搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 72-year old female Parkinson's disease (PD) patient with R1398H variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model provides a good platform for studying the mechanism of PD,and also for drug testing and gene therapy studies. View Publication

textlessptextgreater Hundreds of textlessitalictextgreaterL1CAMtextless/italictextgreater gene mutations have been shown to be associated with congenital hydrocephalus,severe intellectual disability,aphasia,and motor symptoms. How such mutations impair neuronal function,however,remains unclear. Here,we generated human embryonic stem (ES) cells carrying a conditional textlessitalictextgreaterL1CAMtextless/italictextgreater loss-of-function mutation and produced precisely matching control and textlessitalictextgreaterL1CAMtextless/italictextgreater -deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones,we found that deletion of textlessitalictextgreaterL1CAMtextless/italictextgreater dramatically impaired axonal elongation and,to a lesser extent,dendritic arborization. Unexpectedly,we also detected an ∼20–50% and ∼20–30% decrease,respectively,in the levels of ankyrinG and ankyrinB protein,and observed that the size and intensity of ankyrinG staining in the axon initial segment was significantly reduced. Overexpression of wild-type L1CAM,but not of the L1CAM point mutants R1166X and S1224L,rescued the decrease in ankyrin levels. Importantly,we found that the textlessitalictextgreaterL1CAMtextless/italictextgreater mutation selectively decreased activity-dependent Na textlesssuptextgreater+textless/suptextgreater -currents,altered neuronal excitability,and caused impairments in action potential (AP) generation. Thus,our results suggest that the clinical presentations of textlessitalictextgreaterL1CAMtextless/italictextgreater mutations in human patients could be accounted for,at least in part,by cell-autonomous changes in the functional development of neurons,such that neurons are unable to develop normal axons and dendrites and to generate normal APs. textless/ptextgreater View Publication

While in vitro liver tissue engineering has been increasingly studied during the last several years,presently engineered liver tissues lack the bile duct system. The lack of bile drainage not only hinders essential digestive functions of the liver,but also leads to accumulation of bile that is toxic to hepatocytes and known to cause liver cirrhosis. Clearly,generation of bile duct tissue is essential for engineering functional and healthy liver. Differentiation of human induced pluripotent stem cells (iPSCs) to bile duct tissue requires long and/or complex culture conditions,and has been inefficient so far. Towards generating a fully functional liver containing biliary system,we have developed defined and controlled conditions for efficient 2D and 3D bile duct epithelial tissue generation. A marker for multipotent liver progenitor in both adult human liver and ductal plate in human fetal liver,EpCAM,is highly expressed in hepatic spheroids generated from human iPSCs. The EpCAM high hepatic spheroids can,not only efficiently generate a monolayer of biliary epithelial cells (cholangiocytes),in a 2D differentiation condition,but also form functional ductal structures in a 3D condition. Importantly,this EpCAM high spheroid based biliary tissue generation is significantly faster than other existing methods and does not require cell sorting. In addition,we show that a knock-in CK7 reporter human iPSC line generated by CRISPR/Cas9 genome editing technology greatly facilitates the analysis of biliary differentiation. This new ductal differentiation method will provide a more efficient method of obtaining bile duct cells and tissues,which may facilitate engineering of complete and functional liver tissue in the future. View Publication

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However,traces of potentially oncogenic genes remaining in actively transcribed regions of the genome,limit their potential for use in human therapeutic applications. Additionally,non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context. In this video,we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR),which has an advantage over less sensitive techniques previously used to detect gene expression differences. Full conversion into clinical-grade good manufacturing practice (GMP) conditions,allows human clinical relevance. Our protocol offers another methodology--provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications--for deriving GMP-grade hiPSCs,which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions. View Publication

Surface marker expression forms the basis for characterization and isolation of human embryonic stem cells (hESCs). Currently,there are few well-defined protein epitopes that definitively mark hESCs. Here we combine immunotranscriptional profiling of hESC lines with membrane-polysome translation state array analysis (TSAA) to determine the full set of genes encoding potential hESC surface marker proteins. Three independently isolated hESC lines (HES2,H9,and MEL1) grown under feeder and feeder-free conditions were sorted into subpopulations by fluorescence-activated cell sorting based on coimmunoreactivity to the hESC surface markers GCTM-2 and CD9. Colony-forming assays confirmed that cells displaying high coimmunoreactivity to GCTM-2 and CD9 constitute an enriched subpopulation displaying multiple stem cell properties. Following microarray profiling,820 genes were identified that were common to the GCTM-2(high)/CD9(high) stem cell-like subpopulation. Membrane-polysome TSAA analysis of hESCs identified 1,492 mRNAs encoding actively translated plasma membrane and secreted proteins. Combining these data sets,88 genes encode proteins that mark the pluripotent subpopulation,of which only four had been previously reported. Cell surface immunoreactivity was confirmed for two of these markers: TACSTD1/EPCAM and CDH3/P-Cadherin,with antibodies for EPCAM able to enrich for pluripotent hESCs. This comprehensive listing of both hESCs and spontaneous differentiation-associated transcripts and survey of translated membrane-bound and secreted proteins provides a valuable resource for future study into the role of the extracellular environment in both the maintenance of pluripotency and directed differentiation. View Publication

Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of,for example,ion selectivity,gating mechanism,composition,or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs) and their somatic cell source,keratinocytes from plucked human hair. This comparison revealed that 26&x25; of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6&x25; were downregulated. Additionally,iPSCs express a much higher number of ion channels compared to keratinocytes. Further,to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny,namely,neurons and cardiomyocytes derived from iPS cells. To conclude,hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms. View Publication

The human iPSC cell line,RP2-FiPS4F1 (RCPFi001-A),derived from dermal fibroblasts from the patient with retinitis pigmentosa caused by the mutation of the gene PRPF8,was generated by non-integrative reprogramming technology using OCT3/4,SOX2,CMYC and KLF4 reprogramming factors. View Publication

Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However,clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody,we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the alpha2 domain of HLA-A. Here,we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells,lymphocytes,or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors,suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely,2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs,melphalan,or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore,administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I-targeting therapy against multiple myeloma. View Publication

Human interferon (IFN)-alpha is the standard therapy for chronic hepatitis C to prevent its progression to liver cirrhosis and hepatocellular carcinoma. Thrombocytopenia is one of the major adverse effects of IFN-alpha and often leads to dose reduction or treatment discontinuation. However,there is little information on how IFN-alpha inhibits human megakaryopoiesis. In this study,we demonstrated that IFN-alpha did not inhibit colony formation of megakaryocytes from human CD34(+) hematopoietic stem cells. IFN-alpha did not inhibit endomitosis but did inhibit cytoplasmic maturation of megakaryocytes and platelet production in vitro. IFN-alpha suppressed the expression of transcription factors regulating late-stage megakaryopoiesis,such as GATA-1,p45(NF-E2),MafG. IFN-alpha also significantly reduced the number of human platelets but not megakaryocytes,and did not inhibit endomitosis of human megakaryocytes in immunodeficient NOD/Shi-scid/IL-2R gamma(null) (NOG) mice transplanted with human CD34(+) cells (hu-NOG). We also demonstrated that a novel thrombopoietin mimetic,NIP-004,was effective for treating IFN-alpha-induced thrombocytopenia in hu-NOG mice. From ultrastructural study,IFN-alpha inhibited the maturation of demarcation membranes in megakaryocytes,although NIP-004 prevented the inhibitory effects of IFN-alpha. These results defined the pathogenesis of IFN-alpha-induced thrombocytopenia and suggested possible future clinical applications for thrombopoietin mimetics. View Publication

Peripheral blood was collected from a clinically diagnosed 60-year old female patient with multiple schwannoma. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers,and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for further pathological studies of multiple schwannoma. View Publication

UNLABELLED In the classical form of $\$1-antitrypsin deficiency (ATD),aberrant intracellular accumulation of misfolded mutant $\$1-antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain-of-function,proteotoxic" mechanism. Whereas some ATD patients develop severe liver disease (SLD) that necessitates liver transplantation� View Publication

By providing access to affected neurons,human induced pluripotent stem cells (iPSc) offer a unique opportunity to model human neurodegenerative diseases. We generated human iPSc from the skin fibroblasts of children with mucopolysaccharidosis type IIIB. In this fatal lysosomal storage disease,defective α-N-acetylglucosaminidase interrupts the degradation of heparan sulfate (HS) proteoglycans and induces cell disorders predominating in the central nervous system,causing relentless progression toward severe mental retardation. Partially digested proteoglycans,which affect fibroblast growth factor signaling,accumulated in patient cells. They impaired isolation of emerging iPSc unless exogenous supply of the missing enzyme cleared storage and restored cell proliferation. After several passages,patient iPSc starved of an exogenous enzyme continued to proliferate in the presence of fibroblast growth factor despite HS accumulation. Survival and neural differentiation of patient iPSc were comparable with unaffected controls. Whereas cell pathology was modest in floating neurosphere cultures,undifferentiated patient iPSc and their neuronal progeny expressed cell disorders consisting of storage vesicles and severe disorganization of Golgi ribbons associated with modified expression of the Golgi matrix protein GM130. Gene expression profiling in neural stem cells pointed to alterations of extracellular matrix constituents and cell-matrix interactions,whereas genes associated with lysosome or Golgi apparatus functions were downregulated. Taken together,these results suggest defective responses of patient undifferentiated stem cells and neurons to environmental cues,which possibly affect Golgi organization,cell migration and neuritogenesis. This could have potential consequences on post-natal neurological development,once HS proteoglycan accumulation becomes prominent in the affected child brain. View Publication