Ips Cell Technology In Regenerative Medicine

ArticleinAnnals of the New York Academy of Sciences 1192(1):38-44 · March 2010with27 Reads
DOI: 10.1111/j.1749-6632.2009.05213.x · Source: PubMed
The promise of treating human genetic and degenerative diseases through the application of pluripotent cell-based tissue engineering and regenerative medicine has come significantly closer to realization since the isolation of human embryonic stem (ES) cells. While the study of ES cells has greatly increased our fundamental understanding of pluripotency, technical and ethical limitations have been seemingly insurmountable impediments to the application of these cells in the clinic. The recent discovery that somatic mammalian cells can be epigenetically reprogrammed to a pluripotent state through the exogenous expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC has yielded a new cell type for potential application in regenerative medicine, the induced pluripotent stem (iPS) cell. Here we discuss how advances in iPS cell technology have led to the generation of patient-specific cell lines that can potentially be used to model human diseases and ultimately act as therapeutic agents.
    • "Discerning how different splicing factors collaborate to regulate key programs of AS remains a challenge but one that can now be addressed using new genomic technologies. The discovery that exogenous factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) has opened up new fields of investigation that hold great promise for new therapeutic applications (Lengner, 2010). Only recently have roles for AS in pluripotency and reprogramming begun to emerge, including the identification of roles for several splicing regulators such as muscleblind-like splicing factors Mbnl1/2, RBFOX2, Srsf3, and U2af1 (Gabut et al., 2011; Han et al., 2013; Ohta et al., 2013; Salomonis et al., 2010; Venables et al., 2013; Wu et al., 2010; Yeo et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: Alternative splicing (AS) plays a critical role in cell fate transitions, development, and disease. Recent studies have shown that AS also influences pluripotency and somatic cell reprogramming. We profiled transcriptome-wide AS changes that occur during reprogramming of fibroblasts to pluripotency. This analysis revealed distinct phases of AS, including a splicing program that is unique to transgene-independent induced pluripotent stem cells (iPSCs). Changes in the expression of AS factors Zcchc24, Esrp1, Mbnl1/2, and Rbm47 were demonstrated to contribute to phase-specific AS. RNA-binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS during reprogramming by different RNA-binding proteins. Ectopic expression of Esrp1 enhanced reprogramming, in part by modulating the AS of the epithelial specific transcription factor Grhl1. These data represent a comprehensive temporal analysis of the dynamic regulation of AS during the acquisition of pluripotency.
    Full-text · Article · Mar 2016
    • "However, all these promising approaches involve the use of retrovirus for the overexpression of one or more specific transcription factors (Cohen and Melton 2011), leading to several limitations and making cells unsuitable for use in cell therapy and regenerative medicine. To overcome all the problems described above, virus-free protocols for the derivation of iPS cells (Okita et al. 2011; Stadtfeld et al. 2008) or cell transdifferentiation have been proposed but, at present, they are technically demanding, less efficient (Lengner 2010) and do not solve the problems arising from the use of transgenes. "
    [Show abstract] [Hide abstract] ABSTRACT: Different cell types have been suggested as candidates for use in regenerative medicine. Embryonic pluripotent stem cells can give rise to all cells of the body and possess unlimited self-renewal potential. However, they are unstable, difficult to control and have a risk of neoplastic transformation. Adult stem cells are safe but have limited proliferation and differentiation abilities and are usually not within easy access. In recent years, induced pluripotent stem (iPS) cells have become a new promising tool in regenerative medicine. However, the use of transgene vectors, commonly required for the induction of iPS cells, seriously limits their use in therapy. The same problem arising from the use of retroviruses is associated with the use of cells obtained through transdifferentiation. Developing knowledge of the mechanisms controlling epigenetic regulation of cell fate has boosted the use of epigenetic modifiers that drive cells into a 'highly permissive' state. We recently set up a new strategy for the conversion of an adult mature cell into another cell type. We increased cell plasticity using 5-aza-cytidine and took advantage of a brief window of epigenetic instability to redirect cells to a different lineage. This approach is termed 'epigenetic conversion'. It is a simple, direct and safe way to obtain both cells for therapy avoiding gene transfection and a stable pluripotent state.
    Full-text · Article · Mar 2015
    • "The ability to direct ES and induced pluripotent stem (iPS) cell differentiation toward specific tissue fates in vitro provides an excellent opportunity to investigate the gene regulatory networks (GRNs) that operate during organ development [1, 2]. While ES and iPS cells hold promise for cell-based therapies, only in a handful of cases is molecular information detailed enough to guide directed differentiation to specific tissue types. "
    [Show abstract] [Hide abstract] ABSTRACT: Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.
    Full-text · Article · Dec 2014
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