Different pluripotent cell types have been established by capturing pluripotency in different states. Human extended pluripotent stem cells (hEPSCs), recently established by two independent studies, have the capability of differentiating into both embryonic and extraembryonic lineages, as well as forming human blastoids, showing great potential for early human development modeling and regenerative medicine. Considering that X chromosome status in female human pluripotent stem cells is dynamic and heterogeneous, and often leads to functional consequences, we characterized it in hEPSCs. We derived hEPSCs from primed human embryonic stem cells (hESCs) with defined X chromosome status (pre- or post-X chromosome inactivation) using two previously published methods. We showed that hEPSCs derived using both methods had highly similar transcription profiles and X chromosome status. However, the X chromosome status of hEPSCs is largely determined by the primed hESCs from which they were derived, suggesting a lack of complete reprogramming of X chromosome during primed to extended/expanded pluripotency conversion. Furthermore, we found that the X chromosome status of hEPSCs affected their ability to differentiate into embryonic or extraembryonic lineage cells. Taken together, our work characterized the X chromosome status of hEPSCs, providing important information for the future application of hEPSCs.

Pluripotency in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is regulated by three transcription factors-OCT3/4, SOX2, and NANOG. To fully exploit the therapeutic potential of these cells it is essential to have a good mechanistic understanding of the maintenance of self-renewal and pluripotency. In this study, we demonstrate a powerful systems biology approach in which we first expand literature-based network encompassing the core regulators of pluripotency by assessing the behavior of genes targeted by perturbation experiments. We focused our attention on highly regulated genes encoding cell surface and secreted proteins as these can be more easily manipulated by the use of inhibitors or recombinant proteins. Qualitative modeling based on combining boolean networks and in silico perturbation experiments were employed to identify novel pluripotency-regulating genes. We validated Interleukin-11 (IL-11) and demonstrate that this cytokine is a novel pluripotency-associated factor capable of supporting self-renewal in the absence of exogenously added bFGF in culture. To date, the various protocols for hESCs maintenance require supplementation with bFGF to activate the Activin/Nodal branch of the TGFβ signaling pathway. Additional evidence supporting our findings is that IL-11 belongs to the same protein family as LIF, which is known to be necessary for maintaining pluripotency in mouse but not in human ESCs. These cytokines operate through the same gp130 receptor which interacts with Janus kinases. Our finding might explain why mESCs are in a more naïve cell state compared to hESCs and how to convert primed hESCs back to the naïve state. Taken together, our integrative modeling approach has identified novel genes as putative candidates to be incorporated into the expansion of the current gene regulatory network responsible for inducing and maintaining pluripotency.

Peripheral blood mononuclear cells (PBMC) were donated by a healthy 61-year old Chinese Han female. Human OKSM (OCT3/4, KLF4 SOX2, and c-MYC) transcription factors were used to reprogram her PBMCs with the non-integrating episomal vector system. Immunocytochemistry for pluripotency makers confirmed the pluripotency of transgene-free iPSCs and their ability to differentiate spontaneously three germ layers in vitro. The iPSC line displayed a normal karyotype. Our model offers the possibility to be used a control in pathological mechanism studies.

Peripheral blood mononuclear cells (PBMC) were collected from a 65-year old healthy woman with Chinese Han genetic background. The PBMCs were reprogrammed with the human OKSM transcription factors using the non-integrating episomal vector system. The transgene-free iPSC showed pluripotency verified by immunocytochemistry for pluripotency markers and differentiated spontaneously toward the 3 germ layers in vitro. Furthermore, the iPSC line showed normal karyotype. The iPSC line can be used as control in disease mechanism studies. Resource table.

A healthy 31-year-old Chinese Han female donated peripheral blood mononuclear cells (PBMC). Her PBMCs were reprogrammed with human OKSM (OCT3/4, KLF4 SOX2, and c-MYC) transcription factors by the non-integrating episomal vector system. Immunocytochemistry for pluripotency markers confirmed the pluripotency of transgene-free iPSCs. Their ability to differentiate spontaneously three germ layers in vitro is also confirmed. The iPSC line displayed a normal karyotype. This model can be used as a control in pathological mechanism studies.

A 87-year old Alzheimer's Disease(AD) male patient donated his Peripheral blood mononuclear cells (PBMC). The non-integrating episomal vector system was used to reprogram PBMCs with the human OKSM transcription factors. The pluripotency of transgene-free iPSCs was confirmed by immunocytochemistry for pluripotency markers and by the ability of the iPSCs to differentiate spontaneously into 3 germ layers in vitro. In addition, the iPSC line displayed a normal karyotype. Our model might offer a good platform to further study the pathological mechanisms, to identify early biomarkers, and also for drug testing studies in AD.

MicroRNAs are endogenous ~22 nt RNAs that regulate gene expression by translational inhibition and mRNA destabilization. MicroRNA-29b (miR-29b) is essential for progression of mouse embryos past preimplantation development; however, details of the underlying regulatory network remain to be elucidated. Here, we used RNA sequencing to identify changes in the transcriptome of mouse embryos in response to miR-29b inhibition. Morula-stage embryos that had been subject to miR-29b inhibition throughout preimplantation development exhibited significant expression changes in 870 genes compared with controls. Among 405 genes that were downregulated, 30 genes encoded factors with known essential function during early embryonic development, including the pluripotent stem cell factor Nanog. We identified 19 genes encoding putative miR-29b target transcripts. These included Zbtb40, Hbp1, Ccdc117, Ypel2, Klf4, and Tmed9, which are upregulated at the 4-cell state of mouse development concomitant with miR-29b downregulation. Luciferase reporter analysis confirmed that Zbtb40, Hbp1, Ccdc117, Ypel2, and Klf4 transcripts are direct targets of miR-29b. These results suggest that miR-29b decreases the mRNA levels of several target genes during early mouse development, including the gene encoding the reprogramming factor Klf4. We hypothesize that inhibition of miR-29b causes overexpression of its target genes, triggering downstream signaling networks to decrease the expression of genes that are essential for embryonic development. In conclusion, miR-29b controls an extensive regulatory network in early mouse embryos, which comprises reprogramming factors and molecular regulators of post-transcriptional modification processes.

Human amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnostics procedures. Recently, it has been illustrated that these cells may also serve as a valuable model system to study developmental processes and for application in regenerative therapies. Cellular reprogramming is a means of assigning greater value to primary AFCs by inducing self-renewal and pluripotency and, thus, bypassing senescence. Here, we report the generation and characterization of human amniotic fluid-derived induced pluripotent stem cells (AFiPSCs) and demonstrate their ability to differentiate into the trophoblast lineage after stimulation with BMP2/BMP4. We further carried out comparative transcriptome analyses of primary human AFCs, AFiPSCs, fibroblast-derived iPSCs (FiPSCs) and embryonic stem cells (ESCs). This revealed that the expression of key senescence-associated genes are down-regulated upon the induction of pluripotency in primary AFCs (AFiPSCs). By defining distinct and overlapping gene expression patterns and deriving the LARGE (Lost, Acquired and Retained Gene Expression) Principle of Cellular Reprogramming, we could further highlight that AFiPSCs, FiPSCs and ESCs share a core self-renewal gene regulatory network driven by OCT4, SOX2 and NANOG. Nevertheless, these cell types are marked by distinct gene expression signatures. For example, expression of the transcription factors, SIX6, EGR2, PKNOX2, HOXD4, HOXD10, DLX5 and RAXL1, known to regulate developmental processes, are retained in AFiPSCs and FiPSCs. Surprisingly, expression of the self-renewal-associated gene PRDM14 or the developmental processes-regulating genes WNT3A and GSC are restricted to ESCs. Implications of this, with respect to the stability of the undifferentiated state and long-term differentiation potential of iPSCs, warrant further studies.

Otosclerosis is characterized by abnormal bone remodeling in the osseous labyrinth and progressive hearing loss. Although the etiology of otosclerosis is not fully understood, both environmental and genetic factors play important roles in its pathogenesis. Here, we generated an induced pluripotent stem cell line using episomal plasmid vectors from the peripheral blood mononuclear cells of a 48-year-old male with otosclerosis. The morphology and karyotype of the cells were normal. The expression of pluripotency markers was verified by mRNA and protein levels; the pluripotency state of the cell line was verified by successful differentiation into all three germ layers.

An 83-year old Alzheimer's disease (AD) male patient donated his Peripheral blood mononuclear cells (PBMC). The non-integrating episomal vector system used to reprogram PBMCs with the human OKSM transcription factors. The pluripotency of transgene-free iPSCs was confirmed by immunocytochemistry for pluripotency markers and by the ability of the iPSCs to differentiate spontaneously into 3 germ layers in vitro. In addition, the iPSC line displayed a normal karyotype. Our model might offer a good platform to further study the pathological mechanisms, to identify early biomarkers, and also for drug testing studies in AD.

A 32-year old Bipolar Disorder (BD) male patient donated his Peripheral blood mononuclear cells (PBMC). The non-integrating episomal vector system used to reprogram PBMCs with the human OKSM transcription factors. The pluripotency of transgene-free iPSCs was confirmed by immunocytochemistry for pluripotency markers and by the ability of the iPSCs to differentiate spontaneously into 3 germ layers in vitro. In addition, the iPSC line displayed a normal karyotype. Our model might offer a good platform to further study the pathological mechanisms, to identify early biomarkers, and also for drug testing studies in BD. Resource table.

A 62-years old Alzheimer's disease (AD) male patient donated his Peripheral blood mononuclear cells. The non-integrating episomal vector system used to reprogram PBMCs with Oct3/4, Klf4, Sox2 and c-Myc transcription factors. The pluripotency of transgene-free pluripotent stem cell (iPSC) was confirmed by immunocytochemistry for pluripotency markers-SOX2, NANOG, OCT3/4, SSEA4, TRA1-60, and TRA1-81. The differentiation capacity of the iPSCs into endoderm, mesoderm and ectoderm was assessed by AFP, SMA and βIII-TUBULIN, respectively. In addition, the iPSC line displayed a normal karyotype. This iPSC line might offer a good cell model to explore the pathological mechanisms and treatment strategies for AD.

A 76-year old Alzheimer's disease (AD) female patient donated her Peripheral blood mononuclear cells (PBMC). The non-integrating episomal vector system used to reprogram PBMCs with the human OKSM transcription factors. The pluripotency of transgene-free iPSCs was confirmed by immunocytochemistry for pluripotency markers and by the ability of the iPSCs to differentiate spontaneously into 3 germ layers in vitro. In addition, the iPSC line displayed a normal karyotype. Our model might offer a good platform to further study the pathological mechanisms, to identify early biomarkers, and also for drug testing studies in AD.

A 32-year old Obsessive-Compulsive Disorder (OCD) male patient donated his Peripheral blood mononuclear cells (PBMC). The non-integrating episomal vector system used to reprogram PBMCs with the human OKSM transcription factors. The pluripotency of transgene-free iPSCs was confirmed by immunocytochemistry for pluripotency markers and by the ability of the iPSCs to differentiate spontaneously into 3 germ layers in vitro. In addition, the iPSC line displayed a normal karyotype. Our model might offer a good platform to further study the pathological mechanisms, to identify early biomarkers, and also for drug testing studies in OCD. Resource Table.

The genotype of apolipoprotein E (APOE) is closely associated with susceptibility to Alzheimer's disease. Here, we described the generation and characterization of human induced pluripotent stem cells from peripheral blood mononuclear cells (PBMCs) of a 79-year-old female patient with Alzheimer's disease having APOE3/4 genotype. The generated iPSCs expressed pluripotent stem cell markers that were observed using immunocytochemistry. Moreover, they displayed a normal karyotype and had the potential to differentiate spontaneously into three germ layers in vitro. Our model could provide valuable insights into pathological mechanisms, and offer a unique opportunity for developing drugs against specific phenotypes for Alzheimer's disease therapy.

A 62-year old Chinese Han Alzheimer's disease (AD) female patient with ApoE3/4 genetic background donated her Peripheral blood mononuclear cells (PBMC). The non-integrating episomal vector system was used to reprogrammed PBMCs with the human OKSM transcription factors. The pluripotency of transgene-free iPSCs was confirmed by immunocytochemistry for pluripotency markers and by the ability of the iPSCs to differentiate spontaneously into 3 germ layers in vitro. In addition, the iPSC line displayed a normal karyotype. Our model might offer a good platform to further study the pathological mechanisms, to identify early biomarkers, and also for drug testing studies in AD.

Primed and naive human embryonic stem cells (hESCs) do not fully recapitulate the X chromosome status observed in human preimplantation epiblast and fail to initiate random X chromosome inactivation (XCI) upon differentiation. Therefore, an ideal system for studying XCI during early human development is yet to be established. We show that incomplete blocking of autocrine fibroblast growth factor 2 (FGF2) signaling in naive hESCs drives significant heterogeneity in X chromosome and pluripotency status. We derived homozygous XaXa naive hESCs with dual allelic XIST expression and high levels of TFCP2L1, whose transcriptome and X chromosome states are similar to human preimplantation epiblast. Random XCI was initiated upon naive-to-primed conversion of these cells, and both pre- and post-XCI primed hESCs were obtained. We observed random XCI in all cells upon further differentiation of pre-XCI primed hESCs. Together, these findings enable derivation of homogeneous naive hESCs and establish a powerful platform to study human XCI.

Human endometrial epithelia undergo injury repair and regeneration with the menstrual cycle; however, mechanisms underpinning the roles of endometrial epithelial cells in endometrial lesions and regeneration remain incompletely understood, mainly owing to the difficulty in the isolation and expansion of primary endometrial epithelial cells and the lack of reliable models for in vitro and in vivo studies. In this report, we sought to improve methods for the isolation and expansion of human endometrial epithelial cells with a Rho-associated protein kinase (ROCK) inhibitor-modified medium and subsequently characterize endometrial epithelium generated with primary cells cultured in an air-liquid interface (ALI) state. Immunocytochemistry staining revealed the expression of epithelial cellular adhesion molecule (EpCam) and stage-specific embryonic antigen-1 (SSEA-1) but a lack of CD13 in endometrial epithelial cells. Meanwhile, a large number of proliferative Ki67+ cells were observed in isolated epithelial cells. Importantly, the EpCam+/CD13- cells were capable of forming spheroids, a characteristic of epithelial stem/progenitor cells. Interestingly, these cells also exhibited a capacity to reconstitute epithelial layers in an ALI state. Morphological analysis revealed mucosal secretion of differentiated epithelial cells with cilia and microvilli in ALI epithelial cells as determined by electronic microscopy. Immunoblotting assay further demonstrated the expression of endometrial epithelial cell markers keratin 17/19 and EpCam and stem cell marker OCT3/4 but not stromal cell marker Vimentin protein and CD13 in cell expansions. Furthermore, molecular analysis also showed that the exposure of cells to estrogen elevated the expression of estrogen receptor and progesterone receptors in ALI cultures. Our results shed light on the possibility of expanding sufficient numbers of endometrial epithelial cells for stem cell biology studies, and they provide a feasible and alternative model that can recapitulate the characteristics and physiology of endometrial epithelium in vivo.

A 66-year old mild cognitive impairment (MCI) female patient donated her Peripheral blood mononuclear cells (PBMC). PBMC was reprogrammed using non-integrative Sendai viral vectors containing reprogramming factors OCT4, KLF4, SOX2 and C-MYC. The pluripotency of transgene-free iPSCs was confirmed by immunocytochemistry and ability of differentiation spontaneously into 3 germ layers in vitro. Moreover, the iPSC line displayed a normal karyotype. The apolipoprotein E (APOE) ε4 allele, is considered to be the strongest genetic risk factor for Sporadic Alzheimer's disease (AD). Our model might offer a good platform to study the pathological mechanisms and drug testing studies in AD and MCI.

Hybrid anticancer drugs are of great therapeutic interests as they can potentially overcome the deficiencies of conventional chemotherapy drugs and improve the efficacy. Many studies have revealed that the combination of histone deacetylase inhibitors (HDACi) and alkylating agents have synergistic effects. We reported a novel hybrid NL-101, in which the side chain of bendamustine was replaced with the hydroxamic acid of HDACi vorinostat (SAHA). NL-101 exhibited efficient anti-proliferative activity on myeloid leukemia cells especially Kasumi-1 and NB4 cells, accompanied by S phase arrest and caspase-3 dependent apoptosis. Importantly, it presented both the properties of HDAC inhibition and DNA damaging, as assessed by the acetylation of histone H3 and DNA double-strand breaks marker γ-H2AX. NL-101 also down-regulated the expression of anti-apoptotic protein Bcl-xL which was involved in the mitochondrial death pathway. Meanwhile, NL-101 induced apoptosis and DNA damage in primary cells from acute myeloid leukemia (AML) patients. NL-101 treatment could significantly prolong the survival time of t(8;21) leukemia mice with enhanced efficacy than bendamustine. These data demonstrate that NL-101 could be a potent and selective agent for leukemia treatment.