Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment

The mechanism by which angiogenic factors recruit bone marrow (BM)-derived quiescent endothelial and hematopoietic stem cells (HSCs) is not known. Here, we report that functional vascular endothelial growth factor receptor-1 (VEGFR1) is expressed on human CD34(+) and mouse Lin(-)Sca-1(+)c-Kit(+) BM-repopulating stem cells, conveying signals for recruitment of HSCs and reconstitution of hematopoiesis. Inhibition of VEGFR1, but not VEGFR2, blocked HSC cell cycling, differentiation and hematopoietic recovery after BM suppression, resulting in the demise of the treated mice. Placental growth factor (PlGF), which signals through VEGFR1, restored early and late phases of hematopoiesis following BM suppression. PlGF enhanced early phases of BM recovery directly through rapid chemotaxis of VEGFR1(+) BM-repopulating and progenitor cells. The late phase of hematopoietic recovery was driven by PlGF-induced upregulation of matrix metalloproteinase-9, mediating the release of soluble Kit ligand. Thus, PlGF promotes recruitment of VEGFR1(+) HSCs from a quiescent to a proliferative BM microenvironment, favoring differentiation, mobilization and reconstitution of hematopoiesis.     

Modulation of haematopoietic progenitor development by FLT-3 ligand.

The Flt-3 receptor is expressed in primitive haematopoietic cells and its ligand exerts proliferative effects on these cells in vitro in synergy with other cytokines. To increase our knowledge of the functional properties of the human Flt-3 ligand (FL) as relating to in vitro expansion of haematopoietic stem cells, the effects on murine haematopoiesis of FL alone or in combination with other growth factors were studied. Analysis of Flk-2/Flt-3 mRNA expression indicated that Flk-2/Flt-3 was preferentially expressed in primitive haematopoietic cell populations. To examine the expression of the Flk-2/Flt-3 receptor on megakaryocyte progenitors (CFU-Meg), Flk-2/Flt-3 positive and negative CD34(+)populations were separated from human bone marrow and cultured in a plasma clot culture system. CFU-Meg colonies were found in the Flk-2/Flt-3 negative fraction. Myeloid (CFU-GM) derived colonies appeared in the presence of FL alone. Neither FL+IL-3 nor FL+IL-3+IL-6 had any effect on the generation of megakaryocyte colonies (CFU-MK), due to the lack of FL receptor expression on megakaryocyte progenitors. Bone marrow cells remaining after 5-fluorouracil (5-FU) treatment of mice represent a very primitive population of progenitors enriched for reconstituting stem cells. This cell population expressed FL receptors, as revealed by RT-PCR analysis. Addition of FL alone did not enhance the replication of such cells in liquid cultures as compared to controls. However, a significantly greater generation of myeloid progenitors (CFU-GM) in clonogenic assays was observed in the presence of FL+IL-3, FL+GM-CSF or FL+CSF-1. In addition, the effects of FL on in vitro expansion of murine haematopoietic stem cells were studied using lineage-negative (lin(-)) Sca-1 positive (Sca-1(+)) c-kit positive (c-kit(+)) marrow cells from 5-FU treated mice. FL enhanced the survival of primitive murine lin(-)Sca-1(+)c-kit(+)cells. FL and IL-6 were able to significantly expand murine progenitor stem cells in vitro and promote their survival. These studies strongly suggest that FL significantly and selectively enhanced the generation of myeloid progenitors in vitro and increased myeloid progenitor responsiveness to later acting growth factors. In addition, FL synergized with IL-6 to support in vitro expansion of haematopoietic progenitors and promoted the survival of lin(-)Sca-1(+)c-kit(+)cells.     

Kit regulates maintenance of quiescent hematopoietic stem cells

Hematopoietic stem cell (HSC) numbers are tightly regulated and maintained in postnatal hematopoiesis. Extensive studies have supported a role of the cytokine tyrosine kinase receptor Kit in sustaining cycling HSCs when competing with wild-type HSCs posttransplantation, but not in maintenance of quiescent HSCs in steady state adult bone marrow. In this study, we investigated HSC regulation in White Spotting 41 (Kit(W41/W41)) mice, with a partial loss of function of Kit. Although the extensive fetal HSC expansion was Kit-independent, adult Kit(W41/W41) mice had an almost 2-fold reduction in long-term HSCs, reflecting a loss of roughly 10,000 Lin(-)Sca-1(+)Kit(high) (LSK)CD34(-)Flt3(-) long-term HSCs by 12 wk of age, whereas LSKCD34(+)Flt3(-) short-term HSCs and LSKCD34(+)Flt3(+) multipotent progenitors were less affected. Whereas homing and initial reconstitution of Kit(W41/W41) bone marrow cells in myeloablated recipients were close to normal, self-renewing Kit(W41/W41) HSCs were progressively depleted in not only competitive but also noncompetitive transplantation assays. Overexpression of the anti-apoptotic regulator BCL-2 partially rescued the posttransplantation Kit(W41/W41) HSC deficiency, suggesting that Kit might at least in the posttransplantation setting in part sustain HSC numbers by promoting HSC survival. Most notably, accelerated in vivo BrdU incorporation and cell cycle kinetics implicated a previously unrecognized role of Kit in maintaining quiescent HSCs in steady state adult hematopoiesis.     

Detection of murine adult bone marrow stroma-initiating cells in Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells

We attempted to characterize the phenotype of cells which initiate fibroblastic stromal cell formation (stroma-initiating cells: SICs), precursor cells for fibroblastic stromal cells, based on the expression of cell surface antigens. First, we stained adult murine bone marrow cells with several monoclonal antibodies and separated them by magnetic cell sorting. SICs were abundant in the c-kit(+), Sca-1(+), CD34(+), VCAM-1(+), c-fms(+), and Mac-1(-) populations. SICs were recovered in the lineage-negative (Lin(-)) cells but not the Lin(+) cells. When macrophage colony-stimulating factor (M-CSF) was absent from the culture medium, no stromal colony appeared among the populations enriched in SICs. Based on these findings, the cells negative for lineage markers and positive for c-fms (M-CSF receptor) were further divided on the basis of the expression of c-kit, VCAM-1, Sca-1 or CD34 with a fluorescence-activated cell sorter. SICs were found to be enriched in the Lin(-)c-fms(+)c-kit(low) cells and Lin(-)c-fms(+)VCAM-1(+) cells but not in Lin(-)c-fms(+)Sca-1(+) cells and Lin(-)c-fms(+)CD34(low) cells. As a result, the SICs were found to be present at highest frequency in Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells: a mean of 64% of the SICs in the Lin(-) cells were recovered in the population. In morphology and several characteristics, the stromal cells derived from Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells resembled fibroblastic cells. The number of Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells in bone marrow of mice injected with M-CSF was higher than that in control mice. In this study, we identified SICs as Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells and demonstrated that M-CSF had the ability to increase the cell population in vivo.     

Proteomic dataset of Sca-1+ progenitor cells

Embryonic stem cells (ES cells) can differentiate into endothelial cells and smooth muscle cells (SMCs), which participate in vascular angiogenesis. In this study, we differentiated mouse ES cells into Sca-1(+) cells, which have the potential to serve as vascular progenitor cells, and mapped their proteome by 2-DE using a pH 3-10 non-linear gradient and 12% SDS-polyacrylamide gels. A subset of 300 protein spots was analysed and mapped, with 241 protein spots being identified by their PMF using MALDI-TOF MS or by partial amino acid sequencing using MS/MS. Our protein map is the first of Sca-1(+) progenitor cells and will facilitate the identification of proteins differentially expressed during stem cell differentiation. The proteome of adult arterial SMCs is described in an accompanying paper (in this issue, DOI 10.1002/pmic.200402045). All data are made accessible on our website http://www.vascular-proteomics.com.     

Cardiac dysfunction and impaired compensatory response to pressure overload in mice deficient in stem cell antigen-1

Stem cell antigen-1 (Sca-1) has been used to identify cardiac stem cells in the mouse heart. To investigate the function of Sca-1 in aging and during the cardiac adaptation to stress, we used Sca-1-deficient mice. These mice developed dilated cardiomyopathy [end-diastolic left ventricular diameter at 18 wk of age: wild-type (WT) mice, 4.2 mm ± 0.3; Sca-1-knockout (Sca-1-KO) mice, 4.6 mm ± 0.1; ejection fraction: WT mice, 51.1 ± 2.7%; Sca-1-KO mice, 42.9 ± 2.7%]. Furthermore, the hearts of mice lacking Sca-1 demonstrated exacerbated susceptibility to pressure overload [ejection fraction after transaortic constriction (TAC): WT mice, 43.5 ± 3.2%; Sca-1-KO mice, 30.8% ± 4.0] and increased apoptosis, as shown by the 2.5-fold increase in TUNEL(+) cells in Sca-1-deficient hearts under stress. Sca-1 deficiency affected primarily the nonmyocyte cell fraction. Indeed, the number of Nkx2.5(+) nonmyocyte cells, which represent a population of cardiac precursor cells (CPCs), was 2-fold smaller in Sca-1 deficient neonatal hearts. In vitro, the ability of CPCs to differentiate into cardiomyocytes was not affected by Sca-1 deletion. In contrast, these cells demonstrated unrestricted differentiation into cardiomyocytes. Interestingly, proliferation of cardiac nonmyocyte cells in response to stress, as judged by BrdU incorporation, was higher in mice lacking Sca-1 (percentages of BrdU(+) cells in the heart after TAC: WT mice, 4.4 ± 2.1%; Sca-1-KO mice, 19.3 ± 4.2%). These data demonstrate the crucial role of Sca-1 in the maintenance of cardiac integrity and suggest that Sca-1 restrains spontaneous differentiation in the precursor population. The absence of Sca-1 results in uncontrolled precursor recruitment, exhaustion of the precursor pool, and cardiac dysfunction.     

LSK derived LSK- cells have a high apoptotic rate related to survival regulation of hematopoietic and leukemic stem cells

A balanced pool of hematopoietic stem cells (HSCs) in bone marrow is tightly regulated, and this regulation is disturbed in hematopoietic malignancies such as chronic myeloid leukemia (CML). The underlying mechanisms are largely unknown. Here we show that the Lin(-)Sca-1(+)c-Kit(-) (LSK(-)) cell population derived from HSC-containing Lin(-)Sca-1(+)c-Kit(+) (LSK) cells has significantly higher numbers of apoptotic cells. Depletion of LSK cells by radiation or the cytotoxic chemical 5-fluorouracil results in an expansion of the LSK(-) population. In contrast, the LSK(-) population is reduced in CML mice, and depletion of leukemia stem cells (LSCs; BCR-ABL-expressing HSCs) by deleting Alox5 or by inhibiting heat shock protein 90 causes an increase in this LSK(-) population. The transition of LSK to LSK(-) cells is controlled by the Icsbp gene and its downstream gene Lyn, and regulation of this cellular transition is critical for the survival of normal LSK cells and LSCs. These results indicate a potential function of the LSK(-) cells in the regulation of LSK cells and LSCs.     

Lineage(-)Sca1+c-Kit(-)CD25+ cells are IL-33-responsive type 2 innate cells in the mouse bone marrow

IL-33 promotes type 2 immune responses, both protective and pathogenic. Recently, targets of IL-33, including several newly discovered type 2 innate cells, have been characterized in the periphery. In this study, we report that bone marrow cells from wild-type C57BL/6 mice responded with IL-5 and IL-13 production when cultured with IL-33. IL-33 cultures of bone marrow cells from Rag1 KO and Kit(W-sh/W-sh) mice also responded similarly; hence, eliminating the possible contributions of T, B, and mast cells. Rather, intracellular staining revealed that the IL-5- and IL-13-positive cells display a marker profile consistent with the Lineage(-)Sca-1(+)c-Kit(-)CD25(+) (LSK(-)CD25(+)) cells, a bone marrow cell population of previously unknown function. Freshly isolated LSK(-)CD25(+) cells uniformly express ST2, the IL-33 receptor. In addition, culture of sorted LSK(-)CD25(+) cells showed that they indeed produce IL-5 and IL-13 when cultured with IL-33 plus IL-2 and IL-33 plus IL-7. Furthermore, i.p. injections of IL-33 or IL-25 into mice induced LSK(-)CD25(+) cells to expand, in both size and frequency, and to upregulate ST2 and α(4)β(7) integrin, a mucosal homing marker. Thus, we identify the enigmatic bone marrow LSK(-)CD25(+) cells as IL-33 responsive, both in vitro and in vivo, with attributes similar to other type 2 innate cells described in peripheral tissues.     

Role of Flk-1 in mouse hematopoietic stem cells.

It was reported that human hematopoietic stem cells in bone marrow were restricted to the CD34(+)KDR(+) cell fraction. We found that expression levels of Flk-1, a mouse homologue of KDR, were low or undetectable in mouse Lin(-)c-Kit(+)Sca-1(+)CD34(low/-) cells as well as Hoechst33342(-) cells (side population), which have long-term reconstitution capacity. Furthermore, neither Flk-1(+)CD34(low/-) cells nor Flk-1(+)CD34(+) cells had long-term reconstitution capacity in mouse. Taken together with other observations using Flk-1-deficient mice, these results indicate that Flk-1 is essential for the development of hematopoietic stem cells in embryo but not for the function of hematopoietic stem cells in adult mouse bone marrow.     

Mesenchymal stem/stromal cells induce the generation of novel IL-10--dependent regulatory dendritic cells by SOCS3 activation

Suppression of immune response by mesenchymal stem/stromal cells (MSCs) is well documented. However, their regulatory effects on immune cells, especially regulatory dendritic cells, are not fully understood. We have identified a novel Sca-1(+)Lin(-)CD117(-) MSC population isolated from mouse embryonic fibroblasts (MEF) that suppressed lymphocyte proliferation in vitro. Moreover, the Sca-1(+)Lin(-)CD117(-) MEF-MSCs induced hematopoietic stem/progenitor cells to differentiate into novel regulatory dendritic cells (DCs) (Sca-1(+)Lin(-)CD117(-) MEF-MSC-induced DCs) when cocultured in the absence of exogenous cytokines. Small interfering RNA silencing showed that Sca-1(+)Lin(-)CD117(-) MEF-MSCs induced the generation of Sca-1(+)Lin(-)CD117(-) MEF-MSC-induced DCs via IL-10-activated SOCS3, whose expression was regulated by the JAK-STAT pathway. We observed a high degree of H3K4me3 modification mediated by MLL1 and a relatively low degree of H3K27me3 modification regulated by SUZ12 on the promoter of SOCS3 during SOCS3 activation. Importantly, infusion of Sca-1(+)CD117(-)Lin(-) MEF-MSCs suppressed the inflammatory response by increasing DCs with a regulatory phenotype. Thus, our results shed new light on the role of MSCs in modulating regulatory DC production and support the clinical application of MSCs to reduce the inflammatory response in numerous disease states.     

Diabetes alters subsets of endothelial progenitor cells that reside in blood, bone marrow, and spleen

Circulating endothelial progenitor cells (EPCs) derived from the bone marrow (BM) participate in maintaining endothelial integrity and vascular homeostasis. Reduced EPC number and function result in vascular complications in diabetes. EPCs are a population of cells existing in various differentiation stages, and their cell surface marker profiles change during the process of mobilization and maturation. Hence, a generally accepted marker combination and a standardized protocol for the quantification of EPCs remain to be established. To determine the EPC subsets that are affected by diabetes, we comprehensively analyzed 32 surface marker combinations of mouse peripheral blood (PB), BM, and spleen cells by multicolor flow cytometry. Ten subsets equivalent to previously reported mouse EPCs significantly declined in number in the PB of streptozotocin-induced diabetic mice, and this reduction was reversed by insulin treatment. The PI(-)Lin(-)c-Kit(-)Sca-1(+)Flk-1(-)CD34(-)CD31(+) EPC cluster, which can differentiate into mature endothelial cells in vitro, was the highest population in the PB, BM, and spleen and occurred 61 times more in the spleen than in the PB. The cell number significantly decreased in the BM as well as in the PB but paradoxically increased in the spleen under diabetic conditions. Insulin treatment reversed the decrease of EPC subsets in the BM and PB and reversed their increase in spleen. A similar tendency was observed in some of the major cell populations in db/db mice. To the best of our knowledge, we are the first to report spatial population changes in mouse EPCs by diabetes in the blood and in the BM across the spleen. Diminished circulating EPC supply by diabetes may be ascribed to impaired EPC production in the BM and to decreased EPC mobilization from the spleen, which may contribute to vascular dysfunction in diabetic conditions.     

Distinct hemogenic potential of endothelial cells and CD41+ cells in mouse embryos

Definitive hematopoietic progenitor cells have been thought to develop from the vascular endothelium located in the aorta-gonad-mesonephros region of the mouse embryo. However, several recent findings have suggested that most hematopoietic progenitors are derived from non-endothelial precursor cells expressing CD41. We characterized two distinct precursor populations of definitive hematopoietic cell lineages, vascular endothelial (VE)-cadherin(+) CD41(-) CD45(-) endothelial cells and CD41(+) CD45(-) non-endothelial progenitors, both of which are derived from lateral mesoderm. VE-cadherin(+) endothelial cells obtained from cultures of differentiating embryonic stem cells possessed hematopoietic potential encompassing erythroid, myeloid and B lymphoid lineages, whereas CD41(+) progenitors lacked the B lymphopoietic potential. VE-cadherin(+) endothelial cells in the lower trunk of the embryo proper showed a significant potential for initiating B lymphopoiesis in cultures, while endothelial cells in the yolk sac appeared to have a bias for myeloerythropoietic differentiation. CD41(+) progenitors isolated from yolk sac and embryo proper were capable of generating multiple hematopoietic lineages, although mast cell precursors were exclusively enriched in CD41(+) progenitors in the yolk sac. These results suggest that hemogenic endothelial cells and CD41(+) progenitors possess distinct hematopoietic potential depending on the tissues in which they reside.     

CC chemokine receptor 2 expression in donor cells serves an essential role in graft-versus-host-disease

The complete repertoire of cellular and molecular determinants that influence graft-vs-host disease (GVHD) is not known. Using a well-established murine model of GVHD (B6-->bm12 mice), we sought to elucidate the role of the donor non-T cell compartment and molecular determinants therein in the pathogenesis of GVHD. In this model the acute GVHD-inducing effects of purified B6 wild-type (wt) CD4(+) T cells was inhibited by wt non-T cells in a dose-dependent manner. Paradoxically, unlike the chronic GVHD phenotype observed in bm12 mice transplanted with B6wt unfractionated splenocytes, bm12 recipients of B6ccr2-null unfractionated splenocytes developed acute GVHD and died of IFN-gamma-mediated bone marrow aplasia. This switch from chronic to acute GVHD was associated with increased target organ infiltration of activated CD4(+) T cells as well as enhanced expression of Th1/Th2 cytokines, chemokines, and the antiapoptotic factor bfl1. In vitro, ccr2(-/-) CD4(+) T cells in unfractionated splenocytes underwent significantly less activation-induced cell death than B6wt CD4(+) T cells, providing another potential mechanistic basis along with enhanced expression of bfl1 for the increased numbers of activated T cells in target organs of B6ccr2(-/-) splenocyte-->bm12 mice. Collectively, these findings have important clinical implications, as they implicate the donor non-T cell compartment as a critical regulator of GVHD and suggest that ccr2 expression in this cellular compartment may be an important molecular determinant of activation-induced cell death and GVHD pathogenesis.     

Leukemia inhibitory factor induces endothelial differentiation in cardiac stem cells

The importance of interleukin 6 (IL-6)-related cytokines in cardiac homeostasis has been studied extensively; however, little is known about their biological significance in cardiac stem cells. Here we describe that leukemia inhibitory factor (LIF), a member of IL-6-related cytokines, activated STAT3 and ERK1/2 in cardiac Sca-1+ stem cells. LIF stimulation resulted in the induction of endothelial cell-specific genes, including VE-cadherin, Flk-1, and CD31, whereas neither smooth muscle nor cardiac muscle marker genes such as GATA4, GATA6, Nkx-2.5, and calponin were up-regulated. Immunocytochemical examination showed that about 25% of total cells were positively stained with anti-CD31 antibody 14 days after LIF stimulation. Immunofluorescent microscopic analyses identified the Sca-1+ cells that were also positively stained with anti-von Willebrand factor antibody, indicating the differentiating process of Sca-1+ cells into the endothelial cells. IL-6, which did not activate STAT3 and ERK1/2, failed to induce the differentiation of cardiac stem cells into the endothelial cells. In cardiac stem cells, the transduction with dominant negative STAT3 abrogated the LIF-induced endothelial differentiation. And the inhibition of ERK1/2 with the MEK1/2 inhibitor U0126 also prevented the differentiation of Sca-1+ cells into endothelial cells. Thus, both STAT3 and ERK1/2 are required for LIF-mediated endothelial differentiation in cardiac stem cells. Collectively, it is proposed that LIF regulates the commitment of cardiac stem cells into the endothelial cell lineage, contributing to neovascularization in the process of tissue remodeling and/or regeneration.     

Common and distinct features of cytokine effects on hematopoietic stem and progenitor cells revealed by dose-response surface analysis

Recent studies have identified thrombopoietin (TPO), flt-3 ligand (FL), Steel factor (SF), and interleukin-11 (IL-11) as cytokines able to stimulate amplification of the most primitive murine hematopoietic cells in vitro. However, dose-response and interaction parameters that predict how to optimize mixtures of these cytokines have not been previously defined. To obtain this information, Sca-1(+)lin(-) and c-kit(+)Sca-1(+)lin(-) adult mouse bone marrow cells were cultured for 10 and 14 days, respectively, in serum-free medium with varying concentrations of these cytokines. Quantitative assays were performed to determine the influences of the cytokine combinations tested on changes in long-term repopulating hematopoietic stem cells (HSCs), in vitro colony-forming cells (CFCs), and total cell numbers. A two-level factorial design was first used to screen the effects of TPO, SF, FL, and IL-11 as well as two different incubation temperatures. IL-11 and SF were found to be the most significant stimulators of murine HSC expansion. More detailed analyses of the effects on c-kit(+)Sca-1(+)lin(-) cells of IL-11, SF, and FL concentrations and their interactions using response surface methodology showed IL-11 to have a maximal stimulatory effect on HSC expansion at 20 ng/mL with higher concentrations being inhibitory. In contrast, not even high concentration saturation of the effects of either SF or FL was observed as the stimulatory effect of both SF and FL increased beyond 300 ng/mL. A negative interaction between SF and FL on HSCs was discovered. Interestingly, a generally similar pattern of cytokine effects was found to influence the 14-day output of CFCs and total cells from the same c-kit(+)Sca-1(+)lin(-) starting cell population. However, compared with HSCs, the cytokine requirements for maximizing the generation of CFCs and total cells were at much lower cytokine doses. From the information provided by the factorial analysis, mathematical models based on Monod kinetics for inhibitory substrates were developed that allow total cell, CFC, and HSC expansion to be predicted as a function of the IL-11, SF, and FL concentrations in terms of more widely recognized parameters. Overall, these methods should also serve as a guide for the future design and testing of other ex vivo stem cell expansion systems.     

Inhibition of Sca-1-positive skeletal stem cell recruitment by alendronate blunts the anabolic effects of parathyroid hormone on bone remodeling

The anabolic effects of parathyroid hormone (PTH) on bone formation are impaired by concurrent use of antiresorptive drugs. We found that the release of active transforming growth factor (TGF)-β1 during osteoclastic bone resorption is inhibited by alendronate. We showed that mouse Sca-1-positive (Sca-1(+)) bone marrow stromal cells are a skeletal stem cell subset, which are recruited to bone remodeling sites by active TGF-β1 in response to bone resorption. Alendronate inhibits the release of active TGF-β1 and the recruitment of Sca-1(+) skeletal stem cells for the bone formation. The observation was validated in a Tgfb1(-/-) mouse model, in which the anabolic effects of PTH on bone formation are diminished. The PTH-stimulated recruitment of injected mouse Sca-1(+) cells to the resorptive sites was inhibited by alendronate. Thus, inhibition of active TGF-β1 release by alendronate reduces the recruitment of Sca-1(+) skeletal stem cells and impairs the anabolic action of PTH in bone.