Induced pluripotent stem (iPS) cells generated using Yamanaka factors have great potential for use in autologous cell therapy. However, genomic abnormalities exist in human iPS cells, and most mouse iPS cells are not fully pluripotent, as evaluated by the tetraploid complementation assay (TCA); this is most likely associated with the DNA damage response (DDR) occurred in early reprogramming induced by Yamanaka factors. In contrast, nuclear transfer can faithfully reprogram somatic cells into embryonic stem (ES) cells that satisfy the TCA. We thus hypothesized that factors involved in oocyte-induced reprogramming may stabilize the somatic genome during reprogramming, and improve the quality of the resultant iPS cells. To test this hypothesis, we screened for factors that could decrease DDR signals during iPS cell induction. We determined that Zscan4, in combination with the Yamanaka factors, not only remarkably reduced the DDR but also markedly promoted the efficiency of iPS cell generation. The inclusion of Zscan4 stabilized the genomic DNA, resulting in p53 downregulation. Furthermore, Zscan4 also enhanced telomere lengthening as early as 3 days post-infection through a telomere recombination-based mechanism. As a result, iPS cells generated with addition of Zscan4 exhibited longer telomeres than classical iPS cells. Strikingly, more than 50% of iPS cell lines (11/19) produced via this "Zscan4 protocol" gave rise to live-borne all-iPS cell mice as determined by TCA, compared to 1/12 for lines produced using the classical Yamanaka factors. Our findings provide the first demonstration that maintaining genomic stability during reprogramming promotes the generation of high quality iPS cells.

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LIF promotes self-renewal of mouse embryonic stem cells (mESCs), and in its absence, the cells differentiate. LIF binds to the LIF receptor (LIFR) and activates the JAK-STAT3 pathway, but it remains unknown how the receptor complex triggers differentiation or self-renewal. Here, we report that the LIFR cytoplasmic domain contains a self-renewal domain within the juxtamembrane region and a differentiation domain within the C-terminal region. The differentiation domain contains four SPXX repeats that are phosphorylated by MAPK to restrict STAT3 activation; the self-renewal domain is characterized by a 3K motif that is acetylated by p300. In mESCs, acetyl-LIFR undergoes homodimerization, leading to STAT3 hypo- or hyper-activation depending on the presence or absence of gp130. LIFR-activated STAT3 restricts differentiation via cytokine induction. Thus, LIFR acetylation and serine phosphorylation differentially promote stem cell self-renewal and differentiation.

To uncover novel genes potentially involved in embryo development, especially at the midblastula transition (MBT) phase in the developing embryo of zebrafish, Affymetrix zebrafish GeneChip microarray analysis was carried out on the expression of 14,900 gene transcripts. The results of the analysis showed that 360 genes were clearly up-regulated and 119 genes were markedly down-regulated. Many of these genes were involved in transcription factor activity, nucleic acid binding, and cell growth. The present study showed that significant changes in transcript abundance occurred during the MBT phase. The expression of eight of these 479 genes was identified by reverse transcription-polymerase chain reaction analysis, confirming the microarray results. The WSB1 gene, found to be down-regulated by the microarray and reverse transcription-polymerase chain reaction analyses, was selected for further study. Sequence analysis of the WSB1 gene showed that it encodes a protein with 75% identity to the corresponding active human orthologs. In addition, WSB1 gene expression was detected at a higher level at 2 h post fertilization and at a lower level at 4 h post fertilization, consistent with the chip results. Overexpression of the WSB1 gene can result in the formation of abnormalities in embryos, as determined by fluorescence-activated cell sorting. The present study showed unequivocally that the occurrence of WSB1 expression is an important event during the MBT phase in the development of zebrafish embryos.

The introduction of clinical sequencing is dramatically increasing the discovery of variants of uncertain significance (VUSs) in genes linked to inherited cardiomyopathies. We have established a platform for rapid and efficient insertion of TNNT2 gene variants into an induced pluripotent stem cell (iPSC) line to generate an allelic series of isogenic clones for differentiation into cardiomyocytes (iPSC-CMs) for functional annotation of the variants. We first used CRISPR-Cas9 to introduce known pathogenic variants into iPSCs from a healthy person or to correct pathogenic variants in iPSCs from patients with severe cardiomyopathy. Whereas normal/corrected iPSC-CMs responded to isoproterenol treatment with a 50%-70% increase in spontaneous beating rate as assessed by patch-clamp studies, iPSC-CMs with pathogenic variants had minimal responses (close to 0%). Due to the inefficiency of CRISPR-Cas9 in introducing/correcting variants in iPSCs, we next used dual integrase cassette exchange (DICE) to allow for the introduction of a large number of variants in parallel into a pool of cells. In a single pilot use of the DICE platform, we isolated heterozygous clones with 14 unique variants, >10% of all TNNT2 coding variants cataloged in ClinVar. We found that iPSC-CMs with any of 7 VUSs or likely pathogenic variants were impaired in their response to isoproterenol, in contrast to control DICE-treated iPSC-CMs. Finally, we sought to apply the DICE platform to a patient case in real time. A 65-year-old woman with severe hypertrophic cardiomyopathy underwent gene panel testing that identified a single VUS, TNNT2 E251D. Between the first and second clinic visits (~10 weeks), we were able to use DICE to rapidly and efficiently generate iPSC-CMs with the E251D variant and determine they had normal responses to isoproterenol, suggesting that the variant is not pathogenic. Guided by this finding, we recommended that the patient’s children and grandchildren not undergo cascade genetic screening for the E251D variant. In conclusion, this work establishes the feasibility of rapid functional annotation of cardiomyopathy gene variants, which after further validation could be incorporated into clinical practice as a line of evidence to support variant classification.