Mesenchymal stromal cells improve the transplantation outcome of CRISPR-Cas9 gene-edited human HSPCs (NHEJ)
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE206902 | GEO | 2022/09/01
REPOSITORIES: GEO
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Project description:Mesenchymal Stromal Cells (MSCs) have been employed in vitro to support HSPC expansion and in vivo to promote Hematopoietic Stem and Progenitor Cells (HSPCs) engraftment. Based on these studies, we developed an MSC-based co-culture system to optimize transplantation outcome of CRISPR-Cas9 gene-edited (GE) human HSPCs. We show that bone marrow (BM)-MSCs produce several hematopoietic supportive and anti-inflammatory factors capable to alleviate the proliferation arrest and mitigate the apoptotic and inflammatory programs activated in GE-HSPCs, improving their expansion and clonogenic potential in vitro. The use of BM-MSCs resulted in superior human engraftment and increased clonal output of GE-HSPCs contributing to the early phase of hematological reconstitution in the peripheral blood of transplanted mice. In conclusion, our work poses the biological bases for a novel clinical use of BM-MSCs to promote engraftment of GE-HSPCs and improve their transplantation outcome.
2022-09-01 | GSE206900 | GEO
Project description:Mesenchymal Stromal Cells (MSCs) have been employed in vitro to support HSPC expansion and in vivo to promote Hematopoietic Stem and Progenitor Cells (HSPCs) engraftment. Based on these studies, we developed an MSC-based co-culture system to optimize transplantation outcome of CRISPR-Cas9 gene-edited (GE) human HSPCs. We show that bone marrow (BM)-MSCs produce several hematopoietic supportive and anti-inflammatory factors capable to alleviate the proliferation arrest and mitigate the apoptotic and inflammatory programs activated in GE-HSPCs, improving their expansion and clonogenic potential in vitro. The use of BM-MSCs resulted in superior human engraftment and increased clonal output of GE-HSPCs contributing to the early phase of hematological reconstitution in the peripheral blood of transplanted mice. In conclusion, our work poses the biological bases for a novel clinical use of BM-MSCs to promote engraftment of GE-HSPCs and improve their transplantation outcome.
2022-09-01 | GSE168834 | GEO
Project description:The bone marrow (BM) niche regulates multiple HSC processes. Clinical treatment for hematological malignancies, by HSC transplantation often requires preconditioning total body irradiation, which severely and irreversibly impairs the BM niche and HSC regeneration. Novel strategies to enhance HSC regeneration in irradiated BM are needed. We compared the effects of niche factors EGF, FGF2 and PDGFB on HSC hematopoietic regeneration using human MSCs that were transduced with these factors via lentiviral vectors. Among above niche factors tested, PDGFB-MSCs most significantly improved human hematopoietic cell engraftment in immunodeficient mice. PDGFB-MSC-treated BM more efficiently enhanced transplanted human HSC self-renewal in secondary transplantations from primary recipients. Although PDGFB-MSCs did not directly increase HSC expansion in vitro, GSEA revealed anti-apoptotic signaling being increased in PDGFB-MSCs versus GFP-MSCs. PDGFB-MSCs had enhanced survival and expansion after transplantation, leading to an enlarged humanized niche cell pool. Our study demonstrates the efficacy of MSC-mediated niche factors in clinical HSC transplantation for patients.
2020-06-22 | GSE113857 | GEO
Project description:Hematopoietic stem cells (HSC) rely on a unique regulatory machinery that facilitates life-long blood production and enables reconstitution of the entire hematopoietic system upon transplantation. However, the biological processes governing human HSC self-renewal and engraftment ability are poorly understood and challenging to recapitulate ex vivo to facilitate robust human HSC expansion. We discovered a novel HSC regulatory protein, MYCT1 (MYCT target 1), that is selectively expressed in endothelial cells (EC) and undifferentiated human HSPCs but becomes drastically downregulated during HSC culture. Lentiviral knockdown of MYCT1 in human foetal liver and cord blood HSPCs revealed a critical role for MYCT1 in governing human HSPC expansion and engraftment ability. Single cell RNAseq of human CB HSPCs after MYCT1 knockdown and overexpression revealed that MYCT1 governs HSC functional competence and modulates cellular properties essential for HSC stemness, such as low mitochondrial metabolic activity. Indeed, restoring the compromised MYCT1 expression in cultured human CB HSPCs improved ex vivo expansion of the most undifferentiated human HSPCs and enhanced their engraftment ability. We found that MYCT1 is localized in the endosomal membrane and interacts with vesicle trafficking regulators and signalling machinery essential for HSC and EC function. Loss of MYCT1 led to excessive endocytosis and hyperactive signalling responses to cytokines, whereas restoring MYCT1 expression in cultured CB HSPCs balanced the abnormal endocytosis associated with prolonged culture and fine-tuned signalling responses. Our work identifies MYCT1-moderated endocytosis and environmental sensing as an essential regulatory mechanism required to preserve human HSC stemness, and pinpoints silencing of MYCT1 as a critical contributor to the dysfunction of cultured human HSCs that needs to be addressed to improve human HSC culture strategies.
2024-04-15 | PXD042257 | Pride
Project description:Mesenchymal stem cells (MSCs) play important roles in tissue homeostasis by providing a supportive microenvironmental niche for the hematopoietic system. Cigarette smoking induces systemic abnormalities, including an impeded recovery process after hematopoietic stem cell transplantation. However, the role of cigarette smoking-mediated alterations in MSC niche function have not been investigated. In the present study, we asked whether exposure to cigarette smoking extract (CSE) disrupts the hematopoietic niche function of MSCs, and pathways impacted. To investigate the effects on bone marrow (BM)-derived MSCs and support of hematopoietic stem and progenitor cells (HSPCs), we examined the impact of 3R4F as a reference CSE, and HSPC-supporting function was determined. Both direct ex vivo and systemic in vivo MSC exposure to 3R4F resulted in impaired engraftment in a humanized mouse model. Furthermore, transcriptomic profile analysis showed significantly upregulated signaling pathways related to reactive oxygen species (ROS), inflammation, and aging in 3R4F-treated MSCs. 3R4F-exposed MSCs also developed impaired HSPC-supportive function, as confirmed by colony forming unit (CFU) assays and HSPC transplantation into NSG mice, and were further restored by the ROS inhibitor N-acetyl-l-cysteine (NAC). Finally, ingenuity pathway analysis revealed the activation of NLRP3 inflammasome signaling pathway in 3R4F-treated MSCs, and pretreatment with the NLRP3 inhibitor MCC950 rescued the HSPC-supporting ability of 3R4F-treated MSCs. In conclusion, these findings indicate that exposure to CSE reduces the HSPC-supportive function of MSCs by inducing robust ROS production and subsequent NLRP3 activation.
2024-01-17 | GSE253105 | GEO
Project description:Restoration of hematopoiesis upon bone marrow (BM) transplantation relies on the engraftment, expansion and function of transplanted hematopoietic stem cells (HSC). However, the number of donorâderived HSC typically remain much below that present in normal individuals and this likely limits and delays hematopoietic recovery. HSC depend on supportive stromal niches, however, whether the preâconditioning required for BM transplantation damages this stromal niche has not been evaluated extensively. Using mouse models, we now find that that BM stroma cells (BMSC) are severely and permanently damaged by preâconditioning. Transplantation of primary but not cultured BMSC quantitatively reconstitutes stroma function in vivo, which is mediated by a multipotent CD73+ CD105â Sca1+ BMSC subpopulation. BMSC coâtransplantation significantly ameliorates clinical side effects of BM transplantation and doubles expansion of functional, donorâ derived HSC, demonstrating the potential of stroma transplantation to improve HSC transplantation. Purified CD45âTer119âCD73+ CD105â and CD45âTer119âCD73+ CD105+ cells were directly sorted on lysis buffer with FACS Aria 2 in triplicate.
2016-09-01 | E-GEOD-79091 | biostudies-arrayexpress
Project description:The transcriptional regulator signal transducer and activator of transcription 3 (STAT3) has a well-established anti-inflammatory function in mature myeloid cells. This role, however, has precluded an understanding of STAT3 function in hematopoietic stem and progenitor cells (HSPCs), as Stat3 deletion in the hematopoietic system induces systemic inflammation, which can impact HSPC activity. Thus, novel approaches to uncouple STAT3 function in HSPCs from the effects of systemic inflammation are needed. To address this, we established competitive mixed bone marrow (BM) chimeric mice with CreER-mediated conditional Stat3 deletion in 20% of the hematopoietic compartment. After confirming a lack of detectable inflammation in mice with Stat3-deficient BM, we found that, in contrast to Stat3-sufficient controls, Stat3-deficient HSPCs had impaired hematopoietic activity and failed to undergo expansion in BM. Single-cell RNA sequencing of Lin-ckit+Sca1+ (LSKs) BM cells revealed an altered transcriptional landscape in Stat3-deficient hematopoietic stem cells (HSCs) and multipotent progenitors, as evidenced by the intrinsic activation of cell cycle, stress response, and interferon signaling pathways. Consistent with their deregulation, Stat3-deficient LSKs accumulated γH2AX over time. Following secondary BM transplantation, Stat3-deficient HSPCs failed to reconstitute peripheral blood effectively, indicating a severe functional defect in the HSC compartment. Our results reveal essential roles for STAT3 in HSCs and suggest the potential for using targeted synthetic lethal approaches with STAT3 inhibition to remove defective or diseased HSPCs.
2023-06-02 | GSE220466 | GEO
Project description:The identification of small molecules which either increase the number and/or enhance the activity of CD34+ hematopoietic stem and progenitor cells (HSPCs) during ex-vivo expansion has remained challenging. Applying an unbiased in vivo chemical screen in a transgenic (c-myb:EGFP) zebrafish embryo model, histone deacetylase inhibitors (HDACI) (valproic acid, resminostat and entinostat) were shown to significantly amplify the number of phenotypic hematopoietic precursors. The identified HDACIs were confirmed to significantly enhance also the expansion of human HSPCs during ex vivo treatment. Long-term functionality of ex vivo expanded human HSPCs was verified in a xenotransplantation model using NSG mice. However, the HDACI induced proliferation of HSPCs was associated with short-term functional changes. One of the identified hits, valproic acid (VPA), increased the adhesion capacity of CD34+ cells on primary mesenchymal stromal cells and reduced their chemokine-mediated migration capacity in vitro. In line with the reduced migratory potential in vitro, homing as well as early engraftment of VPA treated human CD34+ cells was significantly impaired in the xenotransplantation model. Our data confirms that HDACI treatment leads to a net expansion of HSPCs cells with long-term engraftment potential across different species. However impaired homing and short-term-engraftment has to be kept in mind when designing clinical transplantation protocols. In addition, our gene expression analysis (RNA-Seq) revealed expression of several genes that were altered in CD34+ cells by VPA treatment including cell adhesion molecules and Notch and wnt genes which has been shown to be involved in preservation of stem cell properties.
2017-11-01 | GSE90552 | GEO
Project description:We identified a novel HSC regulatory gene, MYCT1 (MYC target 1), that is selectively expressed in undifferentiated human HSPC and is critical for human HSC expansion and engraftment. MYCT1 knockdown (KD) in human fetal liver and cord blood (CB) HSPCs impaired expansion ex vivo and engraftment after transplantation, whereas restoring the compromised MYCT1 expression via lentiviral overexpression (OE) in cultured human CB HSPCs improved ex vivo expansion of the most undifferentiated human HSPCs (CD34+CD38-CD90+CD45RA-EPCR+ITGA3+) and enhanced their engraftment ability. We performed single cell RNAseq of uncultured human CB HSPCs, as well as 72 hours after MYCT1 KD and OE to identify MYCT1-regulated programs in HSC and understand the role of MYCT1 in governing human HSPC expansion and engraftment ability. Functional enrichment analysis linked the loss of MYCT1 in HLF+ HSCs to a profound dysregulation of multiple cellular programs essential for HSC stemness, such as oxidative phosphorylation or proteostasis, while MYCT1 OE had the opposite effect and showed closer similarity to uncultured HLF+ HSCs. These data indicate that the loss or gain of MYCT1 expression elicits major effects on programs regulating human HSC functional competence.
2024-04-08 | GSE232361 | GEO
Project description:ETS Variant 6 (ETV6) encodes an essential transcriptional repressor abundantly expressed in hematopoietic stem and progenitor cells (HSPCs), where it is required for adult hematopoiesis. Heterozygous pathogenic germline ETV6 variants are associated with Thrombocytopenia 5 (T5), a poorly-understood genetic condition predisposing to thrombocytopenia and hematologic malignancies. To elucidate how germline ETV6 variants impact the HSPC compartment and contribute to disease, we generated a knock-in mouse harboring an Etv6R355X loss-of-function variant, which represents the mouse equivalent to the T5-associated variant ETV6R359X. All HSPC subpopulations are present in the bone marrow (BM) of Etv6R355X/+ mice under homeostatic conditions; however, these animals exhibit subtle shifts in the proportions and/or numbers of specific progenitor subtypes. To examine whether the Etv6R355X/+ mutation impacts HSPC function, we carried out serial competitive transplantation and observed that Etv6R355X/+ lineagesca1+cKit+ (LSK) cells exhibit significantly impaired reconstitution, with near complete failure to repopulate irradiated-recipients by the tertiary transplant. Mechanistic studies incorporating CUT&RUN, ATAC-Seq and Hi-C identify ETV6 binding at inflammatory gene loci, including those within the TNF pathway in Etv6+/+ HSPCs, the mouse BM-progenitor derived HPC5 cell line, and G-CSF-mobilized human CD34+ cells. Further, single-cell RNA sequencing of mouse LSK cells isolated six-weeks post-competitive transplantation reveals upregulation of inflammatory gene pathways. Corroborating these findings, we observe significantly increased production of TNF by Etv6R355X/+ versus Etv6+/+ HSPCs post-transplantation. From these studies, we conclude that ETV6 represses inflammatory response genes within HSPCs under conditions of hematopoietic stress, and that this mechanism may be critical to sustain HSPC function. Functionally, Etv6R355X/+ bone marrow (BM) cells exhibited reduced colony formation in later rounds of serial replating, while Etv6R355X/+ LSKs exhibited significantly impaired engraftment in serial competitive transplantation. RNA-seq of Etv6R355X/+ LSKs and/or sort-purified HSPCs revealed upregulation of genes associated with an active stem cell phenotype and pro-inflammatory signaling, including increased expression of the cytokine Tnf. CUT&RUN of Etv6+/+ LSKs and HPC5 hematopoietic progenitors showed that ETV6 binds to the Tnf locus at regions of open chromatin, while CUT&RUN of human CD34+ cells identified ETV6 targets enriched in TNF signaling. In line with these findings, Etv6R355X/+ HSPCs exhibited increased cell-cycling and TNF expression post-transplantation. Finally, genetic deletion of Tnf restored the serial replating capacity of Etv6R355X/+ BM cells.
2023-08-24 | GSE213596 | GEO