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

BackgroundPhenotypically unstable Schwann cell-like cells (SCLCs),derived from mesenchymal stem cells (MSCs) require intercellular contact-mediated cues for Schwann cell (SCs)-fate commitment. Although rat dorsal root ganglion (DRG) neurons provide contact-mediated signals for the conversion of SCLCs into fate-committed SCs,the use of animal cells is clinically unacceptable. To overcome this problem,we previously acquired human induced pluripotent stem cell-derived sensory neurons (hiPSC-dSNs) as surrogates of rat DRG neurons that committed rat bone marrow SCLCs to the SC fate. In this study,we explored whether hiPSC-dSNs could mimic rat DRG neuron effects to obtain fate-committed SCs from hBMSC-derived SCLCs.MethodshiPSCs were induced into hiPSC-dSNs using a specific chemical small molecule combination. hBMSCs were induced into hBMSC-derived SCLCs in a specific culture medium and then co-cultured with hiPSC-dSNs to generate SCs. The identity of hBMSC-derived SCs (hBMSC-dSCs) was examined by immunofluorescence,western bolt,electronic microscopy,and RNA-seq. Immunofluorescence was also used to detect the myelination capacity. Enzyme-linked immunosorbent assay and neurite outgrowth analysis were used to test the secretion of neurotrophic factors.ResultsThe hBMSC-dSCs exhibited bi-/tri-polar morphology of SCs and maintained the expression of the SC markers S100,p75NTR,p0,GFAP,and Sox10,even after withdrawing the glia-inducing factors or hiPSC-dSNs. Electronic microscopy and RNA-seq analysis provided evidence that hBMSC-dSCs were similar to the original human SCs in terms of their function and a variety of characteristics. Furthermore,these cells formed MBP-positive segments and secreted neurotrophic factors to facilitate the neurite outgrowth of Neuro2A.ConclusionsThese results demonstrated that phenotypically stable and functionally mature hBMSC-dSCs were generated efficiently via the co-culture of hiPSC-dSNs and hBMSC-derived SCLCs. Our findings may provide a promising protocol through which stable and fully developed hBMSC-dSCs can be used for transplantation to regenerate myelin sheath.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04217-5. View Publication

OVERVIEW: We show the existence of adult human mesenchymal progenitor cells (hMPCs) that can proliferate,in a cytokine-dependent manner,as individual cells in stirred suspension cultures (SSC) while maintaining their ability to form functional differentiated mesenchymal cell types. MATERIALS AND METHODS: Ficolled human bone marrow (BM)-derived cells were grown in SSC (and adherent controls) in the presence and absence of exogenously added cytokines. Phenotypic,gene expression,and functional assays for hematopoietic and nonhematopoietic cell populations were used to kinetically track cell production. Limiting-dilution analysis was used to relate culture-produced cells to input cell populations. RESULTS: Cytokine cocktail influenced total and progenitor cell expansion,as well as the types of cells generated upon plating. Flow cytometric analysis of CD117,CD123,and CD45 expression showed that cytokine supplementation influenced SSC output. The concomitant growth of CD45(+) and CD45(-) cells in the cultures that exhibited the greatest hMPC expansions suggests that the growth of these cells may benefit from interactions with hematopoietic cells. Functional assays demonstrated that the SSC-derived cells (input CFU-O number: 1990+/-377) grown in the presence of SCF+IL-3 resulted,after 21 days,in the generation of a significantly greater number (ptextless0.05) of bone progenitors (33,700+/-8763 CFU-O) than similarly initiated adherent cultures (214+/-75 CFU-O). RT-PCR analysis confirmed that the SSC-derived cells grown in osteogenic conditions express bone-specific genes (Cbfa1/Runx2,bone sialoprotein,and osteocalcin). CONCLUSIONS: Our approach not only provides an alternative strategy to expand adult BM-derived nonhematopoietic progenitor cell numbers in a scalable and controllable bioprocess,but also questions established biological paradigms concerning the properties of connective-tissue stem and progenitor cells. View Publication

HIV-1 Tat-interacting protein of 110 kDa [Tip110; p110(nrb)/SART3/p110] is an RNA binding nuclear protein implicated in regulation of HIV-1 gene and host gene transcription,pre-mRNA splicing,and cancer immunology. Recently,we demonstrated a role for Tip110 in regulation of hematopoiesis. Here,we show that TIP110 is also expressed in human embryonic stem cells (hESCs) and expression was decreased with differentiation of these ESCs. TIP110 was found,through up- and down-modulation of expression of Tip110,to be important in maintaining pluripotent factor (NANOG,OCT4,and SOX2) expression in and pluripotency of hESCs,although the mechanisms involved and whether the Tip110 effects are direct remain to be determined. View Publication

PURPOSE:
Human embryonic stem cells (hESC) hold a great potential for regenerative medicine because,in principle,they can differentiate into any cell type found in the human body. In addition,studying the effect of ionizing radiation (IR) on hESC may provide valuable information about the response of human cells to IR exposure in their most naive state,as well as the consequences of IR exposure on the development of organisms. However,the effect of IR,in particular radionuclide uptake,on the pluripotency,proliferation and survival of hESC has not been extensively studied.
METHODS:
In this study we treated cultured hESC with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU),a precursor of DNA synthesis. Then we measured the expansion of colonies and expression of pluripotency markers in hESC.
RESULTS:
We found that uptake of (125)IdU was similar in both hESC and HT1080 human fibrosarcoma cells. However,treatment with 0.1 μCi/ml (125)IdU for 24 hours resulted in complete death of the hESC population; whereas HT1080 cancer cells continued to grow. Treatment with a 10-fold lower dose (125)IdU (0.01 μCi/ml) resulted in colonies of hESC becoming less defined with numerous cells growing in monolayer outside of the colonies showing signs of differentiation. Then we analyzed the expression of pluripotency markers (octamer-binding transcription factor 4 [Oct-4] and stage-specific embryonic antigen-4 [SSEA4]) in the surviving hESC. We found that hESC in the surviving colonies expressed pluripotency markers at levels comparable with those in the non-treated controls.
CONCLUSIONS:
Our results provide important initial insights into the sensitivity of hESC to IR,and especially that produced by the decay of an internalized radionuclide. View Publication

Cellular reprogramming from adult somatic cells into an embryonic cell-like state,termed induced pluripotency,has been achieved in several cell types. However,the ability to reprogram human amniotic epithelial cells (hAECs),an abundant cell source derived from discarded placental tissue,has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs),but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore,AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation,including NEUROD1 and SOX17,markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs,we analyzed global DNA methylation,global histone acetylation,and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts,hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise,quantitative gene expression analyses show that hAECs endogenously express OCT4,SOX2,KLF4,and c-MYC,all four factors used in cellular reprogramming. Thus,hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents. View Publication

Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types,including neurons. However,differentiation of hPSCs with extrinsic factors is a slow,step-wise process,mimicking the protracted timing of human development. Using a small-molecule screen,we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at textgreater75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors,including tetrodotoxin (TTX)-resistant,SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development,suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain. View Publication

Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value,thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity,only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2,PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb,heart and embryonic development related transcription factors and biological processes. Moreover,this study uncovered novel possible mechanisms,such as the inhibition of RANBP1,that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1,GSTA2),that protect the cell from secondary oxidative stress. As a proof of principle,we demonstrated that a combination of transcriptomics and proteomics,along with consistent differentiation of hESCs,enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide. View Publication

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However,dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here,we show that SMAD7,a cell-intrinsic inhibitor of transforming growth factor-β (TGFβ) signaling,is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time course gene expression revealed downregulation of MAPK components,and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. Fibroblast growth factor-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues,pluripotent cells simply revert to a program of neural conversion. Hence,the primed" state of hESCs requires inhibition of the "default" state of neural fate acquisition. This has parallels in amphibians� View Publication

Human embryonic stem cells (hESCs) have an abbreviated G1 phase of the cell cycle that allows rapid proliferation and maintenance of pluripotency. Lengthening of G1 corresponds to loss of pluripotency during differentiation. However,precise mechanisms that link alterations in the cell cycle and early differentiation remain to be defined. We investigated initial stages of mesendodermal lineage commitment in hESCs,and observed a cell cycle pause. Transcriptome profiling identified several genes with known roles in regulation of the G2/M transition that were differentially expressed early during lineage commitment. WEE1 kinase,which blocks entry into mitosis by phosphorylating CDK1 at Y15,was the most highly expressed of these genes. Inhibition of CDK1 phosphorylation by a specific inhibitor of WEE1 restored cell cycle progression by preventing the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal,but not ectodermal,lineages. Functionally,WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. Our findings identify a novel G2 cell cycle pause that is required for endodermal differentiation and provide important new mechanistic insights into early events of lineage commitment. Stem Cells 2016;34:1765-1775. View Publication

It has become increasingly clear that proper cellular control of pluripotency and differentiation is related to the regulation of rRNA synthesis. To further our understanding of the role that the regulation of rRNA synthesis has in pluripotency we monitored rRNA synthesis during the directed differentiation of human embryonic stem cells (hESCs). We discovered that the rRNA synthesis rate is reduced ˜50% within 6 hours of ACTIVIN A treatment. This precedes reductions in expression of specific stem cell markers and increases in expression of specific germ layer markers. The reduction in rRNA synthesis is concomitant with dissociation of the Pol I transcription factor,UBTF,from the rRNA gene promoter and precedes any increase to heterochromatin throughout the rRNA gene. To directly investigate the role of rRNA synthesis in pluripotency,hESCs were treated with the Pol I inhibitor,CX-5461. The direct reduction of rRNA synthesis by CX-5461 induces the expression of markers for all three germ layers,reduces the expression of pluripotency markers,and is overall similar to the ACTIVIN A induced changes. This work indicates that the dissociation of UBTF from the rRNA gene,and corresponding reduction in transcription,represent early regulatory events during the directed differentiation of pluripotent stem cells. View Publication

SHIP is an important regulator of immune cell signaling that functions to dephosphorylate the phosphoinositide phosphatidylinositol 3,4,5-trisphosphate at the plasma membrane and mediate protein-protein interactions. One established paradigm for SHIP activation involves its recruitment to the phospho-ITIM motif of the inhibitory receptor FcγRIIB. Although SHIP is essential for the inhibitory function of FcγRIIB,it also has critical modulating functions in signaling initiated from activating immunoreceptors such as B cell Ag receptor. In this study,we found that SHIP is indistinguishably recruited to the plasma membrane after BCR stimulation with or without FcγRIIB coligation in human cell lines and primary cells. Interestingly,fluorescence recovery after photobleaching analysis reveals differential mobility of SHIP-enhanced GFP depending on the mode of stimulation,suggesting that although BCR and FcγRIIB can both recruit SHIP,this occurs via distinct molecular complexes. Mutagenesis of a SHIP-enhanced GFP fusion protein reveals that the SHIP-Src homology 2 domain is essential in both cases whereas the C terminus is required for recruitment via BCR stimulation,but is less important with FcγRIIB coligation. Experiments with pharmacological inhibitors reveal that Syk activity is required for optimal stimulation-induced membrane localization of SHIP,whereas neither PI3K or Src kinase activity is essential. BCR-induced association of SHIP with binding partner Shc1 is dependent on Syk,as is tyrosine phosphorylation of both partners. Our results indicate that FcγRIIB is not uniquely able to promote membrane recruitment of SHIP,but rather modulates its function via formation of distinct signaling complexes. Membrane recruitment of SHIP via Syk-dependent mechanisms may be an important factor modulating immunoreceptor signaling. View Publication