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

Apoptosis and proliferation are two dynamically and tightly regulated processes that together maintain the homeostasis of renewable tissues. Anoikis is a subtype of apoptosis induced by detachment of adherent cells from the extracellular matrix. By using the defined mTeSR1 medium and collecting freshly detached cells,we found here that human pluripotent stem (PS) cells including embryonic stem (ES) cells and induced pluripotent stem cells are subject to constant anoikis in culture,which is escalated in the absence of basic fibroblast growth factor (bFGF). Withdrawal of bFGF also promotes apoptosis and differentiation of the remaining adherent cells without affecting their cell cycle progression. Insulin-like growth factor 2 (IGF2) has previously been reported to act downstream of FGF signaling to support self-renewal of human ES cells. However,we found that IGF2 cannot substitute bFGF in the TeSR1-supported culture,although endogenous IGF signaling is required to sustain self-renewal of human ES cells. On the other hand,all of the bFGF withdrawal effects observed here can be markedly prevented by the caspase inhibitor z-VAD-FMK. We further demonstrated that the bFGF-repressed anoikis is dependent on activation of ERK and AKT and associated with inhibition of Bcl-2-interacting mediator of cell death and the caspase-ROCK1-myosin signaling. Anoikis is independent of pre-detachment apoptosis and differentiation of the cells. Because previous studies of human PS cells have been focused on attached cells,our findings revealed a neglected role of bFGF in sustaining self-renewal of human PS cells: preventing them from anoikis via inhibition of caspase activation. View Publication

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast,we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34,lineage markers,CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells),endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo,CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials,characterized by the expression of c-Kit and CXCR4 as well as EphB4,EphB2,and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life. View Publication

Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader implicated in the pathogenesis of a number of different cancers and other diseases. Brd4-null mouse embryos die shortly after implantation and are compromised in their ability to maintain the inner cell mass,which gives rise to embryonic stem cells (ESCs). Here we report that BRD4 regulates expression of the pluripotency factor Nanog in mouse ESCs and preimplantation embryos,as well as in human ESCs and embryonic cancer stem cells. Inhibition of BRD4 function using a chemical inhibitor,small interfering RNAs,or a dominant-negative approach suppresses Nanog expression,and abolishes the self-renewal ability of ESCs. We also find that BRD4 associates with BRG1 (brahma-related gene 1,aka Smarca4 (SWI/SNF-related,matrix-associated,actin-dependent regulator of chromatin,subfamily a,member 4)),a key regulator of ESC self-renewal and pluripotency,in the Nanog regulatory regions to regulate Nanog expression. Our study identifies Nanog as a novel BRD4 target gene,providing new insights for the biological function of BRD4 in stem cells and mouse embryos. Knowledge gained from these non-cancerous systems will facilitate future investigations of how Brd4 dysfunction leads to cancers.Cell Death and Differentiation advance online publication,22 August 2014; doi:10.1038/cdd.2014.124. View Publication

Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible,defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex or Matrigel. In addition we have validated an ascorbate free media capable of supporting CD30(low) populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines,MEL1,MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up. View Publication

Four commercially available serum-free and defined culture media tested on 2 human embryonic stem cell (hESC) lines were all found to support undifferentiated growth for textgreater10 continuous passages. For hESC cultured with defined StemPro and mTeSR1 media,the cells were maintained feeder-free on culture dishes coated with extracellular matrices (ECMs) with no requirement of feeder-conditioned media (CM). For xeno-free serum replacer (XSR),HEScGRO,and KnockOut media,mitotically inactivated human foreskin feeders (hFFs) were required for hESC growth. Under the different media conditions,cells continued to exhibit alkaline phosphatase activity and expressed undifferentiated hESC markers Oct-4,stage-specific embryonic antigens 4 (SSEA-4),and Tra-1-60. In addition,hESC maintained the expression of podocalyxin-like protein-1 (PODXL),an antigen recently reported in another study to be present in undifferentiated hESC. The cytotoxic antibody mAb 84 binds via PODXL expressed on hESC surface and kills textgreater90% of hESC within 45 min of incubation. When these cells were spontaneously differentiated to form embryoid bodies,derivatives representing the 3 germ layers were obtained. Injection of hESC into animal models resulted in teratomas and the formation of tissue types indicative of ectodermal,endodermal,and mesodermal lineages were observed. Our data also suggested that StemPro and mTeSR1 media were more optimal for hESC proliferation compared to cells grown on CM because the growth rate of hESC increased by 30%-40%,higher split ratio was thus required for weekly passaging. This is advantageous for the large-scale cultivation of hESC required in clinical applications. View Publication

The corneal epithelium is maintained by stem cells located at the periphery of the cornea in a region known as the limbus. Depletion of limbal stem cells (LSCs) results in limbal stem cell deficiency. Treatments for this disease are based on limbal replacement or transplantation of ex vivo expanded LSCs. It is,therefore,crucial to identify cell surface markers for LSCs that can be used for their enrichment and characterization. Aldehyde dehydrogenases (ALDHs) are enzymes which protect cells from the toxic effects of peroxidic aldehydes. In this manuscript,we show for the first time that ALDH1 is absent from the basal cells of the limbal and corneal epithelium. We separated limbal epithelial cells on the basis of ALDH activity and showed that ALDH(dim) cells expressed significantly higher levels of DeltaNp63 and ABCG2 as well as having a greater colony forming efficiency (CFE) when compared to ALDH(bright) cells. Large scale transcriptional analysis of these two populations led to identification of a new cell surface marker,RHAMM/HMMR,which is located in all layers of corneal epithelium and in the suprabasal layers of the limbal epithelium but is completely absent from the basal layer of the limbus. Our studies indicate that absence of RHAMM/HMMR expression is correlated with properties associated with LSCs. RHAMM/HMMR- limbal epithelial cells are smaller in size,express negligible CK3,have higher levels of DeltaNp63 and have a higher CFE compared to RHAMM/HMMR+ cells. Taken together these results suggest a putative role for RHAMM/ HMMR as a negative marker of stem cell containing limbal epithelial cells. Cell selection based on Hoechst exclusion and lack of cell surface RHAMM/HMMR expression resulted in increased colony forming efficiency compared to negative selection using RHAMM/HMMR alone or positive selection using Hoechst on its own. Combination of these two cell selection methods presents a novel method for LSC enrichment and characterization. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

SHP2,a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene,plays a critical role in developmental hematopoiesis in the mouse,and gain-of-function mutations of SHP2 are associated with hematopoietic malignancies. However,the role of SHP2 in adult hematopoiesis has not been addressed in previous studies. In addition,the role of SHP2 in human hematopoiesis has not been described. These questions are of considerable importance given the interest in development of SHP2 inhibitors for cancer treatment. We used shRNA-mediated inhibition of SHP2 expression to investigate the function of SHP2 in growth factor (GF) signaling in normal human CD34(+) cells. SHP2 knockdown resulted in markedly reduced proliferation and survival of cells cultured with GF,and reduced colony-forming cell growth. Cells expressing gain-of-function SHP2 mutations demonstrated increased dependency on SHP2 expression for survival compared with cells expressing wild-type SHP2. SHP2 knockdown was associated with significantly reduced myeloid and erythroid differentiation with retention of CD34(+) progenitors with enhanced proliferative capacity. Inhibition of SHP2 expression initially enhanced and later inhibited STAT5 phosphorylation and reduced expression of the antiapoptotic genes MCL1 and BCLXL. These results indicate an important role for SHP2 in STAT5 activation and GF-mediated proliferation,survival,and differentiation of human progenitor cells. View Publication

BACKGROUND: Hair bulge progenitor cells (HBPCs) are multipotent stem cells derived from the bulge region of mice vibrissal hairs. The purified HBPCs express CD34,K15 and K14 surface markers. It has been reported that HBPCs could be readily induced to transdifferentiate into adipocytes and osteocytes. However,the ability of HBPCs to transdifferentiate into cardiomyocytes has not yet been investigated. METHODOLOGY/PRINCIPAL FINDINGS: The cardiomyogenic potential of HBPCs was investigated using a small cell-permeable molecule called Cardiogenol C. We established that Cardiogenol C could induce HBPCs to express transcription factors GATA4,Nkx2.5 and Tbx5,which are early specific markers for pre-cardiomyogenic cells. In prolonged cultures,the Cardiogenol C-treated HBPCs can also express muscle proteins,cardiac-specific troponin I and sarcomeric myosin heavy chain. However,we did not observe the ability of these cells to functionally contract. Hence,we called these cells cardiomyocyte-like cells rather than cardiomyocytes. We tried to remedy this deficiency by pre-treating HBPCs with Valproic acid first before exposing them to Cardiogenol C. This pretreatment inhibited,rather than improved,the effectiveness of Cardiogenol C in reprogramming the HBPCs. We used comparative proteomics to determine how Cardiogenol C worked by identifying proteins that were differentially expressed. We identified proteins that were involved in promoting cell differentiation,cardiomyocyte development and for the normal function of striated muscles. From those differentially expressed proteins,we further propose that Cardiogenol C might exert its effect by activating the Wnt signaling pathway through the suppression of Kremen1. In addition,by up-regulating the expression of chromatin remodeling proteins,SIK1 and Smarce1 would initiate cardiac differentiation. CONCLUSIONS/SIGNIFICANCE: In conclusion,our CD34+/K15+ HBPCs could be induced to transdifferentiate into cardiomyocyte-like cells using a small molecule called Cardiogenol C. The process involves activation of the Wnt signaling pathway and altered expression of several key chromatin remodeling proteins. The finding is clinically significant as HBPCs offer a readily accessible and autologous source of progenitor cells for cell-based therapy of heart disease,which is one of major killers in developed countries. View Publication

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro,as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study,we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal,neonatal,and adult fibroblasts through reprogramming with POU5F1,SOX2,NANOG,and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC,H1,H7,H9,H13,and H14). Similar to hESCs,all iPSCs generated CD34(+)CD43(+) hematopoietic progenitors and CD31(+)CD43(-) endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors,iPSC-derived primitive blood cells formed all types of hematopoietic colonies,including GEMM colony-forming cells. Human induced pluripotent cells (hiPSCs)-derived CD43(+) cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43(+)CD235a(+)CD41a(+/-) (erythro-megakaryopoietic),lin(-)CD34(+)CD43(+)CD45(-) (multipotent),and lin(-)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells. Although we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs,the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal,neonatal,or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes,patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks. View Publication

A large number of biologic,technological,and clinical studies await the development of procedures that will allow totipotent hematopoietic stem cells to be expanded in vitro. Previous work has suggested that hematopoiesis can be reconstituted using transplants of cells from long-term marrow cultures. We have used retrovirus mediated gene transfer to demonstrate that marked totipotent hematopoietic stem cells are both maintained and can be amplified in such cultures,and then subsequently regenerate and sustain lympho-myeloid hematopoiesis in irradiated recipients. Marrow cells from 5-fluorouracil-treated male mice were infected with a recombinant virus carrying the neomycin resistence gene and seeded onto irradiated adherent layers of pre-established,long-term marrow cultures of female origin. At 4 weeks,cells from individual cultures were transplanted into single or multiple female recipients. Southern blot analysis of hematopoietic tissue 45 days posttransplantation showed retrovirally marked clones common to lymphoid and myeloid tissues in 14 of 23 mice examined. Strikingly,for 3 of 4 long-term cultures,multiple recipients of cells from a single flask showed marrow and thymus repopulation with the same unique retrovirally marked clone. These results establish the feasibility of retroviral-marking techniques to demonstrate the maintenance of totipotent lympho-myeloid stem cells for at least 4 weeks in the long-term marrow culture system and provide the first evidence of their proliferation in vitro. Therefore,such cultures may serve as a starting point for identifying factors that stimulate totipotent hematopoietic stem cell expansion. View Publication

The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs),we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly,dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus,modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases. View Publication

Injury induces the recruitment of bone marrow-derived cells (BMDCs) that contribute to the repair and regeneration process. The behavior of BMDCs in injured tissue has a profound effect on repair,but the regulation of BMDC behavior is poorly understood. Aberrant recruitment/retention of these cells in wounds of diabetic patients and animal models is associated with chronic inflammation and impaired healing. BMD Gr-1(+)CD11b(+) cells function as immune suppressor cells and contribute significantly to tumor-induced neovascularization. Here we report that Gr-1(+)CD11b(+) cells also contribute to injury-induced neovascularization,but show altered recruitment/retention kinetics in the diabetic environment. Moreover,diabetic-derived Gr-1(+)CD11b(+) cells fail to stimulate neovascularization in vivo and have aberrant proliferative,chemotaxis,adhesion,and differentiation potential. Previously we demonstrated that gene transfer of HOXA3 to wounds of diabetic mice is taken up by and expressed by recruited BMDCs. This is associated with a suppressed inflammatory response,enhanced neovascularization,and accelerated wound healing. Here we show that sustained expression of Hoxa3 in diabetic-derived BMD Gr-1(+)CD11b(+) cells reverses their diabetic phenotype. These findings demonstrate that manipulation of adult stem/progenitor cells ex vivo could be used as a potential therapy in patients with impaired wound healing. View Publication