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

In this study,a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension,only a few aggregated cells were observed. However,after 3 days,culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry,immunocytochemistry and quantitative RT-PCR,and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium,expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore,the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A,BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes,including HCN4,MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes,including pacemakers. Moreover,when cardiac cell sheets were fabricated using differentiated cardiomyocytes,they beat spontaneously and synchronously,indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering. View Publication

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC,these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further,the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial,ventricular (V),and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2,H7,and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4,Rho kinase inhibitor,activin-A,and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16,up to 95% of cells were cTnT(+). Of which,93%,94%,100%,92%,and 92% of cardiac derivatives from HES2,H7,H9,and two iPSC lines,respectively,were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to $\$-adrenergic stimulation. Our simple,cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol,the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors. View Publication

New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4(+) T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here,we adapted this methodology to a high-throughput platform for the efficient,arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner,whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously,enabling studies of interactions among multiple host and viral factors. Finally,in an arrayed screen of 45 genes associated with HIV integrase,we identified several candidate dependency/restriction factors,demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection. View Publication

Recent studies have shown that mesenchymal stem cells (MSC) with the potential for cell-mediated therapies and tissue engineering applications can be isolated from extracted dental tissues. Here,we investigated the collection,processing,and cryobiological characteristics of MSC from human teeth processed under current good tissue practices (cGTP). Viable dental pulp-derived MSC (DPSC) cultures were isolated from 31 of 40 teeth examined. Of eight DPSC cultures examined more thoroughly,all expressed appropriate cell surface markers and underwent osteogenic,adipogenic,and chondrogenic differentiation in appropriate differentiation medium,thus meeting criteria to be called MSC. Viable DPSC were obtained up to 120 h postextraction. Efficient recovery of DPSC from cryopreserved intact teeth and second-passage DPSC cultures was achieved. These studies indicate that DPSC isolation is feasible for at least 5 days after tooth extraction,and imply that processing immediately after extraction may not be required for successful banking of DPSC. Further,the recovery of viable DPSC after cryopreservation of intact teeth suggests that minimal processing may be needed for the banking of samples with no immediate plans for expansion and use. These initial studies will facilitate the development of future cGTP protocols for the clinical banking of MSC. View Publication

BACKGROUND: Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However,the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product,both of which can limit the future clinical application of hESC-ECs. Moreover,to fully understand the beneficial effects of stem cell therapy,investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time. METHODOLOGY: In this study,we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray,and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover,our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods. CONCLUSION: Taken together,we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes,form functional vessels in vivo,and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future. View Publication

Mesenchymal stem cells (MSCs) are highly useful in a variety of cell therapies owing to their multipotential differentiation capability. MSCs derived from umbilical cord blood are generally isolated by their plastic adherence without using specific cell surface markers and examined for their osteogenic,adipogenic,and chondrogenic differentiation properties retrospectively. Here,we report 2 subpopulations of MSCs,separated based on aldehyde dehydrogenase (ALDH) activity. MSCs with a high ALDH activity (Alde-High) proliferated more than those with a low ALDH activity (Alde-Low). Alde-High MSCs had a greater ability to differentiate than Alde-Low MSCs in in vitro culture. Transplantation of Alde-High MSCs into fractured mouse femurs enabled early repair of tissues and rapid bone substitution. Alde-High MSCs were also more responsive to hypoxia than Alde-Low MSCs,with the upregulation of Flt-1,CXCR4,and Angiopoietin-2. Thus,MSCs with a high ALDH activity might serve as an effective therapeutic tool for healing fractures within a short period of time. View Publication

Microfluidic image cytometry (MIC) has been developed to study phenotypes of various hPSC lines by screening several chemically defined serum/feeder-free conditions. A chemically defined hPSC culture was established using 20 ng mL(-1) of bFGF on 20 microg mL(-1) of Matrigel to grow hPSCs over a week in an undifferentiated state. Following hPSC culture,we conducted quantitative MIC to perform a single cell profiling of simultaneously detected protein expression (OCT4 and SSEA1). Using clustering analysis,we were able to systematically compare the characteristics of various hPSC lines in different conditions. View Publication

Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro,but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal,we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray,and tested their ability to perform mature hepatocyte functions (albumin and urea secretion,cytochrome activity). By these measures,ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. View Publication

Introduction Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells/tissues and even mini-brains that are physiologically relevant to model neurological diseases. However,the capacity of signaling factors that regulate 3-D neural tissue patterning in vitro and differential responses of the resulting neural populations to various biomolecules have not yet been fully understood. Methods By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog (SHH) signaling,this study generated different 3-D neuronal cultures that were mainly comprised of either cortical glutamatergic neurons or motor neurons. Results Abundant glutamatergic neurons were observed following the treatment with an antagonist of SHH signaling,cyclopamine,while Islet-1 and HB9-expressing motor neurons were enriched by an SHH agonist,purmorphamine. In neurons derived with different neural patterning factors,whole-cell patch clamp recordings showed similar voltage-gated Na+/K+ currents,depolarization-evoked action potentials and spontaneous excitatory post-synaptic currents. Moreover,these different neuronal populations exhibited differential responses to three classes of biomolecules,including (1) matrix metalloproteinase inhibitors that affect extracellular matrix remodeling; (2) N-methyl-D-aspartate that induces general neurotoxicity; and (3) amyloid ?? (1???42) oligomers that cause neuronal subtype-specific neurotoxicity. Conclusions This study should advance our understanding of hiPSC self-organization and neural tissue development and provide a transformative approach to establish 3-D models for neurological disease modeling and drug discovery. Statement of Significance Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells,tissues and even mini-brains that are physiologically relevant to model neurological diseases. However,the capability of sonic hedgehog-related small molecules to tune different neuronal subtypes in 3-D differentiation from hiPSCs and the differential cellular responses of region-specific neuronal subtypes to various biomolecules have not been fully investigated. By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog signaling,this study provides knowledge on the differential susceptibility of region-specific neuronal subtypes derived from hiPSCs to different biomolecules in extracellular matrix remodeling and neurotoxicity. The findings are significant for understanding 3-D neural patterning of hiPSCs for the applications in brain organoid formation,neurological disease modeling,and drug discovery. View Publication

Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study,after determining the minimum inhibitory level of lactic acid for hiPSCs,a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically,about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture,a final cell density of (1.1 ± 0.1) × 10(6) cells mL(-1) was obtained,with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression,on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method,culture medium refinement by dialysis was established to remove toxic metabolites,recycle autocrine factors as well as other growth factors,and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture. View Publication

The past two decades have witnessed significant strides in leukemia therapies through approval of therapeutic inhibitors targeting oncogene-driving dysregulated tyrosine kinase activities and key epigenetic and apoptosis regulators. Although these drugs have brought about complete remission in the majority of patients,many patients face relapse or have refractory disease. The main factor contributing to relapse is the presence of a small subpopulation of dormant drug-resistant leukemia cells that possess stem cell features (termed as leukemia stem cells or LSCs). Thus,overcoming drug resistance and targeting LSCs remain major challenges for curative treatment of human leukemia. Chronic myeloid leukemia (CML) is a good example,with rare,propagating LSCs and drug-resistant cells that cannot be eradicated by BCR-ABL-directed tyrosine kinase inhibitor (TKI) monotherapy and that are responsible for disease relapse/progression. Therefore,it is imperative to identify key players in regulating BCR-ABL1-dependent and independent drug-resistance mechanisms,and their key pathways,so that CML LSCs can be selectively targeted or sensitized to TKIs. Here,we describe several easily adaptable gene knockdown approaches in CD34+ CML stem/progenitor cells that can be used to investigate the biological properties of LSCs and molecular effects of genes of interest (GOI),which can be further explored as therapeutic modalities against LSCs in the context of human leukemia. View Publication

GATA-2 is an essential transcription factor in the hematopoietic system that is expressed in hematopoietic stem cells (HSCs) and progenitors. Complete deficiency of GATA-2 in the mouse leads to severe anemia and embryonic lethality. The role of GATA-2 and dosage effects of this transcription factor in HSC development within the embryo and adult are largely unexplored. Here we examined the effects of GATA-2 gene dosage on the generation and expansion of HSCs in several hematopoietic sites throughout mouse development. We show that a haploid dose of GATA-2 severely reduces production and expansion of HSCs specifically in the aorta-gonad-mesonephros region (which autonomously generates the first HSCs),whereas quantitative reduction of HSCs is minimal or unchanged in yolk sac,fetal liver,and adult bone marrow. However,HSCs in all these ontogenically distinct anatomical sites are qualitatively defective in serial or competitive transplantation assays. Also,cytotoxic drug-induced regeneration studies show a clear GATA-2 dose-related proliferation defect in adult bone marrow. Thus,GATA-2 plays at least two functionally distinct roles during ontogeny of HSCs: the production and expansion of HSCs in the aorta-gonad-mesonephros and the proliferation of HSCs in the adult bone marrow. View Publication