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

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 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

While distinct stem cell phenotypes follow global changes in chromatin marks,single-cell chromatin technologies are unable to resolve or predict stem cell fates. We propose the first such use of optical high content nanoscopy of histone epigenetic marks (epi-marks) in stem cells to classify emergent cell states. By combining nanoscopy with epi-mark textural image informatics,we developed a novel approach,termed EDICTS (Epi-mark Descriptor Imaging of Cell Transitional States),to discern chromatin organizational changes,demarcate lineage gradations across a range of stem cell types and robustly track lineage restriction kinetics. We demonstrate the utility of EDICTS by predicting the lineage progression of stem cells cultured on biomaterial substrates with graded nanotopographies and mechanical stiffness,thus parsing the role of specific biophysical cues as sensitive epigenetic drivers. We also demonstrate the unique power of EDICTS to resolve cellular states based on epi-marks that cannot be detected via mass spectrometry based methods for quantifying the abundance of histone post-translational modifications. Overall,EDICTS represents a powerful new methodology to predict single cell lineage decisions by integrating high content super-resolution nanoscopy and imaging informatics of the nuclear organization of epi-marks. View Publication

Recent advances in developmental biology and stem cell technology have led to the engineering of functional organs in a dish. However,the limited size of these organoids and absence of a large circulatory system poses limits to its clinical translation. To overcome these issues,decellularized whole kidney scaffolds with native microstructure and extracellular matrix (ECM) are employed for kidney bioengineering,using human-induced pluripotent-stem-cell-derived renal progenitor cells and endothelial cells. To demonstrate ECM-guided cellular assembly,the present work is focused on generating the functional unit of the kidney,the glomerulus. In the repopulated organ,the presence of endothelial cells broadly upregulates the expression level of genes related to renal development. When the cellularized native scaffolds are implanted in SCID mice,glomeruli assembly can be achieved by co-culture of the renal progenitors and endothelial cells. These individual glomerular units are shown to be functional in the context of the whole organ using a simulated bio-reactor set-up with urea and creatinine excretion and albumin reabsorption. Our results indicate that the repopulation of decellularized native kidney using clinically relevant,expandable patient-specific renal progenitors and endothelial cells may be a viable approach for the generation of a functional whole kidney. View Publication

Kit receptor tyrosine kinase and erythropoietin receptor (Epo-R) cooperate in regulating blood cell development. Mice that lack the expression of Kit or Epo-R die in utero of severe anemia. Stimulation of Kit by its ligand,stem cell factor activates several distinct early signaling pathways,including phospholipase C gamma,phosphatidylinositol 3-kinase,Src kinase,Grb2,and Grb7. The role of these pathways in Kit-induced growth,proliferation,or cooperation with Epo-R is not known. We demonstrate that inactivation of any one of these early signaling pathways in Kit significantly impairs growth and proliferation. However,inactivation of the Src pathway demonstrated the most profound defect. Combined stimulation with Epo also resulted in impaired cooperation between Src-defective Kit mutant and Epo-R and,to a lesser extent,with Kit mutants defective in the activation of phosphatidylinositol 3-kinase or Grb2. The impaired cooperation between the Src-defective Kit mutant and Epo-R was associated with reduced transphosphorylation of Epo-R and expression of c-Myc. Remarkably,restoration of only the Src pathway in a Kit receptor defective in the activation of all early signaling pathways demonstrated a 50% correction in proliferation in response to Kit stimulation and completely restored the cooperation with Epo-R. These data demonstrate an essential role for Src pathway in regulating growth,proliferation,and cooperation with Epo-R downstream from Kit. 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

Erythropoietin (EPO) is the hormone necessary for development of erythrocytes from immature erythroid cells. EPO activates Jun N-terminal kinase (JNK),a member of the mitogen-activated protein kinase (MAPK) family in the EPO-dependent murine erythroid HCD57 cells. Therefore,we tested if JNK activity supported proliferation and/or survival of these cells. Treatment with the JNK inhibitor SP600125 inhibited JNK activity and EPO-dependent proliferation of HCD57 cells and the human EPO-dependent cell lines TF-1 and UT7-EPO. SP600125 also increased the fraction of cells in G2/M. Introduction of a dominant-negative form of JNK1 inhibited EPO-dependent proliferation in HCD57 cells but did not increase the fraction of cells in G2/M. Constitutive JNK activity was observed in primary murine erythroid progenitors. Treatment of primary mouse bone marrow cells with the SP600125 inhibitor reduced the number of erythroid burst-forming units (BFU-e's) but not the more differentiated erythroid colony-forming units (CFU-e's),and SP600125 protected the BFU-e's from apoptosis induced by cytosine arabinoside,demonstrating that the SP600125 inhibited proliferation of the BFU-e's. Therefore,JNK activity appears to be an important regulator of proliferation in immature,primary erythroid cells and 3 erythroid cell lines but may not be required for the survival or proliferation of CFU-e's or proerythroblasts. View Publication