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

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development,we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm,then into replicating Nkx2.1+ lung endoderm,and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP,FGF,and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs),creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice. View Publication

BackgroundPathological deposition of hyperphosphorylated tau in the brain closely correlates with the course of Alzheimer’s disease (AD). Tau pathology occurs in axons of affected neurons and tau removal from axons might thus be an early intervention strategy.MethodsWe investigated the role of the RNA-binding protein hnRNP R in axonal localization and local translation of Mapt mRNA in neurons cultured from hnRNP R knockout mice. hnRNP R knockout mice were crossed with 5×FAD mice,an AD mouse model,and the effects of hnRNP R loss on the deposition of phospho-tau and amyloid-? plaques were evaluated. We designed antisense oligonucleotides (MAPT-ASOs) to block the binding of hnRNP R to Mapt mRNA. Cultured mouse and human neurons were treated with MAPT-ASOs and axonal Mapt mRNA and tau protein levels were quantified. MAPT-ASO was injected intracerebroventricularly into 5×FAD mice followed by quantification of phospho-tau aggregates and amyloid-? plaques in their brains. Protein changes in brains of 5×FAD mice treated with the MAPT-ASO were measured by mass spectrometry.ResultsMapt mRNA and tau protein were reduced in axons but not cell bodies of primary neurons cultured from hnRNP R knockout mice. Brains of 5×FAD mice deficient for hnRNP R contained less phospho-tau aggregates and amyloid-? plaques in the cortex and hippocampus. Treatment of neurons with MAPT-ASOs to block hnRNP R binding to Mapt similarly reduced axonal tau levels. Intracerebroventricular injection of a MAPT-ASO reduced the phospho-tau and plaque load and prevented neurodegeneration in the brains of 5×FAD mice,accompanied by rescue of proteome alterations.ConclusionLowering of tau selectively in axons thus represents an innovative therapeutic perspective for treatment of AD and other tauopathies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40035-025-00499-0. View Publication

Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane,because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation,these methods are not standardized,require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA,or EDTA plus mechanical scraping with an electric toothbrush,or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding,and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2,γ1-γ3 chains,α1/α2 and α6 type IV collagen chains,fibronectin,nidogen-2,and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells,immortalized corneal epithelial cells,and induced pluripotent stem cells. This simple,fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients. View Publication

Interleukin-6 (IL-6) is a critical factor in the regulation of stromal function and hematopoiesis. In vivo bromodeoxyuridine incorporation analysis indicates that the percentage of Lin(-)Sca-1(+) hematopoietic progenitors undergoing DNA synthesis is diminished in IL-6-deficient (IL-6(-/-)) bone marrow (BM) compared with wild-type BM. Reduced proliferation of IL-6(-/-) BM progenitors is also observed in IL-6(-/-) long-term BM cultures,which show defective hematopoietic support as measured by production of total cells,granulocyte macrophage-colony-forming units (CFU-GMs),and erythroid burst-forming units (BFU-Es). Seeding experiments of wild-type and IL-6(-/-) BM cells on irradiated wild-type or IL-6-deficient stroma indicate that the hematopoietic defect can be attributed to the stromal and not to the hematopoietic component. In IL-6(-/-) BM,stromal mesenchymal precursors,fibroblast CFUs (CFU-Fs),and stroma-initiating cells (SICs) are reduced to almost 50% of the wild-type BM value. Moreover,IL-6(-/-) stromata show increased CD34 and CD49e expression and reduced expression of the membrane antigens vascular cell adhesion molecule-1 (VCAM-1),Sca-1,CD49f,and Thy1. These data strongly suggest that IL-6 is an in vivo growth factor for mesenchymal precursors,which are in part implicated in the reduced longevity of the long-term repopulating stem cell compartment of IL-6(-/-) mice. View Publication

We investigated the involvement of the urokinase-type plasminogen-activator receptor (uPAR) in granulocyte-colony-stimulating factor (G-CSF)-induced mobilization of CD34+ hematopoietic stem cells (HSCs) from 16 healthy donors. Analysis of peripheral blood mononuclear cells (PBMNCs) showed an increased uPAR expression after G-CSF treatment in CD33+ myeloid and CD14+ monocytic cells,whereas mobilized CD34+ HSCs remained uPAR negative. G-CSF treatment also induced an increase in serum levels of soluble uPAR (suPAR). Cleaved forms of suPAR (c-suPAR) were released in vitro by PBMNCs and were also detected in the serum of G-CSF-treated donors. c-suPAR was able to chemoattract CD34+ KG1 leukemia cells and CD34+ HSCs,as documented by their in vitro migratory response to a chemotactic suPAR-derived peptide (uPAR84-95). uPAR84-95 induced CD34+ KG1 and CD34+ HSC migration by activating the high-affinity fMet-Leu-Phe (fMLP) receptor (FPR). In addition,uPAR84-95 inhibited CD34+ KG1 and CD34+ HSC in vitro migration toward the stromal-derived factor 1 (SDF1),thus suggesting the heterologous desensitization of its receptor,CXCR4. Finally,uPAR84-95 treatment significantly increased the output of clonogenic progenitors from long-term cultures of CD34+ HSCs. Our findings demonstrate that G-CSF-induced upregulation of uPAR on circulating CD33+ and CD14+ cells is associated with increased uPAR shedding,which leads to the appearance of serum c-suPAR. c-suPAR could contribute to the mobilization of HSCs by promoting their FPR-mediated migration and by inducing CXCR4 desensitization. View Publication

The termination of activation signals is a critical step in the control of the immune response; perturbation of inhibitory feedback pathways results in profound immune defects culminating in autoimmunity and overwhelming inflammation. FcgammaRIIB receptor is a well described inhibitory receptor. The ligation of B-cell receptor (BCR) and FcgammaRIIB leads to the inhibition of B-cell activation. Numerous studies have demonstrated that the SH2-domain-containing inositol 5-phosphatase SHIP (referred hereto as SHIP-1) is essential in this process. The cDNA encoding a second SH2-domain-containing inositol 5-phosphatase,SHIP-2,has been cloned [Pesesse,Deleu,De Smedt,Drayer and Erneux (1997) Biochem. Biophys. Res. Commun. 239,697-700]. Here we report the distribution of SHIP-2 in mouse tissues: a Western blot analysis of mouse tissues reveals that SHIP-2 is expressed in both haemopoietic and non-haemopoietic cells. In addition to T-cell and B-cell lines,spleen,thymus and lung are shown to coexpress SHIP-1 and SHIP-2. Moreover,SHIP-2 is detected in fibroblasts,heart and different brain areas. SHIP-2 shows a maximal tyrosine phosphorylation and association to Shc after ligation of BCR to FcgammaRIIB but not after stimulation of BCR alone. Our results therefore suggest a possible role for SHIP-2 in the negative regulation of immunocompetent cells. View Publication

Individually,transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) alter the growth and differentiation of normal and transformed osteoblast-like (OB) cells. Although recent evidence suggests interactions between TGF beta and 1,25(OH)2D3 may occur,little is known of the individual or combined effects of these hormones on the expression of the osteoblast phenotype at the cytochemical and biochemical levels in normal human OB (hOB) cells. Primary cultures of hOBs were treated with TGF beta (0.001-10 ng/ml) and 1,25(OH)2D3 (0.1 pM-100 nM) either alone or in combination. TGF beta and 1,25(OH)2D3 stimulated spindle-shaped cells to become stellate in appearance and increased the number of cytoplasmic processes. TGF beta increased 3H-thymidine incorporation and 1,25(OH)2D3 reduced this effect. Conversely,procollagen type-I synthesis and secretion were increased in a dose-dependent manner in the presence of TGF beta but were not significantly affected in the presence of 1,25(OH)2D3. TGF beta and 1,25(OH)2D3 each marginally increased alkaline phosphatase (ALP) activity,but the combination synergistically increased ALP activity in a dose- and time-dependent manner at the cytochemical and biochemical level (three to tenfold over vehicle controls; n = 12). In contrast,TGF beta reduced 1,25(OH)2D3-stimulated osteocalcin secretion. These data suggest that TGF beta stimulates hOB cells to actively produce collagen matrix and proliferate. The combination of TGF beta and 1,25(OH)2D3,however,produces a synergistic increase in ALP activity and maintenance of collagen synthesis. 1,25(OH)2D3 stimulation may induce cells to advance to an endstage where cell proliferation is reduced and osteocalcin expression is promoted. Interactions between TGF beta and 1,25(OH)2D3 may represent important steps in the regulation of osteoblast differentiation and matrix production. View Publication

The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds,UM171 being the prototype,is independent of suppression of the aryl hydrocarbon receptor,which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy. View Publication

During hematopoietic differentiation of human embryonic stem cells (hESCs),early hematopoietic progenitors arise along with endothelial cells within the CD34(+) population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays,their phenotype has not been defined. Here,using hESC differentiation in coculture with OP9 stromal cells,we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45,persisted on differentiating hematopoietic cells,and reliably separated the hematopoietic CD34(+) population from CD34(+)CD43(-)CD31(+)KDR(+) endothelial and CD34(+)CD43(-)CD31(-)KDR(-) mesenchymal cells. Furthermore,we demonstrated that the first-appearing CD34(+)CD43(+)CD235a(+)CD41a(+/-)CD45(-) cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34(+)CD43(+)CD41a(-)CD235a(-)CD45(-) cells. These cells were negative for lineage-specific markers (Lin(-)),expressed KDR,VE-cadherin,and CD105 endothelial proteins,and expressed GATA-2,GATA-3,RUNX1,C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34(+)CD43(+)CD45(-)Lin(-) cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34(+)CD43(+)CD45(+)Lin(-) cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3(high)GATA3(low)RUNX1(low)PU1(high)MPO(high)IL7RA(high) gene expression profile. View Publication

Hematopoietic stem cells (HSCs) reside in the bone marrow within niches that provide microenvironmental signals in the form of biophysical cues,bound and diffusible biomolecules,and heterotypic cell-cell interactions that influence HSC fate decisions. This study seeks to inform the development of a synthetic culture platform that promotes ex vivo HSC expansion without exhaustion. A library of methacrylamide-functionalized gelatin (GelMA) hydrogels is used to explore remodeling and crosstalk from mesenchymal stromal cells (MSCs) on the expansion and quiescence of murine HSCs. The use of a degradable GelMA hydrogel enables MSC-mediated remodeling,yielding dynamic shifts in the matrix environment over time. An initially low-diffusivity hydrogel for co-culture of hematopoietic stem and progenitor cells to MSCs facilitates maintenance of an early progenitor cell population over 7 days. Excitingly,this platform promotes retention of a quiescent HSC population compared to HSC monocultures. These studies reveal MSC-density-dependent upregulation of MMP-9 and changes in hydrogel mechanical properties ($\Delta$E = 2.61 ± 0.72) suggesting MSC-mediated matrix remodeling may contribute to a dynamic culture environment. Herein,a 3D hydrogel is reported for ex vivo HSC culture,in which HSC expansion and quiescence is sensitive to hydrogel properties,MSC co-culture,and MSC-mediated hydrogel remodeling. View Publication

The study of the regulatory signaling hierarchies of human heart development is limited by a lack of model systems that can reproduce the precise developmental events that occur during human embryogenesis. The advent of human pluripotent stem cell (hPSC) technology and robust cardiac differentiation methods affords a unique opportunity to monitor the full course of cardiac induction in vitro. Here,we show that stage-specific activation of insulin signaling strongly inhibited cardiac differentiation during a monolayer-based differentiation protocol that used transforming growth factor β superfamily ligands to generate cardiomyocytes. However,insulin did not repress cardiomyocyte differentiation in a defined protocol that used small molecule regulators of canonical Wnt signaling. By examining the context of insulin inhibition of cardiomyocyte differentiation,we determined that the inhibitory effects by insulin required Wnt/β-catenin signaling and that the cardiomyocyte differentiation defect resulting from insulin exposure was rescued by inhibition of Wnt/β-catenin during the cardiac mesoderm (Nkx2.5+) stage. Thus,insulin and Wnt/β-catenin signaling pathways,as a network,coordinate to influence hPSC differentiation to cardiomyocytes,with the Wnt/β-catenin pathway dominant to the insulin pathway. Our study contributes to the understanding of the regulatory hierarchies of human cardiomyocyte differentiation and has implications for modeling human heart development. View Publication