Research Article: Molecular Mechanisms of Induced Pluripotency

Date Published: , 2012

Publisher: A.I. Gordeyev

Author(s): I.A. Muchkaeva, E.B. Dashinimaev, V.V. Terskikh, Y.V. Sukhanov, A.V. Vasiliev.



In this review the distinct aspects of somatic cell reprogramming are discussed.
The molecular mechanisms of generation of induced pluripotent stem (iPS) cells
from somatic cells via the introduction of transcription factors into adult
somatic cells are considered. Particular attention is focused on the generation
of iPS cells without genome modifications via the introduction of the mRNA of
transcription factors or the use of small molecules. Furthermore, the strategy
of direct reprogramming of somatic cells omitting the generation of iPS cells is
considered. The data concerning the differences between ES and iPS cells and the
problem of epigenetic memory are also discussed. In conclusion, the possibility
of using iPS cells in regenerative medicine is considered.

Partial Text

Pluripotent stem cells are capable both of self-renewal and generation of all the
cell types that constitute the three germ layers. Until recently, pluripotent stem
cells were derived from cultures obtained from the internal cell mass of the
blastocyst (embryonic stem cells – ESCs) [1, 2]. However, the procedure of
obtaining ESCs was burdened with numerous practical and ethical issues that made it
impossible to use ESCs in clinical practice. Because of this, the global scientific
community pressed on with its active search for an appropriate method for obtaining
cells with characteristics similar to those of ESCs. Certain progress was achieved
in 1997, when Wilmut et al . reprogrammed breast somatic cells via
the transfer of their nuclei into oocytes after the second meiotic division (somatic
cell nuclear transfer, SCNT) [3–6]. In
2001, Tada et al . achieved the same result via the fusion of mouse
thymocytes with ESCs [7]. However, all
attempts aimed at eliminating the technical complexity and low reproducibility of
these methods failed, as did the attempts aimed at using these techniques for
primate cells.

Autoregulatory loop. The equilibrium between Klf4 and c-Myc. The impact of the
Ink4/Arf locus

Low reprogramming efficiency is one of the problems of cell reprogramming through the
addition of transcription factors. In the previous procedure of KMOS transfection,
approximately 0.01–0.1% of the transfected cells were subjected to
reprogramming; this index is considerably lower than that obtained when the cell
fusion or nuclear transfer technique is used. Several hypotheses for explaining such
a low yield of reprogrammed cells have been put forth:

The original technique for the transfection of reprogramming transcription factors
using lentiviral vectors has a number of substantial drawbacks that impede its
application in clinical practice. Virus integration into the host genome (up to 20
insertions per reprogramming procedure) increases the risk of tumor formation, since
virus incorporation into the target cell genome can accidentally activate or
inactivate the host genes, thus increasing the mutagenesis risk. Continuous
transgene overexpression is also problematic because of possible incomplete
transgene suppression during the reprogramming and upon subsequent differentiation.
Even the presence of several pluripotent stem cells in the transplanted tissue may
result in tumor development [47].

The use of low-molecular-weight compounds, the so-called small reprogramming
molecules, is one of the approaches to the reprogramming of human somatic cells.
Combined with the earlier designed methods, these molecules are capable of either
functional substitution of particular reprogramming factors or facilitating the
increase in efficiency of the process. Thus, BIX-01294 (BIX), an inhibitor for the
G9a histone methyltransferase, was used. The application of this agent in addition
to the transfection using the Klf4 , c-Myc , and
Sox2 , as well as the Klf4 and
Oсt4 sets within the lentiviral vectors, considerably
enhanced (by a factor of 6–10) the yield of the reprogrammed cells [45]. This is attributed to the specific
activity of BIX, which facilitates chromatin de-condensing and therefore can
functionally substitute the c-Myc transcription factor [45]. 2-propylvaleric acid (valproic acid, VPA) is another
compound capable of considerably increasing the reprogramming efficiency [62]. It can specifically inhibit DNA
methyltransferases and histone deacetylase. According to [38], the use of this small molecule, in addition to the
standard KMOS set, enhances the reprogramming efficiency by 1–2 orders of
magnitude and allows one to dispense with the c-Myc oncogene. The
positive effect of 5-azacytidine (5-azaC) on the yield of reprogrammed cells was
demonstrated using the same strategy for DNA methyltransferase inhibition [29]. Reprogramming efficiency can also be
increased via the introduction of a small interfering RNA (siRNA), which inhibits
the transcripts of the commitment-associated genes [29]. The positive effect of CHIR99021, a specific inhibitor of glycogen
synthase kinase 3 (GSK-3), on efficiency in the reprogramming of mouse embryonic
fibroblasts has been described. The yield of iPSC colonies was considerably
increased by using CHIR99021. The number of reprogramming factors was reduced to
two, Klf4 and Oct4, thanks to the use of this
reagent in a number of experiments [63].

The so-called direct reprogramming can be attributed to areas that require special
attention. This strategy presupposes the use of various methods for the
transdifferentiation of a specialized cell type into another one, bypassing the
stage of formation of pluripotent stem cells. If the method for direct reprogramming
is designed, it would be possible to use cell technologies in clinical practice.

Despite the fact that many characteristics of iPSCs are rather similar to those of
ESCs, there are also significant differences between these cell types. Among others,
there are differences in the levels of control of pluripotency gene expression and
in the formation of viable organisms after these cells are transplanted into a
developing blastocyst to generate chimeric mice. Evidence has been obtained in
support of the fact that the methylation levels of CpG islands in ESCs and iPSCs are
similar [76]. A full genome analysis of the
CpG islands localized in the functional regions comprising more than 14,000 genes
revealed the difference in the methylation levels of 46 genes. The total CpG
methylation of the promoter regions in pluripotent cells is higher in comparison to
that found in somatic cells. Two ESC and iPSC lines derived from material that was
genetically identical to ESCs were compared [77]. In animal chimera experiments, viable mice were successfully
obtained from two ESC lines, whereas no animals were obtained from iPSCs. After
thorough comparison of the RNA transcript profiles, it was ascertained that the
transcription of the imprinted gene cluster Dlk1-Dio3 in iPSCs is
considerably lower than that in the ESC lines. It was detected that the region on
chromosome 12 containing the key genes for fetal development was silenced in the
iPSC line. Over 60 iPSC-like cell lines were also tested; a similar result was
observed in most cases. It should be noted that this gene cluster was activated in a
number of iPSC lines. Chimeric living mice were subsequently obtained from these
cell lines. Thus, the state of this imprinted cluster allows one to introduce
another characteristic for the adequacy of iPSC reprogramming [77]. iPSCs can be differentiated into definitive endoderm
precursor cells to design approaches to the cell therapy of damaged tissues of
endodermal origin despite the fact that there are some differences between them at
the molecular level [78].

Allogenic organ transplantation is associated with a number of problems, such as
limited tissue engraftment and the necessity to use immunosuppressors. It is
believed that these problems can be overcome by reprogramming the patient’s
own cells, because the cells grafted to the recipient will be genetically identical
to his own cells. The method proposed is undoubtedly superior to the existing
transplantation techniques because of the possibility of in vitro
study and repair of the pathological mutations in the cells. For example, sicklemia
has been successfully repaired using iPSCs on a mouse model [85]. The formation of normal erythrocytes from hematopoietic
precursor cells obtained from completely reprogrammed skin cells was observed in

The use of oncogenes to obtain iPSCs is one of the major problems impeding the
therapeutical use of these cells. The с-Myc oncogene is
hyperexpressed in approximately 70% of human tumors; therefore, the hyperexpression
of an inserted transgene makes the use of iPSCs dangerous [89]. In order to solve this problem, iPSCs obtained from humans
and mice were subjected to study. No postnatal tumor development was observed in
chimeric mice obtained from iPSCs without introduction of c-Myc ,
whereas oncological diseases developed in ~15% of the animals obtained from iPSCs
with exogenous c-Myc [90].
Oct4, Sox2 , and  Klf4 can also be associated
with the emergence of different types of tumors; therefore, researchers increasingly
try to avoid the transduction of these oncogenes [54, 56, 61, 91]. In order to
achieve the necessary results, target cells are selected that would endogenously
express the required factor at an adequate level, hence its introduction would be
rendered unnecessary. Thus, the endogenous Sox2 gene is strongly
expressed in neutral stem cells; these cells were successfully reprogrammed in a
number of experiments by inserting Oct4 and Klf4
only [45, 92] or even Oct4 alone [92, 93]. Meningiocytes
and keratinocytes can be regarded as promising cells for reprogramming because of
their relatively high levels of Sox2 [94], c-Myc, and Klf4
[95, 96] expression. It has also been discovered that it is easier to derive
iPSCs from amniotic fluid cells because of the fact that they are relatively weakly
differentiated [97, 98]. The rate of iPSC formation from amniotic fluid cells is at
least twice faster than that of iPSC formation from fibroblasts, whereas the
reprogramming efficiency in the former case is higher by an order of magnitude. One
of the approaches to reprogramming consists in the replacement of oncogenes for
small molecules [38, 45]. Teratogenicity of iPSCs is a significant issue, since
there may remain a certain amount of undifferentiated iPSCs that are dangerous for
the recipient after these cells are differentiated into the specialized cells
intended for transplantation [99]. The search
for selection methods that would ensure the isolation of iPSCs from the
differentiated cells continues. The karyotypic instability of pluripotent cell lines
has been revealed via the study of the chromosome composition of ESCs and iPSCs
[100], attesting to the necessity for a
thorough cytogenetic analysis of iPSCs and initial cell lines.