VRK3 depletion induces cell cycle arrest and metabolic reprogramming of pontine diffuse midline glioma - H3K27 altered cells.

We previously identified VRK3 as a specific vulnerability in DMG-H3K27M cells in a synthetic lethality screen targeting the whole kinome. The aim of the present study was to elucidate the mechanisms by which VRK3 depletion impact DMG-H3K27M cell fitness. Gene expression studies after VRK3 knockdown emphasized the inhibition of genes involved in G1/S transition of the cell cycle resulting in growth arrest in G1. Additionally, a massive modulation of genes involved in chromosome segregation was observed, concomitantly with a reduction in the level of phosphorylation of serine 10 and serine 28 of histone H3 supporting the regulation of chromatin condensation during cell division. This last effect could be partly due to a concomitant decrease of the chromatin kinase VRK1 in DMG following VRK3 knockdown. Furthermore, a metabolic switch specific to VRK3 function was observed towards increased oxidative phosphorylation without change in mitochondria content, that we hypothesized would represent a cell rescue mechanism. This study further explored the vulnerability of DMG-H3K27M cells to VRK3 depletion suggesting potential therapeutic combinations, e.g. with the mitochondrial ClpP protease activator ONC201.

Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor.

BACKGROUND: hPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages. METHODS: Two hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144+-embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144+, respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other's (e.g. hPSC-ECs vs. hPSC-EB-CD144+) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations. RESULTS: A hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144+ contributed to the generation of CD45+ human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144+. Endothelial differentiation of hPSC-EB-CD144+ yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144+ generated an intermediate population of > 90% CD43+ hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144+, hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144+CD45+, a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors. CONCLUSION: The present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering.

Neurogenic Heterotopic Ossifications Recapitulate Hematopoietic Stem Cell Niche Development Within an Adult Osteogenic Muscle Environment.

Hematopoiesis and bone interact in various developmental and pathological processes. Neurogenic heterotopic ossifications (NHO) are the formation of ectopic hematopoietic bones in peri-articular muscles that develop following severe lesions of the central nervous system such as traumatic cerebral or spinal injuries or strokes. This review will focus on the hematopoietic facet of NHO. The characterization of NHO demonstrates the presence of hematopoietic marrow in which quiescent hematopoietic stem cells (HSC) are maintained by a functional stromal microenvironment, thus documenting that NHOs are neo-formed ectopic HSC niches. Similarly to adult bone marrow, the NHO permissive environment supports HSC maintenance, proliferation and differentiation through bidirectional signaling with mesenchymal stromal cells and endothelial cells, involving cell adhesion molecules, membrane-bound growth factors, hormones, and secreted matrix proteins. The participation of the nervous system, macrophages and inflammatory cytokines including oncostatin M and transforming growth factor (TGF)-ß in this process, reveals how neural circuitry fine-tunes the inflammatory response to generate hematopoietic bones in injured muscles. The localization of NHOs in the peri-articular muscle environment also suggests a role of muscle mesenchymal cells and bone metabolism in development of hematopoiesis in adults. Little is known about the establishment of bone marrow niches and the regulation of HSC cycling during fetal development. Similarities between NHO and development of fetal bones make NHOs an interesting model to study the establishment of bone marrow hematopoiesis during development. Conversely, identification of stage-specific factors that specify HSC developmental state during fetal bone development will give more mechanistic insights into NHO.

Human fetal liver MSCs are more effective than adult bone marrow MSCs for their immunosuppressive, immunomodulatory, and Foxp3+ T reg induction capacity.

BACKGROUND: Mesenchymal stem cells (MSCs) exhibit active abilities to suppress or modulate deleterious immune responses by various molecular mechanisms. These cells are the subject of major translational efforts as cellular therapies for immune-related diseases and transplantations. Plenty of preclinical studies and clinical trials employing MSCs have shown promising safety and efficacy outcomes and also shed light on the modifications in the frequency and function of regulatory T cells (T regs). Nevertheless, the mechanisms underlying these observations are not well known. Direct cell contact, soluble factor production, and turning antigen-presenting cells into tolerogenic phenotypes, have been proposed to be among possible mechanisms by which MSCs produce an immunomodulatory environment for T reg expansion and activity. We and others demonstrated that adult bone marrow (BM)-MSCs suppress adaptive immune responses directly by inhibiting the proliferation of CD4+ helper and CD8+ cytotoxic T cells but also indirectly through the induction of T regs. In parallel, we demonstrated that fetal liver (FL)-MSCs demonstrates much longer-lasting immunomodulatory properties compared to BM-MSCs, by inhibiting directly the proliferation and activation of CD4+ and CD8+ T cells. Therefore, we investigated if FL-MSCs exert their strong immunosuppressive effect also indirectly through induction of T regs. METHODS: MSCs were obtained from FL and adult BM and characterized according to their surface antigen expression, their multilineage differentiation, and their proliferation potential. Using different in vitro combinations, we performed co-cultures of FL- or BM-MSCs and murine CD3+CD25-T cells to investigate immunosuppressive effects of MSCs on T cells and to quantify their capacity to induce functional T regs. RESULTS: We demonstrated that although both types of MSC display similar cell surface phenotypic profile and differentiation capacity, FL-MSCs have significantly higher proliferative capacity and ability to suppress both CD4+ and CD8+ murine T cell proliferation and to modulate them towards less active phenotypes than adult BM-MSCs. Moreover, their substantial suppressive effect was associated with an outstanding increase of functional CD4+CD25+Foxp3+ T regs compared to BM-MSCs. CONCLUSIONS: These results highlight the immunosuppressive activity of FL-MSCs on T cells and show for the first time that one of the main immunoregulatory mechanisms of FL-MSCs passes through active and functional T reg induction.

A kinome-wide shRNA screen uncovers vaccinia-related kinase 3 (VRK3) as an essential gene for diffuse intrinsic pontine glioma survival.

Diffuse intrinsic pontine glioma (or DIPG) are pediatric high-grade gliomas associated with a dismal prognosis. They harbor specific substitution in histone H3 at position K27 that induces major epigenetic dysregulations. Most clinical trials failed so far to increase survival, and radiotherapy remains the most efficient treatment, despite only transient tumor control. We conducted the first lentiviral shRNA dropout screen in newly diagnosed DIPG to generate a cancer-lethal signature as a basis for the development of specific treatments with increased efficacy and reduced side effects compared to existing anticancer therapies. The analysis uncovered 41 DIPG essential genes among the 672 genes of human kinases tested, for which several distinct interfering RNAs impaired cell expansion of three different DIPG stem-cell cultures without deleterious effect on two control neural stem cells. Among them, PLK1, AURKB, CHEK1, EGFR, and GSK3A were previously identified by similar approach in adult GBM indicating common dependencies of these cancer cells and pediatric gliomas. As expected, we observed an enrichment of genes involved in proliferation and cell death processes with a significant number of candidates belonging to PTEN/PI3K/AKT and EGFR pathways already under scrutiny in clinical trials in this disease. We highlighted VRK3, a gene involved especially in cell cycle regulation, DNA repair, and neuronal differentiation, as a non-oncogenic addiction in DIPG. Its repression totally blocked DIPG cell growth in the four cellular models evaluated, and induced cell death in H3.3-K27M cells specifically but not in H3.1-K27M cells, supporting VRK3 as an interesting and promising target in DIPG.

Human Fetal Liver Mesenchymal Stem Cell-Derived Exosomes Impair Natural Killer Cell Function.

Mesenchymal stem cells (MSCs) are powerful immunomodulators that regulate the diverse functions of immune cells involved in allogeneic reactions, such as T cells and natural killer (NK) cells, through cell-cell contact or secreted factors. Exosomes secreted by MSCs may be involved in their regulatory functions, providing new therapeutic tools. Here, we showed that fetal liver (FL) MSC-derived exosomes inhibit proliferation, activation, and cytotoxicity of NK cells. Exosomes bearing latency associated peptide (LAP), TGFß, and thrombospondin 1 (TSP1), a regulatory molecule for TGFß, induced downstream TGFß/Smad2/3 signaling in NK cells. The inhibition of TGFß, using a neutralizing anti-TGFß antibody, restored NK proliferation, differentiation, and cytotoxicity, demonstrating that FL-MSC-derived exosomes exert their inhibition on NK cell function via TGFß. These results suggest that FL-MSC-derived exosomes regulate NK cell functions through exosome-associated TGFß.

VE-Cadherin and ACE Co-Expression Marks Highly Proliferative Hematopoietic Stem Cells in Human Embryonic Liver.

Despite advances to engineer transplantable hematopoietic stem and progenitor cells (HSPCs) for research and therapy, an in-depth characterization of the developing human hematopoietic system is still lacking. The human embryonic liver is at the crossroad of several hematopoietic sites and harbors a complex hematopoietic hierarchy, including the first actively dividing HSPCs that will further seed the definitive hematopoietic organs. However, few are known about the phenotypic and functional HSPC organization operating at these stages of development. In this study, using a combination of four endothelial and hematopoietic surface markers, that is, the endothelial-specific marker vascular endothelial-cadherin (Cdh5, CD144), the pan-leukocyte antigen CD45, the hemato-endothelial marker CD34, and the angiotensin-converting enzyme (ACE, CD143), we identified distinct HSPC subsets, and among them, a population co-expressing the four markers that uniquely harbored an outstanding proliferation potential both ex vivo and in vivo. Moreover, we traced back this population to the yolk sac (YS) and aorta-gonad-mesonephros (AGM) sites of hematopoietic emergence. Taken together, our data will help to identify human HSPC self-renewal and amplification mechanisms for future cell therapies.

Definitive human and mouse hematopoiesis originates from the embryonic endothelium: a new class of HSCs based on VE-cadherin expression.

Hematopoietic stem cells (HSCs) arise first in the third week of human ontogeny inside yolk sac developing blood vessels, and independently, from the wall of the embryonic aorta and vitelline arteries one week later. HSCs produced in the yolk sac and in the embryonic truncal arteries migrate to and transiently colonize the embryonic liver (EL), and thereafter the bone marrow (BM), their permanent site of residence. At the moment, the origin of human HSCs is still controversial; one of the main hypotheses being that they are generated by hemogenic endothelial cells (ECs). To prove definitively the endothelial origin of HSCs that arise within the human embryo, we previously purified ECs from either the yolk sac or the truncal arteries and reported that they were able to produce blood cells in vitro. We then found that some of the HSCs present in the human EL were co-expressing vascular endothelial (VE)-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker, and demonstrated that these HSCs bearing a dual hemato-endothelial phenotype were endowed with remarkably high self renewal and proliferative potentials. Furthermore, a transgenic mouse model based on the VE-cadherin cis-regulating elements that we engineered to trace the fate of the first VE-cadherin expressing cells allowed us to clearly demonstrate that a majority of adult BM HSCs derived from a VE-cadherin ancestor. Altogether our studies strongly suggest that at least a part of both the human and the murine hematopoietic systems arise from an endothelium-like ancestor.

VE-cadherin expression allows identification of a new class of hematopoietic stem cells within human embryonic liver.

Edification of the human hematopoietic system during development is characterized by the production of waves of hematopoietic cells separated in time, formed in distinct embryonic sites (ie, yolk sac, truncal arteries including the aorta, and placenta). The embryonic liver is a major hematopoietic organ wherein hematopoietic stem cells (HSCs) expand, and the future, adult-type, hematopoietic cell hierarchy becomes established. We report herein the identification of a new, transient, and rare cell population in the human embryonic liver, which coexpresses VE-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker. This population displays an outstanding self-renewal, proliferation, and differentiation potential, as detected by in vitro and in vivo hematopoietic assays compared with its VE-cadherin negative counterpart. Based on VE-cadherin expression, our data demonstrate the existence of 2 phenotypically and functionally separable populations of multipotent HSCs in the human embryo, the VE-cadherin(+) one being more primitive than the VE-cadherin(-) one, and shed a new light on the hierarchical organization of the embryonic liver HSC compartment.

Blood-forming endothelium in human ontogeny: lessons from in utero development and embryonic stem cell culture.

During the early weeks of human gestation, hematopoietic cells first emerge within the extraembryonic yolk sac (primitive hematopoiesis) and secondarily within the truncal arteries of the embryo. This second wave includes the stem cells giving rise to adult-type lymphohematopoiesis. In both yolk sac blood islands and embryonic aorta, hematopoietic cells arise in the immediate vicinity of vascular endothelial cells. In vitro hematopoietic differentiation of endothelial cells stringently sorted from human embryonic and fetal blood-forming tissues has demonstrated that primitive endothelium lies at the origin of incipient hematopoiesis. These anatomically and temporally localized blood-forming endothelial cells are ultimately derived from a rare subset of mesodermal angio-hematopoietic stem cells, or hemangioblasts. The evidence for an early progenitor of blood-forming cells within the walls of human embryonic blood vessels concurs with parallel data obtained from lower vertebrate, avian, and murine models. Importantly, converging results have recently been obtained with in vitro differentiated human embryonic stem cells, in which we have modeled primitive and definitive hematopoiesis via an endothelium-like developmental intermediate.

The vascular wall as a source of stem cells.

We have characterized the emerging hematopoietic system in the human embryo and fetus. Two embryonic organs, the yolk sac and aorta, support the primary emergence of hematopoietic stem cells (HSCs), but only the latter contributes lymphomyeloid stem cells for definitive, adult-type hematopoiesis. A common feature of intra- and extraembryonic hematopoiesis is that in both locations hematopoietic cells emerge in close vicinity to vascular endothelial cells. We have provided evidence that a population of angiohematopoietic mesodermal stem cells, marked by the expression of flk-1 and the novel BB9/ACE antigen, migrate from the paraaortic splanchnopleura into the ventral part of the aorta, where they give rise to hemogenic endothelial cells and, in turn, hematopoietic cells. HSCs also appear to develop from endothelium in the embryonic liver and fetal bone marrow, albeit at a much lower frequency. This would imply that the organism does not function during its whole life on a stock of hematopoietic stem cells established in the early embryo, as is usually accepted. We next examined whether the vessel wall can contribute stem cells for other cell lineages, primarily in the model of adult skeletal muscle regeneration. By immunohistochemistry and flow cytometry, we documented the existence in skeletal muscle, besides genuine endothelial and myogenic cells, of a subset of satellite cells that coexpress endothelial cell markers. This suggested the existence of a continuum of differentiation from vascular cells to endothelial cells that was confirmed in long-term culture. The regenerating capacity of these cells expressing both myogenic and endothelial markers is being investigated in skeletal and cardiac muscle, and the results are being compared with those generated by satellite cells. Altogether, these results point to a generalized progenitor potential of a subset of endothelial, or endothelium-like, cells in blood vessel walls, in pre- and postnatal life.

Emergence of hematopoietic stem cells in the human embryo.

We have previously identified a novel site of hematopoietic cell production within the human embryo, which is localised in the ventral wall of the dorsal aorta and vitelline artery. Cells emerging in that territory between 27 and 40 days of gestation exhibit the expected phenotypic, molecular, and functional properties of hematopoietic stem cells and are the first multipotent, lympho-myeloid progenitors that appear in human ontogeny. We have next demonstrated that vascular endothelial cells sorted stringently, by flow cytometry, from the human yolk sac and embryonic aorta exhibit dramatic blood-forming potential in culture. These results suggest a filiation between vascular endothelium and hematopoietic cells in the course of early human ontogeny. More preliminary data indicate that a subpopulation of vascular endothelial cells in the bone marrow may retain this hematogenous potential until adult stages.

Blood-forming potential of vascular endothelium in the human embryo.

Hematopoietic cells arise first in the third week of human ontogeny inside yolk sac developing blood vessels, then, one week later and independently, from the wall of the embryonic aorta and vitelline artery. To address the suggested derivation of emerging hematopoietic stem cells from the vessel endothelium, endothelial cells have been sorted by flow cytometry from the yolk sac and aorta and cultured in the presence of stromal cells that support human multilineage hematopoiesis. Embryonic endothelial cells were most accurately selected on CD34 or CD31 surface expression and absence of CD45, which guaranteed the absence of contaminating hematopoietic cells. Yet, rigorously selected endothelial cells yielded a progeny of myelo-lymphoid cells in culture. The frequency of hemogenic endothelial cells in the yolk sac and aorta reflected the actual blood-forming activity of these tissues, as a function of developmental age. Even less expected, a subset of endothelial cells sorted similarly from the embryonic liver and fetal bone marrow also exhibited blood-forming potential. These results suggest that a part at least of emerging hematopoietic cells in the human embryo and fetus originate in vascular walls.