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

Because neurons are difficult to obtain from humans,generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells,we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation,glutamate receptor function,and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2,resulting in high responsiveness of neural cells to glutamate,N-methyl-d-aspartate (NMDA),α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA),and (S)-3,5-d... View Publication

The intestinal epithelium of human infants is developmentally immature compared to that of adults. Exactly how this immaturity affects key epithelial functions and their interactions with nearby immune cells remains an understudied area of research,partly due to limited access to non-diseased infant gut tissues. Human intestinal organoids,or “mini guts” generated from tissue stem cells,are promising models for investigating intestinal biology and disease mechanisms. These three-dimensional structures closely mimic their tissue of origin,including cellular physiology and genetics. We have also previously shown that neonatal Th17 cells represent a distinct cell population with a cytokine profile skewed toward IL-22 production rather than IL-17A,as seen in adult Th17 cells. In this study,we sought to model the impact of neonatal-derived Th17 cytokine,namely IL-22 and the intestinal epithelium using infant-derived ileal enteroids. We generated enteroids from ileal biopsies from infants (< 6 months old) and cultured them for seven days with standard organoid growth media,organoid media supplemented with conditioned media from cord-blood-derived Th17 cells,or media supplemented with recombinant IL-22. We assessed morphological changes and conducted transcriptomics profiling via RNAseq. Exposing enteroids to neonatal Th17-cells-derived conditioned media led to enhanced growth,maturation,and differentiation as compared to control media. These effects were ablated when an IL-22 neutralizing antibody was used,while conversely,supplementing with recombinant IL-22 mimicked the Th17 effects,increasing intestinal epithelial cell proliferation and inducing marked differentiation of secretory cells. Our transcriptomic profiling similarly demonstrated significant changes in response to IL-22 with downregulation of Wnt and Notch signaling and upregulation of immune pathways,particularly interferon signaling. The transcriptomic data also suggested that IL-22 treatment led to changes in cell type composition with an increase in stem- and progenitor cells at the expense of enterocytes. Taken together,our data suggests that early-life intestinal development is likely influenced by IL-22-dependent crosstalk between the infant epithelium and exposure to neighboring Th17 cells. This promotes epithelial cell maturation and immune readiness,reflected at both the morphological and molecular levels. Our work also provides a relevant framework for studying healthy infant gut development,which can be further leveraged to examine early-life gastrointestinal disorders,model complex human disease,and therapeutic testing while reducing reliance on animal models. View Publication

We describe a new procedure for large-scale CB processing in the collection bag,thus minimizing the risk of CB contamination. A solution of 6% hydroxyethyl starch (HES) was added directly to the CB containing bag. After RBC sedimentation at 4 degrees C,the WBC-rich supernatant was collected in a satellite bag and centrifuged. After supernatant removal,the cell pellet was resuspended and the percent recovery of total WBC,CD34+ progenitor cells,CFU-GM and cobblestone area-forming cells (CAFC) evaluated. Results obtained with three different types of CB collection bags (300,600 and 1000 ml) were analyzed and compared with those of an open system in 50 ml tubes. CB processing procedures in 300 and 1000 ml bags were associated with better WBC,CFU,CD34+ cell and CAFC recovery (83-93%). This novel CB processing procedure appears to be easy,effective and particularly suitable for large-scale banking under GMP conditions. View Publication

The introduction of antibody–drug conjugates represents a significant advancement in targeted therapy of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Our study aims to investigate the role of the DNA damage response pathway and the impact of PARP1 inhibition,utilizing talazoparib,on the response of AML and ALL cells to Gemtuzumab ozogamicin (GO) and Inotuzumab ozogamicin (INO),respectively. AML and ALL cells were treated with GO,INO and γ-calicheamicin in order to induce severe DNA damage and activate the G2/M cell-cycle checkpoint in a dose- and time-dependent manner. The efficacy of PARP1 inhibitors and,in particular,talazoparib in enhancing INO or GO against ALL or AML cells was assessed through measurements of cell viability,cell death,cell cycle progression,DNA damage repair,accumulation of mitotic DNA damage and inhibition of clonogenic capacity. We observed that both ALL and AML cell lines activate the G2/M cell-cycle checkpoint in response to γ-calicheamicin-induced DNA damage,highlighting a shared cellular response mechanism. Talazoparib significantly enhanced the efficacy of INO against ALL cell lines,resulting in reduced cell viability,increased cell death,G2/M cell-cycle checkpoint override,accumulation of mitotic DNA damage and inhibition of clonogenic capacity. Strong synergism was observed in primary ALL cells treated with the combination. In contrast,AML cells exhibited a heterogeneous response to talazoparib in combination with GO. Our findings suggest a potential link between the differential responses of ALL and AML cells to the drug combinations and the ability of talazoparibto override G2/M cell-cycle arrest induced by antibody–drug conjugates. PARP1 emerges as a key player in the response of ALL cells to INO and represents a promising target for therapeutic intervention in this leukemia setting. Our study sheds light on the intricate interplay between the DNA damage response pathway,PARP1 inhibition,and response of γ-calicheamicin-induced DNA damages in AML and ALL. These findings underscore the importance of targeted therapeutic strategies and pave the way for future research aimed at optimizing leukemia treatment approaches. The online version contains supplementary material available at 10.1186/s12967-024-05838-9. View Publication

Coinfection of HIV-1 patients with Plasmodium falciparum,the etiological agent of malaria,results in a raise of viral load and an acceleration of disease progression. The primary objective of this study was to investigate whether the malarial pigment hemozoin (HZ),a heme by-product of hemoglobin digestion by malaria parasites,can affect HIV-1 transmission by monocytes-derived dendritic cells (DCs) to CD4(+) T cells when HZ is initially internalized in monocytes before their differentiation in DCs. We demonstrate in this study that HZ treatment during the differentiation process induces an intermediate maturation phenotype when compared with immature and fully mature DCs. Furthermore,the DC-mediated transfer of HIV-1 is enhanced in presence of HZ,a phenomenon that may be linked with the capacity of HZ-loaded cells to interact and activate CD4(+) T cells. Altogether our findings suggest a new mechanism that could partially explain the increased HIV-1 virus production during a coinfection with P. falciparum. Understanding the multifaceted interactions between P. falciparum and HIV-1 is an important challenge that could lead to the development of new treatment strategies. View Publication

Mesenchymal stem cells have been implicated as playing an important role in stem cell engraftment. Recently,a new pluripotent population of umbilical cord blood (UCB) cells,unrestricted somatic stem cells (USSCs),with intrinsic and directable potential to develop into mesodermal,endodermal,and ectodermal fates,has been identified. In this study,we evaluated the capacity of ex vivo expanded USSCs to influence the homing of UCB-derived CD34(+) cells into the marrow and spleen of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. USSCs induced a significant enhancement of CD34(+) cell homing to both bone marrow and spleen (2.2 +/- 0.3- and 2.4 +/- 0.6-fold,respectively; p textless .05),with a magnitude similar to that induced by USSCs that had been thawed prior to transplantation. The effect of USSCs was dose-dependent and detectable at USSC:CD34(+) ratios of 1:1 and above. Enhanced marrow homing by USSCs was unaltered by extensive culture passaging of the cells,as similar enhancement was observed for both early-passage (passage 5 [p5]) and late-passage (p10) USSCs. The homing effect of USSCs was also reflected in an increased proportion of NOD/SCID mice exhibiting significant human cell engraftment 6 weeks after transplantation,with a similar distribution of myeloid and lymphoid components. USSCs enhanced the homing of cellular products of ex vivo expanded UCB lineage-negative (lin(-)) cells,generated in 14-day cultures by Selective Amplification. The relative proportion of homing CD34(+) cells within the culture-expanded cell population was unaltered by USSC cotransplantation. Production of stromal-derived factor-1 (SDF-1) by USSCs was detected by both gene expression and protein released into culture media of these cells. Knockdown of SDF-1 production by USSCs using lentiviral-SiRNA led to a significant (p textless .05) reduction in USSC-mediated enhancement of CD34(+) homing. Our findings thus suggest a clinical potential for using USSCs in facilitating homing and engraftment for cord blood transplant recipients. View Publication

Differentiation of human pluripotent stem cells (hPSCs) into functional cell types is a crucial step in cell therapy. In the present study,we demonstrate that functional CD34(+) progenitor cells can be efficiently produced from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) by combined modulation of 2 signaling pathways. A higher proportion of CD34(+) cells (∼ 20%) could be derived from hPSCs by inhibition of mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling and activation of bone morphogenic protein-4 (BMP4) signaling. hPSC-derived CD34(+) progenitor cells further developed to endothelial and smooth muscle cells with functionality. Moreover,they contributed directly to neovasculogenesis in ischemic mouse hind limbs,thereby resulting in improved blood perfusion and limb salvage. Our results suggest that combined modulation of signaling pathways may be an efficient means of differentiating hPSCs into functional CD34(+) progenitor cells. View Publication

Liver becomes the predominant site of hematopoiesis by 11.5 dpc (days after coitus) in the mouse and 15 gestational weeks in humans and stays so until the end of gestation. The reason the liver is the major hematopoietic site during fetal life is not clear. In this work,we tried to define which of the fetal liver microenvironmental cell populations would be associated with the development of hematopoiesis and found that a population of cells with mixed endodermal and mesodermal features corresponded to hematopoietic-supportive fetal liver stroma. Stromal cells generated from primary cultures or stromal lines from mouse or human fetal liver in the hematopoietic florid phase expressed both mesenchymal markers (vimentin,osteopontin,collagen I,alpha smooth muscle actin,thrombospondin-1,EDa fibronectin,calponin,Stro-1 antigens,myocyte-enhancer factor 2C) and epithelial (alpha-fetoprotein,cytokeratins 8 and 18,albumin,E-cadherin,hepatocyte nuclear factor 3 alpha) markers. Such a cell population fits with the description of cells in epithelial-to-mesenchymal transition (EMT),often observed during development,including that of the liver. The hematopoietic supportive capacity of EMT cells was lost after hepatocytic maturation,induced by oncostatin M in the cell line AFT024. EMT cells were observed in the fetal liver microenvironment during the hematopoietic phase but not in nonhematopoietic liver by the end of gestation and in the adult. EMT cells represent a novel stromal cell type that may be generated from hepatic endodermal or mesenchymal stem cells or even from circulating hematopoietic stem cells (HSCs) seeding the liver rudiment. View Publication

The mechanisms underlying the decision of a stem or progenitor cell to either self-renew or differentiate are incompletely understood. To address the role of Myc in this process,we expressed different forms of the proto-oncogene Myc in multipotent neural progenitor cells (NPCs) using retroviral transduction. Expression of Myc in neurospheres increased the proportion of self-renewing cells fivefold,and 1% of the Myc-overexpressing cells,but none of the control cells,retained self-renewal capacity even under differentiation-inducing conditions. A Myc mutant (MycV394D) deficient in binding to Miz-1,did not increase the percentage of self-renewing cells but was able to stimulate proliferation of NPCs as efficiently as wild-type Myc,indicating that these two cellular phenomena are regulated by at least partially different pathways. Our results suggest that Myc,through Miz-1,enhances self-renewal of NPCs and influences the way progenitor cells react to the environmental cues that normally dictate the cellular identity of tissues containing self-renewing cells. View Publication

The conventional method of culturing human embryonic stem cells (hESC) is on two-dimensional (2D) surfaces,which is not amenable for scale up to therapeutic quantities in bioreactors. We have developed a facile and robust method for maintaining undifferentiated hESC in three-dimensional (3D) suspension cultures on matrigel-coated microcarriers achieving 2- to 4-fold higher cell densities than those in 2D colony cultures. Stable,continuous propagation of two hESC lines on microcarriers has been demonstrated in conditioned media for 6 months. Microcarrier cultures (MC) were also demonstrated in two serum-free defined media (StemPro and mTeSR1). MC achieved even higher cell concentrations in suspension spinner flasks,thus opening the prospect of propagation in controlled bioreactors. ?? 2009 Elsevier B.V. All rights reserved. View Publication