scholarly journals Oncostatin M Is a Novel Niche Factor That Restrains Hematopoietic Stem Cell Mobilization in Response to G-CSF and CXCR4 Antagonist Plerixafor

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4469-4469
Author(s):  
Kavita Bisht ◽  
Crystal McGirr ◽  
Kylie A Alexander ◽  
Whitney Fleming ◽  
Natalie Sims ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Stromal Cells ◽  
Receptor Gene ◽  
Fold Increase ◽  
Oncostatin M ◽  
Distribution Analysis ◽  
Mesenchymal Progenitor Cells ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem

Granulocyte colony-stimulating factor (G-CSF) and the CXCR4 chemokine receptor antagonist Plerixafor are used to mobilize hematopoietic stem and progenitor cells (HSPC). However, in the autologous setting, prior chemotherapy and radiotherapy impair HSPC mobilizing response to G-CSF and Plerixafor thereby precluding autologous HSC transplantation. We now demonstrate in mice that oncostatin M (OSM) production in the bone marrow (BM) is dramatically increased in response to G-CSF and that OSM counteracts the mobilizing effect of G-CSF and Plerixafor indirectly through the BM stroma. Indeed, by immunohistochemistry, a 4-day G-CSF treatment strongly increased OSM protein expression in cell clusters containing F4/80+ macrophages in the endosteal and central region of the mobilized BM. OSM concentration was 7-fold higher in BM fluids of G-CSF mobilized mice compared to control mice as measured by ELISA. To investigate OSM effects on HSPC mobilization, C57BL/6 mice (WT) and mice lacking the OSM receptor gene (OSMR-/-) were treated with 125 μg/kg human G-CSF bidaily for 2-6 days and/or Plerixafor (10 mg/kg) one hour before harvest. Absence of OSMR increased HSPC mobilization with a 4-fold increase in colony forming cells (CFC), phenotypic Lin-Kit+Sca1+ HSPC (LKS+ cells) and LKS+Flt3-CD48-CD150+ HSC into the blood at days 2 and 4 of G-CSF treatment and increased accumulation of HSPC and HSC in the spleen of OSMR-/- mice at day 6 of G-CSF treatment. In a competitive repopulation assay with serial dilutions of mobilized blood, deletion of OSMR gene enhanced ≈4-fold mobilization of functional reconstituting HSC in response to G-CSF as shown by Poisson's distribution analysis. HSPC mobilization in response to Plerixafor was also significantly enhanced with a doubling of CFC, HSPC and HSC into the blood of OSMR-/- mice compared to WT. OSMR gene deletion also significantly enhanced HSPC mobilization (3-fold) in response to G-CSF plus Plerixafor combination, the most effective mobilization regimen currently approved. To further demonstrate that endogenous OSM restrains HSPC mobilization in response to G-CSF, we produced a recombinant mouse OSM-trap, a dimeric protein chimera made of the extracellular domain of mouse OSMR and mouse gp130 fused with a mutant Fc fragment of mouse IgG2a. Co-administration of this OSM-trap together with G-CSF also increased HSPC and HSC mobilization into the blood in response to G-CSF compared to mice treated with G-CSF and control trap construct. This negative effect of endogenous OSM on HSPC mobilization was indirectly mediated by BM stromal cells. qRT-PCR on BM myeloid and stromal cells sorted from steady-state or mobilized mice showed that Osm mRNA is mostly expressed by CD11b+F4/80+CD169+VCAM1+ macrophages and CD11b+F4/80-Ly6G+ neutrophils. In sharp contrast, Osmr mRNA was undetectable in any sorted BM leukocyte population, but abundantly expressed by CD45-Lin-CD31+Sca1+CD105+ endothelial and CD45-Lin-CD31-CD51+PDGFRα+/-Sca-1+/- mesenchymal progenitor cells. OSMR protein was expressed by vascular beds in the BM as detected by IHC. Confirming the indirect effect of OSM, we found that recombinant OSM had no effect on HSPC chemotaxis in response to CXCL12, or HSPC adhesion to immobilized VCAM-1 or selectins in vitro. However, HSPC freshly isolated from the BM of OSMR-/- mice had enhanced chemotaxis in response to CXCL12 compared to HSPC isolated from WT mice. In conclusion, G-CSF increases OSM expression in the BM, and this appears to restrain HSPC mobilization in response to G-CSF via OSMR-expressing endothelial and mesenchymal cells that decreases HSPC responsiveness to CXCL12 gradient. Disclosures Winkler: GlycoMimetics: Patents & Royalties. Levesque:GlycoMimetics: Equity Ownership.

Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4040-4040
Author(s):  
Szabolcs Fatrai ◽  
Simon M.G.J. Daenen ◽  
Edo Vellenga ◽  
Jan J. Schuringa
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Fold Increase ◽  
Marrow Stromal Cells ◽  
Muc1 Expression ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.

Disruption of CXCR4 Utilizing AMD3100 Bypasses Mobilization Deficiency Exhibited by CD26−/− Mice and Results in Efficient Mobilization of Myeloid Progenitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3492-3492
Author(s):  
Laura A. Paganessi ◽  
Andrew L. Walker ◽  
Stephanie A. Gregory ◽  
Henry C. Fung ◽  
Kent W. Christopherson
Keyword(s):  
Bone Marrow ◽  
Blood Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Genetically Engineered Mice ◽  
Colony Forming Units ◽  
Myeloid Progenitors

Abstract The exopeptidase CD26 (also known as DPPIV/dipeptidylpeptidase IV) cleaves dipeptides from the N-terminus of proteins that contain the required X-Pro or X-Ala motif. We have previously reported that inhibition or loss of CD26 activity results in a deficiency in normal granulocyte-colony stimulating factor (G-CSF) induced mobilization, suggesting that CD26 is a necessary component of mobilization (Christopherson, et al Blood 2003 and Christopherson, et al Exp Hematol 2003). The chemokine CXCL12 (SDF-1, stromal cell derived factor-1) contains the appropriate recognition sequence for CD26 induced cleavage. This combined with the importance of CXCL12 in the trafficking of hematopoietic stem and progenitor cells (HSC/HPC) suggests CXCL12 as a likely functional target of CD26 during G-CSF induced mobilization. For this reason we therefore decided to investigate whether genetically engineered mice lacking CD26 (CD26−/−) could be mobilized utilizing the CXCR4 antagonist, AMD3100. To evaluate this, ten week old C57BL/6 and CD26−/− mice (also on a C57BL/6 background) received a single subcutaneous injection of AMD3100 (1mg/1kg). One hour following injection the mice were euthanized by CO2 inhalation. Peripheral blood was then obtained by heart stick with a 1.2 ml syringe containing EDTA as an anticoagulant. A complete blood count was taken for each peripheral blood sample. Following red blood cell lysis, cells were plated for myeloid colony formation in a standard 1% methylcellulose colony assay containing the appropriate cytokines. Following 7 days of incubation at 5% O2, 5% CO2 and 37°C plates were scored for colony-forming units-granulocyte macrophage (CFU-GM), burst-forming units-erythroid (BFU-E), and colony-forming units-granulocyte, erythroid, macrophage, and megakaryocytic (CFU-GEMM). Data is presented as the number of colonies per femur for the bone marrow and as the number of colonies per ml of whole blood for the peripheral blood. AMD3100 treatment resulted in an increase in white blood cell (WBC) counts from 5.05±0.48 × 106/ml in untreated mice to 10.21±0.88×106/ml in treated mice (p≤0.01). An increase in WBC counts was also observed during AMD3100 treatment in CD26−/− mice from 7.77±1.28×106/ml in untreated mice to 16.7 ±2.11 × 106/ml in treated mice (p<0.01). AMD3100 treatment resulted in an increase in circulating myeloid progenitors in the peripheral blood of C57BL/6 and CD26−/− mice as compared to untreated C57BL/6 and CD26−/− mice respectively (p≤0.01). Specifically, a 2.38, 3.75, 12.33 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of C57BL/6 mice after treatment. A 2.63, 5.48, 14.29 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of CD26−/− mice after treatment. Existing pre-clinical and clinical data suggest that the CXCR4 antagonist, AMD3100, rapidly mobilizes hematopoietic progenitor cells from the bone marrow into the periphery. The results presented here provide pre-clinical evidence that disruption of the interaction between the CXCR4 chemokine receptor and CXCL12, via sub-cutaneous injection of AMD3100, mobilizes significant numbers of myeloid progenitors in mice, even in the absence of CD26. These results support the notion that CD26 is downstream of G-SCF treatment. Additionally, these results support the potential use of AMD3100 to treat patients that may have an altered ability to respond to G-CSF treatment as a result of a reduction or loss in CD26 activity. Future studies are warranted to evaluate potential variations in CD26 levels or activity in the general population, in differing patient populations, and during different treatment regimens.

Mesenchymal Stem and Progenitor Cells In the Mouse Bone Marrow Lack Expression of CD44.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2581-2581
Author(s):  
Hong Qian ◽  
Mikael Sigvardsson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Colony Forming Unit ◽  
Stem And Progenitor Cells ◽  
Cell Fractions

Abstract Abstract 2581 The bone marrow (BM) microenvironment consists of a heterogeneous population including mesenchymal stem cells and as well as more differentiated cells like osteoblast and adipocytes. These cells are believed to be crucial regulators of hematopoetic cell development, however, so far, their identity and specific functions has not been well defined. We have by using Ebf2 reporter transgenic Tg(Ebf2-Gfp) mice found that CD45−TER119−EBF2+ cells are selectively expressed in non-hematopoietic cells in mouse BM and highly enriched with MSCs whereas the EBF2− stromal cells are very heterogenous (Qian, et al., manuscript, 2010). In the present study, we have subfractionated the EBF2− stromal cells by fluorescent activated cell sorter (FACS) using CD44. On contrary to previous findings on cultured MSCs, we found that the freshly isolated CD45−TER119−EBF2+ MSCs were absent for CD44 whereas around 40% of the CD45−TER119−EBF2− cells express CD44. Colony forming unit-fibroblast (CFU-F) assay revealed that among the CD45−LIN−EBF2− cells, CD44− cells contained generated 20-fold more CFU-Fs (1/140) than the CD44+ cells. The EBF2−CD44− cells could be grown sustainably in vitro while the CD44+ cells could not, suggesting that Cd44− cells represents a more primitive cell population. In agreement with this, global gene expression analysis revealed that the CD44− cells, but not in the CD44+ cells expressed a set of genes including connective tissue growth factor (Ctgf), collagen type I (Col1a1), NOV and Runx2 and Necdin(Ndn) known to mark MSCs (Djouad et al., 2007) (Tanabe et al., 2008). Furthermore, microarray data and Q-PCR analysis from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca7,-8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the EBF2−CD44− cells, compared to the CD44+ cells indicating a relatively quiescent state of the CD44− cells ex vivo. This was confirmed by FACS analysis of KI67 staining. Furthermore, our microarray analysis suggested high expression of a set of hematopoietic growth factors and cytokines genes including Angiopoietin like 1, Kit ligand, Cxcl12 and Jag-1 in the EBF2−CD44− stromal cells in comparison with that in the EBF2+ or EBF2−CD44+ cell fractions, indicating a potential role of the EBF2− cells in hematopoiesis. The hematopoiesis supporting activity of the different stromal cell fractions were tested by in vitro hematopoietic stem and progenitor assays- cobblestone area forming cells (CAFC) and colony forming unit in culture (CFU-C). We found an increased numbers of CAFCs and CFU-Cs from hematopoietic stem and progenitor cells (Lineage−SCA1+KIT+) in culture with feeder layer of the EBF2−CD44− cells, compared to that in culture with previously defined EBF2+ MSCs (Qian, et al., manuscript, 2010), confirming a high capacity of the EBF2−CD44− cells to support hematopoietic stem and progenitor cell activities. Since the EBF2+ cells display a much higher CFU-F cloning frequency (1/6) than the CD44−EBF2− cells, this would also indicate that MSCs might not be the most critical regulators of HSC activity. Taken together, we have identified three functionally and molecularly distinct cell populations by using CD44 and transgenic EBF2 expression and provided clear evidence of that primary mesenchymal stem and progenitor cells reside in the CD44− cell fraction in mouse BM. The findings provide new evidence for biological and molecular features of primary stromal cell subsets and important basis for future identification of stage-specific cellular and molecular interaction pathways between hematopoietic cells and their cellular niche components. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Periostin Supports Hematopoietic Stem/Progenitor Cells and Niche-Dependent Myeloblastoma Cells In Vitro

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1494-1494 ◽  
Author(s):  
Akio Maekawa ◽  
Natsumi Hasegawa ◽  
Satowa Tanaka ◽  
Leo Matsubara ◽  
Azusa Imanishi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Line ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Integrin Αvβ3 ◽  
Serum Starvation ◽  
Hematopoietic Stem

Abstract Periostin (POSTN), the fasciclin family extracellular matrix protein also known as osteoblast-specific factor 2 (OSF-2), was previously reported to be required for optimal B lymphopoiesis in vitro. Now, our study first demonstrates the proof that POSTN might be a bona fide niche factor for both normal and malignant myelopoiesis, indicating that it is a niche molecule for hematopoietic stem cells and diverse hematopoietic precursor cells. The Mediator, composed of about 31 subunits, is a master transcriptional coregulator complex that is essential for global transcription governed by RNA polymerase II. Among the Mediator subunits, MED1 acts as a specific coactivator for activators that include nuclear receptors and GATA1. We previously reported that Med1−/− mouse embryonic fibroblasts (MEFs) have a decreased capability to support hematopoietic stem/progenitor cells (HSPCs) relative to wild-type MEFs in vitro, and that the attenuated expression of full-length osteopontin and FGF7 in Med1−/− MEFs is responsible for the observed phenotype. The microarray analyses, showing that the expression of POSTN was also suppressed in Med1−/− MEFs, prompted us to study the role for POSTN in support of both normal and malignant HSPCs in our in vitro niche model. When bone marrow (BM) cells were cocultured with mitomycin C-treated Med1+/+ MEFs, or OP-9 or MS-5 BM stromal cells, in the presence of anti-POSTN blocking antibody, the mitogenicity and growth of BM cells were attenuated. The number of long-term culture-initiating cells (LTC-ICs), i.e., number of both granulo-monocytic and erythroid colonies, was also decreased. When BM cells were cocultured with Med1-/- MEFs in the presence of recombinant POSTN, the mitogenicity and growth of BM cells and the number of LTC-ICs were restored. These results suggest that POSTN mediates mitogenicity of BM cells and HSPCs support. The MB-1 myeloblastoma cell line, originally established from a patient with myeloid crisis chronic myeloid leukemia, is a mesenchymal stromal cell-dependent cell line. These cells are unique in that they grow by forming cobblestone areas in the presence of niche cells but die of apoptosis when detached from stromal cells, thus faithfully conforming to a stochastic model of leukemic stem cells in vitro. Intriguingly, antibody-mediated blockage of stromal cells-derived POSTN markedly reduced the mitogenicity and growth, as well as the cobblestone formation, a leukemic stem cell feature, of MB-1 myeloblastoma cells. Therefore, it appears that niche cell-derived POSTN supports niche-dependent MB-1 myeloblastoma cells. While POSTN was expressed both in BM cells and variably in different BM stromal cells, expression in the latter cells was markedly increased by tactile interaction with hematopoietic cells. Specifically, POSTN was robustly induced 6 hours after BM stromal cells were cocultured with BM cells or MB-1 myeloblastoma cells, and the induction sustained for as long as 24 hours. However, POSTN expression was not enhanced when BM cells were cocultured but physically separated from MS-5 or OP-9 cells using transwell culture wells. Therefore, the major source of POSTN in the coculture appears to be the BM stromal cells associated with hematopoietic cells. The receptor for POSTN, integrin αvβ3, was expressed abundantly in BM stromal cells. Although β3 mRNA was especially prominent in both BM cells and MB-1 cells, in accordance with a previous report that integrin β3/CD61 marks HSPCs, western blot analysis showed that αv and β3 expression levels were below the detection level on BM cells. Hence, integrin αvβ3 is scarce on BM cells compared to BM stromal cells, although it does not exclude the possibility that functional integrin αvβ3 might be enriched on HSPCs as suggested previously. When an excess amount of exogenous POSTN was added to MS-5 or OP-9 BM stromal cells after 24-h serum starvation, FAK (the immediate target of integrin αvβ3) and MAP kinases ERK1/ERK2 (the intermediate hub of various intracellular signals) were robustly phosphorylated as early as 10 min, and the phosphorylation was sustained for over 60 min. Thus, POSTN effectively activates integrin αvβ3 and subsequent intracellular signaling in BM stromal cells. These results suggest that stromal cell POSTN supports both normal HSPCs and leukemia-initiating cells in vitro, at least in part, indirectly by acting on stromal cells in an autocrine or paracrine manner. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mesenchymal stem cell perspective: cell biology to clinical progress

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Mark F. Pittenger ◽  
Dennis E. Discher ◽  
Bruno M. Péault ◽  
Donald G. Phinney ◽  
Joshua M. Hare ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Stromal Cells ◽  
Cell Biology ◽  
Cellular Therapy ◽  
Human Mscs ◽  
Intercellular Signaling ◽  
Mesenchymal Progenitor Cells ◽  
A Cell

AbstractThe terms MSC and MSCs have become the preferred acronym to describe a cell and a cell population of multipotential stem/progenitor cells commonly referred to as mesenchymal stem cells, multipotential stromal cells, mesenchymal stromal cells, and mesenchymal progenitor cells. The MSCs can differentiate to important lineages under defined conditions in vitro and in limited situations after implantation in vivo. MSCs were isolated and described about 30 years ago and now there are over 55,000 publications on MSCs readily available. Here, we have focused on human MSCs whenever possible. The MSCs have broad anti-inflammatory and immune-modulatory properties. At present, these provide the greatest focus of human MSCs in clinical testing; however, the properties of cultured MSCs in vitro suggest they can have broader applications. The medical utility of MSCs continues to be investigated in over 950 clinical trials. There has been much progress in understanding MSCs over the years, and there is a strong foundation for future scientific research and clinical applications, but also some important questions remain to be answered. Developing further methods to understand and unlock MSC potential through intracellular and intercellular signaling, biomedical engineering, delivery methods and patient selection should all provide substantial advancements in the coming years and greater clinical opportunities. The expansive and growing field of MSC research is teaching us basic human cell biology as well as how to use this type of cell for cellular therapy in a variety of clinical settings, and while much promise is evident, careful new work is still needed.

Coagulation Factor Thrombin Regulates Hematopoietic Stem and Progenitor Cell Egress and Mobilization Via PAR-1 & CXCR4 Upregulation, SDF-1 Secretion and EPCR Shedding

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2341-2341 ◽  
Author(s):  
Shiri Gur-Cohen ◽  
Tomer Itkin ◽  
Aya Ludin ◽  
Orit Kollet ◽  
Karin Golan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Progenitor Cell ◽  
Single Injection ◽  
Hematopoietic Stem ◽  
Epcr Shedding

Abstract Abstract 2341 Hematopoietic stem and progenitor cell (HSPC) egress from the bone marrow (BM) to the circulation is tightly regulated and is accelerated during stress conditions. The G-protein-coupled receptor protease-activated receptor-1 (PAR-1) and its activator thrombin play an important role in coagulation following injury and bleeding. We report that a single injection of thrombin induced rapid HSPC mobilization within one hour, increasing circulating leukocytes, predominantly CFU-C and primitive Lin−/Sca-1+/c-Kit+ (SKL) progenitor cells. This rapid mobilization was preceded by a dramatic decrease of SDF-1 (CXCL12) in BM stromal cells, including rare Nestin+ mesenchymal stem cells (MSC) which functionally express PAR-1 and release SDF-1. Thrombin injection also increased expression of PAR-1 and CXCR4 by BM HSPC. These results suggest involvement of the coagulation cascade of thrombin & PAR-1 in rapid SDF-1 secretion from niche supporting BM stromal cells as part of host defense and repair mechanisms. Administration of a PAR-1 specific antagonist (SCH79797) upregulated BM SDF-1 levels and significantly reduced the amounts of circulating CFU-C and primitive SKL progenitor cells. In vitro stimulation of BM mononuclear cells with thrombin for 1 hour led to increased CXCR4 expression by Lin−/c-Kit+ progenitors, accompanied by enhanced spontaneous and SDF-1 induced migration. Of note, specific PAR-1 inhibition in vitro significantly reduced SDF-1-directed migration of Lin-/c-Kit+ progenitors. Mechanistically, we found that thrombin - activated PAR-1 induced the downstream p38 MAPK and eNOS (nitric oxide synthase) signaling pathways. Long term repopulating hematopoietic stem cells (HSC) in murine BM highly express endothelial protein C receptor (EPCRhigh) (Balazs & Mulligan et al Blood 2006; Kent & Eaves et al Blood 2009). EPCR is expressed primarily on endothelial cells (EC) and has anti coagulation and anti inflammatory roles. Surface EPCR expression on EC is downregulated by many factors, including PAR-1 activation by thrombin, a process which is termed shedding and is not fully understood. Importantly, we found that over 90% of BM CD45+/EPCRhigh long-term HSC express PAR-1 and that circulating primitive HSPC in the blood and spleen lack EPCRhigh expression. In addition, in-vivo thrombin administration downregulated EPCR from BM HSC via eNOS signaling, thus allowing the release of stem cells from their BM microenvironment anchorage to the circulation. Correspondingly, in eNOS deficient mice, thrombin failed to induce PAR-1 upregulation, EPCR shedding, and HSPC mobilization. Recently, we reported that the antioxidant NAC inhibits G-CSF induced mobilization (Tesio & Lapidot et al Blood 2011). Co-administration of G-CSF with NAC prevented PAR-1 upregulation, concomitantly with reduced HSPC mobilization and increased levels of EPCRhigh HSC in the BM. Treatment of PAR-1 antagonist with G-CSF inhibited PAR-1 and CXCR4 upregulation on BM leukocytes and immature Lin−/c-Kit+ cells accompanied by increased levels of BM EPCRhigh HSC and reduced HSPC mobilization. Tissue factor (TF) is the main initiator of the coagulation system via the formation of an enzymatic “prothrombinase complex” that converts prothrombin to active thrombin. Unexpectedly, we found a unique structure of cell clusters expressing TF, located preferentially in the trabecular-rich area of the femoral metaphysis in murine bone tips, a region highly exposed to osteoclast/osteoblast bone remodeling. In vitro, immature osteoclasts exhibited increased TF expression in cell fusion areas, suggesting that in vivo osteoclast maturation activates the coagulation thrombin/PAR-1 axis of HSPC migration to the circulation. Finally, mimicking bacterial infection a single injection of Lipopolysaccharide (LPS), rapidly and systemically upregulated TF in the murine BM. LPS treatment prompted an increase in thrombin generation and subsequently HSPC mobilization, which was blocked by the PAR-1 antagonist. In conclusion, our study reveals a new role for the coagulation signaling axis, which acts on both hematopoietic and stromal BM cells to regulate steady state HSPC egress and enhanced mobilization from the BM. This thrombin/PAR-1 signaling cascade involves SDF-1/CXCR4 interactions, immature osteoclast TF activity, Nestin+/PAR-1+ MSC secretion of SDF-1 and EPCR shedding from hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Transcription factor Tlx1 marks a subset of lymphoid tissue organizer-like mesenchymal progenitor cells in the neonatal spleen

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yuta Ueno ◽  
Keiko Fujisaki ◽  
Shoko Hosoda ◽  
Yusuke Amemiya ◽  
Shogo Okazaki ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Lymphoid Tissue ◽  
Stromal Cell ◽  
Lineage Tracing ◽  
Mesenchymal Progenitor Cells ◽  
Reticular Cells

AbstractThe spleen is comprised of spatially distinct compartments whose functions, such as immune responses and removal of aged red blood cells, are tightly controlled by the non-hematopoietic stromal cells that provide regionally-restricted signals to properly activate hematopoietic cells residing in each area. However, information regarding the ontogeny and relationships of the different stromal cell types remains limited. Here we have used in vivo lineage tracing analysis and in vitro mesenchymal stromal cell assays and found that Tlx1, a transcription factor essential for embryonic spleen organogenesis, marks neonatal stromal cells that are selectively localized in the spleen and retain mesenchymal progenitor potential to differentiate into mature follicular dendritic cells, fibroblastic reticular cells and marginal reticular cells. Furthermore, by establishing a novel three-dimensional cell culture system that enables maintenance of Tlx1-expressing cells in vitro, we discovered that signals from the lymphotoxin β receptor and TNF receptor promote differentiation of these cells to express MAdCAM-1, CCL19 and CXCL13, representative functional molecules expressed by different subsets of mature stromal cells in the spleen. Taken together, these findings indicate that mesenchymal progenitor cells expressing Tlx1 are a subset of lymphoid tissue organizer-like cells selectively found in the neonatal spleen.

scholarly journals EBF1 As a Novel Regulator of Bone Marrow Mesenchymal Stromal Progenitors

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 96-96
Author(s):  
Marta Derecka ◽  
Senthilkumar Ramamoorthy ◽  
Pierre Cauchy ◽  
Josip Herman ◽  
Dominic Grun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Myeloid Cells ◽  
Bone Marrow Niche ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Hematopoietic stem and progenitor cells (HSPC) are in daily demand worldwide because of their ability to replenish entire blood system. However, the in vitro expansion of HSPC is still a major challenge since the cues from bone marrow microenvironment remain largely elusive. Signals coming from the bone marrow niche, and specifically mesenchymal stem and progenitor cells (MSPC), orchestrate maintenance, trafficking and stage specific differentiation of HSPCs. Although, it is generally accepted that MSPCs are essential for hematopoietic homeostasis and generating multiple types of stromal cells, the exact transcriptional networks regulating MSPCs are not well established. Early B-cell factor 1 (Ebf1) has been discovered as lineage-specific transcription factor governing B lymphopoiesis. Additionally, it has been shown to play important role in differentiation of adipocytes, which are a niche component supporting hematopoietic regeneration. Thus, in this study we seek to examine if Ebf1 has an alternative function in non-hematopoietic compartment of bone marrow, specifically in mesenchymal stromal cells that maintain proper hematopoiesis. Here, we identified Ebf1 as new transcription regulator of MSPCs activity. Mesenchymal progenitors isolated from Ebf1-/- mice show diminished capacity to form fibroblasticcolonies (CFU-F) indicating reduced self-renewal. Moreover, cells expanded from these colonies display impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. In order to test how this defective MSPCs influence maintenance of HSPCs, we performed long-term culture-initiating cell assay (LTC-IC). After 5 weeks of co-culture of Ebf1-deficient stromal cells with wild type HSPCs we could observe significantly decreased number of cobblestone and CFU colonies formed by primitive HSPCs, in comparison to co-cultures with control stromal cells. Furthermore, in vivo adoptive transfers of wild type HSPCs to Ebf1+/- recipient mice showed a decrease in the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Additionally, Prx1-Cre-mediated deletion of Ebf1 specifically in MSPCs of mice leads to reduced frequency and numbers of HSPCs and myeloid cells in the bone marrow. These results confirm that mesenchymal stromal cells lacking Ebf1 render insufficient support for HSPCs to sustain proper hematopoiesis. Interestingly, we also observed a reduced ability of HSPCs sorted from Prx1CreEbf1fl/fl mice to form colonies in methylcellulose, suggesting not only impaired maintenance but also hindered function of these cells. Moreover, HSPCs exposed to Ebf1-deficient niche exhibit changes in chromatin accessibility with reduced occupancy of AP-1, ETS, Runx and IRF motifs, which is consistent with decreased myeloid output seen in Prx1CreEbf1fl/fl mice. These results support the hypothesis that defective niche can cause epigenetic reprograming of HSPCs. Finally, single cell and bulk transcriptome analysis of MSPCs lacking Ebf1 revealed differences in the niche composition and decreased expression of lineage-instructive signals for myeloid cells. Thus, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs. Disclosures No relevant conflicts of interest to declare.

Cord Blood Hematopoietic Progenitor Cell in Co-Culture with Bone Marrow Mesenchymal Stromal Cells: A Synergic Effect

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4811-4811
Author(s):  
Camillo Almici ◽  
Arabella Neva ◽  
Rosanna Verardi ◽  
Simona Braga ◽  
Andrea Di Palma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Progenitor Cell ◽  
Cord Blood Sample ◽  
Hematopoietic Stem

Abstract Abstract 4811 The number of hematopoietic stem and progenitor cells (HPCs) in cord blood units are limited and this can result in delayed engraftment. In vitro expansion of HPCs provides a perspective to overcome these limitations. Different combinations of cytokines as well as mesenchymal stromal cells (MSC) have been shown to separately support HPCs ex vivo expansion, but the combining effects are under evaluation. Data derived from ex vivo co-culture systems using MSC as a feeder layer suggest that cellular contacts could have a significant impact on expansion. We have evaluated the expansion rate of thawed cord blood samples (n=6) in a medium containing SCF (100 ng/ml) and G-CSF (100 ng/ml) plated over a pre-established bone marrow derived MSC layer in comparison to the absence of either MSC layer or cytokines. After 7 days cultures were demi-depopulated. At 14 days of culture adherent and non-adherent cells were harvested, counted and evaluated for antigens expression and clonogenic capacity. Immunophenotypic analysis was performed using CD34-PE, CD38-FITC, CD45-PE-Cy7, CD133-APC. Clonogenic assay was performed in semisolid methylcellulose culture medium (MethoCult, Stem Cell Technologies), CFU frequencies and total CFU numbers per cord blood sample were determined. After 14 days of culture, in the presence of MSC layer, an 11.2-fold increase (range 4.4–18.4) in total number of cells was observed, in comparison to a 4.8-fold increase (range1.1-10.35) in the absence of MSC layer. The presence of MSC layer generated a 4.3-fold increase (range 1.5–7.2) in the number of CD34 positive cells, compared to a 3.3-fold increase (range 0.9–5.7) in the absence of MSC; when considering the more immature CD34+/CD38− subpopulation the corresponding increase were 26.9-fold vs 2.85-fold, respectively. Moreover, the percentage of the CD34+/CD38− subpopulation was higher in the adherent compared to the non-adherent fraction (76% vs 15%). The selection effect given by the MSC layer was confirmed by the presence of hematopoiesis foci growing onto the MSC layer. Our data show that cord blood HPCs can be expanded in vitro, moreover the co-culture on a MSC layer shows a synergistic effect on TCN, CD34+ cells and on more primitive CD34+/CD38− cells. Therefore, a clinical protocol of cord blood HPCs and MSC co-culture could represent a promising approach for improving engraftment kinetics in cord blood transplant recipients. Disclosures: No relevant conflicts of interest to declare.

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