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

Human mesenchymal stromal cells (hMSCs) have generated significant interest due to their potential use in clinical applications. hMSCs are present at low frequency in vivo,but after isolation can be expanded considerably,generating clinically useful numbers of cells. In this study,we demonstrate the use of a defined embryonic stem cell expansion medium,mTeSR (Stem Cell Technologies),for the expansion of bone-marrow-derived hMSCs. The hMSCs grow at comparable rates,demonstrate tri-lineage differentiation potential,and show similar surface marker profiles (CD29(+),CD44(+),CD49a(+),CD73(+),CD90(+),CD105(+),CD146(+),CD166(+),CD34(-),and CD45(-)) in both the fetal bovine serum (FBS)-supplemented medium and mTeSR. However,expression of early differentiation transcription factors runt-related transcription factor 2,sex-determining region Y box 9,and peroxisome proliferator-activated receptor gamma changed significantly. Both runt-related transcription factor 2 and sex-determining region Y box 9 were upregulated,whereas peroxisome proliferator-activated receptor gamma was downregulated in mTeSR compared with FBS. Although osteogenic and chondrogenic differentiation was comparable in cells grown in mTeSR compared to FBS,adipogenic differentiation was significantly decreased in mTeSR-expanded cells,both in terms of gene expression and absolute numbers of adipocytes. The removal of the FBS from the medium and the provision of a defined medium with disclosed composition make mTeSR a superior study platform for hMSC biology in a controlled environment. Further,this provides a key step toward generating a clinical-grade medium for expansion of hMSCs for clinical applications that rely on osteo- and chondroinduction of MSCs,such as bone repair and cartilage generation. View Publication

High-titer,HIV-1-based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34(+) cells and clonogenic progenitors very poorly (textless 1%),reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier,we developed a simian immunodeficiency virus (SIV)-based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% +/- 1% of rhesus bulk CD34(+) cells and 75% +/- 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector-mediated stem cell gene transfer in vivo,3 rhesus macaques underwent transplantation with transduced,autologous cytokine-mobilized peripheral blood CD34(+) cells following myeloablative conditioning. Hematopoietic reconstitution was rapid,and an average of 18% +/- 8% and 15% +/- 7% GFP-positive granulocytes and monocytes,respectively,were observed 4 to 6 months after transplantation,consistent with the average vector copy number of 0.19 +/- 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models. View Publication

Cardiovascular progenitor cells (CPCs) expressing the ISL1-LIM-homeodomain transcription factor contribute developmentally to cardiomyocytes in all 4 chambers of the heart. Here,we show that ISL1-CPCs can be applied to myocardial regeneration following injury. We used a rapid 3D methylcellulose approach to form murine and human ISL1-CPC spheroids that engrafted after myocardial infarction in murine hearts,where they differentiated into cardiomyocytes and endothelial cells,integrating into the myocardium and forming new blood vessels. ISL1-CPC spheroid-treated mice exhibited reduced infarct area and increased blood vessel formation compared with control animals. Moreover,left ventricular (LV) contractile function was significantly better in mice transplanted with ISL1-CPCs 4 weeks after injury than that in control animals. These results provide proof-of-concept of a cardiac repair strategy employing ISL1-CPCs that,based on our previous lineage-tracing studies,are committed to forming heart tissue,in combination with a robust methylcellulose spheroid-based delivery approach. View Publication

Directed evolution is a process of mutation and artificial selection to breed biomolecules with new or improved activity. Directed evolution platforms are primarily prokaryotic or yeast-based,and stable mammalian systems have been challenging to establish and apply. To this end,we develop PROTein Evolution Using Selection (PROTEUS),a platform that uses chimeric virus-like vesicles to enable extended mammalian directed evolution campaigns without loss of system integrity. This platform is stable and can generate sufficient diversity for directed evolution in mammalian systems. Using PROTEUS,we alter the doxycycline responsiveness of tetracycline-controlled transactivators,generating a more sensitive TetON-4G tool for gene regulation with mammalian-specific adaptations. PROTEUS is also compatible with intracellular nanobody evolution,and we use it to evolve a DNA damage-responsive anti-p53 nanobody. Overall,PROTEUS is an efficient and stable platform to direct evolution of biomolecules within mammalian cells. Subject terms: Synthetic biology,Synthetic biology,Molecular evolution,Next-generation sequencing View Publication

Human pluripotent stem cells (PSCs) are critical in vitro tools forbackslashnunderstanding mechanisms that regulate lineage differentiation inbackslashnthe human embryo as well as a potentially unlimited supply of stembackslashncells for regenerative medicine. Pluripotent human and mouse embryonicbackslashnstem cells (ESCs) derived from the inner cell mass of blastocystsbackslashnshare a similar transcription factor network to maintain pluripotencybackslashnand self-renewal,yet there are considerable molecular differencesbackslashnreflecting the diverse environments in which mouse and human ESCsbackslashnare derived. In the current study we evaluated the role of Proteinbackslashnarginine methyltransferase 5 (PRMT5) in human ESC (hESC) self-renewalbackslashnand pluripotency given its critical role in safeguarding mouse ESCbackslashnpluripotency. Unlike the mouse,we discovered that PRMT5 has no rolebackslashnin hESC pluripotency. Using microarray analysis we discovered thatbackslashna significant depletion in PRMT5 RNA and protein from hESCs changedbackslashnthe expression of only 78 genes,with the majority being repressed.backslashnFunctionally,we discovered that depletion of PRMT5 had no effectbackslashnon expression of OCT4,NANOG or SOX2,and did not prevent teratomabackslashnformation. Instead,we show that PRMT5 functions in hESCs to regulatebackslashnproliferation in the self-renewing state by regulating the fractionbackslashnof cells in Gap 1 (G1) of the cell cycle and increasing expressionbackslashnof the G1 cell cycle inhibitor P57. Taken together our data unveilsbackslashna distinct role for PRMT5 in hESCs and identifies P57 as new target. View Publication

Telomeres as the protective ends of linear chromosomes,are synthesized by the enzyme telomerase (TERT). Critically short telomeres essentially contribute to aging-related diseases and are associated with a broad spectrum of disorders known as telomeropathies. In cardiomyocytes,telomere length is strongly correlated with cardiomyopathies but it remains ambiguous whether short telomeres are the cause or the result of the disease. In this study,we employed an inducible CRISPRi human induced pluripotent stem cell (hiPSC) line to silence TERT expression enabling the generation of hiPSCs and hiPSC-derived cardiomyocytes with long and short telomeres. Reduced telomerase activity and shorter telomere lengths of hiPSCs induced global transcriptomic changes associated with cardiac developmental pathways. Consequently,the differentiation potential towards cardiomyocytes was strongly impaired and single cell RNA sequencing revealed a shift towards a more smooth muscle cell like identity in the cells with the shortest telomeres. Poor cardiomyocyte function and increased sensitivity to stress directly correlated with the extent of telomere shortening. Collectively our data demonstrates a TERT dependent cardiomyogenic differentiation defect,highlighting the CRISPRi TERT hiPSCs model as a powerful platform to study the mechanisms and consequences of short telomeres in the heart and also in the context of telomeropathies. The online version contains supplementary material available at 10.1007/s00018-024-05239-7. View Publication

ETS2 and ERG are transcription factors,encoded on human chromosome 21 (Hsa21),that have been implicated in human cancer. People with Down syndrome (DS),who are trisomic for Hsa21,are predisposed to acute megakaryoblastic leukemia (AMKL). DS-AMKL blasts harbor a mutation in GATA1,which leads to loss of full-length protein but expression of the GATA-1s isoform. To assess the consequences of ETS protein misexpression on megakaryopoiesis,we expressed ETS2,ERG,and the related protein FLI-1 in wild-type and Gata1 mutant murine fetal liver progenitors. These studies revealed that ETS2,ERG,and FLI-1 facilitated the expansion of megakaryocytes from wild-type,Gata1-knockdown,and Gata1s knockin progenitors,but none of the genes could overcome the differentiation block characteristic of the Gata1-knockdown megakaryocytes. Although overexpression of ETS proteins increased the proportion of CD41(+) cells generated from Gata1s-knockin progenitors,their expression led to a significant reduction in the more mature CD42 fraction. Serial replating assays revealed that overexpression of ERG or FLI-1 immortalized Gata1-knockdown and Gata1s knockin,but not wild-type,fetal liver progenitors. Immortalization was accompanied by activation of the JAK/STAT pathway,commonly seen in megakaryocytic malignancies. These findings provide evidence for synergy between alterations in GATA-1 and overexpression of ETS proteins in aberrant megakaryopoiesis. View Publication

We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511,a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component,human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages),after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps,hES cells spread out in a monolayer,maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on alpha6beta1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes. View Publication

OBJECTIVE: We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE(-/-) mice. METHODS AND RESULTS: Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was textgreater25% for HPCs and textgreater70% for macrophages. ApoAI was found in the macrophage culture media,mostly associated with the HDL fraction. Interestingly,a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE(-/-) macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE(-/-) mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls,without influencing plasma HDL-C levels. CONCLUSIONS: Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE(-/-) mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression,exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque. View Publication

Human mesenchymal stem cells (hMSCs) suppress T-cell and dendritic-cell function and represent a promising strategy for cell therapy of autoimmune diseases. Nevertheless,no information is currently available on the effects of hMSCs on B cells,which may have a large impact on the clinical use of these cells. hMSCs isolated from the bone marrow and B cells purified from the peripheral blood of healthy donors were cocultured with different B-cell tropic stimuli. B-cell proliferation was inhibited by hMSCs through an arrest in the G0/G1 phase of the cell cycle and not through the induction of apoptosis. A major mechanism of B-cell suppression was hMSC production of soluble factors,as indicated by transwell experiments. hMSCs inhibited B-cell differentiation because IgM,IgG,and IgA production was significantly impaired. CXCR4,CXCR5,and CCR7 B-cell expression,as well as chemotaxis to CXCL12,the CXCR4 ligand,and CXCL13,the CXCR5 ligand,were significantly down-regulated by hMSCs,suggesting that these cells affect chemotactic properties of B cells. B-cell costimulatory molecule expression and cytokine production were unaffected by hMSCs. These results further support the potential therapeutic use of hMSCs in immune-mediated disorders,including those in which B cells play a major role. View Publication

A method has been described for the long-term culture of human bone marrow cells in liquid medium. Hematopoiesis,as measured by the production of granulocytic-macrophage progenitor cells (CFUc),continued for at least 20 weeks and was dependent upon the presence of a marrow-derived adherent layer of cells. As in the case of murine marrow liquid cultures,the adherent layer consisted of mononuclear phagocytic cells,endothelial cells,and lipid-laden adipocytes,the latter being essential for long-term hematopoiesis. Optimal growth conditions included McCoy's medium supplemented with fetal bovine serum,horse serum,and hydrocortisone and incubation at 33 degrees C. Horse serum in conjunction with hydrocortisone appeared essential for the growth of adipocytes. View Publication

Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells,the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here,we attempted to reprogram E-PZ cells by forced expression of Oct4,Sox2,c-Myc,and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81,SSEA-3,Nanog,Sox2,and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5,CK14,and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal,mesodermal,and endodermal cells in vitro,although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly,E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44,p63,MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR),and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA),a hallmark of secretory cells,suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally,when injected into mice,E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme,E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that iPS-like cells derived from prostatic epithelial cells are pluripotent and capable of prostatic differentiation; therefore,provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification. View Publication