ABSTRACT: CXCL8 and CXCR1 Remodel the Vascular Niche to Promote Hematopoietic Stem and Progenitor Cell Colonization and Engraftment [huvec CXCL8 vs control]
Project description:CXCL8 and CXCR1 Remodel the Vascular Niche to Promote Hematopoietic Stem and Progenitor Cell Colonization and Engraftment [wt vs kdrl:cxcr1]
Project description:The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Interactions between the niche and stem cells have been difficult to track, but recent advances marking fluorescent HSPCs have allowed exquisite visualization in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Sinusoidal endothelial cells interact closely with HSPCs as they colonize this niche. Here we show that the chemokine cxcl8 and its receptor, cxcr1, are abundantly expressed by zebrafish endothelial cells and we identify cxcl8/cxcr1 signaling as a positive regulator of HSPC colonization using genetic gain- and loss-of-function techniques. Single-cell tracking experiments demonstrated that this effect is due to an increase in HSPC “cuddling” by endothelial cells, thereby increasing CHT residency time and allowing more HSPC cell divisions to occur. Enhanced cxcl8/cxcr1 signaling was associated with an increase in the volume of the CHT and induction of cxcl12a expression, favoring HSPC colonization. Finally, using parabiotic zebrafish, we show that cxcr1 acts stem cell non-autonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that cxcl8/cxcr1 signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation.
Project description:The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Interactions between the niche and stem cells have been difficult to track, but recent advances marking fluorescent HSPCs have allowed exquisite visualization in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Sinusoidal endothelial cells interact closely with HSPCs as they colonize this niche. Here we show that the chemokine cxcl8 and its receptor, cxcr1, are abundantly expressed by zebrafish endothelial cells and we identify cxcl8/cxcr1 signaling as a positive regulator of HSPC colonization using genetic gain- and loss-of-function techniques. Single-cell tracking experiments demonstrated that this effect is due to an increase in HSPC “cuddling” by endothelial cells, thereby increasing CHT residency time and allowing more HSPC cell divisions to occur. Enhanced cxcl8/cxcr1 signaling was associated with an increase in the volume of the CHT and induction of cxcl12a expression, favoring HSPC colonization. Finally, using parabiotic zebrafish, we show that cxcr1 acts stem cell non-autonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that cxcl8/cxcr1 signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation.
Project description:Pluripotent stem cells (PSC) represent an alternative source of hematopoietic stem cells (HSCs). Clinical translation is impeded by limited engraftment of human (h)PSC-multipotent progenitor cells (MPP). This barrier suggests that additional cues are required for definitive hematopoiesis. We hypothesized that vascular niche producing Notch ligands Jagged-1 (JAG1) and Delta-like ligand-4 (DLL4) would drive definitive hematopoiesis. To test our hypothesis, hes2 human embryonic stem cells (hESC) 2 and Macaca nemestrina (Mn) iPSC line-7 were differentiated with cytokines ± endothelial cells (EC), which express JAG1 and DLL4. EC co-culture supported emergence of 8-fold more CD34+CD45+ cells compared to co-culture with cytokines ± ECs with JAG1 or DLL4 knockdown. EC-induced cells exhibit Notch activation and express HSC-specific targets of Notch signaling RUNX1 and GATA2. EC-induced PSC-MPP engraft at a higher level in NSG mice compared to cytokine-induced cells (10% >5 months), and selection increased engraftment (30%). Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction is similar to levels achieved for cord blood MPP and up to 20-fold higher than hPSC-MPP engraftment. Our findings identify a previously underappreciated role for endothelial Notch ligands in PSC definitive hematopoiesis and production of long-term engrafting CD34+ cells and suggest they are critical for HSC emergence. Transcriptome sequencing of Macaca nemestrina (Mn) iPSCs
Project description:CXCL8 is produced by many cell types including epithelial, endothelial, fibroblasts and macrophages in response to TLR recognition of microbe-associated molecular patterns (MAMPs) or inflammatory cytokines and recruits phagocytes from the vasculature to sites of infection via interaction with its cognate receptors CXCR1 and CXCR2. In the intestine, CXCL8 has been demonstrated to participate in the migration of neutrophils across the epithelium during acute inflammation. Given the well-recognized role of CXCL8 as an initiator of inflammation and the constant presence of commensal bacteria in the intestinal tract, we hypothesized that in the intestinal epithelium, CXCL8 might be secreted in a vectorial fashion depending on the location and type of stimulus as a mechanism to maintain homeostasis. In addition, we hypothesized that the CXCR1 receptor might control specific functions in polarized IECs depending on its location. We tested these hypotheses using microarray gene expression profiling of IL-8 treated and mock-treated Caco-2 cell lines This study was set up according to a one-treatment, one-control design. It contains individual transcriptional profiles from 3 IL-8-treated and 3 buffer control-treated samples. In total, this study includes data from 3 Caco-2 samples x 2 treatments=6 arrays.
Project description:Generation of abundant engraftable hematopoietic cells from autologous tissues promises new therapies for hematologic diseases. Differentiation of pluripotent stem cells into hematopoietic cells results in emergence of cells that have poor engraftment potential. To circumvent this hurdle, we have devised a vascular niche model to phenocopy the developmental microenvironment of hemogenic cells thereby enabling direct transcriptional reprogramming of human endothelial cells (ECs) into hematopoietic cells. In this approach, transduction of human umbilical vein ECs (HUVECs) or adult human dermal microvascular ECs (hDMECs) with transcription factors (TFs), FOSB, GFI1, RUNX1, and SPI1 (FGRS) and induction with a instructive vascular niche feeder layer in a xenobiotic- and serum-free microenvironment results in generation of long-term engraftable hematopoietic multilineage progenitors (rEC-HMLPs). The rEC-HMLPs had robust proliferative and multilineage colony forming units (CFU) potential, including granulocytic/monocytic, megakaryocytic, erythroid and lymphoid lineages. When transplanted, hDMEC-derived rEC-HMLPs were capable of long-term multilineage primary and secondary hematopoietic engraftment. A subset of engrafted rEC-HMLPs phenotypically and functionally resembled cord blood cells. By conditionally expressing the FGRS TFs, we further optimized reprogramming of ECs into rEC-HMLPs manifesting features of self-renewing multi-potent progenitor populations (MPPs). Our approach replicates critical aspects of hematopoietic development and essential role of vascular niche induction in orchestrating hematopoietic specification and may prove useful for engineering autologous engraftable hematopoietic cells for treatment of inherited and acquired blood disorders. . Transcriptome sequencing of rEC-HMLPs, hDMECs, HUVECs and other cell types
Project description:Pro-inflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution remain yet to be fully elucidated. Previously we identified a critical role for combined constitutive JAK/STAT and aberrant NF-kB pro-inflammatory signaling in myelofibrosis development. Using single-cell transcriptional and cytokine-secretion studies of primary MF patient cells and two separate murine models of myelofibrosis, we extend this previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. MF patient hematopoietic stem/progenitor cells are enriched in a CXCL8/CXCR2 gene signature and display dose-dependent proliferation and fitness in response to exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway in MF patients at risk for continued fibrotic progression.
Project description:Pro-inflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution remain yet to be fully elucidated. Previously we identified a critical role for combined constitutive JAK/STAT and aberrant NF-kB pro-inflammatory signaling in myelofibrosis development. Using single-cell transcriptional and cytokine-secretion studies of primary MF patient cells and two separate murine models of myelofibrosis, we extend this previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. MF patient hematopoietic stem/progenitor cells are enriched in a CXCL8/CXCR2 gene signature and display dose-dependent proliferation and fitness in response to exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway in MF patients at risk for continued fibrotic progression.
Project description:CXCL8 is produced by many cell types including epithelial, endothelial, fibroblasts and macrophages in response to TLR recognition of microbe-associated molecular patterns (MAMPs) or inflammatory cytokines and recruits phagocytes from the vasculature to sites of infection via interaction with its cognate receptors CXCR1 and CXCR2. In the intestine, CXCL8 has been demonstrated to participate in the migration of neutrophils across the epithelium during acute inflammation. Given the well-recognized role of CXCL8 as an initiator of inflammation and the constant presence of commensal bacteria in the intestinal tract, we hypothesized that in the intestinal epithelium, CXCL8 might be secreted in a vectorial fashion depending on the location and type of stimulus as a mechanism to maintain homeostasis. In addition, we hypothesized that the CXCR1 receptor might control specific functions in polarized IECs depending on its location. We tested these hypotheses using microarray gene expression profiling of IL-8 treated and mock-treated Caco-2 cell lines