Differentiation of human pluripotent stem cells into nephron progenitor cells in a serum and feeder free system.
OBJECTIVES Kidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney,stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources,pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However,little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study,we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system. MATERIALS AND METHODS We designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak,intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR,real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility. RESULTS After modification of culture period and concentration of exogenous factors,hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2,GDNF,HOXD11,WT1 and CITED1 in addition to OSR1,PAX2,SALL1 and EYA1. Moreover,NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular,approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems. CONCLUSIONS Our novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases.
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M. V. J. Braham et al. (apr 2019)
Advanced healthcare materials e1801444
A Human Hematopoietic Niche Model Supporting Hematopoietic Stem and Progenitor Cells In Vitro.
Niches in the bone marrow regulate hematopoietic stem and progenitor cell (HSPC) fate and behavior through cell-cell interactions and soluble factor secretion. The niche-HSPC crosstalk is a very complex process not completely elucidated yet. To aid further investigation of this crosstalk,a functional in vitro 3D model that closely represents the main supportive compartments of the bone marrow is developed. Different combinations of human stromal cells and hydrogels are tested for their potential to maintain CD34+ HSPCs. Cell viability,clonogenic hematopoietic potential,and surface marker expression are assessed over time. Optimal HSPC support is obtained in presence of adipogenic and osteogenic cells,together with progenitor derived endothelial cells. When cultured in a bioactive hydrogel,the supportive cells self-assemble into a hypoxic stromal network,stimulating CD34+ CD38+ cell formation,while maintaining the pool of CD34+ 38- HSPCs. HSPC clusters colocalize with the stromal networks,in close proximity to sinusoidal clusters of CD31+ endothelial cells. Importantly,the primary in vitro niche model supports HSPCs with no cytokine addition. Overall,the engineered primary 3D bone marrow environment provides an easy and reliable model to further investigate interactions between HSPCs and their endosteal and perivascular niches,in the context of normal hematopoiesis or blood-related diseases.
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产品类型：
产品号#：
04435
04445
产品名：
MethoCult™H4435富集
MethoCult™H4435富集
文献
Usta S et al. (OCT 2014)
Annals of translational medicine 2 10 97
Chemically defined serum-free and xeno-free media for multiple cell lineages.
Cell culture is one of the most common methods used to recapitulate a human disease environment in a laboratory setting. Cell culture techniques are used to grow and maintain cells of various types including those derived from primary tissues,such as stem cells and cancer tumors. However,a major confounding factor with cell culture is the use of serum and animal (xeno) products in the media. The addition of animal products introduces batch and lot variations that lead to experimental variability,confounds studies with therapeutic outcomes for cultured cells,and represents a major cost associated with cell culture. Here we report a commercially available serum-free,albumin-free,and xeno free (XF) media (Neuro-Pure(TM)) that is more cost-effective than other commercial medias. Neuro-Pure was used to maintain and differentiate various cells of neuronal lineages,fibroblasts,as well as specific cancer cell lines; without the use of contaminants such serum,albumin,and animal products. Neuro-Pure allows for a controlled and reproducible cell culture environment that is applicable to translational medicine and general tissue culture.
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产品类型：
产品号#：
05761
产品名：
用于小鼠和大鼠神经干细胞和祖细胞分化培养的试剂盒
文献
Qin J et al. (NOV 2016)
Scientific reports 6 37388
Connexin 32-mediated cell-cell communication is essential for hepatic differentiation from human embryonic stem cells.
Gap junction-mediated cell-cell interactions are highly conserved and play essential roles in cell survival,proliferation,differentiation and patterning. We report that Connexin 32 (Cx32)-mediated gap junctional intercellular communication (GJIC) is necessary for human embryonic stem cell-derived hepatocytes (hESC-Heps) during step-wise hepatic lineage restriction and maturation. Vitamin K2,previously shown to promote Cx32 expression in mature hepatocytes,up-regulated Cx32 expression and GJIC activation during hepatic differentiation and maturation,resulting in significant increases of hepatic markers expression and hepatocyte functions. In contrast,negative Cx32 regulator 2-aminoethoxydiphenyl borate blocked hESC-to-hepatocyte maturation and muted hepatocyte functions through disruption of GJIC activities. Dynamic gap junction organization and internalization are phosphorylation-dependent and the p38 mitogen-activated protein kinases pathway (MAPK) can negatively regulate Cxs through phosphorylation-dependent degradation of Cxs. We found that p38 MAPK inhibitor SB203580 improved maturation of hESC-Heps correlating with up-regulation of Cx32; by contrast,the p38 MAPK activator,anisomycin,blocked hESC-Heps maturation correlating with down-regulation of Cx32. These results suggested that Cx32 is essential for cell-cell interactions that facilitate driving hESCs through hepatic-lineage maturation. Regulators of both Cx32 and other members of its pathways maybe used as a promising approach on regulating hepatic lineage restriction of pluripotent stem cells and optimizing their functional maturation.
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产品类型：
产品号#：
85850
85857
产品名：
mTeSR™1
mTeSR™1
文献
Domashenko AD et al. (OCT 2010)
Blood 116 15 2676--83
TAT-mediated transduction of NF-Ya peptide induces the ex vivo proliferation and engraftment potential of human hematopoietic progenitor cells.
Retroviral overexpression of NF-Ya,the regulatory subunit of the transcription factor NF-Y,activates the transcription of multiple genes implicated in hematopoietic stem cell (HSC) self-renewal and differentiation and directs HSCs toward self-renewal. We asked whether TAT-NF-Ya fusion protein could be used to transduce human CD34(+) cells as a safer,more regulated alternative approach to gene therapy. Here we show that externally added recombinant protein was able to enter the cell nucleus and activate HOXB4,a target gene of NF-Ya,using real-time polymerase chain reaction RNA and luciferase-based protein assays. After TAT-NF-Ya transduction,the proliferation of human CD34(+) cells in the presence of myeloid cytokines was increased 4-fold. Moreover,TAT-NF-Ya-treated human primary bone marrow cells showed a 4-fold increase in the percentage of huCD45(+) cells recovered from the bone marrow of sublethally irradiated,transplanted NOD-Scid IL2Rγ(null) mice. These data demonstrate that TAT-peptide therapies are an alternative approach to retroviral stem cell therapies and suggest that NF-Ya peptide delivery should be further evaluated as a tool for HSC/progenitors ex vivo expansion and therapy.
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Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells.
Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy,we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters,the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters,suggesting a relative resistance to silencing. Furthermore,an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector,largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.
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EasySep™小鼠TIL（CD45）正选试剂盒
文献
产品手册Hematopoietic Stem and Progenitor Cells - Products for Your Research
产品手册Products for Human Pluripotent Stem Cells
产品手册Products for Human Pluripotent Stem Cells
技术公告Flow Cytometry Methods for Identifying Mouse Hematopoietic Stem and Progenitor Cells
