Abstract
ABSTRACTHuman induced pluripotent stem cell (iPSC) technology has opened exciting opportunities for stem-cell-based therapy. However, its wide adoption is precluded by several challenges including low reprogramming efficiency and potential for malignant transformation. Better understanding of the molecular mechanisms of the changes that cells undergo during reprograming is needed to improve iPSCs generation efficiency and to increase confidence for their clinical use safety. Here, we find that dominant negative mutations in STAT3 in patients with autosomal-dominant hyper IgE (Job's) syndrome (AD-HIES) result in greatly reduced reprograming efficiency of primary skin fibroblasts derived from skin biopsies. Analysis of normal skin fibroblasts revealed upregulation and phosphorylation of endogenous signal transducer and activator of transcription 3 (STAT3) and its binding to the NANOG promoter following transduction with OKSM factors. This coincided with upregulation of NANOG and appearance of cells expressing pluripotency markers. Upregulation of NANOG and number of pluripotent cells were greatly reduced throughout the reprograming process of AD-HIES fibroblasts that was restored by over-expression of functional STAT3. NANOGP8, the human-specific NANOG retrogene that is often expressed in human cancers, was also induced during reprogramming, to very low but detectable levels, in a STAT3-dependent manner. Our study revealed the critical role of endogenous STAT3 in facilitating reprogramming of human somatic cells.
Highlights
Pluripotent cells have the ability to generate all somatic lineages
We find that dominant negative mutations in signal transducer and activator of transcription 3 (STAT3) in patients with autosomal dominant hyper IgE syndrome (AD-HIES; Job’s syndrome) result in greatly reduced reprograming efficiency of primary skin fibroblasts derived from skin biopsies
Upregulation of NANOG and number of pluripotent cells were greatly reduced throughout the reprograming process of AD-HIES fibroblasts that was restored by over-expression of functional STAT3
Summary
Pluripotent cells have the ability to generate all somatic lineages. In vivo, the property of pluripotency exists transiently in the inner cell mass (ICM) of the epiblast, a transient tissue that persist only for a few days. Isolation of cells at this stage and derivation of embryonic stem cells (ESC) lines has made it possible to maintain pluripotency in culture indefinitely as long as they are maintained in a cell culture environment capable of inducing a transcriptional profile and epigenetic states resembling those of pluripotent epiblast cells (Hanna et al, 2010b; Nichols and Smith, 2012; Weinberger et al, 2016) Another source of pluripotent cell lines is the direct in vitro reprograming of somatic cells to pluripotency by ectopic expression of defined factors, yielding induced pluripotent stem cells (iPSCs).(Takahashi et al, 2007; Takahashi and Yamanaka, 2006). Better understanding of the molecular mechanisms of the changes that these cells undergo during reprograming is needed to improve the generation of homogeneous iPSC, mimicking pluripotent cells of preimplantation embryos that can be safely used in clinical practice (Koche et al, 2011; Polo et al, 2012; Takahashi and Yamanaka, 2016)
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