Article

Reprogramming of human somatic cells to pluripotency with defined factors.

Division of Pediatric Hematology/Oncology, Children's Hospital Boston and Dana Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Nature (Impact Factor: 42.35). 02/2008; 451(7175):141-6. DOI: 10.1038/nature06534
Source: PubMed

ABSTRACT 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.

Full-text

Available from: Tan A. Ince, May 31, 2015
3 Followers
 · 
292 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Introduction Bone marrow mesenchymal stem cells (BMSCs), which have the ability to self-renew and to differentiate into multiple cell types, have recently become a novel strategy for cell-based therapies. The differentiation of BMSCs into keratinocytes may be beneficial for patients with burns, disease, or trauma. However, the currently available cells are exposed to animal materials during their cultivation and induction. These xeno-contaminations severely limit their clinical outcomes. Previous studies have shown that the Rho kinase (ROCK) inhibitor Y-27632 can promote induction efficiency and regulate the self-renewal and differentiation of stem cells. In the present study, we attempted to establish a xeno-free system for the differentiation of BMSCs into keratinocytes and to investigate whether Y-27632 can facilitate this differentiation. Methods BMSCs isolated from patients were cultured by using a xeno-free system and characterised by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. Human primary keratinocytes were also isolated from patients. Then, the morphology, population doubling time, and β-galactosidase staining level of these cells were evaluated in the presence or absence of Y-27632 to determine the effects of Y-27632 on the state of the keratinocytes. Keratinocyte-like cells (KLCs) were detected at different time points by immunocytofluorescence analysis. Moreover, the efficiency of BMSC differentiation under different conditions was measured by quantitative real-time-polymerase chain reaction (RT-PCR) and Western blot analyses. Results The ROCK inhibitor Y-27632 promoted the proliferation and lifespan of human primary keratinocytes. In addition, we showed that keratinocyte-specific markers could be detected in BMSCs cultured in a xeno-free system using keratinocyte-conditioned medium (KCM) independent of the presence of Y-27632. However, the efficiency of the differentiation of BMSCs into KLCs was significantly higher in the presence of Y-27632 using immunofluorescence, quantitative RT-PCR, and Western blot analyses. Conclusions This study demonstrated that Y-27632 could promote the proliferation and survival of human primary keratinocytes in a xeno-free culture system. In addition, we found that BMSCs have the ability to differentiate into KLCs in KCM and that Y-27632 can facilitate this differentiation. Our results suggest that BMSCs are capable of differentiating into KLCs in vitro and that the ROCK pathway may play a critical role in this process.
    Stem Cell Research & Therapy 03/2015; 6(1). DOI:10.1186/s13287-015-0008-2 · 4.63 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Induced pluripotent stem cells (iPSCs) are generated through a gradual process in which somatic cells undergo a number of stochastic events. In this study, we examined whether two different doxycycline-inducible iPSCs, slow-forming 4F2A-iPSCs and fast-forming NGFP-iPSCs, have equivalent levels of pluripotency. Multiplex reverse-transcriptase PCR generated gene expression profiles (GEPs) of 13 pluripotency genes in single initially formed-iPSC (if-iPSC) colonies of NGFP and 4F2A group. Assessment of GEP difference using a weighted root mean square deviation (wRMSD) indicates that 4F2A if-iPSCs are more closely related to mESCs than NGFP if-iPSCs. Consistently, Nanog and Sox2 genes were more frequently derepressed in 4F2A if-iPSC group. We further examined 20 genes that are implicated in reprogramming. They were, overall, more highly expressed in NGFP if-iPSCs, differing from the pluripotency genes being more expressed in 4F2A if-iPSCs. wRMSD analysis for these reprogramming-related genes confirmed that the 4F2A if-iPSC colonies were less deviated from mESCs than the NGFP if-iPSC colonies. Our findings suggest that more important in attaining a better reprogramming is the mode of action by the given reprogramming factors, rather than the total activity of them exerting to the cells, as the thin-but-long-lasting mode of action in 4F2A if-iPSCs is shown to be more effective than its full-but-short-lasting mode in NGFP if-iPSCs.
    Stem cell International 01/2015; 2015:1-10. DOI:10.1155/2015/195928 · 2.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Advances in the field of stem cells have led to novel avenues for generating induced pluripotent stem cells (iPSCs) from differentiated somatic cells. iPSCs are typically obtained by the introduction of the four factors OCT4, SOX2, KLF4, and cMYC, via integrating vectors. Here, we report the feasibility of a novel reprogramming process based on vectors derived from the non-integrating vaccine strain of measles virus (MV). We produced a one-cycle MV vector by substituting the viral attachment protein gene with the green fluorescent protein (GFP) gene. This vector was further engineered to encode for OCT4 in an additional transcription unit. After verification of OCT4 expression, we assessed the ability of iPSC reprogramming. The reprogramming vector cocktail with the OCT4-expressing MV vector and SOX2-, KLF4- and cMYC-expressing lentiviral vectors efficiently transduced human skin fibroblasts and formed iPSC colonies. RT-PCR and immunostaining confirmed induction of endogenous pluripotency-associated marker genes, such as SSEA4, TRA-1-60 and Nanog. Pluripotency of derived clones was confirmed by spontaneous differentiation into three germ layers, teratoma formation and guided differentiation into beating cardiomyocytes. MV vectors can induce efficient nuclear reprogramming. Given the excellent safety record of MV vaccines and the translational capabilities recently developed to produce MV-based vectors now used for cancer clinical trials, our MV vector system provides a RNA-based, non-integrating gene transfer platform for nuclear reprogramming that is amenable for immediate clinical translation.
    Stem Cell Research & Therapy 03/2015; 6(1):48. DOI:10.1186/s13287-015-0035-z · 4.63 Impact Factor