The generation of functional skeletal muscle tissues from human pluripotent stem cells (hPSCs) has not been reported. Here, we derive induced myogenic progenitor cells (iMPCs) via transient overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs. In 2D culture, iMPCs readily differentiate into spontaneously contracting multinucleated myotubes and a pool of satellite-like cells endogenously expressing Pax7. Under optimized 3D culture conditions, iMPCs derived from multiple hPSC lines reproducibly form functional skeletal muscle tissues (iSKM bundles) containing aligned multi-nucleated myotubes that exhibit positive force–frequency relationship and robust calcium transients in response to electrical or acetylcholine stimulation. During 1-month culture, the iSKM bundles undergo increased structural and molecular maturation, hypertrophy, and force generation. When implanted into dorsal window chamber or hindlimb muscle in immunocompromised mice, the iSKM bundles survive, progressively vascularize, and maintain functionality. iSKM bundles hold promise as a microphysiological platform for human muscle disease modeling and drug development.

Reprogramming of somatic cells in the enucleated egg made Dolly, the sheep, the first successfully cloned mammal in 1996. However, the mechanism of sheep somatic cell reprogramming has not yet been addressed. Moreover, sheep embryonic stem (ES) cells are still not available, which limits the generation of precise gene-modified sheep. In this study, we report that sheep somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells using defined factors (Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, SV40 large T and hTERT). Our observations indicated that somatic cells from sheep are more difficult to reprogram than somatic cells from other species, in which iPS cells have been reported. We demonstrated that sheep iPS cells express ES cell markers, including alkaline phosphatase, Oct4, Nanog, Sox2, Rex1, stage-specific embryonic antigen-1, TRA-1-60, TRA-1-81 and E-cadherin. Sheep iPS cells exhibited normal karyotypes and were able to differentiate into all three germ layers both in vitro and in teratomas. Our study may help to reveal the mechanism of somatic cell reprogramming in sheep and provide a platform to explore the culture conditions for sheep ES cells. Moreover, sheep iPS cells may be directly used to generate precise gene-modified sheep.

Human embryonic stem (ES) cells possess the potential to differentiate into all the cell types of the human body and provide potential applications in regenerative medicine [1]. However, the concerns of immune rejection hamper transplantation therapies using human ES cells. To avoid the complications of immune rejection, diverse methods, such as somatic nuclear transfer (also called therapeutic cloning) and fusion of somatic cells with human ES cells [2], have been attempted to produce patient-specific pluripotent stem cells. Most of these approaches have resulted in little success. The generation of human iPS cells (induced pluripotent stem cells) from somatic cells with defined transcription factors makes it possible to produce patientspecific ES-like stem cells for therapeutic purposes [3,4]. Two sets of four-factors, OCT4, SOX2, C-MYC, KLF4 reported by Yamanaka's laboratory and OCT4, SOX2, NANOG, LIN28 reported by Thomson's laboratory, have been shown to reprogram human somatic cells to pluripotency with similar efficiency (10-20 iPS cell colonies from 0.1 million initial fibroblasts) [3,4]. We speculated that C-MYC and KLF4 might synergize with Thomson's 4 factors (OCT4, SOX2, NANOG, LIN28) to reprogram the human somatic cells. Our present study shows that a combination of 6 transcription factors, OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4, significantly increases the efficiency of generating iPS cells from human somatic cells.The human genes encoding the transcription factors, OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4, were cloned into a lentiviral vector to produce lentivirus.Half million human newborn foreskin fibroblasts were transduced with the lentivirus carrying GFP (serving as negative control), or a cocktail of lentivirus carrying 4 factors (OCT4, NANOG, SOX2 and LIN28) or 6 factors (OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4). Twenty-four hours later, the cells were dissociated with trypsin, transferred to five flasks (0.1 million initial cells per flask) coated with murine embryonic fibroblast (MEF) feeder, and cultured in human embryonic stem cell media. Colonies with a human ES cell-like morphology (iPS cell colonies) first became visible 12 days after transduction with 4 factors, whereas iPS cell colonies became visible 7 days after transduction with 6 factors. In order to quantify the efficiency of the reprogramming, the cells in one flask for each combination of factors were fixed on day 17 to analyze the alkaline phosphatase expression. A total of 16±3 colonies from 0.1 million initial fibroblast cells transduced with 4 factors were alkaline phosphatase-positive, 166±6 colonies from 0.1 million initial fibroblast cells transduced with 6 factors were alkaline phosphatase-positive, and no colonies were observed in GFP-lentivirus transduced controls ( Figure 1A). The efficiency of 6 factors is 10.4-fold more than that of 4 factors. The iPS colonies generated by transduction of 4 factors were picked on day 26. As reported by Yu et al. [4], these cells expressed alkaline phosphatase and undifferentia...

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Immune rejection hinders the application of human embryonic stem cells (hESCs) in transplantation therapy. Human leukocyte antigens (HLAs) on the cell surface are the major cause of graft rejection. In this study, we generated HLA class I-deficient hESCs via disruption of beta 2-microglobulin (β2m), the light chain of HLA Class I. We found that HLA class I proteins were not present on the cell surface of β2m-null hESCs. These cells showed the same pluripotency as wildtype hESCs and demonstrated hypoimmunogenicity. Thus, HLA class I-deficient hESCs might serve as an unlimited cell source for the generation of universally compatible "off-the-shelf" cell grafts, tissues or organs in the future.

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Human induced pluripotent stem (iPS) cells have great potential in regenerative medicine, but this depends on the integrity of their genomes. iPS cells have been found to contain a large number of de novo genetic alterations due to DNA damage response during reprogramming. Thus, to maintain the genetic stability of iPS cells is an important goal in iPS cell technology. DNA damage response can trigger tumor suppressor p53 activation, which ensures genome integrity of reprogramming cells by inducing apoptosis and senescence. p53 isoform Δ133p53 is a p53 target gene and functions to not only antagonize p53 mediated apoptosis, but also promote DNA double-strand break (DSB) repair. Here we report that Δ133p53 is induced in reprogramming. Knockdown of Δ133p53 results 2-fold decrease in reprogramming efficiency, 4-fold increase in chromosomal aberrations, whereas overexpression of Δ133p53 with 4 Yamanaka factors showes 4-fold increase in reprogamming efficiency and 2-fold decrease in chromosomal aberrations, compared to those in iPS cells induced only with 4 Yamanaka factors. Overexpression of Δ133p53 can inhibit cell apoptosis and promote DNA DSB repair foci formation during reprogramming. Our finding demonstrates that the overexpression of Δ133p53 not only enhances reprogramming efficiency, but also results better genetic quality in iPS cells.

Human embryonic stem cells (hESCs) are a promising model for the research of embryonic development and regenerative medicine. Since the first hESC line was established, many researchers have shown that pluripotent hESCs can be directed into many types of functional adult cells in culture. However, most of the reported methods have induced differentiation through the alteration of growth factors in the culture medium. These methods are time consuming; moreover, it is difficult to obtain a pure population of the desired cells because of the low efficiency of induction. In this study, we used a lentiviral-based inducible gene-expression system in hESCs to control the ectopic expression of MyoD, which is an essential transcription factor in skeletal muscle development. The induction of MyoD can efficiently direct the pluripotent hESCs into mesoderm in 24 h. The cells then become proliferated myoblasts and finally form multinucleated myotubes in vitro. The whole procedure took about 10 days, with an induction efficiency of over 90%. To our knowledge, this is the first time that hESCs have been induced into terminally differentiated cells with only one factor. In the future, these results could be a potential resource for cell therapy for diseases of muscle dysfunction.