Indian Journal of Experimental Biology
Vol. 49, June 2011, pp. 409-415
Review Article
Cellular reprogramming of somatic cells
Huseyin Sumer, Jun Liu & Paul J Verma*
Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, 27-31 Wright Street,
Clayton VIC 3168, Australia
The process of ‘cell reprogramming’ can be achieved by somatic cell nuclear transfer, cell fusion with embryonic stem
cells, exposure to stem cell extracts, or by inducing pluripotentcy mediated by defined factors giving rise to what are termed
induced pluripotent stem cells. More recently, the fate of a somatic cell can be directly induced to uptake other cell fates,
termed lineage-specific reprogramming, without the need to de-differentiate the cells to a pluripotent state. In this review we
will describe the different methods of reprogramming somatic cells.
Keywords: Epigenetic reprogramming, Lineage-specific reprogramming, Nuclear reprogramming, Pluripotency,
Somatic cell, Stem cell
The process of de-differentiating somatic cells to a
pluripotent state whereby they adopt characteristics of
embryonic stem cells is referred to as ‘cellular
reprogramming’. This can be achieved through
somatic cell nuclear transfer (SCNT), cell fusion with
embryonic stem (ES) cells, exposure to stem cell
extracts, or by inducing pluripotentcy mediated by
defined factors giving rise to what are termed induced
pluripotent stem (iPS) cells. The process of
reprogramming and resulting pluripotency in the
resulting cells vary considerably, and the interrogation
of different approaches has aided in elucidation of
reprogramming process. In this review we will
describe the different methods of reprogramming
somatic cells to a pluripotent state and the knowledge
gained from each.
Somatic cell nuclear transfer—Nuclear transfer
typically involves the removal maternal chromosomes
from an oocyte resulting in an enucleated oocyte
this is followed by insertion of the donor cell nucleus
(Fig. 1). Embryonic development is then artificially
triggered by inducing an increase in intracellular
calcium. In mammals the reconstituted embryo can
recapitulate embryogenesis and on transfer into a
recipient animal can resulting in full term
development giving rise to a cloned animal. This
process is known as reproductive cloning.
Alternatively, embryonic stem (ES) cells can be
——————
*
Correspondent author:
Telephone: + 61 3 9594 7000;
Fax: + 61 3 9594 7416:
E-mail: [email protected]
isolated from the cloned blastocysts, and this is
accordingly termed therapeutic cloning. The resulting
NT-ES cells can be used as a tool for biomedical
research or as a source of cells for transplantation
back to the somatic cell donor without the fear of
immune rejection, as the cells are genetically identical
to the donor.
The process of nuclear transfer was pioneered in
frogs. In 1952, Briggs and King1 have shown
that replacing the nucleus of an enucleated frog
(Rana pipiens) oocyte with the embryonic nuclei of
blastula cells do not hinder embryo development2.
The process of somatic cell nuclear transfer (SCNT)
has been first demonstrated by Gurdon and
colleagues2 in 1958 using enucleated oocytes from the
African clawed frog (Xenopus laevis) and
differentiated adult intestinal cells as the somatic cell
donor. They have shown that a somatic cell can be
reprogrammed to a totipotent state which results in
the generation of adult clones2. Ability of an adult
mammalian cell to be reprogrammed has been
demonstrated almost half a century later with the birth
of Dolly the sheep as a result of SCNT using a
mammary epithelial cell3. Collectively these results
demonstrate that adult cells which are programmed to
express a subset of specific genes to the differentiated
cell, can be reprogrammed or de-differentiated to give
rise to an organism genetically identical to the donor
cell. Definitive evidence that terminally differentiated
cells can be reprogrammed has been reported when
mice were cloned from mature lymphocytes4. The
resulting cloned mice and ES cell lines harbor
receptor rearrangements which only occur in
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INDIAN J EXP BIOL, JUNE 2011
terminally differentiated lymphocytes identical to that
of the donor cell line.
The biggest impact of SCNT to date has been its
ability to be translated to the agricultural industry5.
The process of reproductive cloning has been applied
to cattle6, goat7, sheep3, pig8, and horse9. This
technique can be used to create multiple copies of
animals with highly valued or desirable traits, such as
cows with high milk production or bulls with high
quality meat10. It has been shown that there are no
significant difference in biological properties and the
nutritional value of milk and meat obtained from
clones compared with non-cloned animals11,12,
suggesting that products from cloned animals and
their offspring are safe for human consumption and
do not seem to pose any risks to human health as
determined by the United States Food and Drugs
Administration13,14.
Therapeutic cloning is the most applicable form of
SCNT for humans. Ability to generate pluripotent
stem cells from a patient with a disease in
combination with gene therapy has the potential to
treat
diseases
through
autologous
cellular
transplantation. The first proof of principle study on
Fig. 1—Different methods of reprogramming somatic cells.
SUMER et al.: CELLULAR PROGRAMMING OF SOMATIC CELLS
the therapeutic application of SCNT derived cell lines
has been reported in 2002 whereby ES cells were
derived from SCNT blastocysts using donor cells
from immune-deficient Rag2-/- mice15. Following
targeted homologous recombination to correct the
Rag2 recombinase gene in the SCNT derived ES
cells, the cells were differentiated in vitro into
hematopoietic stem cells (HSCs) for transplantation44.
However, there are a number of technical hurdles that
need to be addressed before SCNT can be a viable
source of pluripotent cells for human cell therapy.
SCNT is a very resource intensive procedure
requiring large number of oocytes16. In humans there
have only been three reports of human SCNT
embryos reaching the blastocyst stage17-19, with no
reports of ES cell derivation.
Cell fusion—Reprogramming of somatic cells by
cell fusion involves the hybridization of a somatic cell
with a pluripotent cell resulting in a tetraploid cell
hybrid (Fig. 1). Cell fusion can be induced
experimentally in a number of ways including using
chemicals such as PEG, electrical currents and
virally20. In most of the cell hybrids, the phenotype of
the less-differentiated fusion partner dominates the
phenotype of the more-differentiated partner. The first
study to show that the phenotype of the pluripotent
cell dominates in cell fusion has been performed by
Miller and Ruddle21 who have found that cell hybrids
made between mouse EC and thymocytes take on the
pluripotent characteristics of ECC and can form
tumors containing derivatives of the three germ layers
in the teratoma assay. Furthermore, other pluripotent
stem cells including embryonic germ cells and
embryonic stem cells have been shown to reprogram
somatic cells by cell fusion with the resulting hybrids
having adopting a number of pluripotency
properties22-26.
Although ES-somatic cell hybrids display most
properties of pluripotent stem cells their tetraploid
state renders them incapable of significantly
contributing to the late gestation epiblast26 and
chimeras27. Therefore, in order for cell fusion based
reprogramming of somatic cells to be used as an
alternative method of generating autologous
pluripotent cells the removal of the ES cell derived
DNA
would
be
required
following
reprogramming28,29. One approach that has been
proposed involves the successful reprogramming of a
somatic cell in a heterokaryon before nuclear fusion,
followed by subsequent enucleation of the ES
411
nucleus, which can potentially lead to the generation
of autologous pluripotent cells28,30. This approach
results in only partial reprogramming of the somatic
nucleus30. An alternative approach involves the
removal of the specific ES cell derived chromosomes
responsible for self-recognition in the ES-somatic cell
hybrid resulting in a hybrid aneuploid cell that is
immune matched to the somatic cell31. Despite these
technical advances more advances in the cell fusion
field are required before it can be considered a viable
alternate method for generating autologous
pluripotent stem cells for clinical applications.
Cell extracts—The third process of reprogramming
somatic cells involves the exposure of differentiated
cells or nuclei to cell extracts made from amphibian
oocytes, or more recently to cell extracts made from
mammalian embryonal carcinoma and ES cells32-35.
This technique involves the reversible permeabilisation
of differentiated cells using the chemical streptolysinO (SLO) a member of the family of cholesteroldependent cytolysins, followed by exposure to cell
extracts (Fig. 1). The lesions induced in the plasma
membrane by SLO are repaired/resealed when Ca2+ is
added to the media36. Such cell extract exposure has
been shown to partially reprogram the treated cells
toward an embryonic state, predominantly in
transformed and immortalized cell lines.
The first reprogramming studies using cell extracts
have shown that incubation of somatic cells in
Xenopus egg extracts results in remodeling of
chromatin and changes to gene expression37-39.
Reprogramming of differentiated cells by exposure to
mammalian cell extracts was first demonstrated by
Hakelian et al.32 who have shown that HEK293T
(human embryonic kidney) cells exposed to
stimulated T cell extracts results in direct
reprogramming
toward
a
lymphoid-specific
phenotype. Furthermore, it has been demonstrated
that cell extract based reprogramming involves ATPdependent chromatin remodeling32,40.
The first reprogramming studies using pluripotent
stem cell extracts show that both HEK293T cells and
the immortalized NIH/3T3 mouse fibroblast cells
acquire characteristics of pluripotent stem cells when
exposed to extracts made from a pluripotent human
carcinoma cell line (NCCIT)35. The resulting partially
reprogrammed cells formed colonies (alkaline
phosphatase positive) express pluripotency markers
and switch off differentiation markers, and have
undergone epigenetic changes at the promoters of a
412
INDIAN J EXP BIOL, JUNE 2011
number of pluripotent gene loci35,41. More recently,
human fetal fibroblasts have been shown to form hES
cell-like colonies when treated with a combination of
chromatin inhibitors and hES cell extracts33. In this
study, it has been shown that it is essential to pre-treat
somatic cells with the epigenetic modifiers 5-aza-2′deoxycytosine and trichostatin A, histone deacetylase
and DNA methyltransferase inhibitors respectively,
prior to exposure to hES cell extracts. The resulting
reprogrammed cells have up-regulated a number of
pluripotency genes and although reprogramming to a
complete pluripotent state cannot achieved, the cells
may be trans-differentiated into neurons under
differentiation conditions33. However, an important
point of note on the cell extract technique is that it has
also been demonstrated that pluripotent stem cells that
are the source of the cell extracts can in fact survive
the extract isolation technique/treatment and
constitute a potential source of contamination in
subsequent analysis34.
Induced pluripotent stem cells—The most robust
and exciting advance in the reprogramming field has
been reported by Takahashi and Yamanaka42 who
have accurately hypothesized that factors that are
intrinsically involved in the maintenance of
pluripotency of ES cells also play an important role in
the reprogramming process42. Following a screen of
24 genes associated with pluripotency, they have
identified that the ectopic retroviral expression of four
exogenous transcription factors, Oct3/4, Sox2, Klf4
and c-Myc, can reprogram mouse somatic fibroblast
cells to a pluripotent state and have been termed these
cells ‘induced Pluripotent Stem’ (iPS) cells42 (Fig. 1).
Mouse and human iPS cells have been shown to be
similar to their ES cell counter parts in terms of gene
expression profile, epigenetic state, differentiation
potential both in vitro and in vivo, can be used to
generate autologous cells for transplantation studies,
can reprogram other somatic cell by cell fusion, and
the ability to give rise to germ line competent
chimeras in mouse42-47. iPS cells can be generated
both from primary somatic cell cultures as well as late
passage cells48, as well as from a variety of cell types
at varying efficiencies49. Furthermore, the same 4
factors5,46 or the combination of Oct4, Sox2, Nanog
and Lin2850, have been shown to reprogram human
somatic cells, as well as, monkey51, rat52,53, and
porcine54-56.
Use of viruses and random integration of these
transgenes into the host genome often result in
variable reprogramming and due to the risk off
associated insertional mutagenesis iPS cells generated
in this way may have limited use in a clinical setting.
To circumvent this has been a number of reports
demonstrating that iPS cells can be generated by
reducing the number of viral constructs used47,57
and/or minimize viral integration through substituting
combinations of key reprogramming factors with
either chemical compounds 58,59 or utilizing specific
populations of somatic cells endogenously expressing
one of more of the key pluripotency factors60.
Furthermore, iPS cells can be generated without
genetically modifying the somatic genome by the
expression of the pluripotency factors from nonintegrating viruses61, transposons that can be excised
following successful reprogramming62, episomal
transfection of plasmids63, delivery of reprogramming
proteins, delivery of microRNA64 or mRNA65.
Interestingly, iPS cells appear to have an
‘epigenetic memory’ of the somatic cell type from
which they are derived. When the differentiation
potential into the cells of hematopoietic or osteogenic
lineages are compared for ES cells, blood-derived
iPSCs, fibroblast-derived iPSCs and fibroblast cells
reprogrammed by SCNT, a number of differences
have been observed66. Blood-derived iPSCs are more
efficient
than
fibroblast-derived
iPSCs
at
differentiation toward hematopoietic lineages.
However, fibroblast-derived NT-ES cells are even
more
efficient
at
differentiation
towards
hematopoietic lineages suggesting that SCNT may
result in more complete reprogramming than
transcription factor based reprogramming66. Similarly,
transcriptional profiling of iPS cells derived from tail
tip-derived fibroblasts, splenic B cells, bone marrowderived granulocytes and skeletal muscle precursors
have been shown to continue to express transcripts
from their cell type of origin, and the blood derived
iPS cells are more efficient at in vitro differentiation
hematopoietic lineages67. Furthermore, the epigenetic
and functional differences of iPS cells derived from
different lineages can be abrogated when the iPS cells
are cultured in the presence of chromatin modifying
compounds or after continuous passaging66,67.
The process of transcription factor based
reprogramming has been further optimized to directly
induce somatic cells to uptake other cell fates, termed
lineage-specific reprogramming, without complete dedifferentiation to a pluripotent state (Fig. 1). Using a
similar candidate gene approach to Yamanaka, a
screen of 19 genes that encode transcription factors
involve in neuronal development or function
SUMER et al.: CELLULAR PROGRAMMING OF SOMATIC CELLS
identified that a minimum of the three genes Ascl1,
Brn2 and either Myt1l or Zic1, can convert mouse
fibroblasts directly into neurons68. Similarly, a screen
of 14 candidate genes involved in cardiomyocyte
development and function identified that the three
transcription factors Gata4, Mef2c, and Tbx5 can
convert mouse fibroblasts into cardiomyocytes69.
Interestingly, the trans-differentiation of fibroblasts
into other somatic cell fates is remarkably high when
compared to de-differentiation to a pluripotent state.
Efficiency of transcription factor induced conversion
of mouse fibroblasts into neurons or cardiomyocytes
is around 20% (Refs 68, 69), in contrast to the
inefficiency of generating iPS cells, which is around
0.01-0.5%44,70.
7
8
9
10
11
Summary
The field of reprogramming is rapidly evolving and
the various techniques have aided in the
understanding of both the differentiation and dedifferentiation process. SCNT remains the benchmark
for reprogramming somatic cells to a pluripotent state,
however, the ability to directly program somatic cells
to other cell fates by activating distinct combinations
of lineage-specific factors may negate the need to dedifferentiate and then re-differentiate cells for
therapeutic applications. The added advantage of
direct programming approach is that it reduces the
risk of tumor formation associated with
transplantation
of
pluripotent
cell
derived
therapeutics. Nevertheless all approaches to
programming and reprogramming of somatic cells
need to be explored to increase our understanding of
this incredible phenomenon before meaningful
therapeutic outcomes can be achieved.
12
13
14
15
16
17
18
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