A multistep computational approach reveals a neuro-mesenchymal cell population in the embryonic hematopoietic stem cell niche.

The first hematopoietic stem and progenitor cells (HSPCs) emerge in the Aorta-Gonad-Mesonephros (AGM) region of the mid-gestation mouse embryo. However, the precise nature of their supportive mesenchymal microenvironment remains largely unexplored. Here, we profiled transcriptomes of laser micro-dissected aortic tissues at three developmental stages and individual AGM cells. Computational analyses allowed the identification of several cell subpopulations within the E11.5 AGM mesenchyme, with the presence of a yet unidentified subpopulation characterized by the dual expression of genes implicated in adhesive or neuronal functions. We confirmed the identity of this cell subset as a neuro-mesenchymal population, through morphological and lineage tracing assays. Loss of function in the zebrafish confirmed that Decorin, a characteristic extracellular matrix component of the neuro-mesenchyme, is essential for HSPC development. We further demonstrated that this cell population is not merely derived from the neural crest, and hence, is a bona fide novel subpopulation of the AGM mesenchyme.

Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions.

Portal fibroblasts with mesenchymal stem cell features form a reservoir of proliferative myofibroblasts in liver fibrosis.

BACKGROUND AND AIMS: In liver fibrosis, myofibroblasts derive from HSCs and as yet undefined mesenchymal cells. We aimed to identify portal mesenchymal progenitors of myofibroblasts. APPROACH AND RESULTS: Portal mesenchymal cells were isolated from mouse bilio-vascular tree and analyzed by single-cell RNA-sequencing. Thereby, we uncovered the landscape of portal mesenchymal cells in homeostatic mouse liver. Trajectory analysis enabled inferring a small cell population further defined by surface markers used to isolate it. This population consisted of portal fibroblasts with mesenchymal stem cell features (PMSCs), i.e., high clonogenicity and trilineage differentiation potential, that generated proliferative myofibroblasts, contrasting with nonproliferative HSC-derived myofibroblasts (-MF). Using bulk RNA-sequencing, we built oligogene signatures of the two cell populations that remained discriminant across myofibroblastic differentiation. SLIT2, a prototypical gene of PMSC/PMSC-MF signature, mediated profibrotic and angiogenic effects of these cells, which conditioned medium promoted HSC survival and endothelial cell tubulogenesis. Using PMSC/PMSC-MF 7-gene signature and slit guidance ligand 2 fluorescent in situ hybridization, we showed that PMSCs display a perivascular portal distribution in homeostatic liver and largely expand with fibrosis progression, contributing to the myofibroblast populations that form fibrotic septa, preferentially along neovessels, in murine and human liver disorders, irrespective of etiology. We also unraveled a 6-gene expression signature of HSCs/HSC-MFs that did not vary in these disorders, consistent with their low proliferation rate. CONCLUSIONS: PMSCs form a small reservoir of expansive myofibroblasts, which, in interaction with neovessels and HSC-MFs that mainly arise through differentiation from a preexisting pool, underlie the formation of fibrotic septa in all types of liver diseases.

The crosstalk between hematopoietic stem cells and their niches.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) reside in specific microenvironments also called niches that regulate HSC functions. Understanding the molecular and cellular mechanisms involved in the crosstalk between HSCs and niche cells is a major issue in stem cell biology and regenerative medicine. The purpose of this review is to discuss recent advances in this field with particular emphasis on the transcriptional landscape of HSC niche cells and the roles of extracellular vesicles (EVs) in the dialog between HSCs and their microenvironments. RECENT FINDINGS: The development of high-throughput technologies combined with computational methods has considerably improved our knowledge on the molecular identity of HSC niche cells. Accumulating evidence strongly suggest that the dialog between HSCs and their niches is bidirectional and that EVs play an important role in this process. SUMMARY: These advances bring a unique conceptual and methodological framework for understanding the molecular complexity of the HSC niche and identifying novel HSC regulators. They are also promising for exploring the reciprocal influence of HSCs on niche cells and delivering specific molecules to HSCs in regenerative medicine.

Bistable Epigenetic States Explain Age-Dependent Decline in Mesenchymal Stem Cell Heterogeneity.

The molecular mechanisms by which heterogeneity, a major characteristic of stem cells, is achieved are yet unclear. We here study the expression of the membrane stem cell antigen-1 (Sca-1) in mouse bone marrow mesenchymal stem cell (MSC) clones. We show that subpopulations with varying Sca-1 expression profiles regenerate the Sca-1 profile of the mother population within a few days. However, after extensive replication in vitro, the expression profiles shift to lower values and the regeneration time increases. Study of the promoter of Ly6a unravels that the expression level of Sca-1 is related to the promoter occupancy by the activating histone mark H3K4me3. We demonstrate that these findings can be consistently explained by a computational model that considers positive feedback between promoter H3K4me3 modification and gene transcription. This feedback implicates bistable epigenetic states which the cells occupy with an age-dependent frequency due to persistent histone (de-)modification. Our results provide evidence that MSC heterogeneity, and presumably that of other stem cells, is associated with bistable epigenetic states and suggest that MSCs are subject to permanent state fluctuations. Stem Cells 2017;35:694-704.

CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro-osteogenic and pro-inflammatory cues.

Similar to other adult tissue stem/progenitor cells, bone marrow mesenchymal stem/stromal cells (BM MSCs) exhibit heterogeneity at the phenotypic level and in terms of proliferation and differentiation potential. In this study such a heterogeneity was reflected by the CD200 protein. We thus characterized CD200(pos) cells sorted from whole BM MSC cultures and we investigated the molecular mechanisms regulating CD200 expression. After sorting, measurement of lineage markers showed that the osteoblastic genes RUNX2 and DLX5 were up-regulated in CD200(pos) cells compared to CD200(neg) fraction. At the functional level, CD200(pos) cells were prone to mineralize the extra-cellular matrix in vitro after sole addition of phosphates. In addition, osteogenic cues generated by bone morphogenetic protein 4 (BMP4) or BMP7 strongly induced CD200 expression. These data suggest that CD200 expression is related to commitment/differentiation towards the osteoblastic lineage. Immunohistochemistry of trephine bone marrow biopsies further corroborates the osteoblastic fate of CD200(pos) cells. However, when dexamethasone was used to direct osteogenic differentiation in vitro, CD200 was consistently down-regulated. As dexamethasone has anti-inflammatory properties, we assessed the effects of different immunological stimuli on CD200 expression. The pro-inflammatory cytokines interleukin-1ß and tumour necrosis factor-α increased CD200 membrane expression but down-regulated osteoblastic gene expression suggesting an additional regulatory pathway of CD200 expression. Surprisingly, whatever the context, i.e. pro-inflammatory or pro-osteogenic, CD200 expression was down-regulated when nuclear-factor (NF)-κB was inhibited by chemical or adenoviral agents. In conclusion, CD200 expression by cultured BM MSCs can be induced by both osteogenic and pro-inflammatory cytokines through the same pathway: NF-κB.

Mesenchymal stem cell features of Ewing tumors.

The cellular origin of Ewing tumor (ET), a tumor of bone or soft tissues characterized by specific fusions between EWS and ETS genes, is highly debated. Through gene expression analysis comparing ETs with a variety of normal tissues, we show that the profiles of different EWS-FLI1-silenced Ewing cell lines converge toward that of mesenchymal stem cells (MSC). Moreover, upon EWS-FLI1 silencing, two different Ewing cell lines can differentiate along the adipogenic lineage when incubated in appropriate differentiation cocktails. In addition, Ewing cells can also differentiate along the osteogenic lineage upon long-term inhibition of EWS-FLI1. These in silico and experimental data strongly suggest that the inhibition of EWS-FLI1 may allow Ewing cells to recover the phenotype of their MSC progenitor.

Differential gene expression profiling of human bone marrow-derived mesenchymal stem cells during adipogenic development.

BACKGROUND: Adipogenesis is the developmental process by which mesenchymal stem cells (MSC) differentiate into pre-adipocytes and adipocytes. The aim of the study was to analyze the developmental strategies of human bone marrow MSC developing into adipocytes over a defined time scale. Here we were particularly interested in differentially expressed transcription factors and biochemical pathways. We studied genome-wide gene expression profiling of human MSC based on an adipogenic differentiation experiment with five different time points (day 0, 1, 3, 7 and 17), which was designed and performed in reference to human fat tissue. For data processing and selection of adipogenic candidate genes, we used the online database SiPaGene for Affymetrix microarray expression data. RESULTS: The mesenchymal stem cell character of human MSC cultures was proven by cell morphology, by flow cytometry analysis and by the ability of the cells to develop into the osteo-, chondro- and adipogenic lineage. Moreover we were able to detect 184 adipogenic candidate genes (85 with increased, 99 with decreased expression) that were differentially expressed during adipogenic development of MSC and/or between MSC and fat tissue in a highly significant way (p < 0.00001). Subsequently, groups of up- or down-regulated genes were formed and analyzed with biochemical and cluster tools. Among the 184 genes, we identified already known transcription factors such as PPARG, C/EBPA and RTXA. Several of the genes could be linked to corresponding biochemical pathways like the adipocyte differentiation, adipocytokine signalling, and lipogenesis pathways. We also identified new candidate genes possibly related to adipogenesis, such as SCARA5, coding for a receptor with a putative transmembrane domain and a collagen-like domain, and MRAP, encoding an endoplasmatic reticulum protein. CONCLUSIONS: Comparing differential gene expression profiles of human MSC and native fat cells or tissue allowed us to establish a comprehensive differential kinetic gene expression network of adipogenesis. Based on this, we identified known and unknown genes and biochemical pathways that may be relevant for adipogenic differentiation. Our results encourage further and more focused studies on the functional relevance of particular adipogenic candidate genes.

Novel markers of mesenchymal stem cells defined by genome-wide gene expression analysis of stromal cells from different sources.

Mesenchymal stem cells (MSC) represent a mixture of different cell types, of which only a minority is therapeutically relevant. Surface markers specifically identifying non-differentiated MSC from their differentiated progeny have not been described in sufficient detail. We here compare the gene expression profile of the in vivo bone-forming bone marrow-derived MSC (BM-MSC) with non-bone-forming umbilical vein stromal cells (UVSC) and other non-MSC. Clustering analysis shows that UVSC are a lineage homogeneous cell population, clearly distinct from MSC, other mesenchymal lineages and hematopoietic cells. We find that 89 transcripts of membrane-associated proteins are represented more in cultured BM-MSC than in UVSC. These include previously identified molecules, but also novel markers like NOTCH3, JAG1, and ITGA11. We show that the latter three molecules are also expressed on fibroblast colony-forming units (CFU-F). Both NOTCH3 and ITGA11, but not JAG1, further enrich for CFU-F when combined with CD146, a known marker of cells with MSC activity in vivo. Differentiation studies show that NOTCH3+ and CD146+ NOTCH3+ cells sorted from cultured BM-MSC are capable of adipogenic and osteogenic progeny, while ITGA11-expressing cells mainly show an osteogenic differentiation profile with limited adipogenic differentiation. Our observations may facilitate the study of lineage relationships in MSC as well as facilitate the development of more homogeneous cell populations for mesenchymal cell therapy.

[Human mesenchymal stem cell biology]. / La biologie des cellules souchesmésenchymateuses d'origine humaine.

This brief overview summarises the main characteristics of bone marrow mesenchymal stem cells and of adipose-derived stem cells: methods of obtention, phenotype, differentiation potential, hematopoiesis-supportive (stromal) capacity, and immunosuppressive properties. Two points are discussed in detail: 1) criteria for stemness: multipotency, self-renewal, plasticity, and 2) the repair mechanisms implicated in the different indications of cell therapy using these cells: reconstitution of the tissue functional compartment by repopulation consequent to proliferation and differentiation or reprogrammation, stromal effects by secretion of angiogenic, anti-apoptotic, anti-fibrogenic factors, molecules involved in the regulation of inflammation, etc.

In vivo screen identifies a SIK inhibitor that induces ß cell proliferation through a transient UPR.

It is known that ß cell proliferation expands the ß cell mass during development and under certain hyperglycemic conditions in the adult, a process that may be used for ß cell regeneration in diabetes. Here, through a new high-throughput screen using a luminescence ubiquitination-based cell cycle indicator (LUCCI) in zebrafish, we identify HG-9-91-01 as a driver of proliferation and confirm this effect in mouse and human ß cells. HG-9-91-01 is an inhibitor of salt-inducible kinases (SIKs), and overexpression of Sik1 specifically in ß cells blocks the effect of HG-9-91-01 on ß cell proliferation. Single-cell transcriptomic analyses of mouse ß cells demonstrate that HG-9-91-01 induces a wave of activating transcription factor (ATF)6-dependent unfolded protein response (UPR) before cell cycle entry. Importantly, the UPR wave is not associated with an increase in insulin expression. Additional mechanistic studies indicate that HG-9-91-01 induces multiple signalling effectors downstream of SIK inhibition, including CRTC1, CRTC2, ATF6, IRE1 and mTOR, which integrate to collectively drive ß cell proliferation.

Specific lineage-priming of bone marrow mesenchymal stem cells provides the molecular framework for their plasticity.

Lineage-priming is a molecular model of stem cell (SC) differentiation in which proliferating SCs express a subset of genes associated to the differentiation pathways to which they can commit. This concept has been developed for hematopoietic SCs, but has been poorly studied for other SC populations. Because the differentiation potential of human bone marrow mesenchymal stem cells (BM MSCs) remains controversial, we have explored the theory of lineage-priming applied to these cells. We show that proliferating primary layers and clones of BM MSCs have precise priming to the osteoblastic (O), chondrocytic (C), adipocytic (A), and the vascular smooth muscle (V) lineages, but not to skeletal muscle, cardiac muscle, hematopoietic, hepatocytic, or neural lineages. Priming was shown both at the mRNA (300 transcripts were evaluated) and the protein level. In particular, the master transactivator proteins PPARG, RUNX2, and SOX9 were coexpressed before differentiation induction in all cells from incipient clones. We further show that MSCs cultured in the presence of inducers differentiate into the lineages for which they are primed. Our data point out to a number of signaling pathways that might be activated in proliferating MSCs and would be responsible for the differentiation and proliferation potential of these cells. Our results extend the notion of lineage-priming and provide the molecular framework for inter-A, -O, -C, -V plasticity of BM MSCs. Our data highlight the use of BM MSCs for the cell therapy of skeletal or vascular disorders, but provide a word of caution about their use in other clinical indications.

Inferring Gene Networks in Bone Marrow Hematopoietic Stem Cell-Supporting Stromal Niche Populations.

The cardinal property of bone marrow (BM) stromal cells is their capacity to contribute to hematopoietic stem cell (HSC) niches by providing mediators assisting HSC functions. In this study we first contrasted transcriptomes of stromal cells at different developmental stages and then included large number of HSC-supportive and non-supportive samples. Application of a combination of algorithms, comprising one identifying reliable paths and potential causative relationships in complex systems, revealed gene networks characteristic of the BM stromal HSC-supportive capacity and of defined niche populations of perivascular cells, osteoblasts, and mesenchymal stromal cells. Inclusion of single-cell transcriptomes enabled establishing for the perivascular cell subset a partially oriented graph of direct gene-to-gene interactions. As proof of concept we showed that R-spondin-2, expressed by the perivascular subset, synergized with Kit ligand to amplify ex vivo hematopoietic precursors. This study by identifying classifiers and hubs constitutes a resource to unravel candidate BM stromal mediators.

Comparative proteomic analysis of human mesenchymal and embryonic stem cells: towards the definition of a mesenchymal stem cell proteomic signature.

Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2-DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo-natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.

In vivo osteoprogenitor potency of human stromal cells from different tissues does not correlate with expression of POU5F1 or its pseudogenes.

Expression of "stemness" markers is widely used as a predictor of stem cell properties of mesenchymal stem cells (MSC). Here, we show that bone marrow-derived (BM)-MSC show stem cell-like behavior in vivo; that is, they form ossicles with formation of bone, formation of adipocytes, and establishment of the murine hematopoietic microenvironment. Multipotent umbilical vein-derived stromal cells (UVSC), on the other hand, do not form bone, nor do they give rise to adipocytes in vivo. Despite these differences in stem-cell-like behavior, BM-MSC and UVSC express the two transcripts variants of POU5F1 at a similar level. Also, we found that in BM-MSC and UVSC, POU5F1 is detectable. However, more than 89% of the POU5F1 transcripts correspond to the POU5F1P1, -P3, or -P4 pseudogene. Despite low-level expression of POU5F1, we were unable to precipitate POU5F1 protein in either BM-MSC or UVSC. These results demonstrate that MSC stemness does not correlate to expression of POU5F1 transcripts or its pseudogenes.

Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients.

BACKGROUND AIMS: Advances in bone tissue engineering with mesenchymal stromal cells (MSC) as an alternative to conventional orthopedic procedures has opened new horizons for the treatment of large bone defects. Bone marrow (BM) and trabecular bone are both sources of MSC. Regarding clinical use, we tested the potency of MSC from different sources. METHODS: We obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and trochanter, as well as BM aspirates of the iliac crest and trochanter. The starting material was evaluated by histologic analysis and assessment of colony-forming unit-fibroblasts (CFU-F). The MSC populations were compared for proliferation and differentiation potential, at RNA and morphologic levels. RESULTS: MSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar whatever the starting material. However, the differentiation potential of MSC obtained by bone washing was impaired compared with aspiration; culture-amplified cells showed few Oil Red O-positive adipocytes and few mineralized areas and formed inconsistent Alcian blue-positive high-density micropellets after growth under adipogenic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed better differentiation potential. CONCLUSIONS: Trabecular bone MSC from elderly patients is not good starting material for use in cell therapy for bone repair and regeneration, unless cultured in the presence of FGF-2.

FHL2 mediates dexamethasone-induced mesenchymal cell differentiation into osteoblasts by activating Wnt/beta-catenin signaling-dependent Runx2 expression.

The differentiation of bone marrow mesenchymal stem cells (MSCs) into osteoblasts is a crucial step in bone formation. However, the mechanisms involved in the early stages of osteogenic differentiation are not well understood. In this study, we identified FHL2, a member of the LIM-only subclass of the LIM protein superfamily, that is up-regulated during early osteoblast differentiation induced by dexamethasone in murine and human MSCs. Gain-of-function studies showed that FHL2 promotes the expression of the osteoblast transcription factor Runx2, alkaline phosphatase, type I collagen, as well as in vitro extracellular matrix mineralization in murine and human mesenchymal cells. Knocking down FHL2 using sh-RNA reduces basal and dexamethasone-induced osteoblast marker gene expression in MSCs. We demonstrate that FHL2 interacts with beta-catenin, a key player involved in bone formation induced by Wnt signaling. FHL2-beta-catenin interaction potentiates beta-catenin nuclear translocation and TCF/LEF transcription, resulting in increased Runx2 and alkaline phosphatase expression, which was inhibited by the Wnt inhibitor DKK1. Reduction of Runx2 transcriptional activity using a mutant Runx2 results in inhibition of FHL2-induced alkaline phosphatase expression in MSCs. These findings reveal that FHL2 acts as an endogenous activator of mesenchymal cell differentiation into osteoblasts and mediates osteogenic differentiation induced by dexamethasone in MSCs through activation of Wnt/beta-catenin signaling- dependent Runx2 expression.

Osteogenic differentiation of human bone marrow mesenchymal stem cells seeded on melt based chitosan scaffolds for bone tissue engineering applications.

The purpose of this study was to evaluate the growth patterns and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) when seeded onto new biodegradable chitosan/polyester scaffolds. Scaffolds were obtained by melt blending chitosan with poly(butylene succinate) in a proportion of 50% (wt) each and further used to produce a fiber mesh scaffold. hBMSCs were seeded on those structures and cultured for 3 weeks under osteogenic conditions. Cells were able to reduce MTS and demonstrated increasing metabolic rates over time. SEM observations showed cell colonization at the surface as well as within the scaffolds. The presence of mineralized extracellular matrix (ECM) was successfully demonstrated by peaks corresponding to calcium and phosphorus elements detected in the EDS analysis. A further confirmation was obtained when carbonate and phosphate group peaks were identified in Fourier Transformed Infrared (FTIR) spectra. Moreover, by reverse transcriptase (RT)-PCR analysis, it was observed the expression of osteogenic gene markers, namely, Runt related transcription factor 2 (Runx2), type 1 collagen, bone sialoprotein (BSP), and osteocalcin. Chitosan-PBS (Ch-PBS) biodegradable scaffolds support the proliferation and osteogenic differentiation of hBMSCs cultured at their surface in vitro, enabling future in vivo testing for the development of bone tissue engineering therapies.

In vivo generation of haematopoietic stem/progenitor cells from bone marrow-derived haemogenic endothelium.

It is well established that haematopoietic stem and progenitor cells (HSPCs) are generated from a transient subset of specialized endothelial cells termed haemogenic, present in the yolk sac, placenta and aorta, through an endothelial-to-haematopoietic transition (EHT). HSPC generation via EHT is thought to be restricted to the early stages of development. By using experimental embryology and genetic approaches in birds and mice, respectively, we document here the discovery of a bone marrow haemogenic endothelium in the late fetus/young adult. These cells are capable of de novo producing a cohort of HSPCs in situ that harbour a very specific molecular signature close to that of aortic endothelial cells undergoing EHT or their immediate progenies, i.e., recently emerged HSPCs. Taken together, our results reveal that HSPCs can be generated de novo past embryonic stages. Understanding the molecular events controlling this production will be critical for devising innovative therapies.