1. RUNNING HEAD: IPSC DIFFERENTIATION INTO HSCS 1
Methodology: Comparing Various iPSC Generation Protocols to Optimize
Differentiation Efficiency
Team BLOOD: Michael Amedeo, Sanna Darwish, Aria Jalalian, Prableen Kaur, Sayli
Korde, Eric Lai, Amil Sahai, George Thomas, Akhil Uppalapati, Farah Vejzagic, Abriana Walls,
Elizabeth Zipf
University of Maryland, College Park
Honor Pledge: we pledge on our honor that we have not given or received any
unauthorized aid on this assignment.
2. IPSC DIFFERENTIATION INTO HSCS 2
Table of Contents
Methodology
Phase 1: Method 1
Phase 1: Method 2
Phase 1: Method 3
Phase 2
Data Collection and Analysis
In vitro qualitative analysis
In vivo quantitative analysis
Anticipated Results
References
Appendix A: Detailed Protocols
Appendix B: List of Abbreviations
3. IPSC DIFFERENTIATION INTO HSCS 3
Methodology
Many iPSC production methods exist, but a minute amount of research has actually
compared these methods and determined which techniques are the most efficient for inducing
hematopoiesis (see Appendix B for comprehensive list of abbreviations). The aim of our
research is to answer the following questions:
(1) What is the most qualitatively and quantitatively effective method to produce iPSCs?
(2) What is the most qualitatively and quantitatively effective method to use iPSCs to
induce hematopoiesis?
In order to determine the most effective way to produce iPSCs that differentiate
hematopoietically, various methods of iPSC production must be employed, and a standard
method of differentiation followed. Our testing will occur in two phases outlined below:
Phase 1: This portion of testing will involve transforming somatic cells into iPSCs. This
will be determined using immunofluorescence. Three methods will be tested: PBMNC
generation, PiB transposon generation, and lentiviral gene induction.
Phase 2: This portion of testing will involve differentiating iPSCs using OP9 coculture.
Results will then be tested using in vitro and in vivo methods.
The three Phase 1 methods, which have been studied before, have been selected due to
their efficiency in other studies, including time efficiency and generation efficiency. They have
also been selected for their relative simplicity, making them more promising candidates to use in
a widespread therapy treatment.
4. IPSC DIFFERENTIATION INTO HSCS 4
Of the three methods we will use, we predict that the PBMNC method will be the most
successful, since it has been more extensively studied compared to the other methodologies
presented, with comparatively more success.
Phase 2 will include the process of iPSC coculturing in the OP9 (mouse bone marrow
cell), and then later applying cytokine or collagenase to induce differentiation of specific cells.
Data will be collected on the success of differentiation through immunofluorescence and
engraftment in mice.
Phase 1: Method 1
Peripheral Mononuclear Blood Cell (PBMNC) iPSC generation. Several approaches
have been used by many different groups in order to efficiently induce pluripotency in cells.
Some of these factors include viral integrating vectors (retrovirus, lentivirus), viral non-
integrating vectors (adenovirus), Sendai virus, bacterial artificial chromosome (BAC)
transposons, episomal vectors, proteins or RNA delivery (Meraviglia et al., 2015). Previous
research has shown that usage of viral vectors yields high efficiency reprogramming. However,
viral vectors often integrate themselves into the host genome where a variety of confounding
variables can come into effect, including random insertional mutagenesis, alteration of certain
gene expression, and reactivation of silenced transgenes. Sendai virus also is a method that
achieves high efficiency reprogramming, but its cost prevents it from usage as a therapy.
Starting cell source has remained an issue with many other studies that have been
conducted so far. Currently, skin fibroblasts are the most popular source cell to develop into
iPSCs. Other cell types have been used as well, such as keratinocytes, BM mesenchymal stem
cells, adipose stromal cells, hair follicles, and dental pulp cells (Meraviglia et al., 2015). The
5. IPSC DIFFERENTIATION INTO HSCS 5
isolation of these cells often require surgical procedures, and the cells are often quite
cumbersome to handle. Also, several weeks are needed for in vitro expansion of the cell line into
something usable in a study. Thus, these varying types of cells are often inaccessible, and do not
become iPSCs easily.
Meraviglia et al. (2015) has proposed a PBMNC iPSC generation method to make iPSC
cells easily accessible that could be integrated into our study. To do so, the group changed the
source of cells and tried to induce reprogramming with PBMNCs. First, the group isolated the
PBMNCs and cultured them. Then they transfected the cultured PBMNCs with episomal
plasmid. Finally, the researchers plated the transfected PBMNCs onto MEFs (see Appendix A
for detailed protocols). PBMNCs can be isolated using a minimally invasive procedure and do
not require extensive expansion efforts in order to reprogram. They also represent one of the
most accessible cell sources available at this time. For the purposes of our study, this
methodology will provide us with a solid comparison point to other methodologies that use a
different source to attain iPSC, such as keratinocytes and fibroblasts.
Phase 1: Method 2
Lentiviral gene induction. For the conversion of keratinocytes and fibroblasts to iPSCs,
we will utilize a lentiviral method of inducing genes into these somatic cells. This technique can
serve as a baseline with which we can convert fibroblasts and keratinocytes into iPSCs. In our
process, we will also infect somatic cells with a reverse tetracycline-controlled transactivator
(rtTA) in order to induce gene expression in the presence of a specific antibiotic. It is necessary
that we determine an antibiotic that will provide us with optimal conversion.
6. IPSC DIFFERENTIATION INTO HSCS 6
We have chosen doxycycline as our preferred antibiotic due to its favorable role in the
PI3K-AKT pathway. FOXO3 is a human protein belonging to the class of forkhead transcription
factors and is a major trigger for apoptosis through the upregulation of genes expressing cell
death and inhibition of anti-apoptotic genes like FLIP (Skurk et al., 2004). Protein
kinase (AKT) mediates cell survival through the inhibition of pro-apoptotic proteins like
FOXO3. Doxycycline is an intracellular kinase activator of the PI3K-AKT pathway and is thus
involved in cell renewal, growth, and survival (Chang et al., 2014). Therefore, we will use a
dox-inducible gene expression system in order to promote AKT activation, signaling for cell
survival and obtaining optimal yield for conversion efficiency.
Lentivirus preparation. We will need to produce our lentivirus through preparation of a
10 cm plate of HEK-293 cells. First, we will incubate a mixture containing 30 μl FuGENE 6 and
600 ul serum-free medium (SFDMEM) at approximately 25°C for 5 minutes. Next, we will mix
5 μg lentiviral plasmid (Oct4, Sox2, Klf4, and Nanog) with 4 μg each of viral packaging vectors
(pMDL, pRSV and pVSV-G) and 10 μL rtTA (Wang & McManus, 2009). Vectors will be
packaged by 293 cells through co-transfection by the aforementioned viral packaging vectors. It
is also important to titer the lentiviral product using enzyme-linked immunosorbent assay
(ELISA) in order to determine the concentration of the core antigen and deliver a consistent
amount of viral product to our somatic cells.
For our next step, we will then allow fibroblasts to grow in Dulbecco’s Modified Eagle
Medium (DMEM) with 10% fetal bovine serum (FBS) in addition to glutamine and β-
mercaptoethanol (Maherali et al., 2008). Keratinocytes will be grown on collagen IV in
keratinocyte-free medium and invitrogen. We will add lentiviral stock to each well so that the
7. IPSC DIFFERENTIATION INTO HSCS 7
final volume approximates 600 μL including the DMEM (Nethercot et al., 2011). Our control
well will contain only DMEM and fibroblasts or keratinocytes, with no presence of lentiviral
stock containing transcription factors. We will use live cell staining to determine if conversion
has occurred in our cell cultures every day after day ten.
Phase 1: Method 3
PiB tranposon system. Reprogramming of mouse embryonic fibroblasts via DNA
transfection of factors Oct4, Klf4, Myc-c, and Sox2 has been shown to produce transgene-free
iPSCs (Yusa et. al). One method of transfecting these factors is the PiggyBac (PiB) transposon
system, where delivery of transcription factors is achieved by linking them to the self-cleaving
polypeptides. We will utilize the PiB transposon system to induce pluripotency in our mouse
embryonic fibroblasts (MEFs).
Our first step will be to construct PiB transposon-based
reprogramming vectors. This will be done by combining cDNAs that
encode for Oct4, Myc-c, Sox2, and Klf4 into one reading frame
separated by sequences that encode for 2A peptides and placed under
constitutively active CAG promoter. We will add Lin28 as well as that
has been shown to enhance reprogramming efficiency (Yusa et al.).
Additionally, we will put a pu∆tk cassette in order to serve as a
marker for future transposon removal. The image below serves as a
diagram for what our PiB transposon expression vector will look like
8. IPSC DIFFERENTIATION INTO HSCS 8
(Yusa et al.).
Figure 1.Model PiB transposon expression vector. Oct4, Sox2, Klf4, Myc-c, Lin28 all put into
one reading frame, separated by sequences encoding 2A peptides.
We will then use cationic lipofection via Lipofectamine to deliver 2 µgs of the PiB
transposon and 2 µgs of the PiB Transposase Expression Vector to the MEFs on six-well plates
(Yusa et. al). This will transfect the transcription factors into the genome of the MEFs. One day
after transfection, the cells are plated onto a feeder layer at a 1:18 ratio and cultured in a serum-
free ESC medium free of valproic acid (Yusa et. al). Our cells will be cultured with the F15L
medium, a serum-free medium that has been shown to induce reprogramming in MEFs. After 5
days in culture, immature iPSC colonies will be visible. After 13 days, colonies should be large
enough to be analyzed (Yusa et. al). The expected yield will be approximately 106 cells in about
1000 colonies. The colonies will be recognized via Nanog immunostaining as well as alkaline
phosphatase staining. This staining will test the colonies for Nanog, a gene that is indicative of
successful reprogramming. This methodology has been shown to have an approximate
transfection efficiency rate of 10% in previous studies (Yusa et. al).
9. IPSC DIFFERENTIATION INTO HSCS 9
Transposon removal. Our next step will be to remove the transposons from the iPSC in
order to improve the pluripotency of our stem cell line. In order to do so, we will utilize a
fialuridine (FIAU) selection system to determine which iPSC colonies are FIAU-resistant, as it
has been shown that the FIAU-resistant colonies are transposon-free with this method (Yusa et
al.).
Once the PB transposons are removed from the iPSC colonies, our next objective will be
to test the validity and pluripotency of our cell colonies. This will be done by analyzing the
DNA methylation status at the Oct4 and Nanog promoter regions by bisulphite sequencing (Yusa
et. al). For the cell to be an iPSC, the promoter region must be demethylated. Thus, if we find
that the Oct4 and Nanog promoter regions are not methylated, we can safely conclude that our
cells are pluripotent.
Phase 2
Justification for use of OP9. In order to induce differentiation of iPSCs into
hematopoietic stem cells, direct stromal cell contact is necessary. OP9 is a line of mouse bone
marrow stromal cells that has successfully been used in many studies to produce hematopoietic
stem cells from embryonic stem cells. These stromal cells however, have more recently been
found to be effective in inducing the differentiation of iPSCs as well (Choi et al., 2009; Choi et
al., 2011). We plan to use the OP9 line because it has several key advantages over other methods
of producing hematopoietic stem cells. iPSC/OP9 coculture takes a relatively short time,
developing cells with robust differentiation potential after about 6-10 days, which benefits us by
reducing the amount of time we will need to complete this phase of our research. The OP9 cell
line is also advantageous in that it is sufficient to induce differentiation on its own and does not
10. IPSC DIFFERENTIATION INTO HSCS 10
require exogenous growth factors such as added cytokines. Another potential mouse bone
marrow stromal cell line we could use is S17. The downside of using this cell line, however, is
that it is less effective than OP9, taking more time (8-21 days), and producing less CD34+ and
colony forming cells (Vodyanik et al., 2005 & Qiu et al., 2005). Human fetal liver-derived FH-
B-hTERT cells are another viable option for inducing differentiation of iPSCs and is more
effective than S17. We have ascertained, however, that there is not sufficient research on this cell
line on which to base our methodology and that the acquisition of this cell line is not feasible.
Upon reviewing several other studies’ methods of inducing differentiation, we have come to the
conclusion that OP9 is the best option for our research and therefore plan to use this mouse bone
marrow stromal cell line in our own methodology.
Coculturing protocol. The specific protocol for coculturing OP9 with the cultured iPSCs
has three specific parts: setting up the culture for the OP9 stromal cells, coculturing with the
iPSCs, and harvesting the resulting HSCs.
To set up the OP9 stromal cells culture, we will first prepare the growth medium that
consists of aMEM, FBS and L-glutamine (Lynch et al., 2011). We will then take purchased OP9
stromal cells from ATCC and grow them on the growth medium prepared (ATCC). In order to
properly grow these cells, we will change the media every two days and grow the OP9 cells to
maximum confluency of 70-90%. We will wash the OP9 cells with PBS and add pre-warmed
0.1% Trypsin-Edta from Sigma-Aldrich (Sigma-Aldrich). We will then add these OP9 cells to
the growth medium previously prepared and grow the cells to that maximum 70-90%
confluency. We will plate and grow these cells to this confluency three days prior to actually
using them to co-culture with the cultured iPSCs (Lynch et al., 2011).
11. IPSC DIFFERENTIATION INTO HSCS 11
We will now work to co-culture the iPSCs with the OP9 stromal cells. We will treat the
iPSCs with 1 mg/mL collagenase Invitrogen obtained from Thermo Fisher (Thermo Fisher). We
will then incubate the culture (Choi et al., 2009). To finally harvest the HSCs, we will treat the
cultures with 1 mg/mL collagenase Invitrogen and 0.05% Trypsin-EDTA. We will then wash the
resulting cells with PBS and filter it through a 70-uM strainer, both of which are to be obtained
from Dr. Wang’s Lab. (Choi et al., 2009). These cells are now ready to be tested to ensure that
differentiation did occur and that the initial iPSCs used were indeed effective.
Data Collection and Analysis
We will be using both in vitro and in vivo methods to assess the results of our research
both qualitatively and quantitatively. In vitro methods will be used qualitatively in order to
perform checks when producing iPSCs and HSCs, and in vivo methods will be used
quantitatively to measure the function of HSCs.
The team will be split into two committees, one for in vitro and one for in vivo analysis.
Results will not be shared between the committees until final data comes in, in order to prevent
bias.
In vitro qualitative analysis. Our procedure involves first producing iPSCs from
somatic cells, and then producing HSCs from iPSCs, so we will need to conduct checks to verify
that we are actually producing the cells in question. These checks will be done qualitatively
using immunofluorescence, which utilizes fluorescent-labeled antibodies to target and detect
their respective antigens. When antibodies are introduced to a cell, they initiate an immune
response against their target antigen; if these antibodies are stained with a fluorophore, they can
easily be visualized with a fluorescent microscope.
12. IPSC DIFFERENTIATION INTO HSCS 12
Immunofluorescence is generally broken up into four steps, as outlined on Sigma-
Aldrich. Cells are first prepared by attaching the cells to a solid support, such as a microscope
slide. The cells are then fixed and permeabilized either with an organic solvent, which removes
lipids, dehydrates cells, and precipitates the protein, or with a cross-linking reagent, which link
antigens together. Cells are prepared with the antibody (or antibodies), and are incubated and
washed to remove any unbound antibodies. The stained cells can then be evaluated using
fluorescent microscopy (Immunofluorescence Labeling of Cells).
Throughout our research, immunofluorescence will be used twice. First, when we
generate iPSCs from somatic cells, we will need some verification that iPSCs have actually been
produced. To do this, the iPSCs produced by each method will be stained with the respective
antibodies for the iPSC cell markers. When viewed under the fluorescent microscope, if we are
able to view cells, we can conclude that the antibodies have bound to the antigens on the cells,
and thus infer that the iPSCs have successfully been generated. The protocol for iPSC
immunofluorescence staining of iPSCs, as executed by Wang et al. (2013), is listed in Appendix
A.
Similarly, when we differentiate iPSCs into HSCs, we will need to verify HSCs have
been generated. This will be done by using an antibody which will bind to the CD34+ antigen in
the HSCs and make the cells visible under a fluorescent microscope. Both checks with
immunofluorescence are qualitative, telling only whether or not the cells are produced, and not
giving an actual count of the cells. The protocol for immunofluorescence staining of HSCs
follows a similar procedure to that of iPSCs, executed by Choi, et al. (2011).
13. IPSC DIFFERENTIATION INTO HSCS 13
In vivo quantitative analysis. In order to compare the different methods of producing
iPSCs, we will measure the hematopoietic differentiation of each type of iPSC by transplanting
the isolated HSCs into NSG mice to measure engraftment potential. Although some in vitro
methods exist, they are not effective for measuring HSCs among other precursors. In vivo testing
allows the replication of human biological processes and is important for determining the actual
relevance and effectiveness of the different methods for deriving the iPSCs (Shultz et al., 2007;
Varga et al., 2010). Depending on how many iPSCs are produced by each method, the same
number of HSCs will be injected into each mouse through intraorbital injection. Nine mice will
be used (3 per iPSC production method), as well as 3 additional control mice injected with saline
solution.
After 60 days, the mice will be sacrificed in order to isolate their bone marrow, spleen,
lymph nodes, and peripheral blood, and tested for engraftment potential using a phenotyping
assay (multicolor flow cytometry) that will measure the amount of HSC progenitors by
distinguishing between mouse and human cells. We will do this within 24 hours of procurement
(as opposed to cryopreservation) in order to get a high number of viable cells and to maintain a
high correlation between colony-forming units and CD34 cells. To prepare the cells for flow
cytometry, we will use the Flow-Count-based-Stem Kit. We will use the single platform method
established by the International Society of Hematotherapy and Graft Engineering modified by
Keeney et al. for the flow cytometry using an ammonium chloride lyse, no-wash process in order
to avoid the errors caused by washing our samples. The results of the flow cytometry will tell us
the absolute count of viable CD34 cells in each blood sample. This way we will be able to see
which method for producing iPSCs was the most effective in producing viable HSCs.
14. IPSC DIFFERENTIATION INTO HSCS 14
Statistical analysis will be performed using the ANOVA test in order to compare the
amount of HSC progenitors present in each mouse blood sample and to compare the different
methods to each other.
Anticipated Results
We anticipate the most success with the PBMNC method, because it has been studied
extensively, especially as compared to the other two methodologies. However, it is not expected
that the success will be overwhelming, although it may be statistically significant. All methods
are prone to failure, especially due to contamination. Strict aseptic techniques will be maintained
in order to mitigate the contamination risk. As mentioned in the PBMNC method, lentiviral
induction is subject to a number of other confounding factors that cannot be controlled for due to
the nature of viruses, and fibroblast iPSC generation is inherently less efficient due to the extra
work it takes to obtain fibroblast cells. Overall, PBMNCs have been proven to work, are
inexpensive to access, and have fewer confounding factors, indicating that the method should
succeed in a comparative study as well.
References
ATCC. (2015). OP9 (ATCC® CRL-2749TM). Retrieved from
http://www.atcc.org/Products/All/CRL-2749.aspx
Chang, M.-Y., Rhee, Y.-H., Yi, S.-H., Lee, S.-J., Kim, R.-K., Kim, H., … Lee, S.-H. (2014).
Doxycycline Enhances Survival and Self-Renewal of Human Pluripotent Stem Cells.
Stem Cell Reports, 3(2), 353–364.
15. IPSC DIFFERENTIATION INTO HSCS 15
Choi, K.-D., Vodyanik, M., & Slukvin, I. I. (2011). Hematopoietic differentiation and production
of mature myeloid cells from human pluripotent stem cells. Nature Protocols, 6(3), 296–
313. http://doi.org/10.1038/nprot.2010.184
Choi, K.-D., Yu, J., Smuga-Otto, K., Salvagiotto, G., Rehrauer, W., Vodyanik, M., … Slukvin, I.
(2009). Hematopoietic and Endothelial Differentiation of Human Induced Pluripotent
Stem Cells. STEM CELLS, 27(3), 559–567. http://doi.org/10.1634/stemcells.2008-0922
Flores-Figueroa, E., Varma, S., Montgomery, K., Greenberg, P. L., & Gratzinger, D. (2012).
Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+
mesenchymal stromal cells in benign and myelodysplastic bone marrow. Laboratory
Investigation, 92(9), 1330–1341. http://doi.org/10.1038/labinvest.2012.93
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http://www.sigmaaldrich.com/life-science/cell-biology/antibodies/antibodies-
application/protocols/immunofluorescence.html
Keeney, M., Chin-Yee, I., Weir, K., Popma, J., Nayar, R. and Sutherland, D. R. (1998). Single
platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines.
Cytometry, 34, 61–70.
Lynch, M. R., Gasson, J. C., & Paz, H. (2011). Modified ES / OP9 Co-Culture Protocol Provides
Enhanced Characterization of Hematopoietic Progeny. Journal of Visualized
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Meraviglia, V., Zanon, A., Lavdas, A. A., Schwienbacher, C., Silipigni, R., Di Segni, M., …
Rossini, A. (2015). Generation of Induced Pluripotent Stem Cells from Frozen Buffy
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Nethercott, H. E., Brick, D. J., & Schwartz, P. H. (2011). Derivation of Induced Pluripotent Stem
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Varga, N. L., Bárcena, A., Fomin, M. E., and Muench, M. O. (2010). Detection of human
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ThermoFisher. (2015). Collagenase, Type IV, powder. Retrieved from
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Appendix A
Detailed Protocols
This section includes a detailed description of each method we intend to use. All methods were
obtained from the original paper they were published in. All paper names are indicated next to
the name of the methodology for reference.
Isolation of iPSC using Frozen Buffy Coat sample (Meraviglia et. al, 2015)
A. Isolation
PBMNCs will be obtained from frozen buffy coat samples without density gradient separation.
1. Collect 8mL of venous peripheral blood and combine with sodium citrate buffered plastic
tube. Store at 25°C for 12 hours.
2. Centrifuge tube at 2000 rpm for 15 min. at 4°C. Collect cloudy buffy coat layer which
will exist in the middle phase once the blood has been separated. Store 500 µL into cryovial tube.
3. Re-suspend buffy coat fraction with 500 µL of 2x ice cold 90% FBS and 20% DMSO
(obtain a 1mL volume with 10% DMSO concentration at the end.
4. Freeze vial in -80°C freezer.
B. Thawing and Plating of PBMNCs (DAY 0)
1. Thaw frozen cells in 37°C water bath and dilute mixture with sterile PBS to make total
volume 2 mL
2. Transfer buffy coat solution to 50mL conical tube and combine with 10mL red cell lysis
buffer. Incubate for 10 min. at room temperature. (500 µL red cell lysis buffer can be prepared
by combining 0.5 g of potassium bicarbonate, 4.145 g of ammonium chloride, 100 µl of 0.5 M
EDTA solution to 500 ml of ultrapure water, pH 7.2-7.4.)
19. IPSC DIFFERENTIATION INTO HSCS 19
3. Bring total volume to 50mL by adding sterile PBS. Centrifuge at 300 x g for 10 min. at
room temperature.
4. Re-suspend the cells in 1 ml of PBMNC medium composed of: IMDM and Ham’s F-12
(ratio 1:1), 1% of Insulin-Transferrin-Selenium-Ethanolamine (ITS-X), 1% of Chemically
Defined Lipid Concentrate (CDL), 1% Penicillin/Streptomycin, 0.005% of L-ascorbic acid, 0.5%
of Bovine Serum Albumin (BSA), 1-Thioglycerol (final concentration 200 µM), Stem Cell
Factor SCF (100 ng/ml), Interleukin-3 IL-3 (10 ng/ml), Erythropoietin EPO (2 U/ml), Insulin-
like Growth Factor IGF-1 (40 ng/ml), Dexamethasone (1 µM) and holo-transferrin (100 µg/ml).
5. Transfer them into one well of a 12-well plate with standard tissue culture treated surface,
without coating, at a density of 2 x 10 6 cells/ ml. Incubate the cells at 37 °C, 5% CO2 in a
humidified incubator for 48 hr, until the changing medium step.
C. Culture and Expansion of PBMNCs
1. Collect PBMNCs in suspension culture and centrifuge at 300 x g for 10 minutes at room
temperature.
2. Discard supernatant and re-suspend cells in 1mL fresh PBMNC medium
3. Transfer the cells into 1 well of 12-well plate with standard tissue culture treated surface,
without coating, and incubate them at 37 °C, 5% CO2 in a humidified incubator.
4. Repeat these steps every two days, and split the cells at a ratio 1:2 when they reach the
appropriate confluence (around 80%).
D. Transfection with Episomal Plasmid (Day 14)
1. Centrifuge around 2x106 cells in vial at 300 x g for 10 min. at room temperature.
20. IPSC DIFFERENTIATION INTO HSCS 20
2. Re-suspend 2 x 106 cells in transfection mix containing 100 µl of Resuspension Buffer T
and 1 µg of each plasmid DNA (one plasmid carrying OCT3/4 and shRNA against p53, one
plasmid carrying SOX2 and KLF4, one plasmid carrying L-MYC and LIN28, and one plasmid
carrying EGFP, to check the transfection efficiency).
3. Aspirate the transfection mix containing the cells into a 100 µl tip and insert the pipette
with the sample vertically into the tube, filled with 3 ml of Electrolytic Buffer E2. Efficiently
electroporate cells using the following program: 1,650 V, 10 msec, 3 pulses.
4. Re-suspend the electroporated cells (2 x 106 cells) into 2 ml of pre-warmed PBMNC
medium, adding 0.25 mM Sodium Butyrate (NaB) and count the number of viable cells after
electroporation protocol (on the basis of trypan blue exclusion).
5. Transfer the cells into one well of a 6-well plate without coating, gently rock the plate
and incubate the cells at 37 °C, 5% CO2 in a humidified incubator.
6. The day after electroporation, count the number of GFP-positive cells under fluorescence
microscopy from 8-10 random fields, in order to assess the transfection efficiency.
7. Maintain the transfected cells without splitting for 3 days, until plating them on MEFs.
Replace 2 ml of fresh PBMNCs medium, plus 0.25 mM NaB every two days.
E. Prepare Dishes and Feeder Cells for Co-culture (Day 16)
1. 6 well plate will be coated with basement membrane matrix for at 37 degrees celcius for 15
min.
2. Mouse Embryonic Fibroblasts (MEFs) will be plated in each well at a density of 2 x 105
cells/well with 2 ml of 10% FBS contained DMEM (MEF medium)
F. Plating of Transfected PBMNCs onto MEFs (Day 17-19)
21. IPSC DIFFERENTIATION INTO HSCS 21
1. PBMNCs will be collected in suspension buffer in a 15mL conical tube and centrifuged for
15 min. at 300 x g at RT.
2. Resuspend the pellet in 2 ml of fresh PBMNC medium, plus 0.25 mM NaB.
3. Cells will be plated onto MEF-coated well plates and incubated at 37 °C, 5% CO2 in a
humidified incubator.
4. After two days, the medium will be aspirated and replaced with 2 ml of iPSC medium
composed of knockout DMEM (KO-DMEM), 20% KO-Serum Replacement (KOSR), 1 mM
NEAAs, 1% Penicillin/Streptomycin, 20 mM L-Glutamine, 0.1 mM β-mercaptoethanol, 10
ng/ml FGF (basic bFGF) and 0.25 mM NaB.
5. Replace with fresh iPSC medium every 2 days, and check for iPSCs induction with
immunofluorescence.
PiB Transposon Vector Protocol (Yusa, et. al, 2009)
A. Preparation
1. Obtain MEFs and retroviral vectors (pMXs-Oct3/4, pMXs-Sox2, pMXs-Klf4, pMXs-c-
Myc) from Addgene.
2. Plate MEFs onto 6-well plates one day prior to transfection (5 X 10^5 cells per well).
3. On day of transfection (day 0), transfect piggyBac transposon via Lipofectamine2000
(Invitrogen) according to the instructions of the manufacturer.
4. On day 1, trypsin transfected MEFs and replated onto feeder layers at a split ratio of 1:18
in MEF medium.
5. On day 2, apply F15L medium
22. IPSC DIFFERENTIATION INTO HSCS 22
6. Add VPA to culture medium at 2mM from day 2 to day 7, refreshing the medium every
day.
7. Change medium to K15L on day 7, refreshing the medium every other day
8. Stain colonies using alkaline phosphatase detection kit obtained from Chemicon.
9. Obtain retroviral vectors (Oct3, Sox2, Klf4, Myc-c) from Addgene and infect MEFs with
the vectors.
10. One day after infection, replate cells onto 6-well plates containing feeder layer at 3,000
cells per well.
11. Electroporate piggyBac transposase-expression vector into 2 X 10^6 iPSC; maintain for 3
days.
12. Seed 5 X 10^5 cells onto 10 cm dishes containing feeder cells.
13. Add FIAU to culture medium (0.2 μM) and continue selection for 5 days.
14. Culture without FIAU for 5 days; pick and expand resulting colonies.
15. Perform Bisulphite sequencing using EpiTect Bisulfite Sequence kit to ensure that the
cells are truly pluripotent.
B. Immunofluorescence
1. Fix cells by 4% paraformaldehyde for 15 minutes at room temperature.
2. Allow cells to permeabilized by 0.05% phosphate buffered saline solution (PBS) for 10
minutes at room temperature and then be blocked by 1% PBS at room temperature for 1
hour.
3. Cells washed with PBS and and incubated with anti-Nanog antibody over night at 4° C.
4. Cells labeled with secondary antibodies for 1 hour at room temperature.
23. IPSC DIFFERENTIATION INTO HSCS 23
5. Cells again washed with PBS and nuclei counterstained with Toto-3 at 0.5 μM in PBS for
1 hour at room temperature.
6. After final washing, the cells are analyzed using a fluorescence microscope.
Lentiviral Production (Wang & McManus, 2009)
1. Prepare a plate containing 2.5 X 106 somatic cells in a 10 cm dish
2. Begin diluting your DNA solution by pipetting 30μl FuGENE 6 into 600ul serum-free
medium (SFDMEM)
3. Refrain from allowing FuGENE to touch the side of the test tube as it is delivered into the
medium.
4. Incubate the solution at room temperature for 5 minutes.
5. Mix 4 μg of the lentiviral plasmid that you seek to insert with 4 μg of all three viral
packaging vectors (pMDL, pRSV and pVSV-G).
6. Invert the test tube repeatedly.
7. Incubate at room temperature for 15 minutes.
8. Pippett entire solution onto a plate containing 293 cells and return to incubator for 48-96
hours.
9. Remove the plate from the incubator and use a 10 mL syringe to remove supernatant.
10. Use a .45 μM syringe filter to filter the supernatant, which containts the virus, into a
Beckman Ultracentrifuge tube.
11. Incubate remaining cells in 10% bleach for 45 minutes before discarding.
OP9-iPSC Co-culture (Choi et al., 2009; Lynch et al., 2011)
1. Growth Medium (Lynch et al., 2011)
24. IPSC DIFFERENTIATION INTO HSCS 24
a. The 50mL culture will consist of 39.5mL of aMEM, 10mL 20% FBS (OP-9
tested), and 0.5 mL 2 mM L-glutamine. This culture will be stored at 4°C for at
most one week before use
2. OP9 Stromal Cell Preparation (Lynch et al., 2011)
a. Wash the OP9 cells twice with PBS obtained from Dr. Wang’s lab
b. Add pre-warmed 0.1% Trypsin-EDTA, purchased from Sigma-Aldrich) at 37°C for 5
minutes or until we observe the cells detaching. (Sigma-Aldrich)
c. Add the growth medium previously prepared and then transfer the cells to 50 mL
conical tubes
d. Pellet the cells for 5 minutes at approximately 25°C and discard the supernatant and
then again resuspend the cells in the growth medium previously prepared
e. Plate the cells at a density of at least 104 cells/cm2 on 10 cm dishes and then grow the
cells till the maximum 70-90% confluency
f. Change the media every two days. Grow the cells to the maximum 70-90%
confluency for up to three days prior to coculturing with iPSCs
1. Coculturing with iPSCs (Choi et al., 09)
a. Collect the iPSCs derived and treat them with 1 mg/mL collagenase Invitrogen
obtained from Thermo Fisher (Thermo Fisher)
b. Add this at the density of 1e3cells/cm^2 dish of the OP9 stromal cells prepared 3 days
prior
c. Incubate this co-culture now for 8 days with half-medium changes on day 4 and day 6
1. Harvesting HSCs (Choi et al., 09)
25. IPSC DIFFERENTIATION INTO HSCS 25
a. Treat the cultures with 1 mg/mL collagenase Invitrogen for 20 minutes at 37°C
b. Treat the cultures for 15 minutes at 37°C with 0.05% Trypsin-EDTA
c. Wash resulting cells twice with PBS and then filter through a 70-uM strainer, both of
which is to be obtained from Dr. Wang’s Lab
Immunofluorescence Staining of iPSCs (Wang, et al., 2013)
1. Cells are fixed with 4% paraformaldehyde for 30 minutes, then washed times with
phosphate-buffered saline (PBS).
2. The cells are then permeabilized by treating with 0.5% Triton X-100 at approximately
25℃for 1 hour.
3. Cells are washed again with PBS and then treated with 2% BSA to block for 1 hour at
approximately 25℃.
4. The primary antibody diluted 1:200 with 2% BSA is then applied to the cells, which will
incubate at 4 °C overnight, and then be washed again with PBS.
5. The secondary antibody, again diluted 1:200 with 2% BSA, is applied and cells are
incubated for 1 hour at approximately 25℃.
6. The cells are finally washed again with PBS, stained with 1 mg/mL bisbenzimide, then
observed
Immunofluorescence Staining of HSCs (Flores-Figueroa et. al, 2012)
1. The cells are washed and stained with the primary antibody as listed above.
2. The secondary antibody incubation is done with a 1:200 dilution with 2% BSA for 30
minutes at approximately 25°C.
3. The slides are treated for 30 minutes with 0.1% Sudan Black B dissolved in 70% ethanol.
26. IPSC DIFFERENTIATION INTO HSCS 26
4. The cells are then treated with DAPI (4′,6-diamidino-2-phenylindole) nuclear
counterstain and observed.
Modified ISHAGE protocol for CD34 enumeration (Keeney et al., 1998)
1. Ensure white blood cell count in each sample is less than 10x10^9 per liter, and dilute
sample if necessary with Dulbecco’s phosphate-buffered saline (DPBS).
2. Label four tubes: 45/34 (tubes 1 and 2), WASH (tube 3), and 45/IgG (tube 4 [control])
3. Add 100 microliters to each 3 different tubes labeled with the different antibodies using a
repeater pipette.
4. Add 20 microliters of CD45 FITC/34 PE combination to tubes 1 and 2.
5. Add 20 microliters of CD45 FITC/ISOCLONIC control reagent to the fourth tube.
6. Incubate tubes at room temperature for 15 minutes in the dark.
7. Add 2 microliters ammonium chloride to lyse red cells.
8. Add 2 microliters of 7-AAD to each sample to distinguish viable cells.
9. Gently vortex tubes and incubate at room temperature for 10 minutes in the dark.
10. Add 100 microliters of Stem-CountTM flourospheres to tubes 1, 2, and 4.
11. Keep all samples on ice at 4°C in the dark.
12. Perform flow cytometry within the hour and determine absolute count of CD34 cells.
27. IPSC DIFFERENTIATION INTO HSCS 27
Appendix B
List of Abbreviations
AKT: protein kinase B
AML: acute myeloid leukemia
BAC: bacterial artificial chromosome
BM: bone marrow
BSA: bovine serum albumin
CAG: expression promoter used in mammalian cells
CB: embryonic cord blood
CDL: chemically defined lipid concentrate
CLP: common lymphoid progenitor
CMP: common myeloid progenitor
DAPI: 4′,6-diamidino-2-phenylindole
DMEM: Dulbecco’s modified Eagle’s medium
DMSO: dimethyl sulfoxide
DNA: deoxyribonucleic acid
28. IPSC DIFFERENTIATION INTO HSCS 28
dox: doxycycline
EB: embryoid body
EDTA: ethylenediaminetetraacetic acid
ELISA: enzyme-linked immunosorbent assay
ESC: embryonic stem cell
FBS: fetal bovine serum
FIAU: fialuridine
FOXO: forkhead box O3
F15L: serum-free medium
GFP: green fluorescent protein
GVHD: graft-vs-host disease
HCT: hematopoietic cell transplantation
HEK-293: human embryonic kidney 293 cells
hESC: human embryonic stem cell
hFGF: human fibroblast growth factor
HMC: human kidney mesangial cell
HPC: hematopoietic progenitor cell
HSC: hematopoietic stem cell
IMEM: Iscove’s modified Eagle’s medium
iPSC: induced pluripotent stem cell
ITS-X: insulin-transferrin-selenium-ethanolamine
KLF4: kruppel-like factor 4
