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PRIMER ON PLURIPOTENT STEM CELLS
Gene expression during cell differentiation, such as the formation of neurons or blood cells, has
long been a central issue of developmental biologists [Epigenetic Landscape Model]. For their
pioneering work in this field, Gurdon and Yamanaka received the Nobel Prize in Physiology or
Medicine in 2012 [Photo Gurdon/Yamanaka]. Sir John Gurdon (1962) injected nuclei from
various somatic cells into oocytes, thus showing that the nucleus of e.g. a gut epithelium cell
could support the development of a complete, healthy animal. More than forty years later,
Shinya Yamanaka (2006) reversed the process of cell differentiation in a different way: He
transformed mouse epidermal cells by adding the genes for four transcription factors. Some of
the transformed cells became pluripotent, that is, they developed into any type of differentiated
cell if cultured in appropriate media. These versatile cells were called induced pluripotent stem
cells (iPSC) because they resembled embryonic stem cells (ESC), which had been generated
earlier from embryonic cells. Both iPSC and ESC are pluripotent and divide indefinitely.
The discovery of ESC and iPSC has triggered a flurry of research, which today is driven mainly
by its potential applications in pharmacy and medicine. Cell cultures from biopsies of human
patients are already being used to test drugs for curative effects and toxicity in patients with
different genetic backgrounds and disease histories. In the future, it may be possible to use iPSC
or ESC for cell replacement therapies. For instance, a patient who survived a heart attack could
be saved by injecting the damaged portion of the heart with cardiomyoblasts raised along the
right steps from an iPSC or ESC culture.
In the rush to medical applications, it will be useful to remember the basic questions about the
gene physiology of cell differentiation, from which this line of work started. Following is a
review of the basic phenomena and questions, and how keeping them in mind may to inspire the
translation from bench to bedside.
A.
Adult Stem Cells
1. Development entails formation of differentiated cells from embryonic or other
undifferentiated cells. This process can be monitored by
a. cell morphology and location
b. specific molecular markers, such as transcription products or proteins
2. A large mammal may have trillions of cells, but only about 200 different cell types.
3. Most differentiated cells have limited life spans and do not divide. Instead, worn out
cells are replaced by from reserve cells called “stem cells” [ABD 0311].
4. The definition of adult stem cells uses the following criteria [1PPE, HB 10.6]
a. undifferentiated appearance (cuboidal cell shape, large nuclei)
b. unlimited capacity for mitotoic division
c. Two types of daughter cells: new stem cells and committed progenitor cells
5. Examples of adult stem cell lineages include: epidermal cells, intestinal epithelial
cells, blood cells, mammalian spermatogonia, Drosophila oogonia.
6. Adult stem cells may be
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a. unipotent, i.e. giving rise to only one type of differentiated cells, such as
epidermal cells or spermatogonia.
b. multipotent, i.e. they normally give rise to two or more types of differentiated
cells. Examples: hemangioblast, universal blood stem cell [ABD 20.12]
7. The stem cell niche is defined as the microenvironment of extracellular signals that
maintain “stemness” in a stem cell lineage [HB 10.5]. Such signals may be
a. secreted and diffusible (e.g. TGFß or Wnt families)
b. integral membrane proteins (e.g. Notch and Delta)
c. integrins and extracellular matrix components. Their stiffness and adhesiveness
give them an astounding degree of control over the release of committed
progenitor cells from their niche.
8. The asymmetry of stem cell divisions (production of two types of daughter cells) may
occur in two basic forms
a. “invariant” asymmetry depending on organelles orienting mitotic spindle in
conjunction with localized cytoplasmic determinants or unevenly distributed
external signals). Example: germ line stem cells in Drosophila ovary
b. “populational asymmetry” producing stem cells and committed progenitor cells
stochastically (depending on labile control circuits of transcription factors and
extracellular signals)
9. Under experimental conditions, some adult stem cells have been shown to form
differentiated cells beyond their normal, tissue-specific repertoire.
a. Bjornson et al. (1999, Science 283: 534-537) injected mouse neural stem cells,
which normally form neurons or glia cells, into the blood stream of mice that had
been heavily X-irradiated to destroy their hematopoietic system. Nevertheless,
the recipient mice survived, and newly formed blood cells in their spleens were
shown unequivocally to be derived from the injected brain cells. Circumstantial
evidence suggested that injected neural stem cells had turned into blood stem
cells, which in turn had given rise to the full spectrum of blood cells [ABD
20.22]. However, other interpretations were not excluded.
b. Bone marrow-derived stromal cells, aka mesenchymal stem cells (MSC), gave
rise to cardiomyocytes and epithelial cells in lung, liver, and skin.
c. The possibility of broadening the repertoire of adult stem cells is of great medical
interest for cell replacement therapy. Stem cells from an easily accessible tissue
(such as fat) in a patient may be used to derive isogenic progenitor cells of
different tissues for which adult stem cells are hard to obtain.
B.
Embryonic Stem Cells
1. Mammalian embryonic stem cells (ESC) are defined by the following operational
criteria.
a. they are obtained from the inner cell mass (ICM) of mammalian embryos at the
blastocyst stage [ABD 5.8, 5.11, 5.12]
b. they fulfill the defining criteria used for adult stem cells (section A4)
c. they are highly pluripotent, that is, capable of forming a wide (perhaps unlimited)
range of differentiated cell types. This was shown by the
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2.
3.
4.
5.
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i. formation of teratomas after injection into the skin of normal adult animals
ii. formation of chimeras after addition to genetically marked blastocysts
iii. ability to form, in vitro, differentiated cells normally derived from any one of
the three embryonic germ layers (ectoderm, mesoderm, endoderm)
d. Mouse ESC are totipotent, i.e., capable of forming complete, fertile embryos as
shown by the tetraploid complementation test [HB 10.7]. It is generally assumed
that human and other mammalian ESCs are also totippotent.
ESC not only satisfy the defining criteria of adult stem cells but exceed them in
a. ease of identification
b. robustness and rapid division in culture
c. ease of reprogramming into all kinds of cell types
For basic research, the culture of ESC is an excellent way of studying the signals that
gradually turn embryonic cells into differentiated cells.
For applied research, ES cells are prized because of their
a. use in making transgenic animals by homologous recombination in cultured ESC
followed by somatic nuclear transfer (SNC)
b. potential to generate isogenic human replacement cells in conjunction with gene
therapy (see Section D) and SNC
Human ES cells are derived from blastocysts generated in fertility clinics, which
routinely fertilize more eggs in vitro than can be used for one uterine implantation.
The spare blastocysts are deep-frozen and can be used for additional implantations as
needed. Otherwise these blastocysts are spare and could be used for research.
Research on human embryonic stem cells is politically controversial, and federal
funding in the U.S. has changed under different administrations. The British
Parliament has passed laws including a “14-day rule”. Up to this time limit, approved
research on human embryos is legal. In fact, there have been incentives for British
IVF couples to make their spare embryos available for research.
Regulation and Totipotency in Plants and Animals
1. A fertilized egg (zygote) undergoes cleavage, i.e. a series of cell divisions generating
progressively smaller cells called blastomeres.
2. In many animals including mammals, blastomeres and are totipotent, as shown by
blastomere isolation and monozygotic twin formation [ABD 0114, 0601, 0622]
3. In many animal species, blastomeres can also depart from their normal fates in
development, an amazing ability called regulation. It is shown by the same
blastomere isolation experiments and by fusion of multiple embryos [ABD 0623].
4. The ability of human embryos to undergo regulation is utilized in the procedure
called preimplantation genetic diagnosis, which is offered by fertility clinics as an
adjunct to in vitro fertilization. At the 8-cell stage of development, one blastomere is
removed for genetic testing before the remainder of the embryo is implanted.
Apparently, the procedure does not affect the chances of a successful pregnancy.
5. Attempts to regenerate whole organisms from small body parts have been successful
in sponges, hydrozoans, and planarians but not in higher animals.
6. Attempts to clone whole organisms from single differentiated cells have been
successful in plants [ABD 7.10, 1PPE] but again not in animals. This experience has
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been described as loss of totipotency, that is, the ability to form a complete organism
that can have viable offspring. Loss of totipotency in animal cells has been ascribed
to restrictive effects of animal cytoplasm on gene expression. This hypothesis has
been tested by the strategy of transplanting nuclei of differentiated cells into animal
eggs, which in normal development do support development of a complete organism.
D.
Somatic Nuclear Transfer into Eggs
1. Nuclear transfer into surgically enucleated eggs was first carried out by Briggs and
King (1952) using the leopard frog, Rana pipiens [ABD 0712]. These experiments
were extended by Gurdon (1962) using the African clawed frog, Xenopus laevis, and
improved experimental design features such as serial transplantation. The
investigators’ first question was whether nuclei from advanced donor stages would
support the development of a complete frog, indicating that the transplanted nucleus
was genetically complete.
a. Nuclei from differentiated blood, intestinal, and other adult cells supported the
development of complete organisms, including a few adults [ABD 0715].
b. The success rate was uniformly low, even with spermatogonia which, because of
their function, must carry a full complement of genetic information in each cell.
c. The success rate decreased with the age of the donor cells [ABD 0713].
d. Conclusion: Nuclei from at least some differentiated cells are pluripotent or even
totipotent. However, chromatin from differentiated cells may need some
"remodeling" for DNA replication and new embryonic gene activities.
2. In sheep, a cultured fibroblast was fused with an enucleated egg, which then was
implanted into the uterus of a foster ewe and born as an apparently normal lamb
(“Dolly”) [ABD07.17]. Use of DNA markers confirmed that Dolly did indeed carry
the genome of the nuclear donor cell. Similar experiments were done with cows,
pigs, goats, cats, dogs, mice and Rhesus monkeys, using nuclei from various types of
donor cells.
3. Failure rates were consistently high in sheep [ABD Table 7.1] as well as in other
mammals, and increased rapidly with the age of the cell donating the nucleus [1
PPE]. The miscarried fetuses died of various causes. These high failure rates
absolutely preclude the use of nuclear transfer for human reproductive cloning.
4. However, nuclear transfer may still be pursued for therapeutic cloning, i.e. the
generation of human ESC lines isogenic with patients who may be cured by replacing
a particular lineage of deficient cells.
5. In a futuristic scenario, patients with type I diabetes, Parkinson’s disease, and similar
disorders affecting one particular cell type could be treated by cell replacement
therapy [HB 10.11]. A small biopsy from the patient would be cultured to obtain
cells that can be used as nuclear donors when fused with an enucleated human egg.
When they reach blastocyst stage they are sacrificed to obtain the inner cell mass
(ICM). About 10% of ICM cultures yield embryonic stem cells (ES cells), which are
undifferentiated, multiply indefinitely, and are pluripotent [HB 10.6]. ES cells are
exposed to appropriate growth factors and ECM stimuli to obtain the desired type of
committed progenitor cells. These are transplanted to the patient.
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6. The major advantage of this method is that the replacement cells are isogenic
(genetically identical) with the patient, so that no immune rejection will occur.
Another advantage is that the method could readily be combined with gene therapy in
cases where the human disease is caused by a defect in a known gene. A proof-ofprinciple experiment with mice has already been done [HB11.6].
7. A major concern is that the transplanted progenitor cells may be contaminated with
residual ES cells, which could form tumors. Another problem is that the method
requires large numbers of donated human eggs because only a fraction of re-nucleated
eggs develop into blastocysts and only a fraction of those give rise to ESC cultures.
Such low yields drive up the cost of cell replacement therapy and aggravate ethical
concerns about using human eggs and blastocysts for research.
8. This dilemma highlights the need to better understand why it has been so difficult to
return differentiated mammalian cell or their nuclei to a totipotent or at least
pluripotent state. What is the nature of the “chromatin remodeling” that seems to be
required?
E.
Two Ways of Chromatin Remodeling
1. The basic tenet that all cells of an organism are isogenic is holding up as a general
principle. DNA losses, selective replication, or rearrangements occur in some cell
lineages, but these seem to be exceptions rather than the rule. Cell differentiation
must therefore be ascribed to differential gene expression [ABD 7.2].
2. In accord with the principle of differential gene expression, cell determination and
cell differentiation require the deployment of specific transcription factors.
3. Transcription factors act by binding to specific promoter and enhancer sequences.
However, such binding may be restricted by the way nuclear DNA is packaged with
histones (nucleosomes) and other chromosomal proteins [HB 10.4].
4. The genes that are most actively transcribed in cells, such as globin genes in red
blood cells or ovalbumin genes in oviduct, coincide with those chromosomal regions
that are most sensitive to mild digestion with DNase I.
5. Conceivably, chromatin could be “remodeled” by changing
a. the density of chromatin packaging, which would render the DNA more or less
accessible to transcription factors. (See sections to F to H).
b. the availability of transcription factors, which could displace histone proteins.
This seems to be the case in induced pluripotent stem cells (see Section I and J).
c. both remodeling processes are based on the dynamic nature of protein binding to
DNA.
F.
Genomic Imprinting
Several observations highlight the importance of semi-permanent modifications of DNA
and histones that change the packaging of chromatin in eukaryotic cells.
1. One line of related observations was made in pronuclear transplantation experiments
with newly fertilized mouse eggs [ABD 4.16].
a. Natural parthenogenesis is unknown in mammals.
b. Unfertilized and artificially activated mouse eggs die halfway through gestation.
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c. Experimental transfer of pronuclei [ABD Table 4.2] can generate zygotes with
i. two female pronuclei but no male pronucleus. They develop into bimaternal
embryos, which cease development about the same time as parthenogenetic
embryos do.
ii. two male pronuclei but no female pronucleus. They develop into bipaternal
embryos, which also die midway through gestation but show defects different
from bimaternal embryos.
iii. one male and one female pronucleus, which develop normally
d. These results indicate that male and female pronuclei contribute some gene
activities to the fertilized egg (zygote) that cannot be substituted by the
homologous genes in the pronucleus from the other sex.
2. A related set of observation was made by human geneticists on patients with
uniparental disomy (UPD), who have inherited two copies of a chromosome from
one parent and no homolog from the other parent.
a. patients with maternal UPD 15 (two chromosomes 15 from mother, no
chromosome 15 from father) show Prader-Willi syndrome, which includes severe
obesity, gonadal dysfunction, short stature, and mental retardation.
b. Paternal UPD 15 causes Angelman syndrome, which includes seizures, sleep
disorder, severe mental retardation, and lack of speech.
3. These and other observations indicate that there is a sex-specific and reversible
silencing of certain autosomal genes in mammals known as genomic imprinting or
DNA imprinting [ABD 16.24].
a. Some genes (about 30-40) are imprinted during spermatogenesis while others are
imprinted during oogenesis.
b. The imprinted genes remain silent in early embryonic and in all somatic cells,
including the brain. The homologous genes are then expressed uniparentally.
c. The imprinting is erased in each generation’s primordial germ cells, so that germ
line cells express both alleles of previously imprinted genes.
d. During gametogenesis, each parent imprints anew the sex-specific set of genes.
4. One ultimate cause of genomic imprinting seems to be a conflict between the sexes
over the amount of nutrients that mammalian mothers transfer to their offspring.
Females imprint genes that would otherwise favor a developing fetus at an undue cost
to the mother. Conversely, males imprint genes that would otherwise limit the
amount of maternal nutrients received by the offspring.
G.
DNA Methylation, Histone Acetylation
1. The mechanisms of imprinting are incompletely understood but seem to involve DNA
methylation and histone acetylation.
2. In more than half of the CpG dinucleotides in mammalian DNA, the C residue is
modified with a methyl group to form 5-methyl cytosine. An existing methylation
pattern in CpG dinucleotides is propagated during DNA replication by an enzyme
called DNA (cytosine-5) methyltransferase I (Dnmt 1) [ABD 16.23].
3. DNA methylation in promoter regions generally inhibits gene expression.
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4. At least one way in which DNA methylation exerts its inhibitory effect is by histone
deacetylation. A protein known as methyl CpG binding protein 2 (MeCP2) binds to
methylated CpG and associates with the enzyme histone deacetylase (HDAC).
5. HDAC in turn removes an acetyl group from lysine residues of histone, rendering the
histone more basic and thus more tightly binding to DNA [ABD 16.18]. In turn,
tighter binding to histone makes DNA less accessible to transcription factors.
6. The opposite reaction is catalyzed by histone acetyltransferase (HAT).
H.
Concept and Key Methods of Epigenetics
1. DNA methylation and histone modifications do not change the DNA nucleotide
sequence, but rather the accessibility of chromosomal DNA for gene-regulatory
proteins [Tsankova et al. (2007a, Fig. 1a,b]. Nevertheless, the changed
chromosomal states and the resulting modifications in gene expression are passed on
during cell division. Hence these mechanisms have been called epigenetic (Greek
epi: over, above). Remarkably, epigenetic states can be affected by behavioral cues
and by drugs, and as these are passed on between generations, so are the resulting
epigenetic states. In this sense, epigenetics can amount to a “soft inheritance” of
acquired traits.
2. For example, relaxed rat mothers lick and groom their pups, and their daughters in
turn grow up to be relaxed and high licking/grooming mothers [HB 13.6].
Characteristically, the mothering style is passed on to both biological and adoptive
daughters. Molecular analysis has shown that high-care mothering activates the gene
for glucocorticoid receptor (GR) in the pups’ hippocampus. The activation was found
to be mediated by DNA demethylation and histone acetylation in the promotor region
of the hippocampal GR gene. Most importantly, a drug inhibiting histone deacetylase
(thus promoting histone acetylation) changed the behavior of rats raised by low-care
mothers towards the behavior normally shown by offspring of high-care mothers.
3. Soft inheritance of acquired traits, as exemplified by the mothering style of rats,
illustrates an important advantage of epigenetic inheritance: It allows a quick but
heritable adaptation to environmental variation. In an uncertain environment, rat
mothers will be anxiously guarding their offspring but not lick them. Thus, the
offspring will not have the happiest childhood but will survive. In a relatively safe
environment, mothers will be relaxed, and their offspring will not only survive but
grow up to be active and exploratory.
4. The new appreciation of epigenesis has led to the development of high-throughput
techniques that will generate mountains of epigenomic data on individuals differing
in age, sex, health status, etc.
5. The key method for detecting methylated cytosine residues in genomic DNA is
known as methyl mapping. It uses sodium bisulfite treatment to convert unmethylated
cytosine to uracil, which is detected by automated sequencing. (Methylated cytosine
resists the conversion to uracil.)
6. The key method for detecting histone modifications and DNA binding proteins is
known as chromatin immunoprecipitation (ChIP). It aims to determine whether
certain genomic regions are associated with specific proteins, such as transcription
factors or modified histones. Briefly, protein and DNA are cross-linked before the
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chromatin is sheared. The DNA fragments cross-linked to the protein(s) of interest
are immunoprecipitated, and then the associated DNA fragments are sequenced.
7. These methods are rapidly being developed for high throughput with the goal of
mapping epigenetic changes over the entire genome in various healthy and diseased
tissues at different stages of development, in primary tumors, etc.
I. The Role of Transcription Factors in Cell Differentiation
If normal cell differentiation is controlled by transcription factors, then “flooding” cells
with transcription factors that they do not normally produce may “derail” them from their
normal pathway of differentiation.
1. Cultured fibroblast were transfected with cDNA encoding MyoD, a transcription
factor characteristic of developing skeletal muscle. The treated fibroblasts behaved
like myoblasts, fusing into myotubes, synthesizing myosin, and proceeding to form
skeletal muscle fibers.
2. Similar results were obtained with other cDNAs representing the MyoD family of
transcription factors [ABD 20.18]. These factors share a basic helix-loop-helix
(bHLH) domain, which functions in the formation of dimers, the transcriptionally
active protein structure. The myogenic bHLH proteins, depending on their
heterodimeric partner, can be activating or inhibiting, which makes them well suited
for acting in bistable control circuits [ABD 20.19]. Indeed, the bHLH proteins
interact with many other regulatory proteins in controlling the growth, patterning, and
differentiation of skeletal muscle [ABD 20.20].
3. Zhou et al. (2008, Nature 455: 627-632) reprogrammed pancreatic exocrine cells to
endocrine (β) cells in adult mice [Zhou et al. Fig.1].
a. They caused the formation of new β-cells by injecting into the pancreas a
combination of three β-cell-specific transcription factors (Ngn3, Pdx1, and Mafa).
b. The induced β-cells were indistinguishable from endogenous β-cells in size,
shape, and ultrastructure. They also secreted insulin and reduced hyperglycemia.
However, they did not organize into the islets characteristic of native β-cells.
c. A transient expression of the inducing factors was sufficient to convert exocrine
cells to a stable new β-cell state.
d. Continuous 5-bromodeoxyuridine (BrdU) labeling and other observations
indicated that the induced β-cells did not divide extensively and did not revert to a
dedifferentiated state for an appreciable time.
e. It should be noted that pancreatic exocrine and β-cells differ in their adult
structure and function but are developmentally related cell types. They share their
embryonic origin from pancreatic endoderm as well as many of their epigenomic
markers. Conversion between pancreatic exocrine and β-cells may therefore
require less chromatin remodeling than transitions between other cell types.
f. In conclusion, the authors suggest differentiated cells can be transformed directly
into a developmentally related cell type without reversal to a pluripotent stem cell
state.
4. These and many other experiments indicate that cell differentiation, in the course of
normal development as well as regeneration, typically begins with the activity of an
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appropriate set of key genes, which initiate hierarchic sequences of subordinate gene
activities. Such unfolding patterns of gene expression patterns may be promoted by
a. availability of transcription factors. These would keep the chromatin
conformation in their target regions open due to the dynamic nature of DNAprotein interactions.
b. epigenetic changes in the target chromatin from a closed to an open conformation,
which would facilitate the binding of key transcription factors to their recognition
sequences in nuclear DNA.
J. Induced Pluripotent Stem Cells
If normal cell differentiation is initiated by transcription factors, then one might expect
that “stemness” (i.e. having the characteristics of stem cells) is also controlled by certain
transcription factors. This idea led to the generation of induced pluripotent stem cells, an
experimental approach that has revolutionized many areas in the life sciences.
1. In a pioneering line of work, Shinya Yamanaka and coworkers at Kyoto University
[Photo Yamanaka] transformed differentiated cells into induced pluripotent stem
cells (iPS cells), which resemble embryonic stem cells (ES cells).
a. The researchers screened existing lines of ES cells for commonly expressed genes
encoding transcription factors. Using retroviral vectors, they transformed mouse
fibroblasts in vitro with extra copies of these candidate genes.
b. The fibroblasts had a genetic locus (Fbxo 15) that was specifically expressed in
ES cells. They inserted a dormant drug resistance allele into Fbxo 15 and used it
as a selectable marker for cells that had been transformed to an ES-like state.
c. In one experiment, they tested 24 candidate genes in groups of four. A
combination of transcription factors known as Oct 3/4, Klf4, Sox 2, and c-Myc
was successful in conferring “stemness”. In each of the culture dishes with
fibroblasts, clusters of cells arose showing the characteristics of stem cells:
embryonic morphology, unlimited division, and pluripotency. The new cells were
therefore called induced pluripotent stem cells (iPSC). The four transcription
factors used to make them have become known as OKSM or canonical factors.
2. In recognition of the potential of iPSC human medicine, investigations were extended
to humans, with an emphasis on comparing the properties of human iPSC and ESC.
3. Mouse and human iPSC were shown to be pluripotent in vivo by their ability to form
teratomas when injected into the skin of mice, and in vitro by culture in the presence
of different growth factors. Mouse iPSC were also shown to be totipotent by the
tetraploid complementation test (section B1d).
4. Follow-up experiments were aimed at avoiding the oncogenic effects of some of the
stemness-inducing transcription factors and the disruptive effects of transgene
insertion by viral vectors.
a. Yu et al. (2007) obtained iPSC by transforming human fibroblasts with a
combination of genes (Oct4, Sox2, Nanog, and Lin28) that did not include c-Myc.
This result was significant because c-Myc is notorious as a proto-oncogene.
b. Woltjen et al. (2009) and Kaji et al. (2009) built on the observation that the
stemness-inducing transcription factors need to act only temporarily. Using a
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known inducible recombination enzyme, they seamlessly removed the transgenes
after they had done their stemness-inducing work.
c. Manos et al. (2010) used synthetic RNAs corresponding to the four classical iPSC
transgenes to transform multiple human cell types into ES-like cells. The authors
call these cells RNA-induced pluripotent stem cells, or RiPS cells. The
transforming RNA is quickly degraded.
In human medicine, iPSC hold much promise for several reasons.
a. Patient-specific iPSC cultures can be used as models for studying human diseases
and as preliminary screens for therapeutic drugs [Science 29 Aug. 2008].
b. iPSC are considered to be great candidates for cell replacement therapy because
they can be generated from the patient’s own cells, so that there will be no
immune rejection.
c. If the need for therapy is due to a genetic disorder, the defective gene can be
replaced by homologous recombination in iPSC culture.
d. As compared to making isogenic ES cells by nuclear transfer, iPSC do not depend
on the availability of donated human eggs. Working with IPSC also avoids
ethical concerns about sacrificing human blastocysts.
e. However, because of their more recent discovery, iPSC are currently further away
from clinical applications than ES cells. And some recent observations have
raised concerns.
i. Lister et al. (2011) observed that the methylation patterns of iPSC retained
some “memories” of the adult cells from which they had been derived.
ii. Laurent et al. (2011) found that human ES cells as well as iPS cells acquired
gene duplications and deletions during culture.
Thus, returning differentiated cells, or their nuclei, to a pluripotent state for cell
replacement therapy requires genomic and epigenomic monitoring to ensure stability
and safety.
The relative ease of making iPSC could hasten attempts to derive gametes from such
cells. Experiments to make sperm and oocytes from mouse ES and iPS cells have
already been reported (Okita et al., 2007; Hayashi 2011, 2012). If corresponding
experiments with human ES cells or iPS cells were successful, existing in vitro
fertilization procedures could be used to make human babies, and infertile couples
may be eager to avail themselves of this way to have children.
There is concern that genetically modified human iPSC may be used to generate
gametes, and thus, genetically “enhanced” human babies. This prospect raises great
concern because many human genes are pleiotropic, and the newly created genotypes
would not have been tested in evolution.
At the behest of Shinya Yamanaka, the principal investigator in the original iPS cell
study, the Japanese science ministry has restricted the production of gametes from
human iPS cells and the implantation of embryos made with human iPS cells into
human or animal wombs. International patent applications that would make the
unauthorized use of iPS cell technology illegal are also under way.
K. Mechanisms of Reprogramming Cells to Pluripotency
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The mechanisms of reprogramming differentiated cells to pluripotency are poorly
understood, and they probably differ for ES cells and iPSC. Conceptually, they would
seem to involve inhibition of cell type-specific genes, avoidance of apoptosis and cellular
senescence, perpetuation of the mitotic cycle, inactivation of the transgenes used in
making iPSC, activation of endogenous pluripotency genes, and erasure of “epigenetic
memories” such as DNA methylations and histone acetylations.
1. Not surprisingly, then, cell reprogramming improves with time.
a. In Gurdon’s nuclear transplantation experiments with Xenopus, extending the
reprogramming time by serial transfer from the blastula stage back into eggs
significantly enhanced the yield of complete frog larvae.
b. Likewise, the induction of pluripotency by transcription factors in vitro is a
process that goes on with time, or with additional rounds of cell division.
2. Reprogramming efficiencies have been disappointingly low.
a. For nuclear transfer, the number of complete animals per nuclei transferred have
been 1% or less, as pointed out earlier.
b. For iPSC induction, the number of stem cell colonies per number of input cells
has ranged from 0.01% to 5%, depending on cell type and technique.
c. In “secondary systems”, where the cell population exposed to OKSM is clonal,
meaning that all cells have the same OKSM gene insertions, the efficiency of
fibroblast reprogramming is generally 1-5%. This observation suggests that in
“primary” cultures cells have to overcome more “roadblocks” on their way to the
fully induced to iPSC state.
3. One approach taken to identifying the nature of these roadblocks is to compare ESC
and iPSC, and the same type of stem cells from mice and humans.
a. Analyses of genome-wide expression patterns and global histone modifications
have shown much similarity, but substantial differences have appeared as well
(Stadtfelder and Hochedlinger, 2010). For instance, iPSC-derived neural cells
have shown a greater propensity than ESC-derived neural cells to form tumors
after transplantation into mouse brains. Also, human iPSC-derived blood
progenitor cells seem to undergo premature senescence.
b. iPSC derived from mouse fibroblasts faithfully reactivate their silenced
(maternally or paternally derived) X chromosome, as they normally would in the
female germ line, and than randomly inactivate one X chromosome upon cell
differentiation. In contrast, human iPSC derived from female dermal fibroblasts
fail to reactivate the silenced X chromosome and keep the same X chromosome
inactivated in differentiated cells. Similarly, human iPSC retain an “epigenetic
memory” of their cells of origin, whereas the same memory is attenuated in
mouse iPSC with repeated passage.
c. Clearly, a better understanding of these differences will be important for the basic
science and potential clinical applications of stem cells.
137\PLURIPOTENT STEMCELLS\14Sp
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