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

Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets,a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors,32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3),insulin receptor substrate 2 (IRS2),docking protein 1 (DOK1),Src homology 2 domain containing transforming protein 1 (SHC1),and sprouty homologue 1 (SPRY1) as validating targets,and SPRY2,SH2 domain containing 2A (SH2D2A),and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse,rat,and lower vertebrate genomes. Among tissues and lineages,RHEX was elevated in EPCs,occurred as a plasma membrane protein,was rapidly PY-phosphorylated textgreater20-fold upon EPO exposure,and coimmunoprecipitated with the EPOR. In UT7epo cells,knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation,and RHEX coupling to GRB2. In primary human EPCs,shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development. View Publication

Outgrowth,long-term self-renewal,and terminal maturation of human erythroid progenitors derived from umbilical cord blood in serum-free medium can be modulated by steroid hormones. Homogeneous erythroid cultures,as characterized by flow cytometry and dependence on a specific mixture of physiologic proliferation factors,were obtained within 8 days from a starting population of mature and immature mononuclear cells. Due to previous results in mouse and chicken erythroblasts,the proliferation-promoting effect of glucocorticoids was not unexpected. Surprisingly,however,androgen had a positive effect on the sustained expansion of human female but not male erythroid progenitors. Under optimal conditions,sustained proliferation of erythroid progenitors resulted in a more than 10(9)-fold expansion within 60 days. Terminal erythroid maturation was significantly improved by adding human serum and thyroid hormone (3,5,3'-triiodothyronine [T3]) to the differentiation medium. This resulted in highly synchronous differentiation of the cells toward enucleated erythrocytes within 6 days,accompanied by massive size decrease and hemoglobin accumulation to levels comparable to those in peripheral blood erythrocytes. Thus,obviously,different ligand-activated nuclear hormone receptors massively influence the decision between self-renewal and terminal maturation in the human erythroid compartment. View Publication

Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology,including disease phenotypes after genome editing. In three-dimensional cultures,epiblast-stage hPSCs form spheroids surrounding hollow,amniotic-like cavities. GSK3β inhibition differentiates spheroids into segmented,nephron-like kidney organoids containing cell populations with characteristics of proximal tubules,podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes,and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids,indicating that they are tissue specific. Our findings establish a reproducible,versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications. View Publication

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here,we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistochemistry,live imaging,and single-cell transcriptomics. We find that the cytoarchitecture,cell type composition,and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably,however,live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this,the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity,and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution. View Publication

Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes,both in vitro and in vivo. Beyond uses in cell replacement therapy,patient-specific cardiomyocytes may find applications in drug testing,drug discovery,and disease modeling. Recently,approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs),adult heart-derived cardiac progenitor cells (CPCs),and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts,highlighting potential applications and future challenges. View Publication

The basic helix-loop-helix transcription factor achaete-scute complex homologue 1 (ASCL1) is essential for the development of normal lung neuroendocrine cells as well as other endocrine and neural tissues. Small cell lung cancer (SCLC) and non-SCLC with neuroendocrine features express ASCL1,where the factor may play a role in the virulence and primitive neuroendocrine phenotype of these tumors. In this study,RNA interference knockdown of ASCL1 in cultured SCLC resulted in inhibition of soft agar clonogenic capacity and induction of apoptosis. cDNA microarray analyses bolstered by expression studies,flow cytometry,and chromatin immunoprecipitation identified two candidate stem cell marker genes,CD133 and aldehyde dehydrogenase 1A1 (ALDH1A1),to be directly regulated by ASCL1 in SCLC. In SCLC direct xenograft tumors,we detected a relatively abundant CD133(high)-ASCL1(high)-ALDH1(high) subpopulation with markedly enhanced tumorigenicity compared with cells with weak CD133 expression. Tumorigenicity in the CD133(high) subpopulation depended on continued ASCL1 expression. Whereas CD133(high) cells readily reconstituted the range of CD133 expression seen in the original xenograft tumor,CD133(low) cells could not. Our findings suggest that a broad range of SCLC cells has tumorigenic capacity rather than a small discrete population. Intrinsic tumor cell heterogeneity,including variation in key regulatory factors such as ASCL1,can modulate tumorigenicity in SCLC. View Publication

Focal malformations of cortical development (FMCDs) account for the majority of drug-resistant pediatric epilepsy. Postzygotic somatic mutations activating the phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway are found in a wide range of brain diseases,including FMCDs. It remains unclear how a mutation in a small fraction of cells disrupts the architecture of the entire hemisphere. Within human FMCD-affected brain,we found that cells showing activation of the PI3K-AKT-mTOR pathway were enriched for the AKT3(E17K) mutation. Introducing the FMCD-causing mutation into mouse brain resulted in electrographic seizures and impaired hemispheric architecture. Mutation-expressing neural progenitors showed misexpression of reelin,which led to a non-cell autonomous migration defect in neighboring cells,due at least in part to derepression of reelin transcription in a manner dependent on the forkhead box (FOX) transcription factor FOXG1. Treatments aimed at either blocking downstream AKT signaling or inactivating reelin restored migration. These findings suggest a central AKT-FOXG1-reelin signaling pathway in FMCD and support pathway inhibitors as potential treatments or therapies for some forms of focal epilepsy. View Publication

Gfi-1B is a transcriptional repressor that is crucial for erythroid differentiation: inactivation of the GFI1B gene in mice leads to embryonic death due to failure to produce differentiated red cells. Accordingly,GFI1B expression is tightly regulated during erythropoiesis,but the mechanisms involved in such regulation remain partially understood. We here identify HMGB2,a high-mobility group HMG protein,as a key regulator of GFI1B transcription. HMGB2 binds to the GFI1B promoter in vivo and up-regulates its trans-activation most likely by enhancing the binding of Oct-1 and,to a lesser extent,of GATA-1 and NF-Y to the GFI1B promoter. HMGB2 expression increases during erythroid differentiation concomitantly to the increase of GfI1B transcription. Importantly,knockdown of HMGB2 in immature hematopoietic progenitor cells leads to decreased Gfi-1B expression and impairs their erythroid differentiation. We propose that HMGB2 potentiates GATA-1-dependent transcription of GFI1B by Oct-1 and thereby controls erythroid differentiation. View Publication

Mesenchymal stem cells have been implicated as playing an important role in stem cell engraftment. Recently,a new pluripotent population of umbilical cord blood (UCB) cells,unrestricted somatic stem cells (USSCs),with intrinsic and directable potential to develop into mesodermal,endodermal,and ectodermal fates,has been identified. In this study,we evaluated the capacity of ex vivo expanded USSCs to influence the homing of UCB-derived CD34(+) cells into the marrow and spleen of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. USSCs induced a significant enhancement of CD34(+) cell homing to both bone marrow and spleen (2.2 +/- 0.3- and 2.4 +/- 0.6-fold,respectively; p textless .05),with a magnitude similar to that induced by USSCs that had been thawed prior to transplantation. The effect of USSCs was dose-dependent and detectable at USSC:CD34(+) ratios of 1:1 and above. Enhanced marrow homing by USSCs was unaltered by extensive culture passaging of the cells,as similar enhancement was observed for both early-passage (passage 5 [p5]) and late-passage (p10) USSCs. The homing effect of USSCs was also reflected in an increased proportion of NOD/SCID mice exhibiting significant human cell engraftment 6 weeks after transplantation,with a similar distribution of myeloid and lymphoid components. USSCs enhanced the homing of cellular products of ex vivo expanded UCB lineage-negative (lin(-)) cells,generated in 14-day cultures by Selective Amplification. The relative proportion of homing CD34(+) cells within the culture-expanded cell population was unaltered by USSC cotransplantation. Production of stromal-derived factor-1 (SDF-1) by USSCs was detected by both gene expression and protein released into culture media of these cells. Knockdown of SDF-1 production by USSCs using lentiviral-SiRNA led to a significant (p textless .05) reduction in USSC-mediated enhancement of CD34(+) homing. Our findings thus suggest a clinical potential for using USSCs in facilitating homing and engraftment for cord blood transplant recipients. View Publication

The developmental outcomes of preimplantation mammalian embryos are regulated directly by the surrounding microenvironment,and inappropriate concentrations of amino acids,or the loss of amino acid-sensing mechanisms,can be detrimental and impact further development. A specific role for l-proline in the differentiation of embryonic stem (ES) cells,a cell population derived from the blastocyst,has been shown in culture. l-proline acts as a signalling molecule,exerting its effects through cell uptake and subsequent metabolism. Uptake in ES cells occurs predominantly through the sodium-coupled neutral amino acid transporter 2,Slc38a2 (SNAT2). Dynamic expression of amino acid transporters has been shown in the early mammalian embryo,reflecting functional roles for amino acids in embryogenesis. The expression of SNAT2 and family member Slc38a1 (SNAT1) was determined in mouse embryos from the 2-cell stage through to the early post-implantation pre-gastrulation embryo. Key changes in expression were validated in cell culture models of development. Both transporters showed temporal dynamic expression patterns and changes in intracellular localisation as differentiation progressed. Changes in transporter expression likely reflect different amino acid requirements during development. Findings include the differential expression of SNAT1 in the inner and outer cells of the compacted morula and nuclear localisation of SNAT2 in the trophectoderm and placental lineages. Furthermore,SNAT2 expression was up-regulated in the epiblast prior to primitive ectoderm formation,an expression pattern consistent with a role for the transporter in later developmental decisions within the pluripotent lineage. We propose that the differential expression of SNAT2 in the epiblast provides evidence for an l-proline-mediated mechanism contributing to the regulation of embryonic development. View Publication

Patterning of bioactive enzymes with subcellular resolution is achieved by dispensing droplets of dextran (DEX) onto polyethylene glycol (PEG)-covered cells though a glass capillary needle connected to a pneumatic pump. This technique is applied to purify colonies of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblast (MEF) feeder cultures and inefficiently induced iPSC colonies by selectively dissociating the iPSCs with proteases. View Publication

The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results,NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2,LMX1A,TH,GIRK2,PITX3,EN1,NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue,with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation,the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future. View Publication