive Committee of Tammany. It showed that they were all professional politicians, and that among them were one convicted murderer, three men who had been indicted for murder, felonious assault, and bribery, respectively, four professional gamblers, five ex- keepers of gambling houses, nine who either now or formerly sold liquor, three whose fathers did, three former pugilists, four former rowdies, and six members of the famous Tweed gang. Seventeen of these held office, seven formerly did, and two were favoured contractors.
- 70. By these menNew York was governed down to the year 1894. All the efforts of the reformers seemed in vain. Mr. Godkin reluctantly confessed:— The power of the semi-criminal organisation known as Tammany Hall not only remains unshaken, but grows stronger from year to year. Every year its management descends, with perfect impunity, into the hands of a more and more degraded class. But it is ever the darkest hour before the dawn. Although on the very eve of the November election of 1894 it was declared that “Mr. Croker held almost as despotic a sway over New York as an Oriental potentate over his kingdom,” one month after that statement had been made he was hurled from power by a great outburst of popular indignation. How that was brought about I will now proceed to tell. MR. E. L. GODKIN, EDITOR OF THE “EVENING POST,” NEW YORK. The sworn foe of Tammany.
- 71. CHAPTER IV. THE LEXOWSEARCHLIGHT. Mr. Lowell good-humouredly chaffed John Bull when he declared that He detests the same faults in himself he neglected, When he sees them again in his child’s glass reflected, and we only need to glance at current English criticisms upon American affairs to justify the poet’s remark. Especially is this the case with a vice which of all others is regarded as distinctively English. John Bull has plenty of faults, but of those which render him odious to his neighbours there is none which is quite so loathsome as his “unctuous rectitude.” That phrase, coined by Mr. Rhodes to express the contempt which he and every one who knew the facts felt on contemplating the hypocrisy and Pharisaism displayed in connection with the Jameson Raid, is likely to live long after Mr. Rhodes has vanished from this mortal scene. This tendency to Pharisaism and self-righteous complacency, which thanks God that it is not as other men are, is one of those vices which John Bull’s children seem to have inherited in full measure. We are pretty good at Pharisaism in the Old Country, but we are “not a circumstance,” to use the familiar slang, when we compare ourselves to some of the Pharisees reared across the Atlantic. This has nowhere been brought into such strong relief as when on the very eve of the exposure and discomfiture of Tammany their spokesmen took the stump and talked like very Pecksniffs concerning the immaculate purity of Tammany Hall.
- 72. The same characteristicis observable in all of them. Whether it is Boss Tweed, appealing confidently to the verdict of honest men upon a career of colossal theft and almost inconceivable fraud; or Mr. Croker, who, after surveying his whole life, declares that he has not discovered a single action which he has reason to regret, for he has not done anything but good all his life; or Bourke Cochran, who was at one time the Apollo and the Demosthenes of Tammany, the same unctuous rectitude oozes out of every pore. When Tammany was at its heyday of prosperity and power in 1889, it assembled in its thousands to cheer enthusiastically the impassioned oratory of Mr. Cochran, who declared, as among the self-evident truths which found an echo in every breast, that “if corruption prevails among the people, liberty will become a blighting curse, subversive of order. Corruption once begun, decay is inevitable and irresistible; the destruction of the Republic is immediate, immeasurable, irredeemable; since history does not record a case of a popular government which has been arrested in its downward course.” Tammany listened to this with ecstatic admiration, cheered to the echo their eloquent oracle, and then went on using the proceeds of a system of blackmail for the perfecting of an engine of corruption to which it is difficult to discover a parallel in the annals of mankind. In Mr. Croker’s case, his calm consciousness of incorruptible virtue seems to be based upon a curious inversion of a belief in a Divine Providence. Tammany is not strong in theology, but Mr. Croker, in talking to me, based his argument in favour of the excellence of Tammany on the postulate that the government of the universe was founded on the law of righteousness. This being the case, it was only possible to reconcile the continued existence of Tammany on one of two hypotheses. Either the domination of evil was permitted for a season for some sufficient cause hidden in the inscrutable mysteries of the Divine councils, or we must boldly assert that, all evidence to the contrary notwithstanding, Tammany rule was in accordance with the eternal law, Credo quia impossibile, rather than admit that so great an anomaly as a terrestrial Inferno could be permitted to exist by the good government of God. Mr. Croker, of
- 73. course, adopted thelatter hypothesis. There is much in it, no doubt, especially to those in Mr. Croker’s position. It is, however, open to the fatal objection that the same process of logic would à fortiori secure a certificate of good conduct for the Great Assassin of Stamboul himself. The Ottoman Empire has lasted even longer than Tammany Hall, but even Mr. Croker would shrink from maintaining that Abdul Hamid was on that account the exemplary vicegerent of the Almighty. This Pharisaic panoply in which Tammany was clad, as in a coat of mail, was no small element of its strength. The consciousness of wrong-doing is always an element of weakness. Not until a man can do evil and persuade himself that he is doing good can he silence that conscience which makes cowards of us all. Probably this unctuous rectitude on the part of Tammany and its Boss should be estimated as one of the chief obstacles in the way of the scattered and despairing band of reformers who, five or six years ago, confronted the stronghold of iniquity entrenched in their midst. Its position, indeed, appeared almost impregnable. Tammany Hall commanded an annual revenue large enough to equip and maintain a small army. It had under its orders the whole of the executive force in its police—a body of men practically above the law, armed with powers hardly inferior to those of the police of St. Petersburg. Besides the police, all the persons on the pay-rolls of the City and County were under the thumb of the Boss. There was hardly a city official, from the highest to the lowest, who did not hold office by the sovereign will and pleasure of Tammany. As there are 27,000 names on those pay-rolls, all of whom were voters and were taxable to an almost unlimited extent whenever the Tammany exchequer needed to be replenished, it is obvious how enormous were the odds against the assailants of Tammany.
- 74. Photo by TomReveley, Wantage. RICHARD CROKER IN HIS GARDEN AT WANTAGE, BERKSHIRE. But the unctuous rectitude of its leaders, the prompt obedience of the police Janissaries, and the discipline of the standing army of the twenty-seven thousand Pretorians on the city pay-rolls, were by no means the only difficulties which had to be overcome. Tammany Hall itself might be compared to a central citadel or keep of a Norman fortress. The outworks consisted of all the saloons, gaming hells, and houses of ill-fame in the City of New York. Some of these, no doubt, were by no means enthusiastic in support of the powers that be, but they resembled tribes which, having been subdued by force of arms, are compelled to pay tribute and use their weapons in
- 75. support of theirconquerors. In New York, just before the revolt against Tammany, the number of licences for the sale of intoxicants in New York City was over 6,000. The number of unlicensed drinking places was estimated at from 2,000 to 3,000. Each of these saloons might be regarded as a detached outwork, holding a position in advance of the main citadel, and covering it from the attack of its foes. In those days it used to be said that licences were granted by the Excise Board to anybody who had not served a term in a penitentiary. One indignant divine declared that it was perfectly safe to say that, if the Devil himself should apply to the Excise Board for a licence to set up a branch establishment on the children’s playground in the Central Park, it would be granted. As to the other establishments of even worse fame than the saloon, there was an unwritten contract by which, in return for tribute paid directly or indirectly, they were shielded by the strong arm of Tammany from the enforcement of the law. It was calculated that if all the saloons in New York were placed side by side, averaging them at only twenty feet frontage each, they would form a line of circumvallation twenty miles long. To put it in another way, there was on an average one saloon for every thirty voters. In addition to its control of the saloon, Tammany had two extremely important financial resources which have not yet been mentioned. The first was the control of the city contracts. A great city like New York, with an expenditure that exceeded that of the whole Federal Government of the United States fifty years ago, had an enormous means of influence at its disposal in the mere granting of contracts. But even this was a comparatively trivial element in the financial strength of Tammany. There existed in New York, as in almost every city, great corporations representing enormous capital, and dividing gigantic dividends, which, in the Tammany scheme of the universe, might have been created for the express purpose of furnishing an unfailing supply of revenue to the party chest. The corporations which enjoyed franchises from the city, giving them control of the
- 76. streets, whether forthe purpose of traction, of lighting, or of electrical communication, were Tammany’s milch cows. They all possess monopolies, granted to them in the first instance either by corruption or by negligence, which enable them to plunder the public. These monopolies can only be terminated or modified by the Legislature, and the Legislature can only act in obedience to the party machine. All that needs to be done when the campaign fund runs low is for the Boss to intimate to the various corporations that milking time has come, and that if they do not contribute liberally of their substance to the party treasury, Tammany will no longer be able to give them protection when the usual attack is made next session upon their monopoly or their franchise. Money is the sinews of war, and as the Tammany war chest was always full, Tammany snapped its fingers at all its enemies, and contemptuously declared that the reformers did not amount to a row of pins. THE CHILDREN’S PLAYGROUND, CENTRAL PARK, NEW YORK.
- 77. The outlook undoubtedlywas very gloomy. From the point of view of practical politics it was simply hopeless; nevertheless, in a couple of years the fortress was stormed, and the government of New York placed in the hands of the Reformers. The story of the way in which this was brought about should never be forgotten by all those who are called upon to lead forlorn hopes against immense odds. As long as the world lasts, such narratives are among the most precious cordials which in times of danger and distress restore the courage and revive the faith of man. Dr. Parkhurst’s attack on Tammany is one of the latest of a long series of victories achieved by the leader of an outnumbered handful. When Gideon went forth against the hosts of Midian with only three hundred followers, he left a leading case on record for the encouragement of all who should come after. How many reformers and revolutionists who have helped the world forward in the path of progress have been cheered by the dream in which the Midianitish soldier saw a cake of barley bread smite and overturn the multitudinous camp of the conqueror, history does not record! But if ever a man needed the inspiration of that barley cake it was Dr. Parkhurst, when in 1892 he set himself to the desperate task of wresting New York City from the grasp of Tammany. Dr. Parkhurst was a Massachusetts minister of Puritan ancestry, who, in 1880, at the age of thirty-eight, had been called to Madison Square Church, in New York. For ten years he went in and out among the people, quietly building up his church, ministering to his congregation, and learning at first-hand the real difficulties which offered almost insuperable obstacles to right living in New York. In 1890, on the eve of the November election, he preached a sermon on municipal politics, which, although it failed in influencing the polls, nevertheless marked Dr. Parkhurst out as the man to succeed Dr. Howard Crosby as President of the Society for the Prevention of Crime. He took office in 1891. In less than twelve months he began the campaign from which he never withdrew his hand until the government of the city was wrested from the control of Tammany.
- 78. Nothing is morecharacteristic, both of the state of things in New York and the uncompromising directness of Dr. Parkhurst, than the fact that he had no sooner assumed the control of the Society for the Prevention of Crime than he adopted as his motto the significant watchword, “Down with the Police!” That fact alone speaks volumes as to how utterly New York City had fallen under the control of the Evil One. For a society for the prevention of crime to adopt “Down with the Police!” as its watchword, seems to us of the Old World absolutely inconceivable. The police exist for the prevention of crime, yet here was a society of leading citizens, presided over by a doctor of divinity, putting in the forefront of its programme the formula “Down with the Police!” Strange though it may seem to us, the best people of New York understood and appreciated what Dr. Parkhurst was after. But it was not till the 14th of February, 1892, that he put the trumpet to his lips and blew a blast the echoes of which are still sounding through the world. His sermon was an impeachment of the Government of New York, the like of which had seldom been heard before in a Christian pulpit. If any one questions the justice of the title of this volume, let him read what Dr. Parkhurst said in the sermon, of which the following sentence is a fair sample:— There is not a form under which the Devil disguises himself that so perplexes us in our efforts, or so bewilders us in the devising of our schemes, as the polluted harpies that, under the pretext of governing this city, are feeding day and night on its quivering vitals. They are a lying, perjured, rum-soaked and libidinous lot. That was plain speaking in honest, ringing Saxon, for Dr. Parkhurst knew that there was no better way of spoiling the trump card of the Devil’s game than to refuse to let him keep things mixed. He maintained that the district attorney, or, as we should say, the public prosecutor, was guilty of complicity with vice and crime: that “every effort to make men respectable, honest, temperate, and sexually clean was a direct blow between the eyes of the mayor and his whole gang of drunken and lecherous subordinates, who shielded
- 79. and patronised iniquity.”Criminals and officials, he declared, were hand-and-glove, and he summed up the whole matter in the following concise exposition of the status quo in “Satan’s Invisible World” in New York, 1892:—“It is simply one solid gang of rascals, half of the gang in office and the other half out, and the two halves steadily catering to each other across the official line.” From Frank Leslie’s Weekly. REV. C. H. PARKHURST, D.D., DENOUNCING TAMMANY’S GOVERNMENT OF NEW YORK. Of course there was a great outcry. Some good people were scandalised, while as for the bad ones, they were simply outraged at such “violent and intemperate utterances in the pulpit.” One of the police captains declared “it was a shame for a minister of the Gospel to disgrace the pulpit by such utterances.” Dr. Parkhurst was summoned before the Grand Jury, and solemnly reproved for making
- 80. statements which hecould not for the moment substantiate with chapter and verse. When the Grand Jury condemned him and the judge rebuked him, Tammany was in high glee; but Dr. Parkhurst bided his time. He was not a man to be “downed” by censure. Finding that his general statements were scouted because he could not produce first hand evidence as to the literal accuracy of each particular instance on which he built up his general finding, he took the bold and courageous step of going himself through the houses of ill-fame, gaming hells, and other resorts which were running open under the protection of the police. He was accompanied in his pilgrimage by a detective and a lawyer, and for three weeks every night Dr. Parkhurst, to use his own phrase, “traversed the avenues of our municipal hell.” They entered into no houses not easy of access, went into no places which were not recognised as notorious, and were perfectly well known by the constable on the beat. In one case they succeeded in proving police collusion by getting the policeman on beat to stand guard while they visited the house, ostensibly for an immoral purpose, in order to warn them against any signs of a possible raid. Having thus mastered his facts and obtained incontrovertible evidence at first hand as to the fact of police complicity in the wholesale violation of the law, Dr. Parkhurst stood up in his pulpit on the morning of March 13th, 1892, and once more arraigned the city authorities. This time, however, he was armed with a mass of facts ascertained at first hand, and supported by unimpeachable, independent testimony. He brought forward no fewer than two hundred and eighty-four cases in which the law was flagrantly violated under the noses of the police, who, he maintained, were guilty of corrupt complicity in the violation of the law they were appointed to enforce. It was a great sermon, and one that shook the city to its centre. Some idea of its drift and spirit may be gained from this extract:— There is little advantage in preaching the Gospel to a young fellow on Sunday, if he is going to be sitting on the edge of a Tammany-
- 81. maintained hell therest of the week. Don’t tell me that I don’t know what I am talking about. Many a long, dismal, heart-sickening night, in company with two trusted friends, have I spent since I spoke on this matter before, going down into the disgusting depths of this Tammany-debauched town; and it is rotten with a rottenness that is unspeakable and indescribable, and a rottenness that would be absolutely impossible except by the connivance, not to say the purchased sympathy, of the men whose one obligation before God, men, their own consciences, is to shield virtue and make vice difficult. Now, that I stand by, because before Almighty God I know it, and I will stand by it though buried beneath presentments as thick as autumn leaves in Vallombrosa, or snowflakes in a March blizzard. And stand by it Dr. Parkhurst did. He was promptly summoned again before the Grand Jury, and this time he had his facts at command. Instead of being rebuked, the Grand Jury reported emphatically that it was impossible to reconcile the facts presented by Dr. Parkhurst with any other theory than that of wholesale police corruption. The following month various keepers of disreputable houses were prosecuted upon Dr. Parkhurst’s evidence, when every effort was made to damage Dr. Parkhurst by representing him as the vicious criminal who was responsible for the very evils which he had brought to light. It is the old, old story. As long as you sit still and say nothing you are all right, but the moment you call attention to a hideous wrong or a shameful crime, all those whose iniquities you have disclosed combine with your enemies in order to make a busy public believe that it is you who have exposed the crime who is the real criminal, while they, poor innocents, are the injured parties, for whom a respectable public should have nothing but sympathy, and commiseration. The ferocity of the attacks upon Dr. Parkhurst provoked a reaction in his favour. The City Vigilance Society was formed by the association
- 82. of forty religiousand secular societies of the city. The work of sapping and mining went steadily on. In order to bring odium upon Dr. Parkhurst, the police suddenly decided to close up several houses of ill-fame, so as to turn their unfortunate occupants into the streets on one of the coldest nights of the winter of 1892. Dr. Parkhurst met this by promptly providing homes for all the dispossessed women. Foiled in this cruel manœuvre, the police prosecuted Dr. Parkhurst’s detective for an alleged attempt to levy blackmail. This was Satan reproving sin with a vengeance, and for the moment it had a temporary success. The detective was convicted, in the first instance, but on appeal the verdict was set aside. Undaunted, however, by this reverse, Dr. Parkhurst began to carry the war into the enemy’s camp. He got up cases against forty-five of the sixty- four gambling and disorderly houses which were allowed to run by the police captain of a single precinct. The trials followed with varying results. It was evident that the difficulties in the way of obtaining a full disclosure of police corruption could only be overcome by special measures. Public opinion was now deeply stirred, and the Chamber of Commerce memorialised the Senate of New York City to hold an inquiry into the Police Department of New York. The Senate appointed a Committee of Investigation, and passed a bill providing for the payment of its expenses. This bill was vetoed by Governor Flower, himself a Democrat, whose veto elicited another illustration, if it were wanted, of the marvellous Pharisaism of Tammany and its friends.
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