STEM CELL THERAPY AND ITS CLINICAL APPLICATION

1. Isolation and Maintenance of Stem Cells
The isolation, generation, and maintenance of stem cells pose several
challenges due to the propensity of stem cells to differentiate and for
variations such as chromosomal and epigenetic changes to occur in these
cells during culture. Protocols are continuously evolving and vary for
different types of stem cells.

2. Isolation of Embryonic Stem Cell (ESC) Lines
The majority of ESC lines are mouse, not human. The basic protocol for
generation of ESCs is similar for all species.
ESCs are pluripotent stem cells generated from early-stage embryos. A
fertilized embryo is required for the generation of ESCs. Typically, cells are
harvested from the blastocyst 4–5 days post-fertilization. The outer cell
layer of the blastocyst, called the trophoblast, contains a fluid-filled cavity,
the blastocoele, and an inner cell mass of 10–20 cells. The inner cell mass,
which is also called the embryoblast, is removed for culture. Occasionally,
cells may be obtained from the stage before the formation of the blastocyst,
the morula.
The cells from the inner mass are placed in culture, and those that are
viable are expanded. Generation of ESCs is inefficient; many cells do not
adapt to cell culture and do not survive.
Human, mouse, rat, and other ESC lines are available through commercial
vendors.

3. Somatic Stem Cells
Somatic (adult) stem cells are found in most major organs
and tissues, and are currently being isolated from many
tissues in the body. The methods of isolation and culture
are dependent on the source and lineage. Many isolation
and purification protocols involve flow cytometry and cell
sorting. Positive and negative sorting for cell surface
markers can quickly generate enriched populations.
The number of cells that can be isolated varies greatly
depending upon the cell and tissue type. For example,
cardiac stem cells are quite rare, while hematopoietic
stem cells (HSCs) occur in high enough numbers that they
are routinely isolated and used in bone marrow
transplantation. HSCs for bone marrow transplantation are
collected either directly from bone marrow or by
apheresis, the removal of white blood cells from peripheral
blood. Prior to apheresis, the donor is injected with
granulocyte-colony stimulating factor to mobilize stem
cells from the bone marrow.

4. Mesenchymal stem cells (MSCs) were originally isolated from bone
marrow stroma but were subsequently found to be present in most
tissues of the body including cord blood, adipose tissue, skin, and
periodontal ligaments, which attach teeth to the jaw.
Adipose tissue is becoming an important source of MSCs because
adipocytes are easily accessible and present in relatively high numbers in
the body. It has been estimated that 1 g of adipose tissue yields 5,000
MSCs, whereas bone marrow aspirate contains 100–1,000 MSCs per ml
(Strem et al. 2005).

5. Induced Pluripotent Stem Cells (iPSCs)
Induced pluripotent stem cells (iPSCs) are somatic cells that have been
reprogrammed to become pluripotent. Theoretically, any somatic cell could be
reprogrammed. Practically, it has been found that some are relatively
straightforward and others are more technically challenging.
iPSCs were first generated by the introduction of the transcription factors Oct3/4,
Sox2, Klf4, and c-Myc in cells maintained in culture conditions used for ESC
(Takahashi and Yamanaka 2006). Various combinations of these transcription
factors have since been used by other investigators.

6. Most iPSCs have been generated using retroviral and lentiviral vectors to
introduce transcription factors into stem cells. However, there are concerns with
using these viral vectors. Previous studies of retroviral infection of embryonic cells
had suggested that retroviruses are silenced in these cells. Silencing is an
epigenetic process that suppresses transcription. However, it was demonstrated
that silencing was often incomplete and viral genes could still be expressed. A
major consideration for using retroviruses to generate iPSCs is that the viruses
integrate into the host DNA. Depending on the integration site, integration can
have deleterious effects on the cells, altering gene expression and increasing the
risk of tumor formation. Adenoviruses (Stadfield et al. 2008) and Sendai virus, an
RNA virus (Seki et al. 2012), have been used as alternative vectors to transduce
transcription factors because they do not integrate into the genomic DNA of the
cell.

7. Viral-mediated introduction of transcription factors is very inefficient. A more efficient
method was found to be the combination of lentiviruses and micro RNAs (miRNAs)
to reprogram cells (Anokye-Danso et al. 2012). These small RNAs bind to mRNA
and either inhibit translation or cause degradation of transcripts. miRNA clusters,
including miR-290-295 and miR-302-367, have been shown to enhance
reprogramming of somatic cells into iPSCs.
Another approach was the use of miRNA mimics to enhance viral-mediated
transduction of transcription factors. miRNA mimics are double-stranded modified
RNAs that mimic mature miRNAs (fully processed cellular miRNAs). miRNA mimics
do not require a vector; they can be transfected directly into cells. The combined
use of transcription factors and miRNA mimics produces more homogeneous iPSC
clones (Judson et al. 2009). An advantage of using miRNA mimics with
transcription factors is that the transcription factor c-myc, an oncogene, is not
required.

8. Culture of Stem Cells
The culture conditions and types of media used for
stem cell culture depend on the type of stem cell.
There are a wide range of protocols and products
available for both maintaining stem cells in an
undifferentiated state and for differentiating them into
different lineages and cell types.

9. Feeder Cell Layers
Originally, all ESC cultures were maintained on feeder cell layers. Inactivated
mouse embryonic fibroblasts (MEFs) were used to provide factors and a
substrate that allowed ESCs to grow and divide. There are several problems
associated with MEFs, including the potential for the introduction of mouse-
derived infectious agents, undesirable protein transfer, and lot-to-lot variation
among feeder cells.
MEFs can either be purchased or freshly generated in the lab. Fibroblasts from
~14–15-day-old fetal mice are cultured and expanded for 3–4 days. To be used
as a feeder layer, MEFs must be mitotically inactivated, either by irradiation with
UV light or incubation with mitomycin C. Stocks of MEF cells can be frozen either
before or after inactivation.

10. Preparation of mitotically inactivated mouse embryonic fibroblast (MEF) cells for use as
feeder cells
A consideration in the choice of culture system is that stem cells have
been shown to acquire xenoantigens from culture products derived
from other animal species. This was first shown in hESCs cultured
with mouse feeder cells and animal-derived serum. These stem cells
incorporated non-human sialic acid, against which many humans
already have circulating antibodies (Martin et al. 2005). In addition to
feeder cells, animal-derived products used for culture include serum
and other matrices. The potential for stem cells to cause an
immunogenic response can restrict their clinical use.

11. Feeder-free culture
There are two types of feeder cell-free media: defined media
and conditioned media. These media will support the growth of
stem cells and contain factors that inhibit differentiation. The
components of the media and the supplements vary depending
on the type of stem cell and the animal.
Defined medium is a serum-free medium that has been
supplemented with recombinant growth factors and other
molecules that are required to support the growth and
pluripotency of stem cells.

12. Media formulated for stem cell culture often contain factors such as leukemia inhibitory factor and
bone morphogenetic protein to prevent differentiation. Rho-associated coiled-coil containing protein
kinase (ROCK) inhibitors such as Y-27632 and thiazovivin have been demonstrated to increase the
viability of newly isolated stem cells (Chen et al. 2010). For the culture of hESCs, basic fibroblast
growth factor (bFGF or FGF2) is usually present in the media but not for most other stem cell media.
Some defined media for human cell culture may contain bovine serum albumin (BSA) and is therefore
not completely free of animal protein. Additionally, other culture matrices (described below) are often
derived from animal cells.
Long-term culture in serum-free media has been shown to cause epigenetic changes in cultures as the
cells adapt. For instance, it was shown that hESCs start to express CD30, a marker for certain types of
malignancies, when grown in media with knockout serum replacement but not in media with fetal calf
serum (Chung et al. 2010). Epigenetic changes with different culture conditions have been shown to
include methylation, histone modification, and in female ESCs, X-chromosome inactivation (McKewn
et al. 2013).

13. Cells in culture secrete factors into the media that support cell growth. After cells have grown and
divided for a period of time, the media are removed. This conditioned media can then be used as a
supplement to fresh media. Although there is still concern about the presence of viruses when using
conditioned media, it is much less than when using cross-species feeder cells. An advantage of
conditioned media is that they contain more factors than defined media. Variability between batches of
conditioned media is common, so new lots should be tested.
Human foreskin fibroblasts are the most common cell type used to make conditioned media for human
cells.
Stem cells not growing on a feeder layer need a matrix for attachment and growth. A culture
matrix contains extracellular matrix (ECM) proteins and polysaccharides such as vitronectin and
proteoglycans. There are many matrices available with varying combinations of proteins and
carbohydrates. Different types and sources of stems cells require different matrices, and different
matrix components can either maintain pluripotency or help drive differentiation. Cell matrix products
containing only human ECM are available.

14. Testing of Stem Cells
Frequent testing of stem cultures is necessary due the propensity of
cultured stem cells to undergo genotypic and phenotypic changes or
mosaicism. Initially, when newly isolated cells are at a sufficient density,
they are usually screened for pluripotency, appropriate gene expression,
and a normal karyotype. Thereafter, the stability of the stem cells is
confirmed at regular intervals and after frozen stocks are thawed and
plated. Additionally, particularly in lines that use feeder layers, cultures
must be checked for contamination.

15. Differentiation media
There are many formulations for media for directed differentiation
of stem cells. Usually, there is a reduction in growth factors and an
addition of other factors. Frequently, differentiation medium is
combined with a change of the culture matrix to help promote the
desired path of differentiation.

16. STEM CELLS MARKERS

17. STEM CELLS MARKERS
Coating the surface of every cell in the body are specialized
proteins, called receptors, that have the capability of
selectively binding or adhering to other signaling molecules.
There are many different types of receptors that differ in their
structure and affinity for the signaling molecules. Normally, cells use
these receptors and the molecules that bind to them as a way of
communicating with other cells and to carry out their proper functions
in the body. These same cell surface receptors are the stem cell
markers.

18. Each cell type, for example a liver cell, has a certain combination of
receptors on their surface that makes them distinguishable from other
kinds of cells.
The signaling molecules that selectively adhere to the receptors on the
surface of the cell as a tool that allows them to identify stem cells.
A technique was developed to attach to the signaling molecule another
molecule (or the tag) that has the ability to fluoresce or emit light energy
when activated by an energy source such as an ultraviolet light or laser
beam. Fluorescent tags emit light that differ in color and intensity.

19. Genetic and molecular biology techniques are extensively used to study how
cells become specialized in the organism's development. Genes and
transcription factors (proteins found within cells that regulate a gene's activity)
that are unique in stem cells.
Polymerase chain reaction (PCR) to detect the presence of genes that are active
and play a role guiding the specialization of a cell. This technique has is helpful
to identify genetic markers that are characteristic of stem cells.
Other techniques ?

20. •Example: A gene marker called PDX-1 is specific for a transcription factor protein
that initiates activation of the insulin gene, this marker to identify cells that are
able to develop islet cells in the pancreas.

21. •Example: A special type of hematopoietic stem cell from blood
and bone marrow called side population or SP is described as
(CD34-/low, c-Kit, Sca-1).