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

While distinct stem cell phenotypes follow global changes in chromatin marks,single-cell chromatin technologies are unable to resolve or predict stem cell fates. We propose the first such use of optical high content nanoscopy of histone epigenetic marks (epi-marks) in stem cells to classify emergent cell states. By combining nanoscopy with epi-mark textural image informatics,we developed a novel approach,termed EDICTS (Epi-mark Descriptor Imaging of Cell Transitional States),to discern chromatin organizational changes,demarcate lineage gradations across a range of stem cell types and robustly track lineage restriction kinetics. We demonstrate the utility of EDICTS by predicting the lineage progression of stem cells cultured on biomaterial substrates with graded nanotopographies and mechanical stiffness,thus parsing the role of specific biophysical cues as sensitive epigenetic drivers. We also demonstrate the unique power of EDICTS to resolve cellular states based on epi-marks that cannot be detected via mass spectrometry based methods for quantifying the abundance of histone post-translational modifications. Overall,EDICTS represents a powerful new methodology to predict single cell lineage decisions by integrating high content super-resolution nanoscopy and imaging informatics of the nuclear organization of epi-marks. View Publication

Specialized immune cell subsets are involved in autoimmune disease,cancer immunity,and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However,subset-specific responses may not be detectable in analyses of whole blood samples,and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression. We validate the system's technical performance against standard subset isolation and library construction protocols and demonstrate the importance of subset-specific profiling through in vitro stimulation experiments. We show the ability of this integrated platform to identify subset-specific disease signatures by profiling four immune cell subsets in blood from patients with systemic lupus erythematosus (SLE) and matched control subjects. The platform has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings. View Publication

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs),which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here,we discover that the histone demethylase PHF2 associates with RAD21,a core subunit of cohesin,to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably,the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient,active replication origins,and can act as boundaries to separate adjacent domains. Accordingly,PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore,we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation,and that PHF2’s histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops,where cohesin represses dormant replication origins directly or indirectly,to sustain DNA replication in NSC. View Publication

Apigenin (API),a traditionally sourced flavonoid,is recognized for its anti-neoplastic properties. Despite well-documented effects on tumorigenesis,the detailed therapeutic impact on breast cancer stem cells (BCSCs) and the associated molecular mechanisms are yet to be clarified. The objective of this study is to elucidate the therapeutic effects of API on BCSCs and to uncover its molecular mechanisms through network pharmacology and experimental validation. Interactions of API with candidate targets were examined through target screening,enrichment analysis,construction of protein-protein interaction networks,and molecular docking. MCF-7-derived BCSCs were utilized as a model system to investigate and substantiate the anti-BCSC effects of API and the underlying mechanism. Molecular docking studies have shown that API and TP53 exhibit favorable binding affinity. Compared with the negative control group,API effectively suppressed the expression of BCSC-related proteins such as ALDH1A1,NANOG,EpCAM,and MYC,downregulated p-PI3K and p-AKT,and upregulated p53. This study demonstrates that API can play an anti-BCSC role by regulating the PI3K/AKT/p53 pathway in BCSCs of MCF-7 cells,highlighting its potential as a therapeutic agent for targeting BCSCs. View Publication

To produce pluripotent stem cells from peripheral blood mononuclear cells (PBMCs) of a patient with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and culture and differentiate them into vascular organoids,producing a disease model for cerebral small vessel disease (CSVD). (1) PMBCs from patients clinically diagnosed with CADASIL ( NOTCH3 p.R141C) were induced to differentiate into pluripotent stem cells (iPSCs); the quality and differentiation ability of the iPSCs were determined. (2) CADASIL-derived iPSCs and control iPSCs were cultured and differentiated into vascular organoids. The differences in the morphological structure of the two differentiated groups of vascular organoids were observed,and both were identified. (1) No mycoplasma infections were detected in the iPSCs prepared from the PBMCs of patients with CADASIL. The short tandem repeat (STR) identification verified that the iPSCs originated from the patient,and the karyotype was normal. Flow cytometry and immunofluorescence detection revealed that the iPSCs expressed SSEA4,OCT4,and NANOG stem proteins. Tri-germ differentiation testing confirmed that the iPSCs expressed the endoderm markers SOX17 and FOXA2,the mesoderm markers Brachyury and α-SMA,and the ectoderm markers Pax6 and β-III Tubulin. (2) CADASIL-derived iPSCs and control iPSCs were induced to differentiate and produce endothelial networks and vascular networks,ultimately forming vascular organoids. Compared with control vascular organoids,CADASIL vascular organoids exhibited lower growth density,earlier blood vessel sprouting,longer and thinner vascular filaments,and smaller final vascular organoids. The vascular organoids from the two sources expressed the endothelial cell marker CD31,the vascular smooth muscle marker α-SMA,and the pericyte marker PDGFR-β. Reprogramming technology can be used to induce PBMCs to become iPSCs,and a CSVD disease model can be successfully constructed by culturing and differentiating the iPSCs into CADASIL vascular organoids. The NOTCH3 p.R141C mutation suppresses the vascular differentiation process in CADASIL. View Publication

SummaryTumor-infiltrating regulatory T cells (TI-Tregs) elicit immunosuppressive effects in the tumor microenvironment (TME) leading to accelerated tumor growth and resistance to immunotherapies against solid tumors. Here,we demonstrate that poly-(ADP-ribose)-polymerase-11 (PARP11) is an essential regulator of immunosuppressive activities of TI-Tregs. Expression of PARP11 correlates with TI-Treg cell numbers and poor responses to immune checkpoint blockade (ICB) in human patients with cancer. Tumor-derived factors including adenosine and prostaglandin E2 induce PARP11 in TI-Tregs. Knockout of PARP11 in the cells of the TME or treatment of tumor-bearing mice with selective PARP11 inhibitor ITK7 inactivates TI-Tregs and reinvigorates anti-tumor immune responses. Accordingly,ITK7 decelerates tumor growth and significantly increases the efficacy of anti-tumor immunotherapies including ICB and adoptive transfer of chimeric antigen receptor (CAR) T cells. These results characterize PARP11 as a key driver of TI-Treg activities and a major regulator of immunosuppressive TME and argue for targeting PARP11 to augment anti-cancer immunotherapies. Graphical abstract Highlights•Tumor-derived factors upregulate PARP11 in the tumor-infiltrating Treg cells•PARP11 supports the immunosuppressive properties of Treg cells•Pharmacologic inhibition of PARP11 inactivates intratumoral Treg cells•PARP11 inhibitor augments the efficacy of immunotherapies Basavaraja et al. demonstrate that induction of PARP11 in the intratumoral regulatory T (Treg) cells is required for their regulatory functions and contributes to the immunosuppressive tumor microenvironment. The selective inhibitor of PARP11 ITK7 inactivates tumor Treg cells and improves the efficacy of immunotherapies against tumors. View Publication

AbstractBackgroundNatural killer (NK) cells’ unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However,NK cells have demonstrated only moderate responses against cancer in clinical trials.MethodsAdvanced genome engineering may thus be used to unlock their full potential. Multiplex genome editing with CRISPR/Cas9 base editors (BEs) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here,we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations.ResultsWe observed highly efficient single and multiplex base editing,resulting in significantly enhanced NK cell function in vitro and in vivo. Next,we combined multiplex BE with non-viral TcBuster transposon-based integration to generate interleukin-15 armored CD19 chimeric antigen receptor (CAR)-NK cells with significantly improved functionality in a highly suppressive model of Burkitt’s lymphoma both in vitro and in vivo.ConclusionsThe use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated. View Publication

Umbilical cord blood (UCB) preparation needs to be optimized in order to develop more simplified procedures for volume reduction,as well as to reduce the amount of contaminating cells within the final stem cell transplant. We evaluated a novel filter device (StemQuick((TM))E) and compared it with our routine buffy coat (BC) preparation procedure for the enrichment of hematopoietic progenitor cells (HPCs). Two groups of single or pooled UCB units were filtered (each n = 6),or equally divided in two halves and processed by filtration and BC preparation in parallel (n = 10). The engraftment capacity of UCB samples processed by whole blood (WB) preparation was compared with paired samples processed by filtration in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse animal model. Filtration of UCB units in the two groups with a mean volume of 87.8 and 120.7 ml,respectively,and nucleated cell (NC) content of 9.7 and 23.8 x 10(8) resulted in a sufficient mean cell recovery for mononucleated cells ([MNCs] 74.2%-77.5%),CD34(+) cells (76.3%-79.0%),and colony-forming cells (64.1%-86.3%). Moreover,we detected a relevant depletion of the transplants for RBCs (89.2%-90.0%) and platelets ([PLTs] 77.5%-86.1%). In contrast,the mean depletion rate using BC processing proved to be significantly different for PLTs (10%,p = 0.03) and RBCs (39.6%,p textless 0.01). The NC composition showed a highly significant increase in MNCs and a decrease in granulocytes after filtration (p textless 0.01),compared with a less significant MNC increase in the BC group (p textless 0.05). For mice transplanted with WB-derived progenitors,we observed a mean of 15.3% +/- 15.5% of human CD45(+) cells within the BM compared with 19.9% +/- 16.8% for mice transplanted with filter samples (p = 0.03). The mean percentage of human CD34(+) cells was 4.2% +/- 3.1% for WB samples and 4.5% +/- 3.2% for filter samples (p = 0.68). As the data of NOD/SCID mice transplantation demonstrated a significant engraftment capacity of HPCs processed by filtration,no negative effect on the engraftment potential of filtered UCB cells versus non-volume-reduced cells from WB transplants was found. The StemQuick((TM))E filter devices proved to be a useful tool for Good Manufacturing Practices conform enrichment of HPCs and MNCs out of UCB. Filtration enables a quick and standardized preparation of a volume-reduced UCB transplant,including a partial depletion of granulocytes,RBCs,and PLTs without the need for centrifugation. Therefore,it seems very probable that filter-processed UCB transplants will also result in sufficient hematopoietic reconstitution in humans. View Publication

The development of immunodeficient mouse xenograft models has greatly facilitated the investigation of some human hematopoietic malignancies,but application of this approach to the myelodysplastic syndromes (MDSs) has proven difficult. We now show that cells from most MDS patients (including all subtypes) repopulate nonobese diabetic-severe combined immunodeficient (scid)/scid-beta2 microglobulin null (NOD/SCID-beta2m(-/-)) mice at least transiently and produce abnormal differentiation patterns in this model. Normal marrow transplants initially produce predominantly erythroid cells and later predominantly B-lymphoid cells in these mice,whereas most MDS samples produced predominantly granulopoietic cells. In 4 of 4 MDS cases,the regenerated cells showed the same clonal markers (trisomy 8,n = 3; and 5q-,n = 1) as the original sample and,in one instance,regenerated trisomy 8(+) B-lymphoid as well as myeloid cells were identified. Interestingly,the enhanced growth of normal marrow obtained in NOD/SCID-beta2m(-/-) mice engineered to produce human interleukin-3,granulocyte-macrophage colony-stimulating factor,and Steel factor was seen only with 1 of 7 MDS samples. These findings support the concept that human MDS originates in a transplantable multilineage hematopoietic stem cell whose genetic alteration may affect patterns of differentiation and responsiveness to hematopoietic growth factors. They also demonstrate the potential of this new murine xenotransplant model for future investigations of MDS. View Publication

Constitutive activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway in human cancers is often associated with mutational activation of BRAF or RAS. MAPK/ERK kinase 1/2 kinases lie downstream of RAS and BRAF and are the only acknowledged activators of ERK1/2,making them attractive targets for therapeutic intervention. AZD6244 (ARRY-142886) is a potent,selective,and ATP-uncompetitive inhibitor of MAPK/ERK kinase 1/2. In vitro cell viability inhibition screening of a tumor cell line panel found that lines harboring BRAF or RAS mutations were more likely to be sensitive to AZD6244. The in vivo mechanisms by which AZD6244 inhibits tumor growth were investigated. Chronic dosing with 25 mg/kg AZD6244 bd resulted in suppression of growth of Colo-205,Calu-6,and SW-620 xenografts,whereas an acute dose resulted in significant inhibition of ERK1/2 phosphorylation. Increased cleaved caspase-3,a marker of apoptosis,was detected in Colo-205 and Calu-6 but not in SW-620 tumors where a significant decrease in cell proliferation was detected. Chronic dosing of AZD6244 induced a morphologic change in SW-620 tumors to a more differentiated phenotype. The potential of AZD6244 in combination with cytotoxic drugs was evaluated in mice bearing SW-620 xenografts. Treatment with tolerated doses of AZD6244 and either irinotecan or docetaxel resulted in significantly enhanced antitumor efficacy relative to that of either agent alone. These results indicate that AZD6244 has potential to inhibit proliferation and induce apoptosis and differentiation,but the response varies between different xenografts. Moreover,enhanced antitumor efficacy can be obtained by combining AZD6244 with the cytotoxic drugs irinotecan or docetaxel. View Publication

Phenotypic markers associated with human hematopoietic stem cells (HSCs) were developed and validated using uncultured cells. Because phenotype and function can be dissociated during culture,better markers to prospectively track and isolate HSCs in ex vivo cultures could be instrumental in advancing HSC-based therapies. Using an expansion system previously shown to increase hematopoietic progenitors and SCID-repopulating cells (SRCs),we demonstrated that the rhodamine-low phenotype was lost,whereas AC133 expression was retained throughout culture. Furthermore,the AC133(+)CD38(-) subpopulation was significantly enriched in long-term culture-initiating cells (LTC-IC) and SRCs after culture. Preculture and postculture analysis of total nucleated cell and LTC-IC number,and limiting dilution analysis in NOD/SCID mice,showed a 43-fold expansion of the AC133(+)CD38(-) subpopulation that corresponded to a 7.3-fold and 4.4-fold expansion of LTC-ICs and SRCs in this subpopulation,respectively. Thus,AC133(+)CD38(-) is an improved marker that tracks and enriches for LTC-IC and SRC in ex vivo cultures. View Publication

T cell dysfunction is crucial to the pathogenesis of systemic lupus erythematosus (SLE); however,the molecular mechanisms involved in the deficient expression of the T cell receptor-associated CD3zeta chain in SLE are not clear. SLE T cells express abnormally increased levels of an alternatively spliced isoform of CD3zeta that lacks a 562-bp region in its 3'-untranslated region (UTR). We showed previously that two adenosine/uridine-rich elements (ARE) in this splice-deleted region of CD3zeta transcript are critical for the mRNA stability and protein expression of CD3zeta. In this study we show for the first time that the mRNA-stabilizing protein HuR binds to these two ARE bearing regions of CD3zeta 3'-UTR. Knockdown of HuR resulted in decreased expression of the CD3zeta chain,whereas overexpression led to the increase of CD3zeta chain levels. Additionally,overexpression of HuR in human T cells resulted in increased mRNA stability of CD3zeta. Our results identify the 3'-UTR of CD3zeta as a novel target for the mRNA-stabilizing protein HuR. Thus,the absence of two critical AREs in the alternatively spliced CD3zeta 3'-UTR found in SLE T cells may result in decreased HuR binding,representing a possible molecular mechanism contributing to the reduced stability and expression of CD3zeta in SLE. View Publication