Generation and Differentiation of Human iPS Cells

Pluripotent stem cells (PSCs) have the potential to differentiate into many cell types and therefore can be a valuable source for cell therapy. Embryonic stem cells (ESCs), which are derived from the inner cell mass (ICM) of the blastocyst, are representative of PSCs. However, use of these cells has some limitations, especially ethical restrictions and immune response. As a result, researchers have been looking for other cell sources or strategies to overcome these limitations. One kind of cellular reprogramming is the process of guiding mature cells into a state of gene expression similar to PSCs. It has been demonstrated that somatic cells can be reprogrammed by various methods, including somatic cell nuclear transfer (SCNT) and cell fusion with ESCs or treatment with their extracts. This implies that some factors in oocytes and ESCs are able to initiate the reprogramming process. Accordingly, induced pluripotent stem cells (iPSCs) have been derived from somatic cells by ectopic expression of some transcription factors. This discovery has resulted in raising several important questions about the mechanisms by which these factors influence the reprogramming and epigenetic status of the cells. iPSCs hold great promise for regenerative medicine, developmental biology, and drug discovery because they circumvent problems associated with both ethical issues and immunological rejection. Here we review the experiments involved in the discovery of iPSCs, important factors in their reprogramming, and their future perspectives in cell therapy.

Stem Cell Reviews and Reports, 2012

Cellular reprogramming consists of the conversion of differentiated cells into pluripotent cells; the so-called induced Pluripotent Stem Cells. iPSC are amenable to in vitro manipulation and, in theory, direct production of any differentiated cell type. Furthermore, iPSC can be obtained from sick individuals and subsequently used for disease modeling, drug discovery and regenerative treatments. iPSC production was first achieved by transducing, with the use of retroviral vectors, four specific transcription factors: Oct4, Klf4, Sox2 and c-Myc (OKSM), into primary cells in culture Takahashi and Yamanaka, (Cell 126(4):663-676, 2006). Many alternative protocols have since been proposed: repeated transfections of expression plasmids containing the four pluripotencyassociated genes Okita et al.

Nature, 2012

The field of stem-cell biology has been catapulted forward by the startling development of reprogramming technology. The ability to restore pluripotency to somatic cells through the ectopic co-expression of reprogramming factors has created powerful new opportunities for modelling human diseases and offers hope for personalized regenerative cell therapies. While the field is racing ahead, some researchers are pausing to evaluate whether induced pluripotent stem cells are indeed the true equivalents of embryonic stem cells and whether subtle differences between these cells might affect their research applications and therapeutic potential.

Nature, 2008

Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, we have derived iPS cells from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture.

Differentiated somatic cells can be reprogrammed into pluripotent stem cells by transduction of exogenous reprogramming factors. After induced pluripotent stem (iPS) cells are established, exogenous genes are silenced. In the pluripotent state, retroviral genes integrated in the host genome are kept inactive through epigenetic transcriptional regulation. In this study, we tried to determine whether exogenous genes remain silenced or are reactivated upon loss of pluripotency or on differentiation using an in vitro system. We induced differentiation of iPS cells into neural stem cells (NSCs) in vitro; the NSCs appeared morphologically indistinguishable from brain-derived NSCs and stained positive for the NSC markers Nestin and Sox2. These iPS cellderived NSCs (iPS-NSCs) were also capable of differentiating into all three neural subtypes. Interestingly, iPS-NSCs spontaneously formed aggregates on long-term culture and showed reactivation of the Oct4-GFP marker, which was followed by the formation of embryonic stem cell-like colonies. The spontaneously reverted green fluorescent protein (GFP)-positive (iPS-NSC-GFP 1 ) cells expressed high levels of pluripotency markers (Oct4 and Nanog) and formed germline chimeras, indicating that iPS-NSC-GFP 1 cells had the same pluripotency as the original iPS cells. The reactivation of silenced exogenous genes was tightly correlated with the downregulation of DNA methyltransferases (Dnmts) during differentiation of iPS cells. This phenomenon was not observed in doxycycline-inducible iPS cells, where the reactivation of exogenous genes could be induced only by doxycycline treatment. These results indicate that pluripotency can be regained through reactivation of exogenous genes, which is associated with dynamic change of Dnmt levels during differentiation of iPS cells.

Cell Stem Cell, 2008

Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4 and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox) inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, were only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step towards understanding some of the molecular events governing epigenetic reprogramming.

BioEssays, 2009

The phenomenal proliferation of scientific studies into the nature o nduced pluripotent stem (iPS) cells following publication of the findings of Takahashi and Yamanaka little more than 2 years ago, have significantly expanded our understanding of cellular mechanisms relating to cell lineage, di erentiation, and proliferation. While the full potential o PS cell lineages for both scientific tool and therapeutic applications is as yet unclear, findings from several lines o nvestigation suggests that multipotential and terminally di erentiated cells from an array of cell types are competent to undergo epigenetic reprogramming to a pluripotential state. The nature of this pluripotential state appears to be similar to, but not identical with that previously described for embryonic stem (ES) cells. Understanding the nature of this induced reprogrammed state will be critical to determining the full potential o PS cells. Recently, this issue has been examined through an integrated analysis of the genome in fully and partially reprogrammed iPS cell lineages. These results provide a window onto the temporal components of reprogramming and suggest mechanisms by which the efficacy of reprogramming can be enhanced.

Journal of Tissue Engineering and Regenerative Medicine, 2010

Somatic cells have been reprogrammed into induced pluripotent stem (iPS) cells that recapitulate the pluripotent nature of embryonic stem (ES) cells. Reduced pluripotency and variable differentiation capacities have hampered progress with this technology for applications in regeneration medicine. We have previously shown that Germ Cell Nuclear Factor (Gcnf) is required for the repression of pluripotency genes during ES cell differentiation and embryonic development. Here we report that iPS cell lines, in which the Gcnf gene was properly reprogrammed , allowing expression of Gcnf, repress pluripotency genes during subsequent differentiation. In contrast, iPS clones in which the Gcnf gene was not reprogrammed maintained pluripotency gene expression during differentiation and did not differentiate properly either in vivo or in vitro. These mal-reprogrammed cells re-capitulated the phenotype of Gcnf knock out (Gcnf −/−) ES cells. Re-introduction of Gcnf into either the Gcnf negative iPS cells or the Gcnf −/− ES cells, rescued repression of Oct4 during differentiation. Our findings establish a key role for Gcnf as a regulator of iPS cell pluripotency gene expression. It also demonstrates that reactivation of the Gcnf gene may serve as a marker to distinguish completely reprogrammed iPS cells from incompletely pluripotent cells, which would make therapeutic use of iPS cells safer and more practical as it would reduce the oncogenic potential of iPS cells.

Stem cell reviews, 2012

To provide a comprehensive source of information about the reprogramming process and induced pluripotency. The ability of stem cells to renew their own population and to differentiate into specialized cell types has always attracted researchers looking to exploit this potential for cellular replacement therapies, pharmaceutical testing and studying developmental pathways. While adult stem cell therapy has already been brought to the clinic, embryonic stem cell research has been beset with legal and ethical impediments. The conversion of human somatic cells to human induced pluripotent stem cells (hiPSCs), which are equivalent to human embryonic stem cells (hESCs), provides a system to sidestep these barriers and expedite pluripotent stem cell research for the aforementioned purposes. However, being a very recent discovery, iPSCs have yet to overcome many other obstacles and criticism to be proven safe and feasible for clinical use. This review introduces iPSC, the various methods th...