A Novel Megakaryocyte Subpopulation Poised to Exert the Function of HSC Niche as Possible Driver of Myelofibrosis

Careful morphological investigations, coupled with experimental hematology studies in animal models and in in vitro human cultures, have identified that platelets are released in the circulation by mature megakaryocytes generated by hematopoietic stem cells by giving rise to lineage-restricted progenitor cells and then to morphologically recognizable megakaryocyte precursors, which undergo a process of terminal maturation. Advances in single cell profilings are revolutionizing the process of megakaryocytopoiesis as we have known it up to now. They identify that, in addition to megakaryocytes responsible for producing platelets, hematopoietic stem cells may generate megakaryocytes, which exert either immune functions in the lung or niche functions in organs that undergo tissue repair. Furthermore, it has been discovered that, in addition to hematopoietic stem cells, during ontogeny, and possibly in adult life, megakaryocytes may be generated by a subclass of specialized endothelial precursors. These concepts shed new light on the etiology of myelofibrosis, the most severe of the Philadelphia negative myeloproliferative neoplasms, and possibly other disorders. This perspective will summarize these novel concepts in thrombopoiesis and discuss how they provide a framework to reconciliate some of the puzzling data published so far on the etiology of myelofibrosis and their implications for the therapy of this disease.

Identifying a novel role for the master regulator Tal1 in the Endothelial to Hematopoietic Transition

ABSTRACTRecent progress in the generation of bona-fide Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding in detail this developmental process. Here, we sought to elucidate the function of the hematopoietic regulators Tal1, Lmo2 and Lyl1 in the Endothelial to Hematopoietic Transition (EHT), the process through which HSPCs are generated from endothelial precursors during embryogenesis. We used a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development, and combined gain-of-function experiments in sorted vascular smooth muscle cells (VSM) with multi-omics to obtain mechanistic insights into the mode of action of Tal1, Lmo2 and Lyl1. We found that these factors promote the silencing of the VSM transcriptional program and the activation of the hematopoietic one. Through this approach and the use of a Tet-on system to control the expression of Tal1 during hematopoietic specification from mESCs, we discovered that its expression in endothelial cells is crucial for the EHT to occur.

Distinct Transcriptional Signatures Distinguish the Emergence of Multipotent Progenitors and Hematopoietic Stem Cells from Endothelial Precursors in the Murine Embryo

During embryonic development, blood cells emerge from hemogenic endothelium (HE), producing waves of hematopoietic progenitors prior to the emergence of rare hematopoietic stem cells (HSCs), which have the unique ability to self-renew and generate all cell types of the adult hematopoietic system. HSCs have significant potential for use in cellular therapies and disease modeling. However, efforts to generate HSCs in vitro from pluripotent stem cells (PSCs) have been limited by an incomplete understanding of the unique phenotypic markers and transcriptional programs that distinguish HE with HSC potential. Previous studies have demonstrated that yolk sac-derived erythromyeloid progenitors and HSCs originate from distinct populations of HE. However, it is not known whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from HE with common phenotypic and transcriptional properties. To investigate this, we combined index sorting of single hemogenic precursors with stromal co-culture that enables simultaneous detection of HSC and multilineage hematopoietic potential, to functionally validate surface markers that may distinguish hemogenic precursors with different hematopoietic fates. We previously found that the co-expression of two markers, CD61 and EPCR, identifies a subset of VE-Cadherin+ endothelial cells from the mouse P-Sp/AGM region (para-aortic splanchnopleura/aorta-gonad-mesonephros, where the first HSCs are generated from HE between E9 and E11 in development) enriched phenotypically for arterial endothelial surface markers (e.g. Dll4, CD44) and functionally for hemogenic precursors with HSC potential. However, this population remains heterogeneous, containing clonal hemogenic precursors with the potential for HSC as well as multilineage progenitor-restricted fates. Here, we report that expression of arterial marker CXCR4 further enriched for functional HSC potential in hemogenic precursors in the P-Sp/AGM between E9 and E10, when the first clonal HSC precursors are detected at rare frequency. In contrast, we detected more abundant clonal HE with multilineage hematopoietic potential (producing lymphoid, erythroid, and myeloid progeny in vitro but lacking HSC potential) at the same stage, which are distinguished by comparatively lower CXCR4 expression. To investigate transcriptional differences between HE populations differentially expressing CXCR4, we performed single-cell RNA sequencing of E9 P-Sp-derived VE-Cadherin+CD61+EPCR+ cells. Using an unbiased gene module analysis based on graph autocorrelation in the Monocle 3 platform to identify genes that co-vary over pseudotime, we found that Cxcr4 is uniquely expressed in a subset of cells simultaneously enriched for arterial-specific genes (including Dll4, Efnb2, Hey2, Sox17, Cd44) and genes with established roles in HSC maintenance and self-renewal (including Mecom, Cdkn1c, H19, Txnip, Kmt2a). Conversely, expression of these genes is decreased in cells undergoing the endothelial to hematopoietic transition at this stage based on pseudotemporal ordering, concomitant with increasing expression of hematopoietic-specifying transcription factors Runx1 and Gfi1, and other genes associated with definitive hematopoiesis (egs. Myb, Kit, Hlf, Gata2, Mpl, Lyl1). We also examined the aggregate expression of established HSC-specific signature genes from published data sets across pseudotime, and found that they exhibit similar expression dynamics to that of Cxcr4 and Dll4, reaching peak expression prior to the initiation of Runx1 and Gfi1 expression. Altogether, our studies support a model in which the initial populations of multipotent progenitors and HSCs emerge independently from HE in the P-Sp/AGM. Furthermore, our findings suggest that HE with HSC competence is uniquely defined by co-expression of arterial endothelial genes and genes encoding HSC self-renewal factors, providing insight into the earliest transcriptional programs that must be coordinated to drive HSC fate from endothelial precursors. Future studies will focus on identifying the signal pathways whose integration promotes expression of these HSC-defining transcriptional programs in endothelial cells, with the goal of advancing methods for HSC generation in vitro. Disclosures Bernstein: Lyell Immunopharma: Current equity holder in publicly-traded company, Other: Laboratory Support; Deverra Therapeutics: Current equity holder in publicly-traded company.

Therapeutic Potential of Endothelial Colony Forming Cells in Ischemic Disease: Strategies to Improve Their Regenerative Efficacy

Cardiovascular disease (CVD) comprises a group of heart and circulatory disorders, which are regarded as a global medical issue with high prevalence and mortality rates. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable to surgical revascularization. Therefore, more efficient therapeutic approaches are urgently required to promote neovascularization. Therapeutic angiogenesis represents an emerging strategy that aims at reconstructing the damaged vascular network by stimulating local angiogenesis and/or promoting de novo blood vessel formation according to a process known as vasculogenesis. Circulating endothelial colony forming cells (ECFCs), in turn, represent truly endothelial precursors able to aggregate into bidimensional tube networks and to originate patent vessels. Accordingly, ECFCs provide the most rationale and promising cellular candidate for therapeutic purposes. The current review provides a brief outline on the origin and characterization of ECFCs and a summary of the progress in preclinical studies aiming at assessing their efficacy in a variety of ischemic disorders, including AMI, PAD, ischemic brain disease and retinopathy. We also describe how to enhance the vasoreparative potential of ECFCs by boosting specific pro-angiogenic signalling pathways either pharmacologically or through gene manipulation. Taken together, these observations suggest that ECFCs represent a useful strategy to treat ischemic diseases.

Dermomyotome-derived endothelial cells migrate to the dorsal aorta to support hematopoietic stem cell emergence

Development of the dorsal aorta is a key step in the establishment of the adult blood-forming system, since hematopoietic stem and progenitor cells (HSPCs) arise from ventral aortic endothelium in all vertebrate animals studied. Work in zebrafish has demonstrated that arterial and venous endothelial precursors arise from distinct subsets of lateral plate mesoderm. Earlier studies in the chick showed that paraxial mesoderm generates another subset of endothelial cells that incorporate into the dorsal aorta to replace HSPCs as they exit the aorta and enter circulation. Here we show that a similar process occurs in the zebrafish, where a population of endothelial precursors delaminates from the somitic dermomyotome to incorporate exclusively into the developing dorsal aorta. Whereas somite-derived endothelial cells (SDECs) lack hematopoietic potential, they act as local niche to support the emergence of HSPCs from neighboring hemogenic endothelium. Thus, at least three subsets of endothelial cells (ECs) contribute to the developing dorsal aorta: vascular ECs, hemogenic ECs, and SDECs. Taken together, our findings indicate that the distinct spatial origins of endothelial precursors dictate different cellular potentials within the developing dorsal aorta.

TWIST1-reprogrammed endothelial cell transplantation potentiates neovascularization-mediated diabetic wound tissue regeneration

Hypo-vascularised diabetic non-healing wounds are due to reduced number and impaired physiology of endogenous endothelial progenitor cell (EPC) population that, limits their recruitment and mobilization at the wound site. To enrich the EPC repertoire from non-endothelial precursors, abundantly available mesenchymal stromal cells (MSCs) were reprogrammed into induced-endothelial cells (iECs). We identified cell signaling molecular targets by meta-analysis of microarray datasets. BMP-2 induction leads to the expression of inhibitory Smad 6/7-dependent negative transcriptional regulation of ID1, rendering the latter's reduced binding to TWIST1 during transdifferentiation of WJ-MSC into iEC. TWIST1, in turn, regulates endothelial genes transcription, positively of pro-angiogenic-<i>KDR</i> and negatively, in part, of anti-angiogenic-<i>SFRP4</i>. Twist1 reprogramming enhanced the endothelial lineage commitment of WJ-MSC, increased the vasculogenic potential of reprogrammed EC (rEC). Transplantation of stable <i>TWIST1</i>-rECs into full-thickness type 1 and 2 diabetic-splinted wound healing murine model enhanced the microcirculatory blood flow and accelerated the wound tissue regeneration. An increased or decreased co-localization of GFP with KDR/SFRP4 and CD31 in the regenerated diabetic wound bed with TWIST1 overexpression or silencing (<i>piLenti-TWIST1-shRNA-GFP</i>), respectively further confirmed improved neovascularization. This study depicted the reprogramming of WJ-MSCs into rECs using unique transcription factors, TWIST1 for an efficacious cell transplantation therapy to induce neovascularization–mediated diabetic wound tissue regeneration.

TWIST1-reprogrammed endothelial cell transplantation potentiates neovascularization-mediated diabetic wound tissue regeneration

Hypo-vascularised diabetic non-healing wounds are due to reduced number and impaired physiology of endogenous endothelial progenitor cell (EPC) population that, limits their recruitment and mobilization at the wound site. To enrich the EPC repertoire from non-endothelial precursors, abundantly available mesenchymal stromal cells (MSCs) were reprogrammed into induced-endothelial cells (iECs). We identified cell signaling molecular targets by meta-analysis of microarray datasets. BMP-2 induction leads to the expression of inhibitory Smad 6/7-dependent negative transcriptional regulation of ID1, rendering the latter's reduced binding to TWIST1 during transdifferentiation of WJ-MSC into iEC. TWIST1, in turn, regulates endothelial genes transcription, positively of pro-angiogenic-<i>KDR</i> and negatively, in part, of anti-angiogenic-<i>SFRP4</i>. Twist1 reprogramming enhanced the endothelial lineage commitment of WJ-MSC, increased the vasculogenic potential of reprogrammed EC (rEC). Transplantation of stable <i>TWIST1</i>-rECs into full-thickness type 1 and 2 diabetic-splinted wound healing murine model enhanced the microcirculatory blood flow and accelerated the wound tissue regeneration. An increased or decreased co-localization of GFP with KDR/SFRP4 and CD31 in the regenerated diabetic wound bed with TWIST1 overexpression or silencing (<i>piLenti-TWIST1-shRNA-GFP</i>), respectively further confirmed improved neovascularization. This study depicted the reprogramming of WJ-MSCs into rECs using unique transcription factors, TWIST1 for an efficacious cell transplantation therapy to induce neovascularization–mediated diabetic wound tissue regeneration.