技术资料

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization,but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line,where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter,we screened for textgreater 60 bioactive small molecules that would promote endothelial differentiation,and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated textgreater 50% conversion of hPSCs to endothelial cells (ECs),specifically VEC(+)CD31(+)CD34(+)CD14(-)KDR(high) endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling,in combination with VEGF-A treatment,resulted in efficient formation of EPs via KDR(+) mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods,which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs. View Publication

Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence,comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end,here,we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages,including bone,muscle,and heart. We defined the extrinsic signals controlling each binary lineage decision,enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%???99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in??vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation,a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively,this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. Video Abstract View Publication

Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally,antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails,but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here,we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally,we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example,H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique. View Publication

Human embryonic stem (hES) cell production of heparan sulfate influences cell fate and pluripotency. Human ES cells remain pluripotent in vitro through the action of growth factors signaling,and the activity of these factors depends on interaction with specific receptors and also with heparan sulfate. Here,we tested the hypothesis that matrix-associated heparan sulfate is enough to maintain hES cells under low fibroblast growth factor-2 concentration in the absence of live feeder cells. To pursue this goal,we compared hES cells cultured either on coated plates containing live murine embryonic fibroblasts (MEFs) or on a matrix derived from ethanol-fixed MEFs. hES cells were analyzed for the expression of pluripotency markers and the ability to form embryoid bodies. hES cells cultured either on live mouse fibroblasts or onto a matrix derived from fixed fibroblasts expressed similar levels of Oct-4,SOX-2,Nanog,TRA-1-60 and SSEA-4,and they were also able to form cavitated embryoid bodies. Heparan sulfate-depleted matrix lost the ability to support the adherence and growth of hES cells,confirming that this glycosaminoglycan,bound to the extracellular matrix,is enough for the growth and attachment of hES cells. Finally,we observed that the ethanol-fixed matrix decreases by 30% the levels of Neu5Gc in hES cells,indicating that this procedure reduces xeno-contamination. Our data suggest that matrix-bound heparan sulfate is required for the growth and pluripotency of hES cells and that ethanol-fixed MEFs may be used as a live cell"-free substrate for stem cells." View Publication

AIMS/HYPOTHESIS: Islet transplantation is a promising cell therapy for patients with diabetes,but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However,the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore,we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo.backslashnbackslashnMETHODS: The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte,Laguna Hills,CA,USA) devices into diabetic mice.backslashnbackslashnRESULTS: Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing textgreater80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices,despite lacking direct contact with the host environment,and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells.backslashnbackslashnCONCLUSIONS/INTERPRETATION: Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device,yielding glucose-responsive insulin-producing cells capable of reversing diabetes. View Publication

MicroRNAs (miRNA) are central regulators of diverse biological processes and are important in the regulation of stem cell self-renewal. One of the widely studied miRNA-protein regulators is the Lin28-Let-7 pair. In this study,we demonstrate that contrary to the well-established models of mouse ES cells (mESC) and transformed human cancer cells,the pluripotent state of human ES cells (hESC) involves expression of mature Let-7 family miRNAs with concurrent expression of all LIN28 proteins. We show that mature Let-7 miRNAs are regulated during hESC differentiation and have opposite expression profile with LIN28B. Moreover,mature Let-7 miRNAs fine tune the expression levels of LIN28B protein in pluripotent hESCs,whereas silencing of LIN28 proteins have no effect on mature Let-7 levels. These results bring novel information to the highly complex network of human pluripotency and suggest that maintenance of hESC pluripotency differs greatly from the mESCs in regard to LIN28-Let-7 regulation. View Publication

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. Maxadilan,a 61-amino acid vasodilatory peptide,specifically activates the PACAP type I receptor (PAC1). Although PAC1 has been identified in embryonic stem cells,little is known about its presence or effects in human induced pluripotent stem (iPS) cells. In the present study,we investigated the expression of PAC1 in human iPS cells by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. To study the physiological effects mediated by PAC1,we evaluated the role of maxadilan in preventing apoptotic cell death induced by ultraviolet C (UVC). After exposure to UVC,the iPS cells showed a marked reduction in cell viability and a parallel increase of apoptotic cells,as demonstrated by WST-8 analysis,annexin V/propidium iodide (PI) analysis and the terminal transferase dUTP nick end labeling (TUNEL) assay. The addition of 30 nM of maxadilan dramatically increased iPS cell viability and reduced the percentage of apoptotic cells. The anti-apoptotic effects of maxadilan were correlated to the downregulation of caspase-3 and caspase-9. Concomitantly,immunofluorescence,western blot analysis,real-time quantitative polymerase chain reaction (RT-qPCR) analysis and in vitro differentiation results showed that maxadilan did not affect the pluripotent state of iPS cells. Moreover,karyotype analysis showed that maxadilan did not affect the karyotype of iPS cells. In summary,these results demonstrate that PAC1 is present in iPS cells and that maxadilan effectively protects iPS cells against UVC-induced apoptotic cell death while not affecting the pluripotent state or karyotype. View Publication

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions,the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4,Nanog,Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl(2) (n = 22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (ptextless0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC,E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl(2),at concentrations that selectively block I(K1) (50-100 µM),failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I(K1) in 53% (20/38) of the cells studied,but presence of I(Kr) in all (11/11). Consistent with the electrophysiological data,RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I(K1) in the majority of cells,but robust expression of I(Kr.) In contrast to recently reported studies,our data point to major deficiencies of hiPSC-CM,with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I(Kr) due to the absence of I(K1). Thus,efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications. View Publication

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has provided huge insight into the pathways,mechanisms and transcription factors that control differentiation. Here we use high-throughput RT-PCR technology to take a snapshot of splicing changes in the full spectrum of high- and low-expressed genes during induction of fibroblasts,from several donors,into iPSCs and their subsequent redifferentiation. We uncover a programme of concerted alternative splicing changes involved in late mesoderm differentiation and controlled by key splicing regulators MBNL1 and RBFOX2. These critical splicing adjustments arise early in vertebrate evolution and remain fixed in at least 10 genes (including PLOD2,CLSTN1,ATP2A1,PALM,ITGA6,KIF13A,FMNL3,PPIP5K1,MARK2 and FNIP1),implying that vertebrates require alternative splicing to fully implement the instructions of transcriptional control networks. View Publication