技术资料

Human stem cells are promising sources for bladder regeneration. Among several possible sources,pluripotent stem cells are the most fascinating because they can differentiate into any cell type,and proliferate limitlessly in vitro. Here,we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE),and further specified DE cells into BUCs by treating retinoic acid under a keratinocyte-specific serum free medium. hPSC-derived DE cells showed significantly expressed DE-specific genes,but did not express mesodermal or ectodermal genes. After DE cells were specified into BUCs,they notably expressed urothelium-specific genes such as UPIb,UPII,UPIIIa,P63 and CK7. Immunocytochemistry showed that BUCs expressed UPII,CK8/18 and P63 as well as tight junction molecules,E-CADHERIN and ZO-1. Additionally,hPSCs-derived BUCs exhibited low permeability in a FITC-dextran permeability assay,indicating BUCs possessed the functional units of barrier on their surfaces. However,BUCs did not express the marker genes of other endodermal lineage cells (intestine and liver) as well as mesodermal or ectodermal lineage cells. In summary,we sequentially differentiated hPSCs into DE and BUCs in a serum- and feeder-free condition. Our differentiation protocol will be useful for producing cells for bladder regeneration and studying normal and pathological development of the human bladder urothelium in vitro. View Publication

The genetic reprogramming technology allows one to generate pluripotent stem cells for individual patients. These cells,called induced pluripotent stem cells (iPSCs),can be an unlimited source of specialized cell types for the body. Thus,autologous somatic cell replacement therapy becomes possible,as well as the generation of in vitro cell models for studying the mechanisms of disease pathogenesis and drug discovery. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder that leads to a loss of upper and lower motor neurons. About 10% of cases are genetically inherited,and the most common familial form of ALS is associated with mutations in the SOD1 gene. We used the reprogramming technology to generate induced pluripotent stem cells with patients with familial ALS. Patient-specific iPS cells were obtained by both integration and transgene-free delivery methods of reprogramming transcription factors. These iPS cells have the properties of pluripotent cells and are capable of direct differentiation into motor neurons. View Publication

We measured the dynamics of an essential epigenetic modifier,HP1β,in human cells at different stages of differentiation using Fluorescence Recovery After Photobleaching (FRAP). We found that HP1β mobility is similar in human embryonic stem cells (hES) and iPS cells where it is more mobile compared to fibroblasts; HP1β is less mobile in senescent fibroblasts than in young (dividing) fibroblasts. Introduction of reprogramming factors" View Publication

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work,we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. View Publication

During mammalian embryonic development,the primitive streak initiates the differentiation of pluripotent epiblast cells into germ layers. Pluripotency can be reacquired in committed somatic cells using a combination of a handful of transcription factors,such as OCT3/4,SOX2,KLF4 and c-MYC (hereafter referred to as OSKM),albeit with low efficiency. Here we show that during OSKM-induced reprogramming towards pluripotency in human cells,intermediate cells transiently show gene expression profiles resembling mesendoderm,which is a major component of the primitive streak. Based on these findings,we discover that forkhead box H1 (FOXH1),a transcription factor required for anterior primitive streak specification during early development,significantly enhances the reprogramming efficiency of human fibroblasts by promoting their maturation,including mesenchymal to epithelial transition and the activation of late pluripotency markers. These results demonstrate that during the reprogramming process,human somatic cells go through a transient state that resembles mesendoderm. View Publication

Recent evidence points to extra-telomeric,noncanonical roles for telomerase in regulating stem cell function. In this study,human embryonic stem cells (hESCs) were cultured in 20% or 2% O2 microenvironments for up to 5 days and evaluated for telomerase reverse transcriptase (TERT) expression and telomerase activity. Results showed increased cell survival and maintenance of the undifferentiated state with elevated levels of nuclear TERT in 2% O2-cultured hESCs despite no significant difference in telomerase activity compared with their high-O2-cultured counterparts. Pharmacological inhibition of telomerase activity using a synthetic tea catechin resulted in spontaneous hESC differentiation,while telomerase inhibition with a phosphorothioate oligonucleotide telomere mimic did not. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed variations in transcript levels of full-length and alternate splice variants of TERT in hESCs cultured under varying O2 atmospheres. Steric-blocking of Δα and Δβ hTERT splicing using morpholino oligonucleotides altered the hTERT splicing pattern and rapidly induced spontaneous hESC differentiation that appeared biased toward endomesodermal and neuroectodermal cell fates,respectively. Together,these results suggest that post-transcriptional regulation of TERT under varying O2 microenvironments may help regulate hESC survival,self-renewal,and differentiation capabilities through expression of extra-telomeric telomerase isoforms. View Publication

Werner syndrome (WS) patients exhibit premature aging predominantly in mesenchyme-derived tissues,but not in neural lineages,a consequence of telomere dysfunction and accelerated senescence. The cause of this lineage-specific aging remains unknown. Here,we document that reprogramming of WS fibroblasts to pluripotency elongated telomere length and prevented telomere dysfunction. To obtain mechanistic insight into the origin of tissue-specific aging,we differentiated iPSCs to mesenchymal stem cells (MSCs) and neural stem/progenitor cells (NPCs). We observed recurrence of premature senescence associated with accelerated telomere attrition and defective synthesis of the lagging strand telomeres in MSCs,but not in NPCs. We postulate this aging" discrepancy is regulated by telomerase. Expression of hTERT or p53 knockdown ameliorated the accelerated aging phenotypein MSC View Publication

BACKGROUND Although studies have shown that Oct4,Sox2,Klf4,and c-Myc (OKSM)-mediated induced pluripotent stem cell (iPSC) technology sensitizes cancer cells to drugs,the potential risk of inserting c-Myc and random insertions of exogenous sequences into the genome persists. Several authors,including us,have presented microRNA (miRNA)-mediated reprogramming as an alternative approach. Herein,we evaluated the efficacy of miRNA-mediated reprogramming on hepatocellular carcinoma (HCC) cells. METHODS Among three miRNAs (miR-200c,miR-302s,and miR-369s) that were previously presented for miRNA-mediated reprogramming,miR-302 was expressed at low levels in HCC cells. After transfecting three times with miR-302,the cells were incubated in ES medium for 3 weeks and then characterized. RESULTS iPSC-like spheres were obtained after the 3-week incubation. Spheres presented high NANOG and OCT4 expression,low proliferation,high apoptosis,low epithelial-mesenchymal transition marker expression (N-cadherin,TGFBR2),and sensitization to drugs. Several miRNAs were changed (e.g.,low oncomiR miR-21,high miR-29b). cMyc was decreased,and methylation was elevated on histone 3 at lysine 4 (H3K4). Differentiated cells expressed markers of each germ layer (GFAP,FABP4,and ALB). AOF2 (also known as LSD1 or KDM1),one of the targets for miR-302,was repressed in iPSC-like-spheres. Silencing of AOF2 resulted in similar features of iPSC-like-spheres,including cMyc down-regulation and H3K4 methylation. In drug-resistant cells,sensitization was achieved through miR-302-mediated reprogramming. CONCLUSIONS miR-302-mediated iPSC technology reprogrammed HCC cells and improved drug sensitivity through AOF2 down-regulation,which caused H3K4 methylation and c-Myc repression. View Publication

Emerging studies suggested that murine podoplanin-positive monocytes (PPMs) are involved in lymphangiogenesis. The goal of this study was to demonstrate the therapeutic lymphangiogenesis of human PPMs by the interaction with platelets. Aggregation culture of human peripheral blood mononuclear cells (PBMCs) resulted in cellular aggregates termed hematospheres. During 5-day culture,PPMs expanded exponentially and expressed several lymphatic endothelial cell-specific markers including vascular endothelial growth factor receptor (VEGFR)-3 and well-established lymphangiogenic transcription factors. Next,we investigated the potential interaction of PPMs with platelets that had C-type lectin-like receptor-2 (CLEC-2),a receptor of podoplanin. In vitro coculture of PPMs and platelets stimulated PPMs to strongly express lymphatic endothelial markers and upregulate lymphangiogenic cytokines. Recombinant human CLEC-2 also stimulated PPMs through Akt and Erk signaling. Likewise,platelets in coculture with PPMs augmented secretion of a lymphangiogenic cytokine,interleukin (IL)-1-β,via podoplanin/CLEC-2 axis. The supernatant obtained from coculture was able to enhance the migration,viability,and proliferation of lymphatic endothelial cell. Local injection of hematospheres with platelets significantly increased lymphatic neovascularization and facilitated wound healing in the full-thickness skin wounds of nude mice. Cotreatment with PPMs and platelets augments lymphangiogenesis through podoplanin/CLEC-2 axis,which thus would be a promising novel strategy of cell therapy to treat human lymphatic vessel disease. View Publication

OBJECTIVES Kidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney,stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources,pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However,little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study,we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system. MATERIALS AND METHODS We designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak,intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR,real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility. RESULTS After modification of culture period and concentration of exogenous factors,hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2,GDNF,HOXD11,WT1 and CITED1 in addition to OSR1,PAX2,SALL1 and EYA1. Moreover,NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular,approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems. CONCLUSIONS Our novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases. View Publication

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless,clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally,derivation of hiPSCs should be rapid and efficient in order to minimize manipulations,reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here,we provide an optimized,fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity,purity,stability and safety at a GMP facility and cryopreserved. To our knowledge,as a proof of principle,these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes. View Publication

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) can differentiate into many cell types and are important for regenerative medicine; however,further work is needed to reliably differentiate hESC and hiPSC into neural-restricted multipotent derivatives or specialized cell types under conditions that are free from animal products. Toward this goal,we tested the transition of hESC and hiPSC lines onto xeno-free (XF) / feeder-free conditions and evaluated XF substrate preference,pluripotency,and karyotype. Critically,XF transitioned H9 hESC,Shef4 hESC,and iPS6-9 retained pluripotency (Oct-4 and NANOG),proliferation (MKI67 and PCNA),and normal karyotype. Subsequently,XF transitioned hESC and hiPSC were induced with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to generate neuralized spheres containing primitive neural precursors,which could differentiate into astrocytes and neurons,but not oligoprogenitors. Further neuralization of spheres via LIF supplementation and attachment selection on CELLstart substrate generated adherent human neural stem cells (hNSC) with normal karyotype and high proliferation potential under XF conditions. Interestingly,adherent hNSC derived from H9,Shef4,and iPS6-9 differentiated into significant numbers of O4+ oligoprogenitors (∼20-30%) with robust proliferation; however,very few GalC+ cells were observed (∼2-4%),indicative of early oligodendrocytic lineage commitment. Overall,these data demonstrate the transition of multiple hESC and hiPSC lines onto XF substrate and media conditions,and a reproducible neuralization method that generated neural derivatives with multipotent cell fate potential and normal karyotype. View Publication