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

Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells,including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses,such as Japanese encephalitis virus (JEV),provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition,glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast,only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition,functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover,we discover that vimentin intermediate filament,reported as a putative neurovirulent JEV receptor,is highly expressed in NPCs and glial cells,but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally,we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection. View Publication

The application of stem-cell-based therapies in regenerative medicine is hindered by the tumorigenic potential of residual human pluripotent stem cells. Previously,we identified a human pluripotent stem-cell-specific lectin probe,called rBC2LCN,by comprehensive glycome analysis using high-density lectin microarrays. Here we developed a recombinant lectin-toxin fusion protein of rBC2LCN with a catalytic domain of Pseudomonas aeruginosa exotoxin A,termed rBC2LCN-PE23,which could be expressed as a soluble form from the cytoplasm of Escherichia coli and purified to homogeneity by one-step affinity chromatography. rBC2LCN-PE23 bound to human pluripotent stem cells,followed by its internalization,allowing intracellular delivery of a cargo of cytotoxic protein. The addition of rBC2LCN-PE23 to the culture medium was sufficient to completely eliminate human pluripotent stem cells. Thus,rBC2LCN-PE23 has the potential to contribute to the safety of stem-cell-based therapies. View Publication

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report,we employ electron,immunofluorescence microscopy,and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 $$g/$$g proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 $$g/$$g proteins) reported in human cancer cell lines. Moreover,we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states,similar to those naïve-like hPSCs,with increased glycogen synthesis. Furthermore,we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus,our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions. View Publication

The ability to derive and stably maintain ground-state human pluripotent stem cells (hPSCs) that resemble the cells seen in vivo in the inner cell mass has the potential to be an invaluable tool for researchers developing stem cell-based therapies. To date,derivation of human naive-like pluripotent stem cell lines has been limited to a small number of lineages,and their long-term culturing remains problematic. We describe a protocol for genetic and phenotypic tagging,selecting and maintaining naive-like hPSCs. We tag hPSCs by GFP,expressed by the long terminal repeat (LTR7) of HERVH endogenous retrovirus. This simple and efficient protocol has been reproduced with multiple hPSC lines,including embryonic and induced pluripotent stem cells,and it takes ∼6 weeks. By using the reporter,homogeneous hPSC cultures can be derived,characterized and maintained for the long term by repeated re-sorting and re-plating steps. The HERVH-expressing cells have a similar,but nonidentical,expression pattern to other naive-like cells,suggesting that alternative pluripotent states might exist. View Publication

Adipose tissue is an abundantly available source of proliferative and multipotent mesenchymal stem cells with promising potential for regenerative therapeutics. We previously demonstrated that both human and mouse adipose-derived stem cells (ASCs) can be reprogrammed into induced pluripotent stem cells (iPSCs) with efficiencies higher than those that have been reported for other cell types. The ASC-derived iPSCs can be generated in a feeder-independent manner,representing a unique model to study reprogramming and an important step toward establishing a safe,clinical grade of cells for therapeutic use. In this study,we provide a detailed protocol for isolation,preparation and transformation of ASCs from fat tissue into mouse iPSCs in feeder-free conditions and human iPSCs using feeder-dependent or feeder/xenobiotic-free processes. This protocol also describes how ASCs can be used as feeder cells for maintenance of other pluripotent stem cells. ASC derivation is rapid and can be completed in textless1 week,with mouse and human iPS reprogramming times averaging 1.5 and 2.5 weeks,respectively. View Publication

Our ability to guide differentiation of human pluripotent stem cells (hPSCs) toward desired lineages efficiently and reproducibly in xeno-free conditions is the key to advancing hPSC technology from the laboratory to clinical use. Here we report an engineered biomimetic substrate functionalized with both peptide ligands for α5β1 and α6β1 integrins to support efficient early mesodermal differentiation of human embryonic stem cells (hESCs) when cultured in a differentiation medium containing BMP4. In contrast,mesodermal differentiation is not induced on substrates functionalized with either ligand alone even though the culture medium is identical. Mesodermal differentiation was characterized by immunofluorescent staining,flow cytometric analysis,and RT-PCR analysis of early mesodermal markers Brachyury,Mixl1,and Wnt3. The early mesodermal progenitors derived on the substrate functionalized with both integrin ligands have the normal developmental potential to further differentiate along the hemato-endothelial and cardiac lineages. Immobilized ligands for α5β1 and α6β1 integrins both are permissive,necessary,and sufficient insoluble ligands in this engineered system to support early mesodermal differentiation of hESCs. This synthetic substrate,in conjunction with defined soluble factors,constructs a well-controlled and xeno-free early mesodermal differentiation niche that offers advantages over the previously reported niche constructed with the Matrigel-coated substrate. View Publication

Naturally occurring regulatory T cells (Tregs) maintain self tolerance by dominant suppression of potentially self-reactive T cells in peripheral tissues. However,the activation requirements,the temporal aspects of the suppressive activity,and mode of action of human Tregs are subjects of controversy. In this study,we show that Tregs display significant variability in the suppressive activity ex vivo as 54% of healthy blood donors examined had fully suppressive Tregs spontaneously,whereas in the remaining donors,anti-CD3/CD2/CD28 stimulation was required for Treg suppressive activity. Furthermore,anti-CD3/CD2/CD28 stimulation for 6 h and subsequent fixation in paraformaldehyde rendered the Tregs fully suppressive in all donors. The fixation-resistant suppressive activity of Tregs operated in a contact-dependent manner that was not dependent on APCs,but could be fully obliterated by trypsin treatment,indicating that a cell surface protein is directly involved. By add-back of active,fixed Tregs at different time points after activation of responding T cells,the responder cells were susceptible to Treg-mediated immune suppression up to 24 h after stimulation. This defines a time window in which effector T cells are susceptible to Treg-mediated immune suppression. Lastly,we examined the effect of a set of signaling inhibitors that perturb effector T cell activation and found that none of the examined inhibitors affected Treg activation,indicating pathway redundancy or that Treg activation proceeds by signaling mechanisms distinct from those of effector T cells. View Publication

Encapsulation of donor islets using a hydrogel material is a well-studied strategy for islet transplantation,which protects donor islets from the host immune response. Replacement of donor islets by human embryonic stem cell (hESC) derived islets will also require a means of immune-isolating hESCs by encapsulation. However,a critical consideration of hESC differentiation is the effect of surrounding biophysical environment,in this case capsule biophysical properties,on differentiation. The objective of this study,thus,was to evaluate the effect of capsule properties on growth,viability,and differentiation of encapsulated hESCs throughout pancreatic induction. It was observed that even in the presence of soluble chemical cues for pancreatic induction,substrate properties can significantly modulate pancreatic differentiation,hence necessitating careful tuning of capsule properties. Capsules in the range of 4-7. kPa supported cell growth and viability,whereas capsules of higher stiffness suppressed cell growth. While an increase in capsule stiffness enhanced differentiation at the intermediate definitive endoderm (DE) stage,increased stiffness strongly suppressed pancreatic progenitor (PP) induction. Signaling pathway analysis indicated an increase in pSMAD/pAKT levels with substrate stiffness likely the cause of enhancement of DE differentiation. In contrast,sonic hedgehog inhibition was more efficient under softer gel conditions,which is necessary for successful PP differentiation. Statement of Significance: Cell replacement therapy for type 1 diabetes (T1D),affecting millions of people worldwide,requires the immunoisolation of insulin-producing islets by encapsulation with a semi-impermeable material. Due to the shortage of donor islets,human pluripotent stem cell (hPSC) derived islets are an attractive alternative. However,properties of the encapsulating substrate are known to influence hPSC cell fate. In this work,we determine the effect of substrate stiffness on growth and pancreatic fate of encapsulated hPSCs. We precisely identify the range of substrate properties conducive for pancreatic cell fate,and also the mechanism by which substrate properties modify the cell signaling pathways and hence cell fate. Such information will be critical in driving regenerative cell therapy for long term treatment of T1D. View Publication

Pericytes (PCs) are endothelium-associated cells that play an important role in normal vascular function and maintenance. We developed a method comparable to GMP quality protocols for deriving self-renewing perivascular progenitors from the human embryonic stem cell (hESC),line ESI-017. We identified a highly scalable,perivascular progenitor cell line that we termed PC-A,which expressed surface markers common to mesenchymal stromal cells. PC-A cells were not osteogenic or adipogenic under standard differentiation conditions and showed minimal angiogenic support function in vitro. PC-A cells were capable of further differentiation to perivascular progenitors with limited differentiation capacity,having osteogenic potential (PC-O) or angiogenic support function (PC-M),while lacking adipogenic potential. Importantly,PC-M cells expressed surface markers associated with pericytes. Moreover,PC-M cells had pericyte-like functionality being capable of co-localizing with human umbilical vein endothelial cells (HUVECs) and enhancing tube stability up to 6 days in vitro. We have thus identified a self-renewing perivascular progenitor cell line that lacks osteogenic,adipogenic and angiogenic potential but is capable of differentiation toward progenitor cell lines with either osteogenic potential or pericyte-like angiogenic function. The hESC-derived perivascular progenitors described here have potential applications in vascular research,drug development and cell therapy. View Publication

Organotin compounds,such as tributyltin (TBT),are well-known endocrine disruptors. TBT is also known to cause various forms of cytotoxicity,including neurotoxicity and immunotoxicity. However,TBT toxicity has not been identified in normal stem cells. In the present study,we examined the effects of TBT on cell growth in human induced pluripotent stem cells (iPSCs). We found that exposure to nanomolar concentrations of TBT decreased intracellular ATP levels and inhibited cell viability in iPSCs. Because TBT suppressed energy production,which is a critical function of the mitochondria,we further assessed the effects of TBT on mitochondrial dynamics. Staining with MitoTracker revealed that nanomolar concentrations of TBT induced mitochondrial fragmentation. TBT also reduced the expression of mitochondrial fusion protein mitofusin 1 (Mfn1),and this effect was abolished by knockdown of the E3 ubiquitin ligase membrane-associated RING-CH 5 (MARCH5),suggesting that nanomolar concentrations of TBT could induce mitochondrial dysfunction via MARCH5-mediated Mfn1 degradation in iPSCs. Thus,mitochondrial function in normal stem cells could be used to assess cytotoxicity associated with metal exposure. View Publication

The ability to generate spiral ganglion neurons (SGNs) from stem cells is a necessary prerequisite for development of cell-replacement therapies for sensorineural hearing loss. We present a protocol that directs human embryonic stem cells (hESCs) toward a purified population of otic neuronal progenitors (ONPs) and SGN-like cells. Between 82% and 95% of these cells express SGN molecular markers,they preferentially extend neurites to the cochlear nucleus rather than nonauditory nuclei,and they generate action potentials. The protocol follows an in vitro stepwise recapitulation of developmental events inherent to normal differentiation of hESCs into SGNs,resulting in efficient sequential generation of nonneuronal ectoderm,preplacodal ectoderm,early prosensory ONPs,late ONPs,and cells with cellular and molecular characteristics of human SGNs. We thus describe the sequential signaling pathways that generate the early and later lineage species in the human SGN lineage,thereby better describing key developmental processes. The results indicate that our protocol generates cells that closely replicate the phenotypic characteristics of human SGNs,advancing the process of guiding hESCs to states serving inner-ear cell-replacement therapies and possible next-generation hybrid auditory prostheses. textcopyright Stem Cells Translational Medicine 2017;6:923-936. View Publication

Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind,we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation,of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin,deguelin,patulin and phenethyl caffeate) exhibited high cytotoxicity,with half maximal inhibitory concentrations (IC50) in the low nanomolar range. In particular,the FDA approved drug for the treatment of alcoholism,disulfiram (DSF),was significantly potent across multiple patient samples (IC50 of 31.1 nM). The activity of DSF was potentiated by copper (Cu),which markedly increased GSC death. DSF-Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs,consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier,we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM. View Publication