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

BACKGROUND Cell-based therapy shows promise in treating peripheral arterial disease (PAD); however,the optimal cell type and long-term efficacy are unknown. In this study,we identified a novel subpopulation of adult progenitor cells positive for CD34 and M-cadherin (CD34/M-cad BMCs) in mouse and human bone marrow. We also examined the long-lasting therapeutic efficacy of mouse CD34/M-cad BMCs in restoring blood flow and promoting vascularization in an atherosclerotic mouse model of PAD. METHODS AND FINDINGS Colony-forming cell assays and flow cytometry analysis showed that CD34/M-cad BMCs have hematopoietic progenitor properties. When delivered intra-arterially into the ischemic hindlimbs of ApoE/ mice,CD34/M-cad BMCs alleviated ischemia and significantly improved blood flow compared with CD34/M-cad BMCs,CD34/M-cad BMCs,or unselected BMCs. Significantly more arterioles were seen in CD34/M-cad cell-treated limbs than in any other treatment group 60 days after cell therapy. Furthermore,histologic assessment and morphometric analyses of hindlimbs treated with GFP CD34/M-cad cells showed that injected cells incorporated into solid tissue structures at 21 days. Confocal microscopic examination of GFP CD34/M-cad cell-treated ischemic legs followed by immunostaining indicated the vascular differentiation of CD34/M-cad progenitor cells. A cytokine antibody array revealed that CD34/M-cad cell-conditioned medium contained higher levels of cytokines in a unique pattern,including bFGF,CRG-2,EGF,Flt-3 ligand,IGF-1,SDF-1,and VEGFR-3,than did CD34/M-cad cell-conditioned medium. The proangiogenic cytokines secreted by CD34/M-cad cells induced oxygen- and nutrient-depleted endothelial cell sprouting significantly better than CD34/M-cad cells during hypoxia. CONCLUSION CD34/M-cad BMCs represent a new progenitor cell type that effectively alleviates hindlimb ischemia in ApoE/ mice by consistently improving blood flow and promoting arteriogenesis. Additionally,CD34/M-cad BMCs contribute to microvascular remodeling by differentiating into vascular cells and releasing proangiogenic cytokines and growth factors. View Publication

Endoplasmic reticulum (ER) stress occurs during early embryonic development. The aim of this study is to determine whether ER stress occurs during human embryonic stem cell differentiation induced by retinoic acid (RA). H9 human embryonic stem cells were subjected to RA treatment for up to 29. days to induce differentiation. HEK293 cells were treated with RA as a control. The results demonstrate that several ER stress-responsive genes are differentially regulated in H9 and HEK293 cells in response to 5. days of RA treatment. GRP78/Bip was upregulated in H9 cells but downregulated in HEK293 cells. eIF2?? was downregulated in H9 cells but not in HEK293 cells. Phosphorylation of eIF2?? was downregulated in H9 cells but upregulated in HEK293 cells. XBP-1 was downregulated immediately after RA treatment in H9 cells,but its downregulation was much slower in HEK293 cells. Additionally,two ER-resident E3 ubiquitin ligases,gp78 and Hrd1,were both upregulated in H9 cells following 5. days of exposure to RA. Moreover,the protein Bcl2 was undetectable in H9 cells and H9-derived cells but was expressed in HEK293 cells,and it expression in the two types of cells was unaltered by RA treatment. In H9 cells treated with RA for 29. days,GRP78/Bip,XBP-1 and Bcl2 were all upregulated. These results suggest that ER stress is involved in H9 cell differentiation induced by RA. ?? 2011 Elsevier Inc. View Publication

The differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) towards functional neurons particularly hold great potential for the cell-based replacement therapy in neurodegenerative diseases. Here,we describe a stepwise differentiation protocol that mimics the early stage of neural development in human to promote the generation of neuroprogenitors at a high yield. Both the hESCs and hiPSCs are initially cultured in an optimized feeder-free condition,which offer an efficient formation of aggregates. To specify the neuroectodermal specification,these aggregates are differentiated in a defined neural induction medium to develop into neural rosettes-like structures. The rosettes are expanded into free-floating sphere and can be further propagated or developed into variety of neuronal subtypes. View Publication

Human embryonic stem cells (hESCs) hold great promise for regenerative medicine because they can undergo unlimited self-renewal and retain the capability to differentiate into all cell types in the body. Although numerous genes/proteins such as Oct4 and Gata6 have been identified to play critical regulatory roles in self-renewal and differentiation of hESC,the majority of the regulators in these cellular processes and more importantly how these regulators co-operate with each other and/or with epigenetic modifications are still largely unknown. We propose here a systematic approach to integrate genomic and epigenomic data for identification of direct regulatory interactions. This approach allows reconstruction of cell-type-specific transcription networks in embryonic stem cells (ESCs) and fibroblasts at an unprecedented scale. Many links in the reconstructed networks coincide with known regulatory interactions or literature evidence. Systems-level analyses of these networks not only uncover novel regulators for pluripotency and differentiation,but also reveal extensive interplays between transcription factor binding and epigenetic modifications. Especially,we observed poised enhancers characterized by both active (H3K4me1) and repressive (H3K27me3) histone marks that contain enriched Oct4- and Suz12-binding sites. The success of such a systems biology approach is further supported by experimental validation of the predicted interactions. View Publication

Smooth muscle cells (SMCs) and endothelial cells (ECs) are typically derived separately,with low efficiencies,from human pluripotent stem cells (hPSCs). The concurrent generation of these cell types might lead to potential applications in regenerative medicine to model,elucidate,and eventually treat vascular diseases. Here we report a robust two-step protocol that can be used to simultaneously generate large numbers of functional SMCs and ECs from a common proliferative vascular progenitor population via a two-dimensional culture system. We show here that coculturing hPSCs with OP9 cells in media supplemented with vascular endothelial growth factor,basic fibroblast growth factor,and bone morphogenetic protein 4 yields a higher percentage of CD31(+)CD34(+) cells on day 8 of differentiation. Upon exposure to endothelial differentiation media and SM differentiation media,these vascular progenitors were able to differentiate and mature into functional endothelial cells and smooth muscle cells,respectively. Furthermore,we were able to expand the intermediate population more than a billion fold to generate sufficient numbers of ECs and SMCs in parallel for potential therapeutic transplantations. View Publication

More than 10 years after their first isolation,human embryonic stem cells are finally 'coming of age' in research and biotechnology applications as protocols for their differentiation and undifferentiated expansion in culture become robust and scalable,and validated commercial reagents become available. Production of human cardiomyocytes is now feasible on a daily basis for many laboratories with tissue culture expertise. An additional recent surge of interest resulting from the first production of human iPSCs (induced pluripotent stem cells) from somatic cells of patients now makes these technologies of even greater importance since it is likely that (genetic) cardiac disease phenotypes can be captured in the cardiac derivatives of these cells. Although cell therapy based on replacing cardiomyocytes lost or dysfunctional owing to cardiac disease are probably as far away as ever,biotechnology and pharmaceutical applications in safety pharmacology and drug discovery will probably impact this clinical area in the very near future. In the present paper,we review the cutting edge of this exciting area of translational research. View Publication

Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders that involve defects that preclude the normal function of osteoclasts,which differentiate from hematopoietic precursors. In half of human cases,ARO is the result of mutations in the TCIRG1 gene,which codes for a subunit of the vacuolar proton pump that plays a fundamental role in the acidification of the cell-bone interface. Functional mutations of this pump severely impair the resorption of bone mineral. Although postnatal hematopoietic stem cell transplantation can partially rescue the hematological phenotype of ARO,other stigmata of the disease,such as secondary neurological and growth defects,are not reversed. For this reason,ARO is a paradigm for genetic diseases that would benefit from effective prenatal treatment. Using the oc/oc mutant mouse,a murine model whose osteopetrotic phenotype closely recapitulates human TCIRG1-dependent ARO,we report that in utero transplantation of adult bone marrow hematopoietic stem cells can correct the ARO phenotype in a limited number of mice. Here we report that in utero injection of allogeneic fetal liver cells,which include hematopoietic stem cells,into oc/oc mouse fetuses at 13.5 days post coitum produces a high level of engraftment,and the oc/oc phenotype is completely rescued in a high percentage of these mice. Therefore,oc/oc pathology appears to be particularly sensitive to this form of early treatment of the ARO genetic disorder. 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

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

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

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

HOX genes,notably members of the HOXA cluster,and HOX cofactors have increasingly been linked to human leukemia. Intriguingly,HOXD13,a member of the HOXD cluster not normally expressed in hematopoietic cells,was recently identified as a partner of NUP98 in a t(2;11) translocation associated with t-AML/MDS. We have now tested directly the leukemogenic potential of the NUP98-HOXD13 t(2; 11) fusion gene in the murine hematopoietic model. NUP98-HOXD13 strongly promoted growth and impaired differentiation of early hematopoietic progenitor cells in vitro; this effect was dependent on the NUP98 portion and an intact HOXD13 homeodomain. Expression of the NUP98-HOXD13 fusion gene in vivo resulted in a partial impairment of lymphopoiesis but did not induce evident hematologic disease until late after transplantation (more than 5 months),when some mice developed a myeloproliferative-like disease. In contrast,mice transplanted with bone marrow (BM) cells cotransduced with NUP98-HOXD13 and the HOX cofactor Meis1 rapidly developed lethal and transplantable acute myeloid leukemia (AML),with a median disease onset of 75 days. In summary,this study demonstrates that NUP98-HOXD13 can be directly implicated in the molecular process leading to leukemic transformation,and it supports a model in which the transforming properties of NUP98-HOXD13 are mediated through HOX-dependent pathways. View Publication