Hongran Wang

Induced multipotency in adult keratinocytes through down-regulation of Δnp63 or DGCR8

Significance The p53 family member deltaNp63 (ΔNp63) is required for transcriptional activation of the microprocessor complex subunit DGCR8 in epidermal cells, leading to terminal differentiation of tissues such as the epidermis. We show here that loss of ΔNp63 leads to the generation of cells with self-renewing but limited differentiation capacity. When DGCR8 is reexpressed in cells deficient for ΔNp63, these cells can terminally differentiate into all three germ layers. We dubbed these cells induced multipotent stem cells because of their remarkable plasticity and ability to differentiate into multiple cell lineages. Based on our results using human keratinocytes, we predict that epidermal cells can be extracted from patient skin biopsies and reprogrammed into multipotent stem cells by knockdown of ΔNp63 or DGCR8. The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ΔNp63, is a transcriptional activator of a cofactor critical for miRNA processing (DGCR8). This regulation gives rise to a unique miRNA signature resulting in reprogramming cells to multipotency. Strikingly, ΔNp63−/− epidermal cells display profound defects in terminal differentiation and express a subset of markers and miRNAs present in embryonic stem cells and fibroblasts induced to pluripotency using Yamanaka factors. Moreover, ΔNp63−/− epidermal cells transduced with an inducible DGCR8 plasmid can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ΔNp63 or DGCR8 can be reprogrammed in 6 d and express a unique miRNA and gene expression signature that is similar but not identical to human induced pluripotent stem cells. Our data reveal a role for ΔNp63 in the transcriptional regulation of DGCR8 to reprogram adult somatic cells into multipotent stem cells.

Generation of Viable Male and Female Mice from Two Fathers

In sexual species, fertilization of oocytes produces individuals with alleles derived from both parents. Here we use pluripotent stem cells derived from somatic cells to combine the haploid genomes from two males to produce viable sons and daughters. Male (XY) mouse induced pluripotent stem cells (Father #1) were used to isolate subclones that had spontaneously lost the Y chromosome to become genetically female (XO). These male-derived XO stem cells were used to generate female chimeras that were bred with genetically distinct males (Father #2), yielding progeny possessing genetic information that was equally derived from both fathers. Thus, functional oocytes can be generated from male somatic cells after reprogramming and spontaneous sex reversal. These findings have novel implications for mammalian reproduction and assisted reproductive technology.

Localization of angiotensin II in pig ovary and its effects on oocyte maturation in vitro

The renin-angiotensin system (RAS) has been found in mammalian ovarian tissue; however, its physiological role is unclear. This study examined the content of angiotensin II (Ang II) in porcine follicular fluid (pFF), Ang II localization and its receptors in ovary, and the effects of Ang II on porcine oocyte maturation. The concentrations of Ang II were 6951.82 +/- 1295.83, 3502.99 +/- 679.10, 3147.89 +/- 690.60, and 2545.92 +/- 407.01 pg/ml in pFF from small, medium, large, and extra-large follicles, respectively. In addition, Ang II was found on zona pellucidae (ZP) and granulosa cells by immunoreactive staining. The distribution of AT1, an Ang II receptor subtype, was in accordance with that of Ang II. However, AT2, another Ang II receptor, was mainly distributed in the stroma and thecal layers of follicles. When oocytes were cultured in media containing various concentrations of Ang II, a higher (P<0.05) proportion of oocytes reached metaphase II (MII) in the medium with 100 ng/ml (87.0%) than without Ang II (61%). When oocytes from different sizes of follicles were separately cultured in media containing 100 ng/ml Ang II, maturation rates were significantly higher in oocytes from small (61.5%) and medium (85.1%) follicles than that of their controls (45.1 and 72.6%, respectively). However, addition of Ang II inhibited nuclear maturation in oocytes from large follicles (77.8% versus 87.3%). Fertilization and male pronuclear (MPN) formation rates of oocytes matured in medium containing 100 or 1000 ng/ml of Ang II were higher (P<0.05) than that of oocytes matured in medium containing 0 or 10 ng/ml Ang II. Glutathione content in oocytes cultured for 44 h in medium containing 100 or 1000 ng/ml of Ang II was also higher (P<0.01) than that of oocytes cultured in medium containing 0 or 10 ng/ml Ang II. In conclusion, Ang II was present in porcine ovaries and may regulate follicle growth and oocyte maturation.

Epigenetic reprogramming of the germ cell nuclear factor gene is required for proper differentiation of induced pluripotent cells.

Somatic cells have been reprogrammed into induced pluripotent stem (iPS) cells that recapitulate the pluripotent nature of embryonic stem (ES) cells. Reduced pluripotency and variable differentiation capacities have hampered progress with this technology for applications in regeneration medicine. We have previously shown that germ cell nuclear factor (Gcnf) is required for the repression of pluripotency genes during ES cell differentiation and embryonic development. Here we report that iPS cell lines, in which the Gcnf gene was properly reprogrammed, allowing expression of Gcnf, repress pluripotency genes during subsequent differentiation. In contrast, iPS clones in which the Gcnf gene was not reprogrammed maintained pluripotency gene expression during differentiation and did not differentiate properly either in vivo or in vitro. These mal‐reprogrammed cells recapitulated the phenotype of Gcnf knockout (Gcnf−/−) ES cells. Reintroduction of Gcnf into either the Gcnf negative iPS cells or the Gcnf−/− ES cells rescued repression of Oct4 during differentiation. Our findings establish a key role for Gcnf as a regulator of iPS cell pluripotency gene expression. It also demonstrates that reactivation of the Gcnf gene may serve as a marker to distinguish completely reprogrammed iPS cells from incompletely pluripotent cells, which would make therapeutic use of iPS cells safer and more practical as it would reduce the oncogenic potential of iPS cells. STEM Cells 2013;31:2659–2666

Germ cell nuclear factor (GCNF) represses Oct4 expression and globally modulates gene expression in human embryonic stem (hes) cells

Oct4 is considered a key transcription factor for pluripotent stem cell self-renewal. It binds to specific regions within target genes to regulate their expression and is downregulated upon induction of differentiation of pluripotent stem cells; however, the mechanisms that regulate the levels of human Oct4 expression remain poorly understood. Here we show that expression of human Oct4 is directly repressed by germ cell nuclear factor (GCNF), an orphan nuclear receptor, in hES cells. Knockdown of GCNF by siRNA resulted in maintenance of Oct4 expression during RA-induced hES cell differentiation. While overexpression of GCNF promoted repression of Oct4 expression in both undifferentiated and differentiated hES cells. The level of Oct4 repression was dependent on the level of GCNF expression in a dose-dependent manner. mRNA microarray analysis demonstrated that overexpression of GCNF globally regulates gene expression in undifferentiated and differentiated hES cells. Within the group of altered genes, GCNF down-regulated 36% of the genes, and up-regulated 64% in undifferentiated hES cells. In addition, GCNF also showed a regulatory gene pattern that is different from RA treatment during hES cell differentiation. These findings increase our understanding of the mechanisms that maintain hES cell pluripotency and regulate gene expression during the differentiation process.

GCNF-dependent activation of cyclin D1 expression via repression of Mir302a during ESC differentiation.

Cyclin D1 plays an important role in the regulation of cellular proliferation and its expression is activated during gastrulation in the mouse; however, it remains unknown how cyclin D1 expression is regulated during early embryonic development. Here, we define the role of germ cell nuclear factor (GCNF) in the activation of cyclin D1 expression during embryonic stem cell (ESC) differentiation as a model of early development. During our study of GCNF knockout (GCNF−/−) ESC, we discovered that loss of GCNF leads to the repression of cyclin D1 activation during ESC differentiation. This was determined to be an indirect effect of deregulation Mir302a, which is a cyclin D1 suppressor via binding to the 3′UTR of cyclin D1 mRNA. Moreover, we showed that Mir302 is a target gene of GCNF that inhibits Mir302 expression by binding to a DR0 element within its promoter. Inhibition of Mir302a using Mir302 inhibitor during differentiation of GCNF−/− ESCs restored cyclin D1 expression. Similarly over‐expression of GCNF during differentiation of GCNF−/− ESCs rescued the inhibition of Mir302a expression and the activation of cyclin D1. These results reveal that GCNF plays a key role in regulating activation of cyclin D1 expression via inhibition of Mir302a. Stem Cells 2014;32:1527–1537

Generation of electrophysiologically functional cardiomyocytes from mouse induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells can efficiently differentiate into the three germ layers similar to those formed by differentiated embryonic stem (ES) cells. This provides a new source of cells in which to establish preclinical allogeneic transplantation models. Our iPS cells were generated from mouse embryonic fibroblasts (MEFs) transfected with the Yamanaka factors, the four transcription factors (Oct4, Sox2, Klf4 and c-Myc), without antibiotic selection or MEF feeders. After the formation of embryoid bodies (EBs), iPS cells spontaneously differentiated into Flk1-positive cardiac progenitors and cardiomyocytes expressing cardiac-specific markers such as alpha sarcomeric actinin (α-actinin), cardiac alpha myosin heavy chain (α-MHC), cardiac troponin T (cTnT), and connexin 43 (CX43), as well as cardiac transcription factors Nk2 homebox 5 (Nkx2.5) and gata binding protein 4 (gata4). The electrophysiological activity of iPS cell-derived cardiomyocytes (iPS-CMs) was detected in beating cell clusters with optical mapping and RH237 a voltage-sensitive dye, and in single contracting cells with patch-clamp technology. Incompletely differentiated iPS cells formed teratomas when transplanted into a severe combined immunodeficiency (SCID) mouse model of myocardial infarction. Our results show that somatic cells can be reprogrammed into pluripotent stem cells, which in turn spontaneously differentiate into electrophysiologically functional mature cardiomyocytes expressing cardiac-specific makers, and that these cells can potentially be used to repair myocardial infarction (MI) in the future.

Chromatin Changes in Dicer-Deficient Mouse Embryonic Stem Cells in Response to Retinoic Acid Induced Differentiation

Loss of Dicer, an enzyme critical for microRNA biogenesis, results in lethality due to a block in mouse embryonic stem cell (mES) differentiation. Using ChIP-Seq we found increased H3K9me2 at over 900 CpG islands in the Dicer-/-ES epigenome. Gene ontology analysis revealed that promoters of chromatin regulators to be among the most impacted by increased CpG island H3K9me2 in ES (Dicer-/-). We therefore, extended the study to include H3K4me3 and H3K27me3 marks for selected genes. We found that the ES (Dicer-/-) mutant epigenome was characterized by a shift in the overall balance between transcriptionally favorable (H3K4me3) and unfavorable (H3K27me3) marks at key genes regulating ES cell differentiation. Pluripotency genes Oct4, Sox2 and Nanog were not impacted in relation to patterns of H3K27me3 and H3K4me3 and showed no changes in the rates of transcript down-regulation in response to RA. The most striking changes were observed in regards to genes regulating differentiation and the transition from self-renewal to differentiation. An increase in H3K4me3 at the promoter of Lin28b was associated with the down-regulation of this gene at a lower rate in Dicer-/-ES as compared to wild type ES. An increase in H3K27me3 in the promoters of differentiation genes Hoxa1 and Cdx2 in Dicer-/-ES cells was coincident with an inability to up-regulate these genes at the same rate as ES upon retinoic acid (RA)-induced differentiation. We found that siRNAs Ezh2 and post-transcriptional silencing of Ezh2 by let-7g rescued this effect suggesting that Ezh2 up-regulation is in part responsible for increased H3K27me3 and decreased rates of up-regulation of differentiation genes in Dicer-/-ES.

Generation of integration-free induced pluripotent stem cells from a patient with spina bifida

A skin biopsy was obtained from a 14-year-old female patient with a history of Myelomeningocele. Dermal fibroblasts were isolated and reprogrammed with Sendai virus (SeV) vectors encoding OCT3/4, SOX2, KLF4, and c-MYC. The generated induced Pluripotent Stem Cell (iPSC) clones NTDi4_09A were free of genomically integrated reprogramming genes, had a stable normal karyotype and expressed pluripotency markers. The iPSCs formed teratomas in mice, which were differentiated towards derivatives of the three germ layers in vivo. This iPSC line offers a useful resource to study a genetic profile of a patient with spina bifida.

Reciprocity of Action of Increasing Oct4 and Repressing p53 in Transdifferentiation of Mouse Embryonic Fibroblasts into Cardiac Myocytes

p53 is a barrier to somatic cell reprogramming. Deletion or transient suppression of p53 increases the efficiency of reprogramming of somatic cells into induced pluripotent stem cells. Whether p53 represents an obstacle to a similar process transdifferentiation of somatic cells is unknown. However, it is predicted that inhibition of p53 would promote transdifferentiation of fibroblasts into cardiomyocytes. In this study, the effect of p53 on the capacity of cardiogenic transdifferentiation is evaluated using p53 wild-type (p53+/+), p53 heterozygous mutant (p53+/-), and p53 homozygous mutant (p53-/-) mouse embryonic fibroblasts (MEFs). Repression of p53 in MEFs increases the expression level of mesoderm transcription factors Brachyury (T) and MESP1. The cardiac-specific markers, Myh6 (Myosin, Heavy Chain 6), Myh7 (Myosin, Heavy Chain 7), and cTnI (cardiac muscle troponin I), show elevated expression in p53+/- and p53-/- MEFs compared with wild-type MEFs, but cardiac muscle troponin T (cTnT) showed a lower expression level when p53 was inhibited. After induction to cardiac differentiation, cTnT expression increased and markers of endoderm and ectoderm decreased in p53+/- and p53-/- MEFs. The effect of an important reprogramming factor Oct4 on cardiac transdifferentiation was also evaluated in the allelic series of p53 MEFs. We found that overexpression of Oct4 significantly enhanced Mesp1, Tbx5, and Isl1 expression in p53+/+ and p53+/- MEFs. Oct4 also enhanced cTnT expression in all three cell lines, especially in p53+/- MEFs. Thus, inhibition of p53 expression and viral expression of Oct4 both promote transdifferentiation of MEFs into cardiomyocytes, establishing reciprocity of action in the process.