Project description:To construct comprehensive competing endogenous RNA (ceRNA) networks , depleting ribosomal RNA strand-specific libraries were constructed from the ADO2-iPSCs and normal control human iPSCs (NC-iPSCs) for deep RNA sequencing.
Project description:To identify potential miRNA–mediated targets associated with osteopetrosis at the genome-wide level, two small RNA libraries and a degradome library were constructed from the ADO2-iPSCs and normal control human iPSCs (NC-iPSCs) for deep sequencing.
Project description:To identify potential miRNA–mediated targets associated with osteopetrosis at the genome-wide level, two small RNA libraries and a degradome library were constructed from the ADO2-iPSCs and normal control human iPSCs (NC-iPSCs) for deep sequencing.
Project description:Corneal endothelial cells (CECs) are critical to maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cells (PBMC)-originated induced pluripotent stem cells (iPSCs)-derived CECs. We isolated PBMC and programmed the mononuclear cells to generate iPSCs. Subsequently, the PBMC-originated iPSCs were differentiated to CECs. The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent, and CECs-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSCs-derived CECs and human corneal endothelium (CE) were examined by mass spectrometry-based proteome sequencing. The PBMC-originated iPSCs expressed pluripotent-specific markers at levels similar to expression in H9 human embryonic stem cells (hESCs). Phase contrast microscopy illustrated that iPSCs-derived CECs are tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CECs-associated markers were expressed at many orders of magnitude higher in iPSCs-derived CECs at days 13, 20, and 30 compared to their respective levels in iPSCs. Importantly, only residual expression levels of pluripotency markers were detected in iPSCs-derived CECs. Mass spectrometry-based proteome profiling identified 10,575 proteins in iPSCs-derived CECs. In parallel, we completed proteome profiling of the human CE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSCs-derived CECs suggesting a 90.82% overlap between the iPSCs-derived CECs and human CE proteomes. Importantly, cryopreservation of iPSCs-derived CECs did not affect the tight adherence of CECs, and their hexagonal-like shape while expressing high levels of CECs-associated markers. We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the human CE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSCs-derived CECs.
Project description:To elucidate the function of 30Kc19α-Lin28A protein in osteogenic differentiation of urine-derived stem cells, we established urine-derived stem cell lines differentiated with or without protein treatment. We then performed gene expression profiling analysis using data obtained from RNA-seq of osteogenic differentiated urine-derived stem cells with or without 30Kc19α-Lin28A protein treatment, undifferentiated urine-derived stem cells, and human osteoblasts
Project description:We generated iPSCs from human urine cells (hUCs) with the aid of small molecules and autologous hUC feeders. A compound cocktail including Cyclic Pifithrin-a, a p53 inhibitor and other compounds known for benefiting reprogramming like A-83-01, CHIR99021, Thiazovivin, NaB and PD0325901 was used to aid hUC reprogramming (Plan B). Aided by this cocktail, we achieved significantly improved efficiency (170 folds more) for hUC reprogramming and iPSC generation. In addition, to enable iPSC generation in some cases that massive cell death occurred during delivering reprogramming factors, we replaced Matrigel with autologous hUCs as feeder for reprogramming and iPSC generation (Plan C). Replacing Matrigel with autologous feeder not only enhanced reprograming, but also avoided concern using animal components for human iPSC generation. These were efficient approaches to enable iPSC generation from hUCs that were otherwise difficult for reprogramming, which would be valuable for banking patient’s specific iPSCs.
Project description:Human urine is now recognized as a non-invasive source of stem cells with regeneration potential. These cells are mesenchymal stem cells but their detailed molecular and cellular identities are poorly defined. Furthermore, unlike the mouse, the gene regulatory network driving stemness and differentiation into functional renal cells in vitro remain unresolved. In this study, we isolated urine mesenchymal stem cells from 10 individuals of both genders and distinct ages, these cells express pluripotency- associated proteins- TRA-1-160, TRA-1-181, SSEA4, C-KIT and CD133. Expression of pluripotency-associated proteins enabled rapid reprogramming into iPSCs using episomal-based plasmids without pathway purtabations. Transcriptome analysis revealed expression of a plethora of nephrogenesis-related genes such as SIX2, OSR1, CITED1, NPHS2, NPHS1, PAX2, SALL1, AQP2, EYA1, SLC12A1 and UMOD. As expected the cells transport Albumin by endocytosis. Based on this we refer to these cells as urine derived renal progenitor cells- UdRPCs. Associated GO-term analysis of UdRPCs and UdRPC-iPSCs underlined their renal identity and functionality. Upon differentiation by WNT activation using the GSK3β inhibitor (CHIR99021), transcriptome and KEGG pathway analysis revealed upregulation of WNT-associated genes-AXIN2, JUN and NKD1. Protein interaction network identified JUN,- a downstream target of the WNT pathway in association with STAT3, ATF2 and MAPK1 as a putative key regulator of self-renewal and differentiation in UdRPCs. Furthermore, like pluripotent stem cells, self-renewal is maintained by FGF2-driven TGFβ-SMAD2/3 pathway. Ultimately, our in vitro model and the data presented in this study should lay the foundation for studying nephrogenesis in man.