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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

Nature Methods 8,293 (2011). doi:10.1038/nmeth0411-293 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

The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA,nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently,there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions,we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription View Publication

Aim Reactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine,due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut,and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut. Results NoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer,NoxO1 has a protective role and may influence the population of natural killer cells. Conclusion NoxO1 affects colon epithelium homeostasis and prevents inflammation. View Publication

Durable response to chimeric antigen receptor T (CART) cell therapy remains limited in part due to CART cell exhaustion. Here,we investigate the regulation of CART cell exhaustion with three independent approaches including: a genome-wide CRISPR knockout screen using an in vitro model for exhaustion,RNA and ATAC sequencing on baseline and exhausted CART cells,and RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the ZUMA-1 clinical trial. Each of these approaches identify interleukin (IL)-4 as a regulator of CART cell dysfunction. Further,IL-4-treated CD8+ CART cells develop signs of exhaustion independently of the presence of CD4+ CART cells. Conversely,IL-4 pathway editing or the combination of CART cells with an IL-4 monoclonal antibody improves antitumor efficacy and reduces signs of CART cell exhaustion in mantle cell lymphoma xenograft mouse models. Therefore,we identify both a role for IL-4 in inducing CART exhaustion and translatable approaches to improve CART cell therapy. The application and therapeutic success of CAR-T cell approaches are limited by the development of T cell exhaustion. Here,Stewart et al discover a role for IL-4 in driving CD8+ CAR-T cell exhaustion and demonstrate the improvement of CAR-T cell effectivity with interruption of IL-4 signalling. View Publication

Heme oxygenase-1 (HO-1,HMOX1 ) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions,heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here,we investigate whether HO-1 safeguards cells against replication stress. Experiments were conducted in control and HMOX1 -deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1,the effect that was further enhanced in response to δ-aminolevulinic acid (ALA),a substrate in heme synthesis. This was associated with replication stress,as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1 -deficient patient. Interestingly,in the absence of HO-1,the speed of fork progression was higher,and the response to DNA conformational hindrance less stringent,indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead,we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53,an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin,which in turn induces strong lipid peroxidation. Our results indicate that protection against replication stress is a universal feature of HO-1,presumably contributing to its widely recognized cytoprotective activity. View Publication

AbstractIntroduction
Preeclampsia and other placental pathologies are characterized by a lack of spiral artery remodeling associated with insufficient invasion by extravillous trophoblast cells (EVT). Because trophoblast invasion occurs in early pregnancy when access to human placental tissue is limited,there is a need for model systems for the study of trophoblast differentiation and invasion. Human embryonic stem cells (hESC) treated with BMP4- differentiate to trophoblast,and express HLA-G,a marker of EVT. The goals of the present study were to further characterize the HLA-G+ cells derived from BMP4-treated hESC,and determine their suitability as a model.
Methods
HESC were treated with BMP4 under 4% or 20% oxygen and tested in Matrigel invasion chambers. Both BMP4-treated hESC and primary human placental cells were separated into HLA-G+ and HLA-G−/TACSTD2+ populations with immunomagnetic beads and expression profiles analyzed by microarray.
Results
There was a 10-fold increase in invasion when hESC were BMP4-treated. There was also an independent,stimulatory effect of oxygen on this process. Invasive cells expressed trophoblast marker KRT7,and the majority were also HLA-G+. Gene expression profiles revealed that HLA-G+,BMP4-treated hESC were similar to,but distinct from,HLA-G+ cells isolated from first trimester placentas. Whereas HLA-G+ and HLA-G− cells from first trimester placentas had highly divergent gene expression profiles,HLA-G+ and HLA-G− cells from BMP4-treated hESC had somewhat similar profiles,and both expressed genes characteristic of early trophoblast development.
Conclusions
We conclude that hESC treated with BMP4 provide a model for studying transition to the EVT lineage. View Publication

Recently,induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer,both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore,the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front,although donor cell types for generating iPSCs are wide-ranging,T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells,which can be applied to novel immunotherapies. In the present study,we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining,had a normal karyotype,and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined,animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application. View Publication

IntroductionRetinal ganglion cells (RGCs) are susceptible to degenerative conditions such as glaucoma and traumatic optic neuropathies,which lead to vision loss. MicroRNA-21-5p has demonstrated potential neuroprotective effects,but its mechanisms in optic nerve injury remain underexplored. This study evaluates the neuroprotective role of microRNA-21-5p derived from induced pluripotent stem cells (iPSCs) in an optic nerve crush (ONC) model.Materials and methodsIn vitro qPCR demonstrated that the expression of microRNA-21-5p was increased in the co-culture medium of RGCs and iPSCs. Subsequently,in the in vivo experiments,we used a microRNA-21-5p agonist to assess its protective effects on RGCs. RNA sequencing was then performed in a mouse ONC model after treatment with a microRNA-21-5p agonist to explore the mechanisms underlying its neuroprotective effects on RGCs.ResultsAs demonstrated in our previous experiments,the RGCs-iPSCs co-culture group led to a higher survival rate of RGCs,as indicated by live/dead cell staining,compared to the RGCs-only group. Quantitative PCR (qPCR) results revealed a significant increase in the expression of microRNA-21-5p in the medium of the RGCs-iPSCs co-culture group. Furthermore,the survival rate of mouse retinal RGCs treated with a microRNA-21-5p agonist was significantly greater than that of the control group. Lastly,RNA sequencing of the retina from microRNA-21-5p agonist-treated mice indicated that microRNA-21-5p plays a protective role in RGCs by downregulating the expression of several genes,including Irf1,Ccl4,Itk,Cxcr2,Dclre1c,Traf1,Traf2,Rbl1,Cxcl5,Cxcl3,Cxcl1,Cxcl9,Il2rg,Cd3e,Cd3d,Cxcl10,Ccl5,Ccl12,Tap1,and Cxcr4.ConclusionMicroRNA-21-5p derived from iPSCs can enhance the survival rate of RGCs in the ONC model. This suggests that microRNA-21-5p may represent a novel and effective strategy for repairing RGC damage. Such a strategy could potentially be realized through the modulation of apoptosis,T-cell regulatory pathways,or TNF-? signaling.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12886-025-04244-z. View Publication

APOBEC3G (A3G) is an intrinsic antiviral factor that inhibits the replication of human immunodeficiency virus (HIV) by deaminating cytidine residues to uridine. This causes guanosine-to-adenosine hypermutation in the opposite strand and results in inactivation of the virus. HIV counteracts A3G through the activity of viral infectivity factor (Vif),which promotes degradation of A3G. We report that viral protein R (Vpr),which interacts with a uracil glycosylase,also counteracted A3G by diminishing the incorporation of uridine. However,this process resulted in activation of the DNA-damage–response pathway and the expression of natural killer (NK) cell–activating ligands. Our results show that pathogen-induced deamination of cytidine and the DNA-damage response to virus-mediated repair of the incorporation of uridine enhance the recognition of HIV-infected cells by NK cells. View Publication

Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease,as well as for future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture,differentiation,isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2-3 weeks: this involves differentiation (10 d),isolation (1 d) and 4 or 10 d of expansion of ECs and pericytes,respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon coculture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs. View Publication