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

Recombinant human GM-CSF has been expressed as a fusion protein in E. coli in the form of inclusion bodies. Using denaturing agents,acid cleavage and sulfitolysis,the biologically inactive GM-CSF protein could be highly purified and additionally renaturated under suitable reoxidizing conditions. The thorough repair of the two disulfide bridges could be confirmed by sequencing fragments obtained by tryptic digestion. Refolding of the molecule has been studied by CD spectrometry and identity by Western blotting and SDS-PAGE analysis. As could be demonstrated,full biological activity (colony-forming assay with fresh human bone marrow cells) was restored during renaturation of the GM-CSF protein. Further proof of biological equivalence of the E. coli-derived protein with a yeast-derived biologically active rh GM-CSF has been published elsewhere. View Publication

Neural stem cells (NSCs) have the ability to self-renew and to differentiate into various cell types of the central nervous system. This potential can be recapitulated by human induced pluripotent stem cells (hiPSCs) in vitro. The differentiation capacity of hiPSCs is characterized by several stages with distinct morphologies and the expression of various marker molecules. We used the monoclonal antibodies (mAbs) 487(LeX),5750(LeX) and 473HD to analyze the expression pattern of particular carbohydrate motifs as potential markers at six differentiation stages of hiPSCs. Mouse ESCs were used as a comparison. At the pluripotent stage,487(LeX)-,5750(LeX)- and 473HD-related glycans were differently expressed. Later,cells of the three germ layers in embryoid bodies (hEBs) and,even after neuralization of hEBs,subpopulations of cells were labeled with these surface antibodies. At the human rosette-stage of NSCs (hR-NSC),LeX- and 473HD-related epitopes showed antibody-specific expression patterns. We also found evidence that these surface antibodies could be used to distinguish the hR-NSCs from the hSR-NSCs stages. Characterization of hNSCs(FGF-2/EGF) derived from hSR-NSCs revealed that both LeX antibodies and the 473HD antibody labeled subpopulations of hNSCs(FGF-2/EGF). Finally,we identified potential LeX carrier molecules that were spatiotemporally regulated in early and late stages of differentiation. Our study provides new insights into the regulation of glycoconjugates during early human stem cell development. The mAbs 487(LeX),5750(LeX) and 473HD are promising tools for identifying distinct stages during neural differentiation. View Publication

In guiding hES cell technology toward the clinic,one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days). Basic fibroblast growth factor (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So,we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture,including the developmental potential to differentiate into representative tissues of all three embryonic germ layers,unlimited and undifferentiated proliferative ability,and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF,insulin-like growth factor 2 (IGF-2),and transforming growth factor β (TGF-β),thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together,bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically. View Publication

BACKGROUND AIMS: We evaluated hematopoietic stem cells according to CD34 expression and aldehyde dehydrogenase (ALDH) activity in peripheral blood and apheresis product samples from patients after mobilization with granulocyte-colony-stimulating factor (G-CSF) alone or G-CSF after high-dose cyclophosphamide (4 g/m²) once daily,intravenously on day 1). We also investigated the relationship between the number of SSC(lo) CD45(dim) CD34(hi) cells,SSC(lo) ALDH(br) cells and engraftment. METHODS: Thirty patients (20 males and 10 females),who were candidates for autologous peripheral blood stem cell transplantation,were included in the study. Cyclophosphamide + G-CSF was used for 17 and G-CSF alone for 24 mobilizations. Primary diagnoses were multiple myeloma (n = 14),Hodgkin's lymphoma (n = 7),non-Hodgkin's lymphoma (n = 2),acute myloid leukemia (n = 2),chronic lymphocytic leukemia (n = 1) and germ cell testis tumor (n = 1). RESULTS: Numbers of SSC(lo) CD45(dim) CD34(hi) cells and SSC(lo) ALDH(br) cells were highly correlated in both peripheral blood and apheresis products (P textless 0.001). We could not find a relationship between the transplanted SSC(lo) CD45(dim) CD34(hi) cell dose or SSC(lo) ALDH(br) cell dose and platelet or neutrophil recovery. The optimal thresholds for SSC(lo) CD45(dim) CD34(hi) cells were 5.40 × 10�?�/kg for neutrophil recovery and 7.22 x 10�?�/kg for platelet recovery. The optimal thresholds for SSC(lo) ALDH(br) cells were 6.53 x 10�?�/kg for neutrophil recovery and 8.72 x 10�?�/kg platelet recovery. CONCLUSIONS: According to our data,numbers of SSC(lo) ALDH(br) cells are in very good agreement with numbers of SSC(lo) CD45(dim) CD34(hi) cells and can be a predictor of stem cell mobilization. View Publication

Acute myeloid leukemia (AML) is a malignant disorder derived from neoplastic myeloid progenitor cells characterized by abnormal proliferation and differentiation. Although novel therapeutics have recently been introduced,AML remains a therapeutic challenge with insufficient cure rates. In the last years,immune-directed therapies such as chimeric antigen receptor (CAR)-T cells were introduced,which showed outstanding clinical activity against B-cell malignancies including acute lymphoblastic leukemia (ALL). However,the application of CAR-T cells appears to be challenging due to the enormous molecular heterogeneity of the disease and potential long-term suppression of hematopoiesis. Here we report on the generation of CD33-targeted CAR-modified natural killer (NK) cells by transduction of blood-derived primary NK cells using baboon envelope pseudotyped lentiviral vectors (BaEV-LVs). Transduced cells displayed stable CAR-expression,unimpeded proliferation,and increased cytotoxic activity against CD33-positive OCI-AML2 and primary AML cells in vitro. Furthermore,CD33-CAR-NK cells strongly reduced leukemic burden and prevented bone marrow engraftment of leukemic cells in OCI-AML2 xenograft mouse models without observable side effects. View Publication

The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture,they display low permissivity to the vector,requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation,we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays,we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer,allowing the reach of very high levels of vector integration in their progeny in vivo. Thus,LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly,cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors,highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy. View Publication

While distinct stages of natural killer (NK) cell development have been defined,the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which,through contact-dependent mechanisms,promote the generation of mature,functional human NK cells from CD34(+) precursors. We show that developing NK cells undergo unique,developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells,and that the relative motility of NK cells increases as they move through development in vitro and ex vivo. These interactions include the formation of a synapse between developing NK cells and stromal cells,which we term the developmental synapse. Finally,we identify a role for CD56 in developmental synapse structure,NK cell motility and NK cell development. Thus,we define the developmental synapse leading to human NK cell functional maturation. View Publication

Although adipose tissue is an expandable and readily attainable source of proliferating,multipotent stem cells,its potential for use in regenerative medicine has not been extensively explored. Here we report that adult human and mouse adipose-derived stem cells can be reprogrammed to induced pluripotent stem (iPS) cells with substantially higher efficiencies than those reported for human and mouse fibroblasts. Unexpectedly,both human and mouse iPS cells can be obtained in feeder-free conditions. We discovered that adipose-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF,TGFbeta,fibronectin,and vitronectin and can serve as feeders for both autologous and heterologous pluripotent cells. These results demonstrate a great potential for adipose-derived cells in regenerative therapeutics and as a model for studying the molecular mechanisms of feeder-free iPS generation and maintenance. View Publication

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes,mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture,hES cells expressing cell-surface NGFR protein (CD271,p75NTR) were isolated by immunoaffinity adsorption,and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression,examined by quantitative RT-PCR,found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR,SNAI1,NTRK3,SOX9,and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer,mRNAs typifying adult stromal stem cells were detected,including BMI1,KIT,NES,NOTCH1,and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1,B3GNT7,PTDGS,and ALDH3A1 were upregulated. mRNA for keratocan (KERA),a cornea-specific proteoglycan,was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate,a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells,therefore,may provide a renewable source of material for development of treatment of corneal stromal opacities. View Publication

Although aldehyde dehydrogenase (ALDH) activity has become a surrogate of hematopoietic stem and progenitor cells (HSPCs),its function during hematopoiesis was unclear. Here,we examined its role in zebrafish hematopoiesis based on pharmacological inhibition and morpholino (MO) knockdown. Zebrafish embryos were treated with diethylaminobenzaldehyde (DEAB,1 μmol/l) between 0- and 48 hour-post-fertilization (hpf). MOs targeting aldhs were injected between 1 and 4-cell stage. The effects on hematopoiesis were evaluated at different stages. DEAB treatment between 0 and 18 hpf increased gene expression associated with HSPC (scl,lmo2),erythropoiesis (gata1,α- and β-eHb) and myelopoiesis (spi1) as well as gfp(+) cells in dissociated Tg(gata1:gfp) embryos. The effects were ameliorated by all-trans retinoic acid (1 nmol/l). Definitive hematopoiesis and the erythromyeloid precursors were unaffected. In all,14 out of 15 zebrafish aldhs were detectable by reverse transcription PCR in 18 hpf embryos,of which only aldh1a2 and aldh16a1 were expressed in sites pertinent to hematopoiesis. Molecular targeting by MOs was demonstrated for 15 aldhs,but none of them,even in combined aldh1a2 and aldh1a3 knockdown,recapitulated the hematopoietic expansion in DEAB-treated embryos. In conclusion,DEAB expands HSPC population during primitive hematopoiesis through inhibition of aldh and retinoic acid synthesis. The specific aldh isoform(s) remains to be determined. View Publication

p18 INK4 C (CDKN2C,encoded by p18 INK4c or Cdkn2c ) is an early G1-phase cyclin-dependent kinase inhibitor protein. Previous studies demonstrated enhanced self-renewal capacity of hematopoietic stem cells (HSCs) in p18 −/− mice compared to wild-type (WT) mice. Given the critical role of bone marrow niche cells-particularly mesenchymal stromal cells (MSCs)-in hematopoiesis,this study investigated the functional alterations of p18 −/− MSCs and their impact on hematopoietic support. Bone marrow derived MSCs were isolated from p18 −/− and WT mice. Their proliferation and differentiation capacities were assessed,followed by evaluation of hematopoietic support using cobblestone area-forming cell assay and long-term culture-initiating cell assay. RNA sequencing was performed to analyze the transcriptional profile of p18 −/− MSCs,with a focus on differentially expressed genes (DEGs). Key pathways associated with hematopoietic support were identified using Ingenuity Pathway Analysis. A candidate protein was quantified by ELISA,and its functional role in hematopoietic support was validated via a modified coculture system. p18 −/− MSCs displayed an increased proliferation rate,preferential differentiation toward osteogenesis over adipogenesis,and enhanced hematopoietic support. RNA sequencing analysis identified 137 DEGs,with secreted phosphoprotein 1 ( Spp1,encoding osteopontin,Opn) being significantly upregulated in p18 −/− MSCs. Elevated Opn levels were confirmed in both bone marrow and MSC-conditioned media from p18 −/− mice. Functional validation further demonstrated that Opn enhanced the hematopoietic supportive capacity of MSCs in vitro. p18 deficiency promotes osteogenic differentiation and enhances the hematopoietic supportive function of MSCs,likely mediated by Opn upregulation. These findings suggest a potential therapeutic strategy for improving bone regeneration and HSC expansion. The online version contains supplementary material available at 10.1186/s13287-025-04402-6. View Publication

Administration of bone marrow-derived mesenchymal stem cells (MSCs) is an innovative approach for the treatment of a range of diseases that are not curable by current therapies including heart failure. A number of clinical trials have been completed and many others are ongoing; more than 2,000 patients worldwide have been administered with culture-expanded allogeneic or autologous MSCs for the treatment of various diseases,showing feasibility and safety (and some efficacy) of this approach. However,protocols for isolation and expansion of donor MSCs vary widely between these trials,which could affect the efficacy of the therapy. It is therefore important to develop international standards of MSC production,which should be evidence-based,regulatory authority-compliant,of good medical practice grade,cost-effective,and clinically practical,so that this innovative approach becomes an established widely adopted treatment. This review article summarizes protocols to isolate and expand bone marrow-derived MSCs in 47 recent clinical trials of MSC-based therapy,which were published after 2007 onwards and provided sufficient methodological information. Identified issues and possible solutions associated with the MSC production methods,including materials and protocols for isolation and expansion,are discussed with reference to relevant experimental evidence with aim of future clinical success of MSC-based therapy. View Publication