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

The prevalence of hearing loss after damage to the mammalian cochlea has been thought to be due to a lack of spontaneous regeneration of hair cells,the primary receptor cells for sound. Here,we show that supporting cells,which surround hair cells in the normal cochlear epithelium,differentiate into new hair cells in the neonatal mouse following ototoxic damage. Using lineage tracing,we show that new hair cells,predominantly outer hair cells,arise from Lgr5-expressing inner pillar and third Deiters cells and that new hair cell generation is increased by pharmacological inhibition of Notch. These data suggest that the neonatal mammalian cochlea has some capacity for hair cell regeneration following damage alone and that Lgr5-positive cells act as hair cell progenitors in the cochlea. View Publication

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin,glucagon,somatostatin,pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm,gut-tube endoderm,pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells,they release C-peptide in response to multiple secretory stimuli,but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy. View Publication

UNLABELLED The differentiation capacity of human embryonic stem cells (hESCs) holds great promise for therapeutic applications. We report a novel three-stage method to efficiently direct the differentiation of human embryonic stem cells into hepatic cells in serum-free medium. Human ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment. Next,the presence of fibroblast growth factor-4 and bone morphogenetic protein-2 in the culture medium for 5 days induced efficient hepatic differentiation from definitive endoderm cells. Approximately 70% of the cells expressed the hepatic marker albumin. After 10 days of further in vitro maturation,these cells expressed the adult liver cell markers tyrosine aminotransferase,tryptophan oxygenase 2,phosphoenolpyruvate carboxykinase (PEPCK),Cyp7A1,Cyp3A4 and Cyp2B6. Furthermore,these cells exhibited functions associated with mature hepatocytes including albumin secretion,glycogen storage,indocyanine green,and low-density lipoprotein uptake,and inducible cytochrome P450 activity. When transplanted into CCl4 injured severe combined immunodeficiency mice,these cells integrated into the mouse liver and expressed human alpha-1 antitrypsin for at least 2 months. In addition,we found that the hESC-derived hepatic cells were readily infected by human immunodeficiency virus-hepatitis C virus pseudotype viruses. CONCLUSION We have developed an efficient way to direct the differentiation of human embryonic stem cells into cells that exhibit characteristics of mature hepatocytes. Our studies should facilitate searching the molecular mechanisms underlying human liver development,and form the basis for hepatocyte transplantation and drug tests. View Publication

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia,haploid spermatocytes,or spermatids. Here,we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages,including postmeiotic,spermatid-like cells,in vitro without genetic manipulation. Furthermore,our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-,PLZF-,and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin,transition protein 1,and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro View Publication

Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed,we report that the cells survive and differentiate upon transplant into newborn rat brain. View Publication

Numerous studies have shown that somite development is a necessary stage of myogenesis chondrogenesis and osteogenesis. Our previous study has established a stable presomitic mesoderm progenitor cell line (UiPSM) in vitro. Naturally,we wanted to explore whether UiPSM cell can develop bone and myogenic differentiation. Selective culture conditions yielded PAX3 and PAX7 positive skeletal muscle precursors from UiPSM cells. The skeletal muscle precursors undergo in vitro maturation resulting in myotube formation. MYOD effectively promoted the maturity of the skeletal myocytes in a short time. We found that UiPSM and MYOD mediated UiPSM cell-derived skeletal myocytes were viable after transplantation into the tibialis anterior muscle of MITRG mice,as assessed by bioluminescence imaging and scRNA-seq. Lack of teratoma formation and evidence of long-term myocytes engraftment suggests considerable potential for future therapeutic applications. Moreover,UiPSM cells can differentiate into osteoblast and chondroblast cells in vitro. UiPSM differentiation has potential as a developmental model for musculoskeletal development research and treatment of musculoskeletal disorders. The online version contains supplementary material available at 10.1186/s13578-024-01274-w. View Publication

The groundbreaking discovery of induced pluripotent stem cells (iPS cells) provides a new source for cell therapy. However,whether the iPS derived functional lineages from different cell origins have different immunogenicity remains unknown. It had been known that the cells isolated from extra-embryonic tissues,such as umbilical cord mesenchymal cells (UMCs),are less immunogenic than other adult lineages such as skin fibroblasts (SFs). In this report,we differentiated iPS cells from human UMCs and SFs into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with allologous peripheral blood mononuclear cells (PBMCs),we showed that UMCs were indeed less immunogenic than skin cells to simulate proliferation of PBMCs. Surprisingly,we found that the NPCs differentiated from UMC-iPS cells retained low immunogenicity as the parental UMCs based on the PBMC proliferation assay. In cytotoxic expression assay,reactions in most kinds of immune effector cells showed more perforin and granzyme B expression with SF-NPCs stimulation than that with UMC-NPCs stimulation in PBMC co-culture system,in T cell co-culture system as well. Furthermore,through whole genome expression microarray analysis,we showed that over 70 immune genes,including all members of HLA-I,were expressed at lower levels in NPCs derived from UMC-iPS cells than that from SF-iPS cells. Our results demonstrated a phenomenon that the low immunogenicity of the less immunogenic cells could be retained after cell reprogramming and further differentiation,thus provide a new concept to generate functional lineages with lower immunogenicity for regenerative medicine. View Publication

Standardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast,induced pluripotent stem cells (iPSCs) assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs,which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology,immunophenotype,in vitro differentiation potential,and gene expression profiles as primary MSCs. However,iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm) profiles of iPSCs maintained donor-specific characteristics,whereas tissue-specific,senescence-associated,and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion,but they remained rejuvenated with regard to age-related DNAm. Overall,iPS-MSCs are similar to MSCs,but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns - particularly of DNAm patterns associated with tissue type and aging. View Publication

The present study describes and compares functional properties of Nuli-1 cells and primary human nasal epithelial cells (HNEC) including TLR expression and function. Differences in gene expression were identified for non-TLR genes that play a role in TLR response pathways. However,experiments comparing TLR gene expression for both Nuli-1 cells and HNECs indicated conserved expression in both cell types. Stimulation of the two cell types resulted in a conserved response to TLR3 agonists,but in differences in response to agonists for TLR5 and TLR6/2. HNECs were much more susceptible to infection with Staphylococcus aureus than NuLi-1 cells. Furthermore,when cultured at air-liquid interface (ALI),NuLi-1 cells possessed much lower trans-epithelial resistance than primary HNEC and did not exhibit maintenance of cell morphology or mucous production which was observed in HNECs. Nor did they produce the characteristic interconnecting pattern of tight junction complexes at the apicolateral margin of adjacent cells. Caution should therefore be exercised when selecting cell lines for immunological studies and a thorough screen of properties relevant to the study should always be carried out prior to commencement. View Publication

Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation,two hESCs lines were cultured on mixed feeder cells (MFCs,MEFs: HFFs = 1:1) and HFFs feeder,respectively,and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry,quantitative fluorescent real-time PCR,transmission and scanning electron microscopy,and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However,compared to hESCs line on MFCs feeder,hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2,PITX3,NURR1,and TH genes. In addition,the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion,HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons,but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore,feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines,but also electrophysiological properties of hESCs-derived DA neurons. View Publication

Differentiation approaches to obtain mesenchymal stem cells (MSCs) have gradually developed over the last few decades. The problem is that different protocols give different MSC types,making further research difficult. Here,we tried three different approaches to differentiate embryonic stem cells (ESCs) from early mesoderm to MSCs using serum-containing or xeno-free differentiation medium and observed differences in the cells’ morphology,doubling rate,ability to form colonies,surface marker analysis,and multilineage differentiation potential of the obtained cell lines. We concluded that the xeno-free medium best fits the criteria of MSCs’ morphology,growth kinetics,and surface marker characterization. In contrast,the serum-containing medium gives better potential for further MSC differentiation into osteogenic,chondrogenic,and adipogenic lineages. View Publication

SUMMARY Alveolar epithelial type I cells (AT1s) line the gas exchange barrier of the distal lung and have been historically challenging to isolate or maintain in cell culture. Here,we engineer a human in vitro AT1 model system via directed differentiation of induced pluripotent stem cells (iPSCs). We use primary adult AT1 global transcriptomes to suggest benchmarks and pathways,such as Hippo-LATS-YAP/TAZ signaling,enriched in these cells. Next,we generate iPSC-derived alveolar epithelial type II cells (AT2s) and find that nuclear YAP signaling is sufficient to promote a broad transcriptomic shift from AT2 to AT1 gene programs. The resulting cells express a molecular,morphologic,and functional phenotype reminiscent of human AT1 cells,including the capacity to form a flat epithelial barrier producing characteristic extracellular matrix molecules and secreted ligands. Our results provide an in vitro model of human alveolar epithelial differentiation and a potential source of human AT1s. In brief Kotton and colleagues generate human alveolar epithelial type I cells (AT1s) from induced pluripotent stem cells (iPSCs). The resulting cells can be grown as 3D organoids or in 2D air-liquid interface cultures,displaying many of the molecular,morphologic,and functional phenotypes of primary AT1s. Graphical abstract View Publication