搜索结果: 'gmp t cell activator'

In order to develop a novel method of visualizing possible Ca(2+) signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters,we expressed the bioluminescent Ca(2+) reporter,apo-aequorin,in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca(2+) transients (generated by release from intracellular stores) were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2,indicating that holo-f-aequorin was functional in these cells. Furthermore,following the addition of exogenous ATP,an inositol trisphosphate receptor (IP3R) agonist,small Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) release from functional IP3Rs was demonstrated by treatment with 2-APB,a known IP3R antagonist. In contrast,following treatment with caffeine,a ryanodine receptor (RyR) agonist,a minimal Ca(2+) response was observed at day 8 of differentiation only. Thus,our data indicate that unlike RyRs,IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation. View Publication

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH,however,lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore,other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs),characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming,and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation,the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients. View Publication

Pluripotent stem cells (PSCs) can self-renew or differentiate from naive or more differentiated,primed,pluripotent states established by specific culture conditions. Increased intracellular α-ketoglutarate (αKG) was shown to favor self-renewal in naive mouse embryonic stem cells (mESCs). The effect of αKG or αKG/succinate levels on differentiation from primed human PSCs (hPSCs) or mouse epiblast stem cells (EpiSCs) remains unknown. We examined primed hPSCs and EpiSCs and show that increased αKG or αKG-to-succinate ratios accelerate,and elevated succinate levels delay,primed PSC differentiation. αKG has been shown to inhibit the mitochondrial ATP synthase and to regulate epigenome-modifying dioxygenase enzymes. Mitochondrial uncoupling did not impede αKG-accelerated primed PSC differentiation. Instead,αKG induced,and succinate impaired,global histone and DNA demethylation in primed PSCs. The data support αKG promotion of self-renewal or differentiation depending on the pluripotent state. View Publication

Human embryonic stem cells (hESCs) can differentiate into all somatic lineages including stratified squamous epithelia. Thus,efficient methods are required to direct hESC differentiation to obtain a pure subpopulation for tissue engineering. The study aimed to assess the effects of retinoic acid (RA),bone morphogenetic protein-4 (BMP4),and ascorbic acid (AA) on the differentiation of hESCs into keratinocyte progenitors in vitro. The first media contained AA and BMP4; the second contained RA,AA,and BMP4; the third was commercial-defined keratinocyte serum-free medium,which was used to differentiate H9 hESCs (direct approach) or embryoid bodies (EBs) (indirect approach) into keratinocyte progenitors. Real-time RT-PCR,immunofluorescence,and flow-cytometry were used to characterize the differentiated cells. Cells induced by AA + BMP4 + RA showed the typical epithelial morphology,while cells induced by AA + BMP4 showed multiple appearances. CK14 and p63 messenger RNA (mRNA) expressions in the AA + BMP4 + RA-treated cells were higher than those of the AA + BMP4-treated cells (CK14: 22.4-fold; p63: 84.7-fold). Epithelial marker CK18 mRNA expressions at 14 d of differentiation and keratinocyte marker CK14 and transcription factor p63 mRNA expressions at 35 d of differentiation were higher in cells differentiated from hESCs compared with those differentiated from EBs (CK18 10.51 ± 3.26 vs. 6.67 ± 1.28; CK14 9.27 ± 3.61 vs. 5.32 ± 1.86; p63 0.73 ± 0.06 vs. 0.44 ± 0.12,all P textless 0.05) After hESC induction by AA+BMP4+RA,CK14 mRNA expression was upregulated after day 21,peaking by 35 d of differentiation. Combined RA,BMP4,and AA could effectively induce differentiation of hESCs into keratinocyte progenitors in vitro. These keratinocytes could be used for oral mucosa and skin tissue engineering. View Publication

Inefficient neural differentiation of human induced pluripotent stem cells (hiPSCs) motivates recent investigation of the influence of biophysical characteristics of cellular microenvironment,in particular nanotopography,on hiPSC fate decision. However,the roles of geometry and dimensions of nanotopography in neural lineage commitment of hiPSCs have not been well understood. The objective of this study is to delineate the effects of geometry,feature size and height of nanotopography on neuronal differentiation of hiPSCs. HiPSCs were seeded on equally spaced nanogratings (500 and 1000nm in linewidth) and hexagonally arranged nanopillars (500nm in diameter),each having a height of 150 or 560nm,and induced for neuronal differentiation in concert with dual Smad inhibitors. The gratings of 560nm height reduced cell proliferation,enhanced cytoplasmic localization of Yes-associated protein,and promoted neuronal differentiation (up to 60% βIII-tubulin(+) cells) compared with the flat control. Nanograting-induced cell polarity and cytoplasmic YAP localization were shown to be critical to the induced neural differentiation of hiPSCs. The derived neuronal cells express MAP2,Tau,glutamate,GABA and Islet-1,indicating the existence of multiple neuronal subtypes. This study contributes to the delineation of cell-nanotopography interactions and provides the insights into the design of nanotopography configuration for pluripotent stem cell neural lineage commitment. View Publication

Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology,including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells-based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity. One of the well-known mechanisms that are activated by chemically-induced oxidative stress is the Nrf2 signaling pathway. Therefore,in the current study,we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)-derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone-induced oxidative stress mediated by complex I inhibition of mitochondrial respiration. Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM),as shown by Nrf2 nuclear translocation and the up-regulation of the Nrf2-downstream antioxidant enzymes,NQO1 and SRXN1. Interestingly,exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover,rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH(+)) cells was observed. The obtained results suggest that hiPSC-derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation. View Publication

Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study,after determining the minimum inhibitory level of lactic acid for hiPSCs,a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically,about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture,a final cell density of (1.1 ± 0.1) × 10(6) cells mL(-1) was obtained,with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression,on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method,culture medium refinement by dialysis was established to remove toxic metabolites,recycle autocrine factors as well as other growth factors,and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture. View Publication

Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is a rare,early-onset retinal dystrophy characterised by distinct bands of circumferential pigmentary degeneration in the peripheral retina and developmental eye defects. ADVIRC is caused by mutations in the Bestrophin1 (BEST1) gene,which encodes a transmembrane protein thought to function as an ion channel in the basolateral membrane of retinal pigment epithelial (RPE) cells. Previous studies suggest that the distinct ADVIRC phenotype results from alternative splicing of BEST1 pre-mRNA. Here,we have used induced pluripotent stem cell (iPSC) technology to investigate the effects of an ADVIRC associated BEST1 mutation (c.704T textgreater C,p.V235A) in patient-derived iPSC-RPE. We found no evidence of alternate splicing of the BEST1 transcript in ADVIRC iPSC-RPE,however in patient-derived iPSC-RPE,BEST1 was expressed at the basolateral membrane and the apical membrane. During human eye development we show that BEST1 is expressed more abundantly in peripheral RPE compared to central RPE and is also expressed in cells of the developing retina. These results suggest that higher levels of mislocalised BEST1 expression in the periphery,from an early developmental stage,could provide a mechanism that leads to the distinct clinical phenotype observed in ADVIRC patients. View Publication

Long noncoding RNAs (lncRNAs) exhibit diverse functions,including regulation of development. Here,we combine genome-wide mapping of SMAD3 occupancy with expression analysis to identify lncRNAs induced by activin signaling during endoderm differentiation of human embryonic stem cells (hESCs). We find that DIGIT is divergent to Goosecoid (GSC) and expressed during endoderm differentiation. Deletion of the SMAD3-occupied enhancer proximal to DIGIT inhibits DIGIT and GSC expression and definitive endoderm differentiation. Disruption of the gene encoding DIGIT and depletion of the DIGIT transcript reveal that DIGIT is required for definitive endoderm differentiation. In addition,we identify the mouse ortholog of DIGIT and show that it is expressed during development and promotes definitive endoderm differentiation of mouse ESCs. DIGIT regulates GSC in trans,and activation of endogenous GSC expression is sufficient to rescue definitive endoderm differentiation in DIGIT-deficient hESCs. Our study defines DIGIT as a conserved noncoding developmental regulator of definitive endoderm. View Publication

Functional hepatocytes derived from human pluripotent stem cells (hPSCs) have potential as tools for predicting drug-induced hepatotoxicity in the early phases of drug development. However,the propensity of hPSC lines to differentiate into specific lineages is reported to differ. The ability to predict low propensity of hPSCs to differentiate into hepatocytes would facilitate the selection of useful hPSC clones and substantially accelerate development of hPSC-derived hepatocytes for pharmaceutical research. In this study,we compared the expression of genes associated with hepatic differentiation in five hPSC lines including human ES cell line,H9,which is known to differentiate into hepatocytes,and an hPSC line reported with a poor propensity for hepatic differentiation. Genes distinguishing between undifferentiated hPSCs,hPSC-derived hepatoblast-like differentiated cells,and primary human hepatocytes were drawn by two-way cluster analysis. The order of expression levels of genes in undifferentiated hPSCs was compared with that in hPSC-derived hepatoblast-like cells. Three genes were selected as predictors of low propensity for hepatic differentiation. Expression of these genes was investigated in 23 hPSC clones. Review of representative cells by induction of hepatic differentiation suggested that low prediction scores were linked with low hepatic differentiation. Thus,our model using gene expression ranking and bioinformatic analysis could reasonably predict poor differentiation propensity of hPSC lines. View Publication

The ability of human embryonic stem cells (hESCs) to proliferate indefinitely is attributed to its high telomerase activity and associated long telomere. However,factors regulating telomere length in hESCs remain largely uncharacterized. The aims of this study were,to identify factors which modulate telomere length of hESCs,and to determine if the telomere length of hESCs influences cellular senescence of its differentiated progeny cells. Telomerase reverse transcriptase (TERT) gene expression,telomerase activity and telomere length of hESCs cultured in different culture systems were compared. Genetically identical hESCs of different telomere lengths were differentiated into fibroblasts simultaneously,and the population doubling and cellular senescence levels were determined. We found that telomere lengths were significantly different in different culture systems and Fibroblast growth factor-2 (FGF-2) upregulated TERT expression,telomerase activity and telomere length via Wnt/β-catenin signaling pathway in hESCs in a significant manner. We also provide evidence that fibroblast differentiated from hESCs with longer telomere exhibited significant more population doublings and longer life span than those derived from hESCs with shorter telomeres. Thus,FGF-2 levels in hESCs culture systems can be manipulated to generate cells with longer telomere which would be advantageous in the applications of hESCs in regenerative medicine. View Publication

OBJECTIVE To elucidate the genetic background of a patient with neonatal-onset multisystem inflammatory disease (NOMID) with no NLRP3 mutation. METHODS A Japanese male child diagnosed as having NOMID was studied. The patient did not have any NLRP3 mutation,even as low-frequency mosaicism. We performed whole-exome sequencing on the patient and his parents. Induced pluripotent stem cells (iPSCs) were established from the patient's fibroblasts. The iPSCs were then differentiated into monocyte lineage to evaluate the cytokine profile. RESULTS We established multiple iPSC clones from a patient with NOMID and incidentally found that the phenotypes of monocytes from iPSC clones were heterogeneous and could be grouped into disease and normal phenotypes. Because each iPSC clone was derived from a single somatic cell,we hypothesized that the patient had somatic mosaicism of an interleukin-1β-related gene. Whole-exome sequencing of both representative iPSC clones and the patient's blood revealed a novel heterozygous NLRC4 mutation,p.T177A (c.529AtextgreaterG),as a specific mutation in diseased iPSC clones. Knockout of the NLRC4 gene using the clustered regularly interspaced short palindromic repeat/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype. CONCLUSION Our findings indicate that the patient has somatic mosaicism of a novel NLRC4 mutation. To our knowledge,this is the first case showing that somatic mutation of NLRC4 causes autoinflammatory symptoms compatible with NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC-based phenotype dissection for individualized diagnoses. View Publication