Endothelial origin of mesenchymal stem cells

Mesenchymal stem/stromal cells (MSCs) are fibroblastoid cells capable of long-term expansion and skeletogenic differentiation. While MSCs are known to originate from neural crest and mesoderm, immediate mesodermal precursors that give rise to MSCs have not been characterized. Recently, using human embryonic stem cells (hESCs), we demonstrated that mesodermal MSCs arise from APLNR⁺ precursors with angiogenic potential, mesenchymoangioblasts, which can be identified by FGF2-dependent colony-forming assay in serum-free semisolid medium. In this overview we provide additional insights on cellular pathways leading to MSC establishment from mesoderm, with special emphasis on endothelial-mesenchymal transition as a critical step in MSC formation. In addition, we highlight an essential role of FGF2 in induction of angiogenic cells with potential to transform into MSCs (mesenchymoangioblasts) or hematopoietic cells (hemangioblasts) from mesoderm, and discuss correlations of our in vitro findings with the course of angioblast development during embryogenesis.Key words: mesenchymoangioblast, hemangioblast, human embryonic stem cells, endothelial-mesenchymal transition, epithelial-mesenchymal transition, mesenchymal stem cells, endothelial cells, apelin receptor, FGFMesenchymal stem/stromal cells (MSCs) are defined as multipotent fibroblastoid cells that give rise to cells of the skeletal connective tissue including osteoblasts, chondrocytes and adipocytes.^1–4 Although MSCs were described more than 40 years ago and are widely used for cellular therapies, very little knowledge exists regarding the developmental origins of MSCs in the embryo, the hierarchy of MSC progenitors or heterogeneity of MSCs within tissues. It has been demonstrated that during embryonic development, MSCs arise from a two major sources: neural crest and mesoderm.^5–7 Using Cre-recombinase lineage tracing experiments, Takashima et al. identified Sox1⁺ neuroepithelium as pre-cursors of MSCs of neural crest origin. However, direct precursors of mesoderm-derived MSCs were unknown. To identify these precursors, we employed human embryonic stem cells (hESCs) directed toward mesendodermal differentiation in coculture with mouse bone marrow stromal cells OP9,⁸ using the experimental approach depicted in Figure 1. As shown in this differentiation system, mesoderm reminiscent of lateral plate/extraembryonic mesoderm in the embryo can be identified by expression of apelin receptor (APLNR), otherwise known as angiotensin receptor like-1 receptor. Because we observed a positive selective effect of FGF2 on production of mesenchymal cells from hESCs in OP9 coculture, we decided to test whether FGF2 can induce the formation of colonies with mesenchymal potential from APLNR⁺ mesodermal cells. Indeed, when we isolated APLNR⁺ cells from hESCs differentiated on OP9 for 2 days and placed them in serum-free semisolid medium containing FGF2, we observed the formation of sharply-circumscribed spheroid colonies formed by tightly packed cells with a gene expression profile representative of embryonic mesenchyme originating from lateral plate/extraembryonic mesoderm and CD140a⁺CD146⁺C D90⁺CD56⁺CD166⁺CD31⁻CD43⁻CD45⁻ phenotype typical of mesenchymal cells. Based on cellular composition, we designated these colonies as mesenchymal (MS) colonies and cells forming these colonies as MS colony-forming cells (MS-CFCs). MS colony formation required serum-free medium and was solely dependent on FGF2 as a colony-forming factor. MS colonies were significantly enhanced by PDGF-BB, but suppressed by VEGF, TGFβ1 and Activin A. When transferred to the adherent cultures in serum-free medium with FGF2, individual MS colonies gave rise to multi-potential mesenchymal cell lines with typical phenotype (CD146⁺ CD105⁺ CD73⁺ CD31⁻ CD43/45⁻), differentiation (chondro-, osteo- and adipogenesis) and robust proliferation (>80 doublings) potentials. Using single cell deposition assay, chimeric hESC lines and time-lapse studies we demonstrated the clonality/single cell origin of MS colonies.Open in a separate windowFigure 1Schematic diagram of the experimental approach used to identify precursors and cellular events leading to formation of mesoderm-derived MSCs. hESCs were committed to mesendodermal differentiation through coculture with OP9 for 2 days. APLNR⁺ mesodermal cells were selected using magnetic sorting. In serum-free semisolid medium, APLNR⁺ cells grew into FGF2-dependent compact spheroid colonies composed of mesenchymal cells. MS colonies were formed through establishment of tightly-packed single cell-derived cores (day 3 of clonogenic culture), which expanded into spheroid colonies (days 6 and 12 of clonogenic culture). To evaluate differentiation potential, MS colonies were collected at different stages of clonogenic culture and placed on OP9. The presence of endothelial and mesenchymal cells after coculture of MS colonies with OP9 was evaluated by flow cytometry and immunofluorescence. In addition, colonies at core stage (day 3 of clonogenic culture) and mature colonies (day 12 of clonogenic cultures) were collected for molecular profiling studies. To generate clonal MSC lines, individual mature colonies were plated on the collagen/fibronectin-coated plastic and cultured in presence of FGF2.MS-CFCs could be detected only transiently, with a major peak on day 2 of hESC differentiation and disappeared after 4 days of differentiation. Notably, MS-CFC activity was developed prior to the expression of CD73 and CD105 MSC markers and upregulation of MSC-related genes, i.e., before onset of mesenchymogenesis. APLNR⁺ cells isolated from hESC cultures differentiated for 3 days also formed colonies in response to FGF2; however, the vast majority of these colonies were composed of blood cells and had a morphology similar to the previously described blast (BL) or hemangioblast colonies, which identify a common precursor for hematopoietic and endothelial cells.^9,10To fully evaluate the differentiation potential of MS colonies, we collected these colonies from semisolid cultures and placed them back on OP9 feeders, which are known to support development of a broad range of mesodermal lineage cells, including hematopoietic, vascular and cardiac.^11–13 Using this approach, we confirmed that individual BL colonies possess hemangioblastic potential, i.e., generate both hematopoietic and endothelial cells. When MS colonies were picked from clonogenic cultures and cultured on OP9, we found that the majority of cells differentiated into CD146⁺CD31⁻CD43/CD45⁻ mesenchymal cells as expected. However, we also discovered that MS colonies gave rise to CD31/VE-cadherin⁺CD43/45⁻ endothelial cells, indicating that MS colonies similar to BL colonies possess endothelial potential. The endothelial potential of MS colonies was also confirmed by demonstration of tube formation by MS colonies grown on Matrigel. In contrast, MSC lines derived from MS colonies did not produce any endothelial cells after coculture with OP9 indicating a progressive restriction of differentiation potential following MSC formation. Because single MS-CFC shows potential to form endothelium and MSCs, we designated the MSC precursor identified by this colony-forming assay as mesenchymoangioblast.To define more precisely the cellular events leading to establishing MSCs, we examined the formation of MS colonies using time-lapse cinematography and analyzed the kinetic of their angiogenic potential. As demonstrated by time-lapse studies, APLNR⁺ mesodermal cells placed in semisolid medium possessed a high motility, which was more pronounced before and during the first cell division. Following several divisions, single APLNR⁺ cells formed a core, an immotile structure composed of a small number of tightly packed cells. While APLNR⁺ mesodermal cells lacked endothelial gene expression, molecular profiling of MS colonies at the core stage revealed that these cells acquired angioblastic gene expression profile as indicated by upregulation of FLT1, TEK, CDH5 (VE-cadherin), PECAM1 (CD31), FLI1, SELE (ELAM-1) and ICAM2 endothelial genes. When we collected MS cores (day 3 of clonogenic culture) and placed them on OP9, they formed predominantly VE-cadherin⁺ endothelial clusters, strongly indicating the endothelial nature of the core-forming cells. Subsequently, cells at the periphery of the core underwent endothelial-mesenchymal transition (EndMT) and formed a shell of tightly packed spindle-like cells around the core. When we picked colonies at this stage (day 6 of colony-forming culture) and placed them on OP9, most of the colonies (>70%) grew cell clusters composed of endothelial and mesenchymal cells. In contrast, mature MS colonies collected on day 12 of clonogenic culture formed predominantly clusters of mesenchymal cells, indicating a progressive loss of endothelial potential following colony maturation. Although no CD31 expression was detected in the mesenchymal cells composing mature MS colonies, these cells retained several endothelial traits including surface expression of endothelial tyrosine kinase (TEK or TIE2), FLT1 (VEGFR1) and endomucin. The critical role of EndMT in MS colony formation and MSC development was also congruous with our observation of the suppressive effect of VEGF, a known inhibitor of EndMT,^14,15 on MS colonies. When VEGF was added to MS clonogenic cultures, hESC-derived mesodermal cells were capable of forming angiogenic cores; however, these cores did not transform into mesenchymal cells, indicating that VEGF abrogates MS colony development at the core stage through inhibition of EndoMT. The schematic diagram demonstrating development of mesodermal MSCs is presented in Figure 2.Open in a separate windowFigure 2A model of mesoderm-derived MSC development from hESCs. Coculture with OP9 stromal cells predominantly induces hESC differentiation toward APLNR⁺ mesoderm. APLNR⁺ population contains angiogenic mesodermal precursors with either mesenchymal (mesenchymoangioblast) or hematopoietic (hemangioblast) potentials. Mesenchymoangioblasts and hemangioblasts arise sequentially during differentiation and can be revealed by MS and BL colony formation in response to FGF2. Development of MS and BL colonies in semisolid media proceed through a core stage at which APLNR⁺ cells form clusters of tightly packed cells with angiogenic potential. Subsequently, core-forming cells undergo EndMT giving rise to mesenchymal cells, which form a shell around the core developing into a mature MS colony. VEGF, EndMT inhibitor, blocks MS colony-formation at core stage. The ability of MS-CFCs to generate mesenchymal and endothelial cells can be revealed by coculture of individual colonies with OP9. Similar to MS colonies, BL colonies are formed through establishment of angiogenic core. However, hemangioblast core-forming cells undergo endothelial-hematopoietic transition and grew hematopoietic cells around the core.The close relationship between endothelial and hematopoietic cell development was recognized more than 130 years ago (reviewed by ref. ¹⁶) and confirmed in multiple modern studies.^9,17–22 However, the association between endothelial pre-cursors and MSCs during development was not well established, although cells with endothelial and mural cell potential were identified²³ and the critical role of EndMT in the formation of endocardial cushion²⁴ and testicular cords²⁵ in the embryo was acknowledged. Our hESC-based in vitro studies indicated that formation of mesodermal MSCs proceed through the endothelial stage and likely included at least two successive cycles of cell transitions. Initially APLNR⁺ mesoderm, which consists of fibroblast-like migratory cells, give rise to core structures composed of tightly packed endothelial cells in response to FGF2. Subsequently, endothelial cells forming cores undergo epithelial-mesenchymal transition, i.e., EndMT and form MSCs. The question remains how well this in vitro model reflects in vivo development. Although only sparse data exist regarding MSC precursors in the embryo, development of angiogenic hematopoietic precursors, hemangioblasts was studied more extensively in mammals and birds, and therefore parallels between in vivo and in vitro studies can be drawn. As we demonstrated,⁸ APLNR⁺ mesodermal cells collected from hESCs differentiated on OP9 for 3 days formed disperse BL colonies that identify hemangioblasts in vivo and in vitro.^9,26 Similar to MS colonies, the development of BL colonies required FGF2 and proceeded through angiogenic core formation. However, in contrast to MS cores, BL cores transformed into blood cells, i.e., underwent endothelial-hematopoietic transformation (see Fig. 2). Importantly, in vivo studies identified FGF2 as the essential factor in hemangioblast induction²⁷ analogous to our in vitro observation. In chicken embryo, the activation of FGF signaling leads to aggregation of migrating mesodermal cells adjacent to the endoderm, upregulation of VEGFR2 (KDR) expression, and subsequent formation of angioblasts and hemangioblasts.^28–30 This sequence of events leading to hemangioblast development in vivo considerably resembles what we observed in vitro, and highly suggests accurate recapitulation of embryonic development by our hESC differentiation model. Therefore, searching for an in vivo equivalent of mesenchymonagioblast would be a reasonable next step.In addition to embryonic development, EndMT is also implicated in several pathologies including cancer progression and development of cardiac and renal fibrosis.^31–34 Recently, Olsen group revealed that endothelial cells could be transformed directly into MSCs through overexpression of ALK2 or its activation by TGFβ2 or BMP4,¹⁵ indicating that endothelial cells could be an important source of MSCs in postnatal life. Conversely, the transition from MSCs to endothelial cells, has been also described in reference ³⁵. Based on these observations, a cycle of cell-fate transition from endothelium to MSCs and back to endothelium was proposed as a circuit controlling stem cell state.³⁶ Since multiple parallels could be drawn between EndMT described in adult tissues and during hESC differentiation, one may wonder whether bipotential cells with endothelial and MSC potential similar to embryonic mesenchymoangioblasts are present and constitute an important element of EndMT circuit in adults.In conclusion, the identification of mesenchymoangioblast as a clonogenic precursor of mesoderm-derived MSCs is an important step toward defining pathways of MSC development and specification. In addition, the demonstration of MSC formation from mesoderm through EndMT provides new insights into the mechanisms involved in establishment of MSCs.     

Deciphering the hierarchy of angiohematopoietic progenitors from human pluripotent stem cells

Identification of sequential progenitors leading to blood formation from pluripotent stem cells (PSCs) will be essential for understanding the molecular mechanisms of hematopoietic lineage specification and for development of technologies for in vitro production of hematopoietic stem cells (HSCs). It is well established that during development, blood and endothelial cells in the extraembryonic and embryonic compartments are formed in parallel from precursors with angiogenic and hematopoietic potentials. However, the identity and hierarchy of these precursors in human PSC (hPSC) cultures remain obscure. Using developmental stage-specific mesodermal and endothelial markers and functional assays, we recently identified discrete populations of angiohematopoietic progenitors from hPSCs, including mesodermal precursors and hemogenic endothelial cells with primitive and definitive hematopoietic potentials. In addition, we discovered a novel population of multipotent hematopoietic progenitors with an erythroid phenotype, which retain angiogenic potential. Here we introduce our recent findings and discuss their implication for defining putative HSC precursor and factors required for activation of self-renewal potential in hematopoietic cells emerging from endothelium.     

BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages

BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.     

Effect of aging on behaviour of mesenchymal stem cells

Organs whose source is the mesoderm lineage contain a subpopulation of stem cells that are able to differentiate among mesodermal derivatives (chondrocytes, osteocytes, adipocytes). This subpopulation of adult stem cells, called “mesenchymal stem cells” or “mesenchymal stromal cells (MSCs)”, contributes directly to the homeostatic maintenance of their organs; hence, their senescence could be very deleterious for human bodily functions. MSCs are easily isolated and amenable their expansion in vitro because of the research demanding to test them in many diverse clinical indications. All of these works are shown by the rapidly expanding literature that includes many in vivo animal models. We do not have an in-depth understanding of mechanisms that induce cellular senescence, and to further clarify the consequences of the senescence process in MSCs, some hints may be derived from the study of cellular behaviour in vivo and in vitro, autophagy, mitochondrial stress and exosomal activity. In this particular work, we decided to review these biological features in the literature on MSC senescence over the last three years.     

Specification and maintenance of the spinal cord stem zone

Epiblast cells adjacent to the regressing primitive streak behave as a stem zone that progressively generates the entire spinal cord and also contributes to paraxial mesoderm. Despite this fundamental task, this cell population is poorly characterised, and the tissue interactions and signalling pathways that specify this unique region are unknown. Fibroblast growth factor (FGF) is implicated but it is unclear whether it is sufficient and/or directly required for stem zone specification. It is also not understood how establishment of the stem zone relates to the acquisition of spinal cord identity as indicated by expression of caudal Hox genes. Here, we show that many cells in the chick stem zone express both early neural and mesodermal genes; however, stem zone-specific gene expression can be induced by signals from underlying paraxial mesoderm without concomitant induction of an ambivalent neural/mesodermal cell state. The stem zone is a site of FGF/MAPK signalling and we show that although FGF alone does not mimic paraxial mesoderm signals, it is directly required in epiblast cells for stem zone specification and maintenance. We further demonstrate that caudal Hox gene expression in the stem zone also depends on FGF and that neither stem zone specification nor caudal Hox gene onset requires retinoid signalling. These findings thus support a two step model for spinal cord generation - FGF-dependent establishment of the stem zone in which progressively more caudal Hox genes are expressed, followed by the retinoid-dependent assignment of spinal cord identity.     

Neuroepithelial cells supply an initial transient wave of MSC differentiation

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and are able to give rise to multiple mesenchymal lineages. Although MSCs are already used in regenerative medicine little is known about their in vivo behavior and developmental derivation. Here, we show that the earliest wave of MSC in the embryonic trunk is generated from Sox1+ neuroepithelium but not from mesoderm. Using lineage marking by direct gfp knock-in and Cre-recombinase mediated lineage tracing, we provide evidence that Sox1+ neuroepithelium gives rise to MSCs in part through a neural crest intermediate stage. This pathway can be distinguished from the pathway through which Sox1+ cells give rise to oligodendrocytes by expression of PDGFRbeta and A2B5. MSC recruitment from this pathway, however, is transient and is replaced by MSCs from unknown sources. We conclude that MSC can be defined as a definite in vivo entity recruited from multiple developmental origins.     

Heparan sulfate facilitates FGF and BMP signaling to drive mesoderm differentiation of mouse embryonic stem cells

Heparan sulfate (HS) has been implicated in regulating cell fate decisions during differentiation of embryonic stem cells (ESCs) into advanced cell types. However, the necessity and the underlying molecular mechanisms of HS in early cell lineage differentiation are still largely unknown. In this study, we examined the potential of EXT1(-/-) mouse ESCs (mESCs), that are deficient in HS, to differentiate into primary germ layer cells. We observed that EXT1(-/-) mESCs lost their differentiation competence and failed to differentiate into Pax6(+)-neural precursor cells and mesodermal cells. More detailed analyses highlighted the importance of HS for the induction of Brachyury(+) pan-mesoderm as well as normal gene expression associated with the dorso-ventral patterning of mesoderm. Examination of developmental cell signaling revealed that EXT1 ablation diminished FGF and BMP but not Wnt signaling. Furthermore, restoration of FGF and BMP signaling each partially rescued mesoderm differentiation defects. We further show that BMP4 is more prone to degradation in EXT1(-/-) mESCs culture medium compared with that of wild type cells. Therefore, our data reveal that HS stabilizes BMP ligand and thereby maintains the BMP signaling output required for normal mesoderm differentiation. In summary, our study demonstrates that HS is required for ESC pluripotency, in particular lineage specification into mesoderm through facilitation of FGF and BMP signaling.     

Loss of FGF-Dependent Mesoderm Identity and Rise of Endogenous Retinoid Signalling Determine Cessation of Body Axis Elongation

The endogenous mechanism that determines vertebrate body length is unknown but must involve loss of chordo-neural-hinge (CNH)/axial stem cells and mesoderm progenitors in the tailbud. In early embryos, Fibroblast growth factor (FGF) maintains a cell pool that progressively generates the body and differentiation onset is driven by retinoid repression of FGF signalling. This raises the possibility that FGF maintains key tailbud cell populations and that rising retinoid activity underlies cessation of body axis elongation. Here we show that sudden loss of the mesodermal gene (Brachyury) from CNH and the mesoderm progenitor domain correlates with FGF signalling decline in the late chick tailbud. This is accompanied by expansion of neural gene expression and a similar change in cell fate markers is apparent in the human tailbud. Fate mapping of chick tailbud further revealed that spread of neural gene expression results from continued ingression of CNH-derived cells into the position of the mesoderm progenitor domain. Using gain and loss of function approaches in vitro and in vivo, we then show that attenuation of FGF/Erk signalling mediates this loss of Brachyury upstream of Wnt signalling, while high-level FGF maintains Brachyury and can induce ectopic CNH-like cell foci. We further demonstrate a rise in endogenous retinoid signalling in the tailbud and show that here FGF no longer opposes retinoid synthesis and activity. Furthermore, reduction of retinoid signalling at late stages elevated FGF activity and ectopically maintained mesodermal gene expression, implicating endogenous retinoid signalling in loss of mesoderm identity. Finally, axis termination is concluded by local cell death, which is reduced by blocking retinoid signalling, but involves an FGFR-independent mechanism. We propose that cessation of body elongation involves loss of FGF-dependent mesoderm identity in late stage tailbud and provide evidence that rising endogenous retinoid activity mediates this step and ultimately promotes cell death in chick tailbud.     

Bone marrow mesenchymal stem cells: agents of immunomodulation and neuroprotection

Mesenchymal stromal cells (MSC) are part of the bone marrow stem cells repertoire which also includes the main stem cells population of the bone marrow, the hematopoietic stem cells. The main role of MSCs is to support hematopoiesis but they can also give rise to cells of the mesodermal layers. Recently, significant interactions between MSCs and cells from the immune system have been demonstrated: MSCs were found to downregulate T and B lymphocytes, natural killer cells (NK) and antigen presenting cells through various mechanisms, including cell-to cell interaction and soluble factor production. Besides the immunomodulatory effects, MSCs were shown to possess additional stem cells features, such as the self-renewal potential and multipotency. Their debatable transdifferentiation potential to cells of the endo- and exo-dermal layer, including cells of the CNS, may explain in part their reported neuroprotective effects. Studies in vitro and in vivo (in cells cultures and in animal models) have indicated neuroprotective effects. MSCs are believed to promote functional recovery following CNS injury or inflammation, by producing trophic factors that may facilitate the mobilization of endogenous neural stem cells and promote the regeneration or the survival of the affected neurons. These immunomodulatory and neuroprotective features could make MSCs potential candidates for future therapeutic modalities in immune-mediated and neurodegenerative diseases.     

FGF8-like1 and FGF8-like2 encode putative ligands of the FGF receptor Htl and are required for mesoderm migration in the Drosophila gastrula

BACKGROUND: Mesoderm migration in the Drosophila gastrula depends on the fibroblast growth factor (FGF) receptor Heartless (Htl). During gastrulation Htl is required for adhesive interactions of the mesoderm with the ectoderm and for the generation of protrusive activity of the mesoderm cells during migration. After gastrulation Htl is essential for the differentiation of dorsal mesodermal derivatives. It is not known how Htl is activated, because its ligand has not yet been identified. RESULTS: We performed a genome-wide genetic screen for early zygotic genes and identified seven genomic regions that are required for normal migration of the mesoderm cells during gastrulation. One of these genomic intervals produces upon its deletion a phenocopy of the htl cell migration phenotype. Here we present the genetic and molecular mapping of this genomic region. We identified two genes, FGF8-like1 and FGF8-like2, that encode novel FGF homologs and were only partially annotated in the Drosophila genome. We show that FGF8-like1 and FGF8-like2 are expressed in the neuroectoderm during gastrulation and present evidence that both act in concert to direct cell shape changes during mesodermal cell migration and are required for the activation of the Htl signaling cascade during gastrulation. CONCLUSIONS: We conclude that FGF8-like1 and FGF8-like2 encode two novel Drosophila FGF homologs, which are required for mesodermal cell migration during gastrulation. Our results suggest that FGF8-like1 and FGF8-like2 represent ligands of the Htl FGF receptor.     

Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny

Quail-chick intracoelomic grafts of organ rudiments were used to study the origin of endothelia and haemopoietic cells during avian organogenesis in conjunction with the monoclonal antibody QH1 which recognizes the quail haemangioblastic lineage. Results differed according to the germ-layer constitution of the grafted rudiments. In the case of the limb buds, endothelial cells from the host invaded the graft through an angiogenic process. Haemopoietic progenitors from the host also colonized the grafted bone marrow. In contrast, rudiments of internal organs provided their own contingent of endothelial precursors, a process termed vasculogenesis. Nevertheless, haemopoietic cells in these organs were all derived from the host. In the lung, this extrinsic cell population appeared regularly scattered around the parabronchi and had a macrophage-like phenotype. In the pancreas, the granulocytes which differentiate as dense aggregates located in the wall of the largest vessels were extrinsic. Similarly in the spleen, a mesodermal primordium that develops in close association with the pancreatic endoderm, endothelial cells were intrinsic and haemopoietic cells host-derived. This study demonstrates that, in ontogeny, vascularization obeys different rules depending on which germ layer the mesoderm is associated with: in mesodermal/ectodermal rudiments angiogenesis is the rule; in mesodermal/endodermal rudiments, vasculogenesis occurs. However, in these internal organs undergoing vasculogenesis, endothelial and haemopoietic cells have separate origins. We put forward the hypothesis that the endoderm induces the emergence of endothelial cells in the associated mesoderm. Formation of blood stem cells may also involve interactions between endoderm and mesoderm, but in this case the responding capacity of the mesoderm appears restricted to the paraaortic region.     

Human mesenchymal stem cells stimulated by TNF-alpha, LPS, or hypoxia produce growth factors by an NF kappa B- but not JNK-dependent mechanism

Understanding the mechanisms by which adult stem cells produce growth factors may represent an important way to optimize their beneficial paracrine and autocrine effects. Components of the wound milieu may stimulate growth factor production to promote stem cell-mediated repair. We hypothesized that tumor necrosis factor-alpha (TNF-alpha), endotoxin (LPS), or hypoxia may activate human mesenchymal stem cells (MSCs) to increase release of vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), insulin-like growth factor 1 (IGF-1), or hepatocyte growth factor (HGF) and that nuclear factor-kappa B (NF kappa B), c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) mediates growth factor production from human MSCs. To study this, human MSCs were harvested, passaged, divided into four groups (100,000 cells, triplicates) and treated as follows: 1) with vehicle; 2) with stimulant alone [24 h LPS (200 ng/ml), 24 h TNF-alpha (50 ng/ml), or 24 h hypoxia (1% O2)]; 3) with inhibitor alone [NF kappa B (PDTC, 1 mM), JNK (TI-JIP, 10 microM), or ERK (ERK Inhibitor II, 25 microM)]; and 4) with stimulant and the various inhibitors. After 24 h incubation, MSC activation was determined by measuring supernatants for VEGF, FGF2, IGF-1, or HGF (ELISA). TNF-alpha, LPS, and hypoxia significantly increased human MSC VEGF, FGF2, HGF, and IGF-1 production versus controls. Stem cells exposed to injury demonstrated increased activation of NF kappa B, ERK, and JNK. VEGF, FGF2, and HGF expression was significantly reduced by NF kappa B inhibition (50% decrease) but not ERK or JNK inhibition. Moreover, ERK, JNK, and NF kappa B inhibitor alone did not activate MSC VEGF expression over controls. Various stressors activate human MSCs to increase VEGF, FGF2, HGF, and IGF-1 expression, which depends on an NFkB mechanism.     

Secretome from mesenchymal stem cells induces angiogenesis via Cyr61

It is well known that bone marrow‐derived mesenchymal stem cells (MSCs) are involved in wound healing and regeneration responses. In this study, we globally profiled the proteome of MSCs to investigate critical factor(s) that may promote wound healing. Cysteine‐rich protein 61 (Cyr61) was found to be abundantly present in MSCs. The presence of Cyr61 was confirmed by immunofluorescence staining and immunoblot analysis. Moreover, we showed that Cyr61 is present in the culture medium (secretome) of MSCs. The secretome of MSCs stimulates angiogenic response in vitro, and neovascularization in vivo. Depletion of Cyr61 completely abrogates the angiogenic‐inducing capability of the MSC secretome. Importantly, addition of recombinant Cyr61 polypeptides restores the angiogenic activity of Cyr61‐depleted secretome. Collectively, these data demonstrate that Cyr61 polypeptide in MSC secretome contributes to the angiogenesis‐promoting activity, a key event needed for regeneration and repair of injured tissues. J. Cell. Physiol. 219: 563–571, 2009. © 2009 Wiley‐Liss, Inc.     

Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation

The hematopoietic and endothelial lineages derive from mesoderm and are thought to develop through the maturation of a common progenitor, the hemangioblast. To investigate the developmental processes that regulate mesoderm induction and specification to the hemangioblast, we generated an embryonic stem cell line with the green fluorescent protein (GFP) targeted to the mesodermal gene, brachyury. After the in vitro differentiation of these embryonic stem cells to embryoid bodies, developing mesodermal progenitors could be separated from those with neuroectoderm potential based on GFP expression. Co-expression of GFP with the receptor tyrosine kinase Flk1 revealed the emergence of three distinct cell populations, GFP(-)Flk1(-), GFP(+)Flk1(-) and GFP(+)Flk1(+) cells, which represent a developmental progression ranging from pre-mesoderm to prehemangioblast mesoderm to the hemangioblast.     

BMP and FGF regulate the differentiation of multipotential pericardial mesoderm into the myocardial or epicardial lineage

Proepicardial cells give rise to epicardium, coronary vasculature and cardiac fibroblasts. The proepicardium is derived from the mesodermal lining of the prospective pericardial cavity that simultaneously contributes myocardium to the venous pole of the elongating primitive heart tube. Using proepicardial explant cultures, we show that proepicardial cells have the potential to differentiate into cardiac muscle cells, reflecting the multipotency of this pericardial mesoderm. The differentiation into the myocardial or epicardial lineage is mediated by the cooperative action of BMP and FGF signaling. BMP2 is expressed in the distal IFT myocardium and stimulates cardiomyocyte formation. FGF2 is expressed in the proepicardium and stimulates differentiation into the epicardial lineage. In the base of the proepicardium, coexpression of BMP2 and FGF2 inhibits both myocardial and epicardial differentiation. We conclude that the epicardial/myocardial lineage decisions are mediated by an extrinsic, inductive mechanism, which is determined by the position of the cells in the pericardial mesoderm.     

Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol promotes angiogenesis in a mouse model

Several studies of stem cell-based gene therapy have indicated that long-lasting regeneration following vessel ischemia may be stimulated through VEGFA gene therapy and/or MSC transplantation for reduction of ischemic injury in limb ischemia and heart failure. The therapeutic potential of MSC transplantation can be further improved by genetically modifying MSCs with genes which enhance angiogenesis following ischemic injury. In the present study, we aimed to develop an approach in MSC-based therapy for repair and mitigation of ischemic injury and regeneration of damaged tissues in ischemic disease. HSP70 promoter-driven VEGFA expression was induced by resveratrol (RSV) in MSCs, and in combination with known RSV biological functions, the protective effects of our approach were investigated by using ex vivo aortic ring coculture system and a 3D scaffolds in vivo model. Results of this investigation demonstrated that HSP promoter-driven VEGFA expression in MSC increased approximately 2-fold over the background VEGFA levels upon HSP70 promoter induction by RSV. Exposure of HUVEC cells to medium containing MSC in which VEGFA had been induced by cis-RSV enhanced tube formation in the treated HUVEC cells. RSV-treated MSC cells differentiated into endothelial-like phenotypes, exhibiting markedly elevated expression of endothelial cell markers. These MSCs also induced aortic ring sprouting, characteristic of neovascular formation from pre-existing vessels, and additionally promoted neovascularization at the MSC transplantation site in a mouse model. These observations support a hypothesis that VEGFA expression induced by cis-RSV acting on the HSP70 promoter in transplanted MSC augments the angiogenic effects of stem cell gene therapy. The use of an inducible system also vastly reduces possible clinical risks associated with constitutive VEGFA expression.