Fibroblastic Potential Of Cd41~+Cells In The Mouse Agm Region And Yolk Sac At E11.0

Embryonic development of the mammalian haematopoietic system is complex and,in many aspects, poorly understood. Generation of blood cells in the vertebrate embryo isa dynamic process, with different anatomical locations transiently hosting different wavesof hematopoiesis. The predominant sites and patterns of hematopoiesis change duringmurine ontogeny. The first blood cells, generated in the YS beginning at embryonic day(E7.0), are a unique population of primitive erythroid progenitors (EryP) that mature intolarge primitive erythroblasts expressing both embryonic and adult hemoglobins. This firstprimitive erythroid progenitors phase of yolk sac (YS) blood cell production byconvention has been termed primitive hematopoiesis. The sites of origin of definitivehematopoiesis have been a subject of longstanding debate and intense investigation. Thereported results demonstrate the complexity of Hematopoietic stem cells (HSCs)formation and suggest that the generation of mammalian HSCs may occur de novo inseveral embryonic locations. HSCs emerge de novo from mesoderm precursors withinYS and paraaortic splanchnopleura (P-Sp) and possibly the allantois and placenta, leadingto marking of the descendant definitive hematopoietic cells in the fetal liver. DefinitiveHSCs/HPCs expand in the YS but are found in the embryo proper soon after the onset ofcardiac contractions at E8.25.The P-Sp contains multilineage short-term hematopoieticpotential beginning at E8.5, rather than the yolk sac, the first long-term, high-level,multilineage repopulating HSCs are autonomously generated in theaorta-gonads-mesonephros (AGM) region and specifically the aorta subregion. Becausethe accurate determination of the sites of origin of hematopoietic progenitor cells iscomplicated by the development of the cardiovascular function, finding a markerexpressed by nascent HSCs to denote the onset of hematopoietic fate is very necessary.CD41(also known as αIIb Integrin), a previous well known specific marker formegakaryocytes and platelets, has been recently found to mark the initiation of murineprimitive and definitive hematopoiesis. And CD41has been suggested as a markerexpressed by nascent HSCs, denoting the onset of hematopoietic fate. In the mouseembryo, expression of CD41was detected in yolk sac blood islands, AGM region andplacenta. However, it is still unclear the full properties of total CD41⁺population inmouse embryo. And mesenchymal, endothelial and hematopoietic cells have closedevelopmental relationships. It has found these embryonic CD41⁺hematopoietic cells possess no endothelial potential in vitro. Herein, we studied the differentiation abilityincluding hematopoietic, mesenchymal potential, and migration ability of isolated CD41⁺cells by FACS at E11.0to underlying hematopoiesis and ontogeny development.Firstly, we indicated AGM, YS and embryonic circulating blood (CB)-derivedCD41⁺cells of E11.0all have hematopoietic colony formation in vitro. We found thatAGM and YS-derived CD41⁺cells possess the mesenchymal potential in vitro and can bedifferentiated into vimentin⁺/α-SMA⁺/epimorphin⁺cells in vivo, whereas CB-derivedCD41⁺cells may not harbor the potential in vivo. Some difference including in vitrogrowth rate and α-SMA expression level were also observed in differentiatedmyofibroblast/fibroblast cells derived from AGM CD41⁺or YS CD41⁺cells. Meanwhile,we found that AGM-derived CD41⁺cells respond to the chemotaxin of circulating bloodplasma more strongly than that of YS-derived CD41⁺cells. To test which sub-fractioningof the CD41cell populations contain mesenchymal potential, we sorted cells based onCD41expression levels. In AGM and YS of E11.0, the frequency of CD41^intcells isgreater than that of CD41highcells. We found almost exclusively in the AGM andYS-derived CD41(intermediate)intfraction of E11.0cells have the potential to differentiateinto mesenchymal-like cells. And then we sorted cells coexpression with CD34. Wefound both YS derived CD41^intCD34-and CD41^intCD34⁺have the potential todifferentiate into mesenchymal-like cells, while only almost exclusively in AGM derivedCD41^intCD34^-.Taken together, our data revealed that at E11.0(I) In addition to hematopoieticpotential, AGM and YS-derived CD41⁺cells may have the potential to differentiate intomesenchymal-like cells varied on growth rate and α-SMA expression level, assumed tobe myofibroblasts/fibroblasts, in vitro and in vivo.(II) It may possess a higher capacity ofAGM-derived CD41⁺cells than that of YS-derived CD41⁺cells in migrating intocirculation, whereas CD41⁺cells with mesenchymal potential may rarely migrate into thebloodstream. CB-derived CD41⁺cells have little α-SMA⁺or vimentin⁺cells in cultureand also have no capacity to differentiate into α-SMA⁺mesenchymal cells intransplantation assay.(â…¢) The mesenchymal potential of CD41⁺cells in AGM and YSare almost exclusively in CD41(intermediate)intfraction. We also found both YS derivedCD41^intCD34-and CD41^intCD34⁺have the potential to differentiate intomesenchymal-like cells, while only almost exclusively in AGM derived CD41^intCD34-. Large numbers of patients worldwide suffer from ischemic damage diseases,including myocardial ischemia and critical limb ischemia. Promoting angiogenesis byusing angiogenic factors, or through implantation of endothelial progenitor cells, is aviable therapeutic option to repair ischemic tissue. EPCs can augment neovascularizationin ischemia diseases for which ischemic tissues require reperfusion around occludedvessels to recover their function. Several studies have shown that EPCs from cord bloodor bone marrow promote angiogenesis in damaged tissues and increase blood flow inanimal models of hind limb ischemia. The identification of EPCs in the peripheral blood(PB) derived from the bone marrow (BM) and the demonstration of their promptmobilization, incorporation, and differentiation to the sites of injury have suggested thatEPCs in PB could serve as an endothelial reserve with the capacity to repair damagedvascular endothelium. It is believed that EPCs home to sites of lesion after ischemiainsult, where they differentiate into endothelial cells. However, under steady-stateconditions, EPCs circulate in the PB at very low frequencies that are insufficient forischemic tissue repair. An efficient therapeutic strategy is to mobilize more EPCs intoperipheral circulation with mobilizing agents, which would promote the incorporation ofmore EPCs into the ischemic tissue to enhance angiogenesis and tissue regeneration. Themechanism by which EPCs are mobilized from BM are complex, but EPCs are believedto interact with specific molecules expressed on extracellular matrix and then roll alongthe vessel wall until they encounter chemokines trapped in the endothelial glycocalyx.The majority of this recruitment process is regulated by stromal cell-derivedfactor-1alpha (SDF-1α, which is also named CXCL12) and its receptor CXCR4, whichperform critical functions in the bone marrow. Perturbing the interaction between bonemarrow-derived SDF-1α and CXCR4expressed on EPCs allows EPCs to escape the bonemarrow niche via transendothelial migration. Multiple studies have suggested thatdecreased SDF-1α levels in BM or a CXCR4antagonist can mobilize EPCs. IncreasingSDF-1α in the peripheral circulation promoted angiogenesis of ischemic tissue inexperimental animals.Recent reports have suggested that local or systemic administration of cytokines canmobilize EPCs, enhance ischemic angiogenesis and improve the function of ischemictissues in animals with ischemic hind limbs or myocardial ischemia. EPCs are known tomobilize from the bone marrow (BM) in response to various stimuli, including cytokines (G-CSF, VEGF, and estradiol), CXCR4antagonists (ADM3100), chemokines (CXCL2,CXCL8, and CXCL12), nitric oxide (NO) and angiopoietin. Ischemic environmentsappear to promote the release of SDF-1α, which leads to a change in the concentrationgradient between ischemic tissue and bone marrow. Until now, several EPC-mobilizingagents have been reported to exert therapeutic effects in ischemic tissue repair; however,their therapeutic effect needs to be improved. Given the high morbidity associated withischemic diseases, it is very important to develop novel effective non-toxic smallmolecules that promote the mobilization of EPCs and angiogenesis in ischemic tissue.In this study, we provide the first evidence that a novel small molecule, Me6,induced long-lasting and effective mobilization of EPCs from the BM. Increased numbersof Flk-1+Sca-1+cells were detected by flow cytometry in the peripheral blood and thespleen of mice injected with Me6. Compared to control mice, a significant increase ofFlk-1+Sca-1+cells mobilized to peripheral blood at about12h (Me61.98±0.13%vs.vehicle control0.71±0.09%). Until72h, EPCs in PB recovered to normal level. Inaddition, we also evaluated Sca-1+Flk-1+cell distribution in the spleen using FACS. AfterMe6injection, flow cytometry analysis showed that there were more Flk-1+Sca-1+cellsdetected in spleen compared with control mice (Me67.3±1.2%vs. vehicle control3.3±0.6%). We also observed much more CD34+in PBMNCs during the mobilizationperiod (Me619.5±1.4vs. vehicle control14.8±0.76). FACS analysis revealed that Me6treatment did have some effect on MSCs markers, such as CD29, but did not affectCD105expression in PBMNCsCell transplantation experiments suggested that Me6-mobilized EPCs have canenhance the restoration of blood perfusion in ischemic hind limbs Laser Dopplerperfusion imager (LDPI) analysis. First we observed more Sca-1+Flk-1+cells in PB usingFACS during the mobilization period at7d in HLI model(Me63.09±0.5%; AMD31003.17±0.4%; vehicle control1.49±0.1%). More importantly, we demonstrated that Me6mobilizes autologous bone marrow-derived EPCs and promotes angiogenesis in ischemichind limbs. We also found that decreasing concentrations of SDF-1α in BM andupreguation of CXCR4are involved in Me6-mediated mobilization of EPCs. Andsignificant up-regulation of CXCR4expression was observed on BM cells from micetreated Me6from29.1±4.3%to54.9±6.1%(p <0.05) at12h after a single injection ofMe6. This up-regulation was persistent till to48h after Me6administration, when the percentage of the BM cells expressing CXCR4reached to49.7%±2.6%, during themobilization period.Our results suggest that Me6may be a promising therapeutic option for ischemicdiseases and peripheral arterial diseases to enhancing angiogenesis and repairingischemic tissue. Furthermore, this strategy may have implications for potentially clinicaltrials in patients with refractory ischemic cardiovascular diseases.