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

Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study,we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4,hSox2,hKlf4,and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci,a hallmark of DM1,were detected in DM1 iPSCs,neural stem cells (NSCs),and terminally differentiated neurons and astrocytes. In conclusion,we have successfully established disease-specific human DM1 iPSC lines,NSCs,and neuronal lineages with pathognomonic intranuclear RNA foci,which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development. View Publication

Regulatory mechanisms governing adhesion of hematopoietic progenitor cells to the stromal nische are poorly understood. Growth factors such as stem cell factor (SCF),granulocyte-macrophage colony-stimulating factor,and thrombopoietin were reported to upregulate the adhesion of hematopoietic progenitors to immobilized fibronectin through activation of integrin alpha4beta1 and alpha5beta1. Macrophage inflammatory protein (MIP)-1alpha is a C-C chemokine that suppresses colony formation by stem/progenitor cells in vitro. We asked if MIP-1alpha would modulate the adhesive phenotype of colony-forming cells (CFCs) obtained from healthy donor bone marrow (BM),cord blood (CB),and mobilized peripheral blood (mPB) CD34+ cells,in comparison with SCF,using immobilized fibronectin. SCF significantly increased the level of adhesion of CFCs from BM,CB,and mPB. On the other hand,MIP-1alpha significantly increased the level of adhesion of CFCs from BM and CB,but less so from mPB. The effects of MIP-1alpha were inhibited by blocking antibodies to integrin alpha4,alpha5,or beta1,and polymerization plus rearrangement of F-actin were observed in affected cells by labeling with rhodamine-conjugated phalloidine. These data indicate that the effect of MIP-1alpha on the adhesive phenotype of CFCs is mediated by modulation of the organization of integrin. The amount of MIP-1alpha receptor on mPB was less than for BM or CB,which may explain the distinct characteristics in the adhesive response induced by MIP-1alpha. We suggest that hematopoietic progenitor cells from different sources may be heterogeneous with respect to maturation,integrin affinity,MIP-1alpha receptor expression,and regulation of MIP-1alpha signaling. Our data indicate that MIP-1alpha may affect migration,homing,and mobilization of hematopoietic progenitors by modulating the adhesive phenotype of these cells. View Publication

The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology--in particular,in the identification of induced pluripotent stem (iPS) cells during the reprogramming process. Based on the selective expression of stem cell surface markers,a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts,allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES) cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells,but that specific genes,including positive and negative selection markers,regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies. View Publication

The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen,we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure,while cell surface marker analyses revealed a VE-cadherin+CD31+CD34+KDR+CD43???putative endothelial progenitor population. Furthermore,molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL+multi-cellular modules and a VEGFR3+sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation. View Publication

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here,through the sequential in vitro targeting of selected signaling pathways,we have developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex,as an extracellular matrix,could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP,SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL,and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells,1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally,ES-DBCs were responsive to high glucose in static incubation and perifusion studies,and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion,targeting selected signaling pathways for 25-30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs,small molecules or genes that may have potential to influence beta-cell function. View Publication

Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining,leading to reading frame disruption. The approach is applicable to dominant negative disorders,which can be treated simply by knocking out the mutant allele,while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB),which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation,c.80688084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed,respectively,into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting,90% of the iPSCs were edited,and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition,we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders. View Publication

Various systems for differentiating hematopoietic cells from human pluripotent stem cells (PSCs) have been developed,although none have been fully optimized. In this report,we describe the development of a novel three-dimensional system for differentiating hematopoietic cells from PSCs using collagen sponges (CSs) reinforced with poly(ethylene terephthalate) fibers as a scaffold. PSCs seeded onto CSs were differentiated in a stepwise manner with appropriate cytokines under serum-free and feeder-free conditions. This process yielded several lineages of floating hematopoietic cells repeatedly for more than 1 month. On immunohistochemical staining,we detected CD34+ cells and CD45+ cells in the surface and cavities of the CS. Taking advantage of the portability of this system,we were able to culture multiple CSs together floating in medium,making it possible to harvest large numbers of hematopoietic cells repeatedly. Given these findings,we suggest that this novel three-dimensional culture system may be useful in the large-scale culture of PSC-derived hematopoietic cells. View Publication

iPSC-derived cells (from induced pluripotent stem cells) are a useful source that provide a powerful and widely accepted tool for the study of various types of human cells in vitro. Indeed,iPSC-derived cells from patients with hereditary diseases have been shown to reproduce the hallmarks of these diseases in vitro,phenotypes that can then also be manipulated in vitro. Quantitative reverse transcription PCR (qRT-PCR) is often used to characterize the progress of iPSC differentiation,validate mature cell types and to determine levels of pathological markers. Quantitative reverse transcription PCR (qRT-PCR) is used to quantify mRNA levels. This method requires some way of normalizing the data,typically by relating the obtained levels of gene expression to the levels of expression of a house keeping gene"� View Publication

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels,which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2-8 weeks,the gels contain bilayered mammary epithelial structures,including luminal and myoepithelial cells,their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations. View Publication

The mechanisms ensuring the long-term self-renewal of human embryonic stem cells are still only partly understood,limiting their use in cellular therapies. Here we found that increased activity of the RB cell cycle inhibitor in human embryonic stem cells induces cell cycle arrest,differentiation and cell death. Conversely,inactivation of the entire RB family (RB,p107 and p130) in human embryonic stem cells triggers G2/M arrest and cell death through functional activation of the p53 pathway and the cell cycle inhibitor p21. Differences in E2F target gene activation upon loss of RB family function between human embryonic stem cells,mouse embryonic stem cells and human fibroblasts underscore key differences in the cell cycle regulatory networks of human embryonic stem cells. Finally,loss of RB family function promotes genomic instability in both human and mouse embryonic stem cells,uncoupling cell cycle defects from chromosomal instability. These experiments indicate that a homeostatic level of RB activity is essential for the self-renewal and the survival of human embryonic stem cells. View Publication

In this study,we focused on two biological products as ideal tools for toxicological assessment: long non-coding RNAs (lncRNAs) and human-induced pluripotent stem cells (hiPSCs). lncRNAs are an important class of pervasive non-protein-coding transcripts involved in the molecular mechanisms associated with responses to cellular stresses. hiPSCs possess the capabilities of self-renewal and differentiation into multiple cell types,and they are free of the ethical issues associated with human embryonic stem cells. Here,we identified six novel lncRNAs (CDKN2B-AS1,MIR22HG,GABPB1-AS1,FLJ33630,LINC00152,and LINC0541471v2) that respond to model chemical stresses (cycloheximide,hydrogen peroxide,cadmium,or arsenic) in hiPSCs. Our results indicated that the lncRNAs responded to general and specific chemical stresses. Compared with typical mRNAs such as p53-related mRNAs,the lncRNAs highly and rapidly responded to chemical stresses. We propose that these lncRNAs have the potential to be surrogate indicators of chemical stress responses in hiPSCs. View Publication

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin,+ROCKi/-spin,-ROCKi/+spin,and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions,including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation,elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment,and low-cost scalability,which will directly support automated,large-scale production of hEBs and hESC-derived cells needed for clinical,research,or therapeutic applications. View Publication