Michaela Close
Genetics
November 1, 2012
Inducing Pluripotent Stem Cells and the Potential of Stem Cells in Medicine
Stem Cells
Stem cells are unique in that they are undifferentiated, have the ability to
proliferate for long periods of time, and can differentiate into specialized cells and
tissues. There are two naturally occurring types of stem cells, embryonic and somatic.
Embryonic stem cells (ES cells) and somatic stem cells differ in their pluripotency and in
their ability to proliferate. ES cells are completely pluripotent, while somatic stem cells
can usually only generate the cell types of a specific organ. Also, ES cells have the
ability to proliferate for a long time, while somatic stem cells have limited potential for
proliferation. (Stem Cell Basics)
ES cells’ pluripotency allows them to differentiate into all of the cells and tissues
of the body. For instance, during the first trimester of fetal development, an embryo or
early fetus has the ability to heal serious injuries without scarring, and even regenerate
lost limbs. This is because the ES cells within the fetus still have the capacity for
pluripotency. However, after the initial stages of development, the ES cells’ pluripotency
deteriorates, and the fetus’s capacity to heal itself declines (Yannas et al. 2007).
The process of differentiation is regulated by gene signals. Current research
suggests that the potential for pluripotency is associated with at least four known genes
that code for the transcription factors: Oct4, Klf4, Sox2 and c-Myc (Lorenzo et al. 2012).
It is believed that the reason for the decline in pluripotency and regenerative ability
during fetal development is due to silencing of the genes that signal for differentiation.
The idea that gene silencing prevents differentiation suggests that somatic cells still have
the potential to differentiate if the necessary transcription factors are re-introduced.
(Russell 2007)
Induced pluripotent stem cells (iPSCs) are scientifically manufactured stem cells
that originate from adult somatic cells and are reprogrammed to achieve pluripotency. In
iPSCs the transcription factors Oct4, Klf4, Sox2 and c-Myc are reintroduced. These
transcription factors change the gene regulation that occurs at the transcriptional level,
and allow for pluripotency. (Russell 2007)
Inducing Pluripotent Stem Cells
Creating iPSCs involves acquiring and reprogramming adult somatic cells. The
somatic cell types used are most commonly derived from dermal fibroblasts, as well as
teeth, adipose tissues, or blood cells. Ono et al. (2012) were able to use human nasal
epithelial cells, which proved to be an effective yet less invasive method for harvesting
cells.
Once the somatic cells are obtained, they are transferred to a matrix so that they
can proliferate. The four transcription factors, also known as reprogramming factors,
Oct4, Klf4, Sox2 and c-Myc must then be introduced into the somatic cells using a
vector. Most commonly, the reprogramming factors are transduced through lentiviral or
Sendai virus vectors (Lorenzo et al. 2012). This process of introducing foreign DNA into
a cell using a virus vector is called transduction.
There is concern over the use of viral vectors due to the chance that some of the
viral DNA will be integrated into the DNA of the cell. For this reason, the multiplicity of
infection (MOI) must be considered. The MOI refers to the number of viral particles that
infect each cell, and it is important that this number be as small as possible when using a
virus. (Lorenzo et al. 2012)
With viral vectors the MOI must be balanced with the reprogramming efficiency.
The reprogramming efficiency is the number of iPSC colonies formed per number of
infected cells. While the reprogramming efficiency needs to be sufficiently high, the MOI
must be small to prevent viral integration into the DNA (Ono et al. 2012).
Once the somatic cells are infected with the reprogramming factors, they
proliferate to form colonies on the matrix. The colonies are then analyzed to check for ES
cell-like morphology. The optimal balance between MOI and reprogramming efficiency
is then determined by selecting the smallest MOI that will effectively induce transgenes
and form iPSC colonies (Figure 1). (Ono et al. 2012)
The colonies must be tested for integration of the viral genome. To analyze the
structural variations in the iPSC colonies high-density SNP genotyping must be
conducted. The SNP genotyping of the derived colonies generated by Ono et al. (2012)
revealed that the concordance between the genotype of the original parent cells and the
derived cells was greater than 99.9%. This high percentage suggests that the genome was
in the same state as the parent cells, and therefore the iPSCs did not introduce the viral
genome.
Figure 1. This chart shows a comparison of the cell morphology and the MOI. Ono et al. (2012)
determined that the MOI of 3 or 4 was sufficient. The efficiency of iPSC generation was .75%
for MOI 3 and .1% for MOI 4.
The colonies of iPSCs must then be tested to check for variation between iPSCs
and ES cells. Ono et al. (2012) analyzed the iPSCs and ES cells for similarities in
epigenetic behavior by performing a DNA methylation analysis. The analysis revealed
that the methylation of the promoter region in the iPSCs was more similar to the
methylation of an ES cell than the methylation of the original somatic cell. This
similarity in methylation patterns implies that the iPSCs took on similar epigenetic
qualities as an ES cell. (Figure 2)
Figure 2. This figure shows a methylation analysis of the promoter region. This figure shows
that the iPSCs and the ES cells have much more similar methylation regions than the human nasal
epithelial cells (HNECs) and ES cells. The significance of this data is that the iPSC now has
similar epigenetic behavior to the ES cell. The methylation was determined using a bisulfite
sequencing analysis.
Finally, the iPSCs and the ES cells were compared based on the level of
expression of the four transcription factors. The level of expression was tested using RTPCR. The iPSC colonies showed similar levels of Oct4, Klf4, Sox2 and c-Myc as the ES
cells. RT-PCR also revealed that the viral vector did not cause the level of expression of
the four factors. (Ono et al. 2012)
After iPSCs show a genotype that is significantly similar to ES cells’ genotype,
and significantly different from the viral vector’s genotype, the differentiation capacity of
the iPSCs must be tested in vivo. Ono et al. (2012) tested the differentiation potential of
the iPSCs by injecting immunocompromised with derived iPSC lines. They looked for
the formation of teratomas, or a tumor comprised of multiple non-native tissues. They
then conducted a histological examination of the teratomas to look for different cell
types. Ono et al. (2012) found that the teratomas were comprised of endoderm,
mesoderm, and ectoderm tissues; also, the teratomas had neural and epithelial tissues,
muscle, cartilage, bone, gut-like structures, and glandular structures. The formation of
the three germ layers and the various other tissue structures proved that the somatic cells
were effectively reprogrammed to differentiate and achieved pluripotency.
Limitations of Induced Pluripotent Stem Cells
Despite our ability to effectively induce pluripotent stem cells, there are many
aspects of iPSCs that hinder their use in a clinical setting. For one, it is very challenging
to generate a pure population of iPSCs due to the low reprogramming efficiencies (Rao et
al. 2012). For example, Ono et al. achieved an efficiency of .1% and .75% for MOI 3 and
4 respectively.
Furthermore, the risk of viral genome integration through insertional mutagenesis
is still a serious concern. One recent advance in delivery mechanisms has been the
creation of nonintegrating episomal vectors that do not contaminate the iPSC with the
vector DNA because the episome is removed after transduction. However, even if the
vector’s DNA does not contaminate the genome, there is still the risk that there is
variation between the iPSCs and ES cells. The difference between the ES cell genotype
and the iPSC genotype is inevitable because the cells must be grown in vitro, not in vivo
(Lorenzo et al. 2012). Even though a 99.9% concordance rate seems high, a 0.1%
difference in the genome is significant, and could lead to new problems in vivo.
Overall, we still do not know whether all the differences that will inevitably arise
between ES cells and iPSCs are significant in a clinical setting (Stem Cell Basics). There
are already some clinical applications of stem cells in use, but if this field is to progress,
we must further our understanding of stem cells, and refine our methods of inducing
them.
The Future of Stem Cells
The potential uses for induced stem cells are extensive. Stem cells offer hope for
medicine in issues such as skin and tissue regeneration in burn victims, pancreatic repair
in individuals with diabetes, and neuronal repair after nerve or brain injury. Already
stem cells have been used to repair damaged tissues in clinical situations, and lab-grown
tissues have been transplanted into human patients. In 2006, doctors and scientists were
able to repair seven diseased bladders by using the patients’ cells to generate tissue and
then transplanting the tissue (Donn 2006). However, doctors and scientists have not yet
been able to transplant whole organs that are entirely derived from induced stem cells.
Through iPSCs we may eventually be able to grow and manufacture organs for
transplant.
Furthermore, we are currently using iPSCs to model the progression of diseases,
and may eventually use them to develop pharmaceuticals (Rao et al. 2012). Scientists
can take cells from a diseased patient, generate iPSCs, allow the tissues to differentiate in
vitro, and observe how the disease affects the development and function of various
tissues. Through this process, scientists can learn about how a disease progresses, and can
use actual human tissue to examine the effects of various treatments or pharmaceuticals.
(Underbayev et al. 2012)
Many other beneficial uses of iPSCs and ES cells may soon be discovered.
Scientific research is on the path to realizing the full potential of stem cells and how they
can be applied to better human lives.
Works Cited
1. Rao L, Tang W, Wei Y, Bao L, Chen J, et al. 2012. Highly Efficient Derivation of
Skeletal Myotubes from Human Embryonic Stem Cells. Stem Cell Reviews and
Reports 8.
http://www.ncbi.nlm.nih.gov.pallas2.tcl.sc.edu/pubmed?term=Highly Efficient
Derivation of Skeletal Myotubes from Human Embryonic Stem Cells
2. Lorenzo LM, Fleishcer A, Bachiller D. 2012. Generation of Mouse and Human
Induced Pluripotent Stem Cells (iPSC) from Primary Somatic Cells. Stem Cell
Reviews and Reports 8.
http://www.ncbi.nlm.nih.gov.pallas2.tcl.sc.edu/pubmed/23104133
3. Ono M, Hamada Y, Horiuchi Y, Matsuo-Takasaki M, Imoto Y, et al. 2012.
Generation of Induced Pluripotent Stem Cells from Human Nasal Epithelial Cells
Using a Sendai Virus Vector. PLoS ONE 7(8).
http://www.ncbi.nlm.nih.gov.pallas2.tcl.sc.edu/pubmed?term=Generation of
Induced Pluripotent Stem Cells from Human Nasal Epithelial Cells
4. Yannas IV, Kwan MD, Longaker MT. 2007. Early Fetal Healing as a Model for
Adult Organ Regeneration. Tissue Engineering 13(8):1789-98.
http://www.ncbi.nlm.nih.gov.pallas2.tcl.sc.edu/pubmed/17518739
5. Stem Cell Basics: Introduction. In Stem Cell Information [World Wide Web site].
Bethesda, MD: National Institutes of Health, U.S. Department of Health and
Human Services, 2009 [cited Thursday, November 01, 2012]
http://stemcells.nih.gov/info/basics/basics1
6. Russell, Alan. 2007. Alan Russell: The Potential of Regenerative Medicine.
TED. N.p., Web. 1 November 2012.
http://www.ted.com/talks/alan_russell_on_regenerating_our_bodies.html
7. Donn, Jeff. 2006 Organ Re-Engineered for the First Time in Bladder Transplants.
USA Today. 1 November 2012.
http://usatoday30.usatoday.com/tech/science/genetics/2006-04-03-bladderregrown_x.htm
8. Underbayev C, Kasar S, Yuan Y, and E. Raveche. 2012. MicroRNAs and Induced
Pluripotent Stem Cells for Human Disease Mouse Modeling. Journal of
Biomedicine and Biotechnology 2012.
http://www.hindawi.com.pallas2.tcl.sc.edu/journals/jbb/2012/758169/