Human Molecular Genetics 2008 17(R1):R37-R41; doi:10.1093/hmg/ddn053
© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Generation of isogenic pluripotent stem cells
James A. Byrne*
Center for Human Embryonic Stem Cell Research and Education, Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University, Palo Alto, CA 94304, USA
* To whom correspondence should be addressed at: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 1050 Arastradero Road, Palo Alto, CA 94304, USA. Tel: +1 6504987303; Fax: +1 6507362961; Email: byrnej{at}stanford.edu
Received December 27, 2007; Accepted February 15, 2008
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ABSTRACT
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The ability to reprogram somatic cell nuclei back into a pluripotent
epigenetic state provides exciting new possibilities for
in vitro research and cell transplantation therapy. There has been
a significant quantity of recent research studies demonstrating
that this epigenetic reprogramming process is possible with
human and non-human primate somatic cells. In this review, various
methodologies for reprogramming primate somatic cells into pluripotent
stem cells are examined, epigenetic reprogramming following
somatic cell nuclear transfer and normal primate embryonic development
is compared, and future potential methods to induce direct reprogramming
without using genetic modification are discussed.
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INTRODUCTION
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Every cell in an adult organism, with very few exceptions, contains
the same genetic DNA code. Cell fate is dictated by the specific
epigenetic code of DNA methylation and histone modifications
established during development and cellular differentiation.
In this way, early embryonic cells, which are pluripotent (able
to form any cell type) become gradually restricted, through
epigenetic modifications, to a specific and generally irreversible
cell fate. The principle goal of the isogenic pluripotent stem
cell (PSC) research field is to reverse or reprogram this differentiated
(somatic) epigenetic code back into a pluripotent embryonic
stem cell (ESC) like state. Once epigenetically reprogrammed,
these isogenic PSCs would be capable of proliferating indefinitely
in culture and differentiating into any of the 200 plus cell-types
found in the adult body. These PSCs would be isogenic (genetically
identical) to the donor individual/patient, so if the patient
suffered from a degenerative disease then the differentiated
therapeutic derivatives of the isogenic PSCs could theoretically
be transferred back into the patient (an autologous transfer
or autograft) to cure or alleviate the symptoms of the specific
disease without eliciting an immune response (Fig.
1).
Neural precursor cells could be created for patients with spinal
cord injury, and dopamine-secreting cells could be created for
patients with Parkison's disease. Almost any physical injury
or degenerative disease could theoretically be treated in this
manner, either directly from isogenic PSC derivatives (
1), or
following some form of gene therapy to correct the patient's
original genetic defect (
2,
3). Nuclear transfer-derived ESCs
have been differentiated into dopaminergic neurons that have
alleviated the phenotype of a mouse model of Parkinson's disease
(
1), and mice with an immunodeficiency or sickle cell phenotype
have been treated with genetically repaired hemotopoietic precursors
derived from isogenic PSCs generated through either nuclear
transfer (
2) or direct reprogramming (
3). This isogenic cellular
therapy remains the long-term goal of the isogenic PSC field.
However, the more immediate benefit from isogenic PSC derivation
research would be to generate isogenic PSCs carrying the same
genetic defect as a patient with a genetic disease for
in vitro research purposes. These disease-specific isogenic PSCs could
be used for
in vitro research into understanding the mechanisms
of poorly understood diseases, drug screening, developmental
biology and other forms of basic research. For example, an isogenic
PSC line derived from a patient with motor neuron disease could
be differentiated into motor neurons
in vitro and various drug
and chemical combinations could be screened to search for drugs
preventing or significantly decreasing the destruction of motor
neurons. Both the long-term cellular transplantation and short-term
in vitro research applications of isogenic PSCs require that
the epigenetic state of the differentiated somatic cell nucleus,
which is typically very stable, be fully reprogrammed back into
a pluripotent state. There are currently four different methodologies
capable, to a greater or lesser extent, of reprogramming primate
somatic cell nuclei back into a pluripotent state: cell fusion
(
4), extract-based reprogramming (
5), direct reprogramming (
6)
and somatic cell nuclear transfer (SCNT) (
7).
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Figure 1. There are multiple potential uses for human isogenic pluripotent stem cells (PSCs) including: investigating the mechanisms of disease, drug screening, human genetics, developmental biology and autologous transfer back into the donor patient.
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METHODS FOR REPROGRAMMING SOMATIC CELL NUCLEI
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It has been demonstrated both in the mouse (
8) and in the human
(
4) when a differentiated somatic cell is fused with a pluripotent
ESC, unknown factors within the pluripotent cell are capable
of modifying the epigenetic state of the somatic nucleus back
into a pluripotent state, with reactivation of key pluripotency
markers such as
OCT4,
SOX2 and
NANOG and differentiation of
the somatic/ESC hybrid cells into representatives of all three
germ layers following
in vivo teratoma formation (
4). This fusion-based
approach provides an important
in vitro research tool and helps
in the identification of enhancers of reprogramming. However,
the hybrid cells resulting from this fusion-based reprogramming
possess both somatic chromosomes and ESC chromosomes, and there
is currently no known methodology capable of removing the ESC
chromosomal complement, significantly limiting the utilization
of the hybrid cells themselves (Fig.
2A). The second method
for reprogramming primate somatic cells involves creating a
crude cell extract from a pluripotent population of cells (either
embryonic stem or embryonic carcinoma cells) and then transferring
this crude extract, with its associated reprogramming factors,
directly into the somatic cells. This extract-based reprogramming
approach has been used to reprogram human somatic cells to re-express
pluripotency markers (
5). However, cells produced in this manner
possess only a partially reprogrammed transcriptional state,
are not pluripotent and no extract reprogrammed cell has yet
demonstrated any
in vivo differentiation capacity (Fig.
2B).
The third method for reprogramming a primate somatic cell into
a pluripotent epigenetic state involves using virus-mediated
transduction to transfer specific reprogramming factors directly
into multiple random points throughout the somatic chromatin.
The expression of the integrated factors is induced by strong
constitutively active promoters to produce large amounts of
the reprogramming proteins that over time induce the somatic
cell nucleus into a pluripotent epigenetic state via an unknown
mechanism. Human somatic cells have been successfully reprogrammed
into isogenic PSCs using genetic integration of various factors,
combinations of which consistently include
OCT4 and
SOX2 (
6,
9–
11)
and these directly reprogrammed cells have demonstrated pluripotency
with the capacity to differentiate into representatives of all
the three germ layers following
in vivo teratoma formation.
However, isogenic PSCs produced using this direct reprogramming
approach require genetic integrations of strongly expressed
factors into multiple random locations throughout the genome.
This presents a problem, from a therapeutic standpoint, as the
initial integration of these factors could potentially inactivate
various genes resulting in negative downstream effects, and
the possible future reactivation of potentially oncogenic factors,
such as
OCT4 (
12), significantly limits the potential therapeutic
applications of these directly reprogrammed isogenic PSCs (Fig.
2C). While the integration of genetic factors may preclude the
usage of directly reprogrammed PSCs for cell transplantation
therapies, they would almost certainly be extremely valuable
for
in vitro applications such as disease research, drug discovery
and human genetics. The fourth methodology to reprogram a primate
somatic cell nucleus into a pluripotent epigenetic state involves
transferring a differentiated nucleus into a metaphase II oocyte
(ovum) that has been enucleated (had its DNA either destroyed
or removed). The oocyte cytoplasm is capable of epigenetically
reprogramming the primate somatic nucleus into a pluripotent
state capable of generating teratomas containing representatives
of all the three germ layers (
7). The SCNT methodology is the
gold standard for epigenetic reprogramming as it produces a
PSC that is isogenic to the donor, diploid, fully pluripotent
and not genetically modified in any way, making it ideal for
both the short-term
in vitro research applications and the long-term
cellular transplantation options (Fig.
2D).
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SOMATIC CELL NUCLEAR TRANSFER: OOCYTE-BASED REPROGRAMMING
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The first demonstration proving that a vertebrate somatic cell
nucleus could be reprogrammed back into an embryonic state was
in 1962, when John Gurdon transferred the nucleus from a differentiated
intestinal epithelial cell into an enucleated
Xenopus laevis egg, generating a cloned embryo that developed into a fertile
adult frog (
13). This oocyte-based nuclear reprogramming approach
was successfully repeated by Ian Wilmut and Keith Campbell with
an adult mammalian somatic nucleus in 1997 (
14). To date a large
number of animals have been cloned through SCNT (
15) and PSCs
have been derived from mouse (
16), bovine (
17) and rabbit (
18)
SCNT embryos. However, deriving PSCs from primate SCNT embryos
proved unachievable with the standard methodologies (
19,
20)
and it was speculated that biological barriers, specific to
the primate, may prevent successful reprogramming following
SCNT (
20). Recently, it was demonstrated that a modification
to the SCNT methodology—the usage of polarized light during
the oocyte enucleation step rather than the usual Hoechst staining
and UV visualization—significantly improved the rate of
SCNT blastocyst formation (
21,
22). It is possible that the UV-based
enucleation methodology may be particularly damaging to primate
oocytes due to their relative transparency in comparison with
similar sized oocytes from other species, including cows, pigs
and sheep. Using the modified polarized light SCNT protocol
it was demonstrated that fibroblast nuclei derived from an adult
rhesus macaque male could be reprogrammed into PSCs following
SCNT. The rhesus monkey SCNT-produced PSC lines exhibited a
normal ESC morphology, expressed key stem-cell markers, were
transcriptionally similar to control ES cells and differentiated
into multiple cell types
in vitro and
in vivo (
7). This work
succeeded as the first successful nuclear reprogramming of primate
somatic cell nuclei into a fully pluripotent epigenetic state.
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IMPAIRED EPIGENETIC REPROGRAMMING FOLLOWING SOMATIC CELL NUCLEAR TRANSFER
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While it is truly remarkable that the cytoplasmic content of
an oocyte is capable of reprogramming a somatic nucleus back
into a pluripotent epigenetic state, the efficiency of this
reprogramming process is extremely low. In total, 304 primate
oocytes were required to produce only two isogenic PSC lines
(
7) and this low reprogramming efficiency has been demonstrated
across all mammalian species cloned to date (
23). There is significant
empirical evidence from comparison analysis of primate embryos
produced through normal fertilization and SCNT, that in most
cases, following nuclear transfer, the somatic nucleus is incompletely
reprogrammed to a pluripotent epigenetic state (
21,
22,
24). Following
both human and non-human primate fertilization, the paternal
(sperm) nucleus is actively demethylated by the oocyte cytoplasm
during the one cell stage, while the maternal (oocyte) nucleus
becomes gradually and passively demethylated through multiple
rounds of cell division (
24,
25). On the third day of normal
development the primate embryo undergoes embryonic genome activation
with a large number of embryonic genes getting significantly
expressed at this stage (
26). Between developmental days 5 and
7, the primate embryo divides into a ball of
50–100 cells
and forms an inner cavity. This cavitated embryo is referred
to as a blastocyst and contains an inner cell mass (ICM) of
embryonic cells that express the pluripotency marker
OCT4 (
27).
It is from these
OCT4 positive ICM cells that PSC lines can
be derived (
28). Following primate SCNT, the DNA methylation
is usually not efficiently removed from the somatic chromatin
(
24) and on day 3 of embryonic development the primate SCNT
embryos typically demonstrate a significantly higher level of
DNA methylation and a significantly lower level of histone (H3K9)
acetylation, suggesting an impaired embryonic genome activation
(
24). The small number of SCNT embryos that do successfully
reach the blastocyst stage can still demonstrate an
OCT4 expression
pattern that is aberrant, reduced or absent (
21,
22), which may
explain why the ESC-derivation efficiency from primate SCNT
embryos (
7) is lower than from fertilized primate embryos (
28).
Recent research in the mouse (
29) and rabbit (
30) has demonstrated
that histone acetylation inhibitors such as trichostatin A and
sodium butyrate can induce epigenetic modifications that result
in an improved developmental efficiency following SCNT (
29,
30).
It would be very interesting to discover if these and other
epigenetic modification approaches can improve the developmental
efficiency following human and non-human primate SCNT. However,
even if the efficiency of SCNT is increased, the human SCNT
reprogramming methodology is still limited by the requirement
for fresh human oocytes and the restricted access to this material.
The ideal approach would be to recapitulate the epigenetic reprogramming
ability of an oocyte, which can reprogram a somatic cell nucleus
without genetically modifying it, without actually requiring
the usage of an oocyte, which is limited in supply. The solution
to this problem may lie in modifying the human direct reprogramming
protocol (
6,
9) so that the epigenetic reprogramming processes
can occur without genetic modification of the somatic chromatin.
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DIRECT REPROGRAMMING: AVOIDING GENETIC INTEGRATION
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Recent research has demonstrated in both the mouse (
3,
31–
33)
and the human (
6,
9–
11) that it is possible to use genetic
integration of various factors into somatic cell chromatin to
induce the somatic cell to reprogram back into a PSC. These
randomly incorporated factors become transcriptionally silenced
over time through de-novo DNA methylaton (
33). However, these
factors can and do get spontaneously reactivated, resulting
in negative downstream effects (
32). Mice derived from PSCs
produced though direct reprogramming have a propensity to develop
tumors, principally through the re-activation of
cMYC (
32).
While
cMYC itself may not be necessary for reprogramming to
occur (
10), all current reprogramming protocols still use
OCT4,
a dose dependent oncogene (
12), and the act of genetic integration
itself may inactivate tumor suppressor genes resulting in oncogenesis.
The possibility these genetically modified PSCs may become malignant
significantly reduces the potential applications of these cells
for cell transplantation therapy. Unfortunately, as the integration
of the factors is at multiple locations randomly dispersed throughout
the genome, direct physical extraction of the integrated factors
following the reprogramming process is not possible with current
technology. Therefore, the only way to significantly increase
the feasibility of isogenic PSC-based therapies would be to
reprogram the somatic cells without the initial incorporation
of the reprogramming factors into the somatic cell chromatin.
All of the theoretical methodologies for directly reprogramming human somatic cells into PSCs without genetic integration seek to maintain a high level of the various reprogramming proteins within the somatic cell for sufficient time to allow for the reprogramming events to occur. There are currently three general approaches to achieve this: add the reprogramming proteins themselves, add the nucleic acids (DNA or mRNA) coding for the reprogramming proteins or introduce external signals or small molecules capable of activating the expression of the reprogramming proteins. It is not yet clear which of these methodologies will succeed in reprogramming human somatic cells without genetic integration. However, the most similar approach to the current successful direct reprogramming methodology would be the introduction of non-integrated vectors containing the same strongly induced reprogramming factors. While retroviruses and lentiviruses incorporate into the host chromatin, the vast majority of introduced adenoviruses and plasmids do not integrate and remain epichromosomal. These non-integrated epichromosomal vectors utilize the nuclear machinery to continually transcribe and translate the genes they contain, making them ideal for expressing large quantities of the reprogramming proteins inside the transfected cells. The principle disadvantage with the epichromosomal vector approach is that as the cells divide, the vector number per cell decreases, potentially removing or eliminating the expression of the relevant factors before the reprogramming process is complete. One possible solution to this dilution issue is to utilize the serum concentration in the cell culture medium as a variable to control cell division. The concentration of serum in the cell culture medium demonstrates a dose-dependent effect on the cell population doubling time of primary human fibroblasts, with serum concentrations of 10, 4, 2 and 1% giving a respective population doubling time (in hours) of 17.5 ± 0.7, 21.5 ± 0.5, 28.7 ± 1.7 and 57.4 ± 10.8 (Byrne, Chang and Reijo Pera, unpublished data) opening up the possibility of using reduced serum concentrations to significantly slow cell growth and thereby reduce the rate at which the non-integrated vectors are diluted through cell division. Additionally, primary human fibroblasts can be induced to stop dividing altogether (enter quiescence) through exposure to a 0.5% serum concentration and those quiescent primary human fibroblasts can be induced to recommence cell division again by re-exposing the cells to higher serum concentrations (Byrne, Chang and Reijo Pera, unpublished data). By using non-dividing quiescent cells, very high intra-cellular concentrations of the reprogramming factors could be produced, potentially increasing the rate of epigenetic reprogramming without factor integration into the chromatin, an exciting, albeit as-of-yet untested hypothesis. When human nuclei are injected into Xenopus oocytes (34) or into mouse myotubes (35) they undergo significant nuclear reprogramming in the absence of DNA replication or cell division, providing supporting evidence for the hypothesis that direct reprogramming of human somatic cell nuclei into a pluripotent epigenetic state may be possible in non-dividing cells.
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CONCLUDING STATEMENT
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Producing human PSCs genetically identical to a patient generates
exciting new possibilities for investigating the mechanisms
of disease, drug screening, human genetics, developmental biology
and other forms of
in vitro research. Once their
in vivo safety,
functionality and long-term survival is established, it opens
the door for allowing these cells to be used for autologous
transfer back into the donor patient. Eventually, it may be
possible to use human PSCs and their derivatives to replace
our cells and regenerate our bodies as they age, mutate and
die, allowing us cure our injuries, diseases and disorders,
and live longer, healthier lives.
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FUNDING
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J.B. is supported by comprehensive grant to R Reijo Pera (California
Institute for Regenerative Medicine; #RC1-00137-1).
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ACKNOWLEDGEMENTS
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I am very much indebted to Jessica Byrne for painting both Figures
1 and
2, and for her editorial review of the manuscript. I would
also like to thank Renee Reijo Pera for her support, advice
and valuable scientific discussions.
Conflict of Interest statement. None declared.
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ABSTRACT
INTRODUCTION