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.

Aging of the Hematopoietic Stem Cell Niche: New Tools to Answer an Old Question

The hematopoietic stem cell (HSC) niche is a specialized microenvironment, where a complex and dynamic network of interactions across multiple cell types regulates HSC function. During the last years, it became progressively clearer that changes in the HSC niche are responsible for specific alterations of HSC behavior. The aging of the bone marrow (BM) microenvironment has been shown to critically contribute to the decline in HSC function over time. Interestingly, while upon aging some niche structures within the BM are degenerated and negatively affect HSC functionality, other niche cells and specific signals are preserved and essential to retaining HSC function and regenerative capacity. These new findings on the role of the aging BM niche critically depend on the implementation of new technical tools, developed thanks to transdisciplinary approaches, which bring together different scientific fields. For example, the development of specific mouse models in addition to coculture systems, new 3D-imaging tools, ossicles, and ex-vivo BM mimicking systems is highlighting the importance of new technologies to unravel the complexity of the BM niche on aging. Of note, an exponential impact in the understanding of this biological system has been recently brought by single-cell sequencing techniques, spatial transcriptomics, and implementation of artificial intelligence and deep learning approaches to data analysis and integration. This review focuses on how the aging of the BM niche affects HSCs and on the new tools to investigate the specific alterations occurring in the BM upon aging. All these new advances in the understanding of the BM niche and its regulatory function on HSCs have the potential to lead to novel therapeutical approaches to preserve HSC function upon aging and disease.

YAP/TAZ Promote Hematopoietic Regeneration Via Accelerating the Recovery of Bone Marrow Niche

Adult hematopoietic stem cells (HSCs) reside and are protected in a unique bone marrow (BM) microenvironment, termed the HSC niche, which consists mainly of vascular endothelial cells (EC) and EC-associated mesenchymal stromal cells (MSC). Myeloablative stresses, such as ionizing radiation (IR) and chemotherapy, induce not only depletion of hematopoietic cells but also disruption of HSC niche components, as exemplified by dilation and leakiness of BM vasculature and depletion and dysfunction of BM MSCs. These structural and functional changes in the HSC niche restrain efficient hematopoietic recovery, which often compromises the efficacy of HSC transplantation (HSCT) and chemotherapy. YAP/TAZ are the two transcriptional coactivators normally repressed by LATS kinases downstream of the Hippo pathway. Although cumulative evidence has established a critical role of YAP/TAZ activation in tissue regeneration of various solid organs, their role in BM regeneration remains poorly understood. Our quantitive RT-PCR revealed that YAP/TAZ are abundantly expressed in steady-state mouse ECs (CD45 -Ter119 -CD31 +Sca1 +CD105 hi) and MSCs (CD45 -Ter119 -CD31 -PDGFRα +CD51 +LepR +) but scarcely in hematopoietic cells including HSCs (Lin -cKit +Sca1 +Flk2 -CD150 +CD48 -CD34 lo), which was confirmed by reanalysis of the published single cell RNA-seq datasets (GSE128423). Immunofluorescent imaging of BM sections revealed that YAP/TAZ are distributed mainly in the cytoplasm of ECs but evenly in the cytoplasm and nuclei of MSCs, indicating their differential basal activity in these two HSC niche components. Kinetic transcriptome analysis revealed that YAP/TAZ activity is transiently activated in ECs at 24 hours and returns to a basal repressive state by day 3 after sublethal IR. This transient activation of endothelial YAP/TAZ was critical for vascular integrity, as conditional deletion of YAP/TAZ in ECs (Cdh5-Cre ERT2Yap1 f/fTaz f/f) caused 100% lethality of mice within 10 days following sublethal IR. In sharp contrast, the kinetic expression analysis of a YAP/TAZ target gene CTGF indicated their transient inhibition in MSCs after sublethal IR, and the conditional YAP/TAZ deletion in BM MSCs (Ebf3-Cre ERT2Yap1 f/fTaz f/f) led to their reduced colony forming ability when assessed by colony forming unit fibroblast (CFU-F) assay. Recently, we discovered a novel and potent LATS inhibitor GA-003 that selectively induces mouse and human YAP/TAZ activation in vitro (IC 50 against LATS1 = 1.06 ± 0.08 nM). To analyze the effect of pharmacological YAP/TAZ activation on BM regeneration in vivo, we treated mice with intraperitoneal injection of GA-003 (50 mg/kg per day, for 8 days) following sublethal IR. Remarkably, we observed an accelerated recovery of hematopoiesis, with the absolute numbers of BM cellularity, GMP (Lin -cKit +Sca1 -FcγR +CD34 +) and HSC EPCR (Lin -cKit +Sca1 +CD150 +EPCR +) on day 14 increased by 3.50-fold (p=0.0002), 6.49-fold (p=0.0022) and 11.41-fold (p=0.022), respectively in the GA-003-treated group compared to vehicle-treated group. In addition, GA-003 also promoted hematopoietic recovery after 5-FU injection (150 mg/kg) and HSCT. Nonetheless, consistent with the scarce expression of YAP/TAZ in hematopoietic stem and progenitor cells (HSPC), in vitro GA-003 treatment did not enhance HSPC growth, suggesting niche-mediated effects by GA-003. Indeed, in vitro tube formation assay indicated accelerated angiogenesis by GA-003-treated human umbilical vein ECs, and CFU-F assays revealed significant enhancement of colony formation by mouse BM-derived MSCs upon GA-003 treatment. To reveal the effect of GA-003 on the HSC niche components in vivo, we performed whole BM immunofluorescent imaging at various time points following sublethal IR and GA-003 treatment. We observed alleviated vascular dilation and leakiness and earlier restoration of vascular damage in GA-003-treated group compared to vehicle-treated group, which was associated with increased VE-Cadherin expression in ECs. These results suggest that reinforcing YAP/TAZ activity upon myelosuppression promotes HSC niche integrity and recovery and accelerates hematopoietic regeneration. Taken together, our results establish YAP/TAZ as novel regulators of HSC niche and highlight YAP/TAZ as promising therapeutic targets to boost hematopoietic recovery after myeloablative interventions such as chemotherapy and HSCT. Disclosures Aihara: Nissan Chemical Corporation: Current Employment. Iwawaki: Nissan Chemical Corporation: Current Employment. Nishino: Nissan Chemical Corporation: Current Employment. Iwama: Nissan Chemical Corporation: Research Funding.

Mechanisms of stem cells action: reality and hypotheses

Despite the long history of stem cells studying, the mechanisms of their action are still not fully understood. A classic example is the described non-hematological effect of hematopoietic stem cells (HSC), presumably due to the ability to transdifferentiation and cell fusion. The focus of clinical application in the field of regenerative medicine is mesenchymal stromal cells (MSC), and the understanding of the ways of implementfnion of their regenerative potential has evolved significantly. MSC were discovered and described as a structural unit of the HSC niche responsible for the repair of connective tissue through differentiation. However, it later turned out that they are a regulator of various processes in the body as a whole. These processes include anti-inflammatory, antifibrotic, and immunomodulatory effects, which are realized in various ways. Among these pathways, the paracrine mechanism has already been identified – the release of various growth factors, exosomes and microvesicles, the mechanism of direct transfer of mitochondria and other cellular organelles from cell to cell using tunneling nanotubes, the mechanism of efferocytosis. One of the latest discoveries in this field was the immunomodulatory effect of apoptotic MSC.In general, the study of the stem cells mechanisms of action is a living, developing field of science in which the last word will not be said soon.

p57Kip2 regulates embryonic haematopoietic stem cell numbers by controlling the size of the sympathoadrenal progenitor pool

ABSTRACTHaematopoietic stem cells (HSCs) are of major clinical importance, and finding methods for their in vitro generation is a prime research focus. We demonstrate that the cell cycle inhibitor p57Kip2/Cdkn1c limits HSC numbers by restricting the size of the sympathetic nervous system (SNS) and the amount of HSC-supportive catecholamines secreted by these cells, specifically in the aorta-gonads-mesonephros (AGM) region via β2-adrenergic receptor signalling. This regulation occurs at the SNS progenitor level and is in contrast to the cell-intrinsic function of p57Kip2 in maintaining adult HSCs. Using single-cell RNA-Seq we dissect the differentiation pathway of neural crest cells into SNS cells in the AGM and reveal that they are able to take an alternative differentiation pathway, giving rise to a subset of mesenchymal cells expressing HSC-supportive factors. Neural crest cells thus appear to contribute to the AGM HSC niche via two different mechanisms: SNS-mediated catecholamine secretion and HSC-supportive mesenchymal cell production.

Spatial transcriptome profiling by MERFISH reveals fetal liver hematopoietic stem cell niche architecture

The hematopoietic stem cell (HSC) niche has been extensively studied in bone marrow, yet a more systematic investigation into the microenvironment regulation of hematopoiesis in fetal liver is necessary. Here we investigate the spatial organization and transcriptional profile of individual cells in both wild type (WT) and Tet2−/− fetal livers, by multiplexed error robust fluorescence in situ hybridization. We find that specific pairs of fetal liver cell types are preferentially positioned next to each other. Ligand-receptor signaling molecule pairs such as Kitl and Kit are enriched in neighboring cell types. The majority of HSCs are in direct contact with endothelial cells (ECs) in both WT and Tet2−/− fetal livers. Loss of Tet2 increases the number of HSCs, and upregulates Wnt and Notch signaling genes in the HSC niche. Two subtypes of ECs, arterial ECs and sinusoidal ECs, and other cell types contribute distinct signaling molecules to the HSC niche. Collectively, this study provides a comprehensive picture and bioinformatic foundation for HSC spatial regulation in fetal liver.

Single-Cell Analysis of Ploidy and Transcriptome Reveals Functional and Spatial Divergency in Murine Megakaryopoiesis

Megakaryocytes (MKs), the platelet progenitor cell, play important roles in hematopoietic stem cell (HSC) maintenance and immunity. However, it is not known whether these diverse programs are executed by a single population or by distinct subsets of cells. Here, we manually-isolated primary CD41+ MKs from the bone marrow (BM) of mice and human donors based on ploidy (2N-32N), performed single-cell RNA sequencing analysis. We found that cellular heterogeneity existed within three distinct subpopulations possessing gene signatures related to platelet-generation, HSC niche interaction, and inflammatory responses, respectively. In situ immunostaining of mouse BM demonstrated that platelet-generation and HSC-niche related MKs were physically in close proximity to blood vessels and HSCs, respectively. Proplatelets, which could give rise to platelets under the blood shear forces, were predominantly formed on platelet-generation subset. Remarkably, the inflammatory responses subpopulation, consisting generally of low-ploidy LSP1+ and CD53+ MKs (≤8N), represented approximately 5% of total MKs in the BM. These MKs could specifically respond to pathogen infections in mice. Rapid expansion of this population was accompanied by strong upregulation of a pre-existing PU.1 and IRF-8-associated monocytic-like transcriptional program involved in pathogen recognition and clearance, as well as antigen presentation. Consistently, isolated primary CD53+ cells were capable to engulf and digest bacteria and to stimulate T cells in vitro. Together, our findings uncover new molecular, spatial, and functional heterogeneity within MKs in vivo and demonstrate the existence of a specialized MK subpopulation that may act as a new type of immune cell.

Single-Cell Atlas Reveals Fatty Acid Metabolites Regulate the Functional Heterogeneity of Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (MSCs) are widely used clinically due to their versatile roles in multipotency, immunomodulation, and hematopoietic stem cell (HSC) niche function. However, cellular heterogeneity limits MSCs in the consistency and efficacy of their clinical applications. Metabolism regulates stem cell function and fate decision; however, how metabolites regulate the functional heterogeneity of MSCs remains elusive. Here, using single-cell RNA sequencing, we discovered that fatty acid pathways are involved in the regulation of lineage commitment and functional heterogeneity of MSCs. Functional assays showed that a fatty acid metabolite, butyrate, suppressed the self-renewal, adipogenesis, and osteogenesis differentiation potential of MSCs with increased apoptosis. Conversely, butyrate supplement significantly promoted HSC niche factor expression in MSCs, which suggests that butyrate supplement may provide a therapeutic approach to enhance their HSC niche function. Overall, our work demonstrates that metabolites are essential to regulate the functional heterogeneity of MSCs.

The parathyroid hormone-dependent activation of osteoblasts enhances hematopoietic stem cell migration and reduces their engraftment abilities

Hematopoietic stem cells (HSCs) in the bone marrow (BM) reside in HSC niches ensuring their maintenance. The HSC niche is made up of perivascular and trabecular cells including osteoblasts whose role on HSCs remains to be clearly defined. Increased numbers of osteoblasts have been observed in the CL2 transgenic mouse expressing a constitutively activated form of the parathyroid hormone (PTH)/PTH-related peptide receptor. This mouse model mimicking PTH anabolic effect has also been described to exhibit increased numbers of the BM stem/progenitor population. Furthermore, PTH is known to induce BM stem/progenitor cell migration into blood circulation. However PTH role on long-term repopulating HSCs (LT-HSCs) is incompletely known. Here we show that CL2 BM contains a regular proportion of LT-HSCs, suggesting that osteoblasts may not be a determinant of LT-HSC numbers but act mainly on more mature progenitors. We further show increased LT-HSC migration in CL2 mice correlated with higher granulocyte colony-stimulating factor (G-CSF) serum levels, supporting the idea that PTH can enhance the migration of LT-HSCs. Finally, we found a defect in the ability of CL2 BM HSCs to reconstitute irradiated BM suggesting that PTH activation of osteoblasts negatively influences abilities of HSC population to engraft and reconstitute irradiated BM. In summary, our study highlights new insights into the role of the PTH-dependent activation of osteoblasts on LT-HSC migration and their BM repopulation abilities. Our findings will be useful to improve treatments on hematological disorders, especially therapies involving HSC harvest and transplantation.

3D Scaffolds to Model the Hematopoietic Stem Cell Niche: Applications and Perspectives

Hematopoietic stem cells (HSC) are responsible for the production of blood and immune cells during life. HSC fate decisions are dependent on signals from specialized microenvironments in the bone marrow, termed niches. The HSC niche is a tridimensional environment that comprises cellular, chemical, and physical elements. Introductorily, we will revise the current knowledge of some relevant elements of the niche. Despite the importance of the niche in HSC function, most experimental approaches to study human HSCs use bidimensional models. Probably, this contributes to the failure in translating many in vitro findings into a clinical setting. Recreating the complexity of the bone marrow microenvironment in vitro would provide a powerful tool to achieve in vitro production of HSCs for transplantation, develop more effective therapies for hematologic malignancies and provide deeper insight into the HSC niche. We previously demonstrated that an optimized decellularization method can preserve with striking detail the ECM architecture of the bone marrow niche and support HSC culture. We will discuss the potential of this decellularized scaffold as HSC niche model. Besides decellularized scaffolds, several other methods have been reported to mimic some characteristics of the HSC niche. In this review, we will examine these models and their applications, advantages, and limitations.