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

BackgroundSeveral approaches are being explored for engineering off-the-shelf chimeric antigen receptor (CAR) T cells. In this study,we engineered chimeric Fcγ receptor (FcγR) T cells and tested their potential as a versatile platform for universal T cell therapy.MethodsChimeric FcγR (CFR) constructs were generated using three distinct forms of FcγR,namely CD16A,CD32A,and CD64. The functionality of CFR T cells was evaluated through degranulation assays,specific target lysis experiments,in vitro cytokine production analysis,and assessment of tumor xenograft destruction specificity in mouse models using different monoclonal antibodies (MoAbs).ResultsThree types of CFR T cells were engineered,16s3,32-8a,64-8a CFR T cells. In the presence of rituximab (RTX),cytotoxicity of all three types of CFR T cells against CD20+ Raji-wt,K562-CD20+,and primary tumor cells was significantly higher than that of the mock T cells (P < 0.001). When herceptin was used,all three types of CFR T cells exhibited significant cytotoxicity against HER2+ cell lines of SK-BR-3,SK-OV-3,and HCC1954 (P < 0.001). The cytotoxicity of 64-8a CFR T cells was significantly inhibited by free human IgG at a physiological dose (P < 0.001),which was not observed in 16s3,32-8a CFR T cells. Compared to 32-8a CFR T cells,16s3 CFR T cells exhibited more prolonged cytotoxicity than 32-8a CFR T cells (P < 0.01). In in vivo assays using xenograft models,16s3 CFR T cells significantly prolonged the survival of mice xenografted with Raji-wt cells in the presence of RTX (P < 0.001),and effectively reduced tumor burden in mice xenografted with SK-OV-3 cells in the presence of herceptin (P < 0.05). No significant non-specific cytotoxicity of CFR T cells was found in vivo.ConclusionThe anti-tumor effects of the CFR T cells in vitro and in xenograft mouse models are mediated by specific MoAbs such as RTX and herceptin. The CFR T cells therefore have the features of universal T cells with specificity directed by MoAbs. 16s3 CFR T cells are chosen for clinical trials.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40164-025-00595-x. View Publication

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells,and overexpression of Hoxa-9 markedly expands hematopoietic stem cells,suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-kit(+)flk-2(-)Sca-1+ [KLFS] cells). However,sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia,indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays,Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability,a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures,Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation,with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo. View Publication

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha,we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally,enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus,the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling,and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution. View Publication

Prenatal lethality associated with mouse knockout of Mettl16,a recently identified RNA N6-methyladenosine (m 6 A) methyltransferase,has hampered characterization of the essential role of METTL16-mediated RNA m 6 A modification in early embryonic development. Here,using cross-species single-cell RNA sequencing analysis,we found that during early embryonic development,METTL16 is more highly expressed in vertebrate hematopoietic stem and progenitor cells (HSPCs) than other methyltransferases. In Mettl16-deficient zebrafish,proliferation capacity of embryonic HSPCs is compromised due to G1/S cell cycle arrest,an effect whose rescue requires Mettl16 with intact methyltransferase activity. We further identify the cell-cycle transcription factor mybl2b as a directly regulated by Mettl16-mediated m 6 A modification. Mettl16 deficiency resulted in the destabilization of mybl2b mRNA,likely due to lost binding by the m 6 A reader Igf2bp1 in vivo. Moreover,we found that the METTL16-m 6 A- MYBL2 -IGF2BP1 axis controlling G1/S progression is conserved in humans. Collectively,our findings elucidate the critical function of METTL16-mediated m 6 A modification in HSPC cell cycle progression during early embryonic development. View Publication

To investigate intestinal health and its potential disruptors in vitro,representative models are required. Human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cells (IECs) more closely resemble the in vivo intestinal tissue than conventional in vitro models like human colonic adenocarcinoma Caco-2 cells. However,the potential of IECs to study immune-related responses upon external stimuli has not been investigated in detail yet. The aim of the current study was to evaluate immune-related effects of IECs by challenging them with a pro-inflammatory cytokine cocktail. Subsequently,the effects of Lactiplantibacillus plantarum WCFS1 were investigated in unchallenged and challenged IECs. All exposures were compared to Caco-2 cells and in vivo data where possible. Upon the inflammatory challenge,IECs and Caco-2 cells induced a pro-inflammatory response which was strongest in IECs. Heat-killed L. plantarum exerted the strongest effect on immune parameters in the IEC model,while L. plantarum in the stationary growth phase had most pronounced effects on immune-related gene expression in Caco-2 cells. Unfortunately,comparison to in vivo transcriptomics data showed limited similarities,which could be explained by essential differences in the study setups. Altogether,hiPSC-derived IECs show a high potential as a model to study immune-related responses in the intestinal epithelium in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-74802-w. View Publication

Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells,only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study,we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1,a critical transcription factor for pancreatic development,leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore,in the presence of biochemical factors,200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin,glucagon,or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ,suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells. View Publication

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-,atrial-,and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives,which in turn promoted cardiomyocyte differentiation. Moreover,a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation,improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation,measuring action potentials,and intracellular Ca(2+) dynamics. These findings are relevant for improving our understanding on human heart development,and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes,similar to those observed in adult cardiac myocytes. View Publication

Stable pluripotent feeder-free propagation of human embryonic stem cells (hESCs) prior to their therapeutic applications remains a major challenge. Matrigel™ (BD Singapore) is a murine sarcoma-derived extracellular matrix (ECM) widely used as a cell-free support combined with conditioned or chemically defined media; however,inherent xenogenic and immunological threats invalidate it for clinical applications. Using human fibrogenic cells to generate ECM is promising but currently suffers from inefficient and time-consuming deposition in vitro. We recently showed that macromolecular crowding (MMC) accelerated ECM deposition substantially in vitro. In the current study,we used dextran sulfate 500 kDa as a macromolecular crowder to induce WI-38 fetal human lung fibroblasts at 0.5% serum condition to deposit human ECM in three days. After decellularization,the generated ECMs allowed stable propagation of H9 hESCs over 20 passages in chemically-defined medium (mTEsR1) with an overall improved outcome compared to Matrigel in terms of population doubling while retaining teratoma formation and differentiation capacity. Of significance,only ECMs generated by MMC allowed the successful propagation of hESCs. ECMs were highly complex and in contrast to Matrigel,contained no vitronectin but did contain collagen XII,ig-h3 and novel for hESC-supporting human matrices,substantial amounts of transglutaminase 2. Genome-wide analysis of promoter DNA methylation states revealed high overall similarity between human ECM- and Matrigel-cultured hESCs; however,distinct differences were observed with 49 genes associated with a variety of cellular functions. Thus,human ECMs deposited by MMC by selected fibroblast lines are a suitable human microenvironment for stable hESC propagation and clinically translational settings. View Publication

A major road-block in stem cell therapy is the poor homing and integration of transplanted stem cells with the targeted host tissue. Human induced pluripotent stem (hiPS) cells are considered an excellent alternative to embryonic stem (ES) cells and we tested the feasibility of using small,physiological electric fields (EFs) to guide hiPS cells to their target. Applied EFs stimulated and guided migration of cultured hiPS cells toward the anode,with a stimulation threshold of textless30 mV/mm; in three-dimensional (3D) culture hiPS cells remained stationary,whereas in an applied EF they migrated directionally. This is of significance as the therapeutic use of hiPS cells occurs in a 3D environment. EF exposure did not alter expression of the pluripotency markers SSEA-4 and Oct-4 in hiPS cells. We compared EF-directed migration (galvanotaxis) of hiPS cells and hES cells and found that hiPS cells showed greater sensitivity and directedness than those of hES cells in an EF,while hES cells migrated toward cathode. Rho-kinase (ROCK) inhibition,a method to aid expansion and survival of stem cells,significantly increased the motility,but reduced directionality of iPS cells in an EF by 70-80%. Thus,our study has revealed that physiological EF is an effective guidance cue for the migration of hiPS cells in either 2D or 3D environments and that will occur in a ROCK-dependent manner. Our current finding may lead to techniques for applying EFs in vivo to guide migration of transplanted stem cells. View Publication

Avian species are important model animals for developmental biology and disease research. However,unlike in mice,where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function,clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs),the first nonmammalian iPSCs,which were clonally isolated and propagated,important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes,indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes,oligodendrocytes,and neurons. Ultimately,qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers,extraembryonic tissues,and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals. View Publication

Embryonic stem cells (ESCs) are unique cells,which have the ability to differentiate into all cell types that comprise the adult organism. Furthermore,ESCs can infinitely self-renew under optimized conditions. These features place human ESCs (hESCs) in a position where these cells can be exploited for tissue engineering and regenerative medicine approaches in treating human degenerative disorders. However,cell therapy approaches will require large amounts of clinically useable cells,not typically achievable using standard static cell culture methods. Here,we describe a method wherein clinically relevant numbers of hESCs can be generated in a cost and time effective manner. View Publication