Hematopoietic progenitors polarize in contact with bone marrow stromal cells by engaging CXCR4 receptors

AbstractHematopoietic stem and progenitor cells (HSPCs) are located in the bone marrow, where they regulate the permanent production and renewal of all blood-cell types. HSPC proliferation and differentiation is locally regulated by their interaction with cells forming specific microenvironments close to the bone matrix or close to blood vessels. However, the cellular mechanisms underlying HSPC’s interaction with these cells and their potential impact on HSPC polarity is still poorly understood. Here we modelled the bone-marrow niche using microfluidic technologies in a bone-marrow on a chip device, and evaluated long-duration cell-cell contacts between single HSPCs and stromal cells or endothelial cells in a custom-designed microwell cell-culture system. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of their cytoskeleton architectures. As in the case with immune synapses formed by lymphocytes, the centrosome was located in proximity of the cell-cell contact. The entire microtubule network emanated from the centrosome, and the nucleus was confined to the side opposite of the cell-cell contact. The capacity of the HSPC to polarize appeared specific as it was not observed in contact with skin fibroblasts. The receptors ICAM, VCAM and CXCR4 were identified in the polarizing contact, and were all independently capable of inducing morphological polarization. However, only CXCR4 was independently capable of inducing the polarization of the centrosome-microtubule network. Altogether these results revealed a novel mechanism of HSPC polarization associated with its anchorage to specific cells in the bone-marrow, which might be instrumental in the regulation of their fate.

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Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression

Scientific Reports ◽

10.1038/s41598-021-85122-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marie-Theresa Weickert ◽

Judith S. Hecker ◽

Michèle C. Buck ◽

Christina Schreck ◽

Jennifer Rivière ◽

...

Keyword(s):

Bone Marrow ◽

Cell Fate ◽

Stromal Cells ◽

Adipogenic Differentiation ◽

Cell Types ◽

Bone Marrow Niche ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Bm Niche

AbstractMyelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73+ CD105+ CD271+ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.

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Hematopoietic progenitors polarize in contact with bone marrow stromal cells in response to SDF1

The Journal of Cell Biology ◽

10.1083/jcb.202005085 ◽

2021 ◽

Vol 220 (11) ◽

Author(s):

Thomas Bessy ◽

Adrian Candelas ◽

Benoit Souquet ◽

Khansa Saadallah ◽

Alexandre Schaeffer ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Contact Site ◽

Cellular Mechanisms ◽

Microtubule Network ◽

Hematopoietic Stem ◽

Discrete Contact ◽

Stem And Progenitor Cells ◽

Potential Impact ◽

The Impact

The fate of hematopoietic stem and progenitor cells (HSPCs) is regulated by their interaction with stromal cells in the bone marrow. However, the cellular mechanisms regulating HSPC interaction with these cells and their potential impact on HSPC polarity are still poorly understood. Here we evaluated the impact of cell–cell contacts with osteoblasts or endothelial cells on the polarity of HSPC. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of its cytoskeleton architecture. Notably, the centrosome was located in proximity to the contact site. The capacity of HSPCs to polarize in contact with stromal cells of the bone marrow appeared to be specific, as it was not observed in primary lymphoid or myeloid cells or in HSPCs in contact with skin fibroblasts. The receptors ICAM, VCAM, and SDF1 were identified in the polarizing contact. Only SDF1 was independently capable of inducing the polarization of the centrosome–microtubule network.

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Aging of Bone Marrow Mesenchymal Stromal Cells: Hematopoiesis Disturbances and Potential Role in the Development of Hematologic Cancers

Cancers ◽

10.3390/cancers13010068 ◽

2020 ◽

Vol 13 (1) ◽

pp. 68

Author(s):

Fulvio Massaro ◽

Florent Corrillon ◽

Basile Stamatopoulos ◽

Nathalie Meuleman ◽

Laurence Lagneaux ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Progression ◽

Cancer Progression ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Cell Communication ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Wide Range ◽

Major Player

Aging of bone marrow is a complex process that is involved in the development of many diseases, including hematologic cancers. The results obtained in this field of research, year after year, underline the important role of cross-talk between hematopoietic stem cells and their close environment. In bone marrow, mesenchymal stromal cells (MSCs) are a major player in cell-to-cell communication, presenting a wide range of functionalities, sometimes opposite, depending on the environmental conditions. Although these cells are actively studied for their therapeutic properties, their role in tumor progression remains unclear. One of the reasons for this is that the aging of MSCs has a direct impact on their behavior and on hematopoiesis. In addition, tumor progression is accompanied by dynamic remodeling of the bone marrow niche that may interfere with MSC functions. The present review presents the main features of MSC senescence in bone marrow and their implications in hematologic cancer progression.

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Adult blood stem cell localization reflects the abundance of reported bone marrow niche cell types and their combinations

Blood ◽

10.1182/blood.2020006574 ◽

2020 ◽

Vol 136 (20) ◽

pp. 2296-2307 ◽

Cited By ~ 1

Author(s):

Konstantinos D. Kokkaliaris ◽

Leo Kunz ◽

Nina Cabezas-Wallscheid ◽

Constantina Christodoulou ◽

Simon Renders ◽

...

Keyword(s):

Bone Marrow ◽

Cell Types ◽

Bone Marrow Niche ◽

Functional Heterogeneity ◽

Hematopoietic Stem ◽

Native Bone ◽

Cell Localization ◽

Exact Localization ◽

Stem And Progenitor Cells ◽

Multiple Cell

Abstract The exact localization of hematopoietic stem cells (HSCs) in their native bone marrow (BM) microenvironment remains controversial, because multiple cell types have been reported to physically associate with HSCs. In this study, we comprehensively quantified HSC localization with up to 4 simultaneous (9 total) BM components in 152 full-bone sections from different bone types and 3 HSC reporter lines. We found adult femoral α-catulin-GFP+ or Mds1GFP/+Flt3Cre HSCs proximal to sinusoids, Cxcl12 stroma, megakaryocytes, and different combinations of those populations, but not proximal to bone, adipocyte, periarteriolar, or Schwann cells. Despite microanatomical differences in femurs and sterna, their adult α-catulin-GFP+ HSCs had similar distributions. Importantly, their microenvironmental localizations were not different from those of random dots, reflecting the relative abundance of imaged BM populations rather than active enrichment. Despite their functional heterogeneity, dormant label-retaining (LR) and non-LR hematopoietic stem and progenitor cells both had indistinguishable localization from α-catulin-GFP+ HSCs. In contrast, cycling juvenile BM HSCs preferentially located close to Cxcl12 stroma and farther from sinusoids/megakaryocytes. We expect our study to help resolve existing confusion regarding the exact localization of different HSC types, their physical association with described BM populations, and their tissue-wide combinations.

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Endothelial cell-derived angiopoietin-like protein 2 supports hematopoietic stem cell activities in bone marrow niches

Blood ◽

10.1182/blood.2021011644 ◽

2021 ◽

Author(s):

Zhuo Yu ◽

Wenqian Yang ◽

Xiaoxiao He ◽

Chiqi Chen ◽

Wenrui Li ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Endothelial Cell ◽

Cell Biology ◽

Ex Vivo ◽

Cell Types ◽

Ex Vivo Expansion ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Bone Marrow Niches

Bone marrow niche cells have been reported to fine-tune HSC stemness via direct interaction or secreted components. Nevertheless, how niche cells control HSC activities remains largely unknown. We previously showed that angiopoietin-like protein 2 (ANGPTL2) can support the ex vivo expansion of HSCs by binding to human leukocyte immunoglobulin-like receptor B2 (LILRB2). However, how ANGPTL2 from specific niche cell types regulates HSC activities under physiological conditions is still not clear. Herein, we generated an Angptl2-flox/flox transgenic mouse line and conditionally deleted Angptl2 expression in several niche cells, including Cdh5+ or Tie2+ endothelial cells, Prx1+ mesenchymal stem cells and Pf4+ megakaryocytes, to evaluate its role in the regulation of HSC fate. Interestingly, we demonstrated that only endothelial cell-derived ANGPTL2 and not ANGPTL2 from other niche cell types plays important roles in supporting repopulation capacity, quiescent status and niche localization. Mechanistically, ANGPTL2 enhances PPARD expression to transactivate G0s2 to sustain the perinuclear localization of nucleolin to prevent HSCs from entering the cell cycle. These findings reveal that endothelial cell-derived ANGPTL2 serves as a critical niche component to maintain HSC stemness, which may benefit the understanding of stem cell biology in bone marrow niches and the development of a unique strategy for the ex vivo expansion of HSCs.

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Extracellular Vesicles Isolated from Mesenchymal Stromal Cells Primed with Hypoxia: Novel strategy in Regenerative Medicine

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15999200918110638 ◽

2020 ◽

Vol 15 ◽

Author(s):

Shalmali Pendse ◽

Vaijayanti Kale ◽

Anuradha Vaidya

Keyword(s):

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Therapeutic Potential ◽

Ex Vivo ◽

Cell Types ◽

Molecular Profile ◽

Cell Contact ◽

Hematopoietic Stem

: Mesenchymal stromal cells (MSCs) regulate other cell types through a strong paracrine component called the secretome, comprising of several bioactive entities. The composition of the MSCs’ secretome is dependent upon the microenvironment in which they thrive, and hence, it could be altered by pre-conditioning the MSCs during in vitro culture. The primary aim of this review is to discuss various strategies that are being used for pre-conditioning of MSCs, also known as “priming of MSCs”, in the context of improving their therapeutic potential. Several studies have underscored the importance of extracellular vesicles (EVs) derived from primed MSCs in improving their efficacy in the treatment of various diseases. We have previously shown that co-culturing hematopoietic stem cells (HSCs) with hypoxiaprimed MSCs improves their engraftment potential. Now the question we pose is would priming of MSCs with hypoxiafavorably alter theirsecretome and would this altered secretome work as effectively as the cell to cell contact did? Here we review the current strategies of using the secretome, specifically the EVs (microvesicles and exosomes), collected from the primed MSCs with the intention of expanding HSCs ex vivo. We speculate that an effective priming of MSCs in vitrocould modulate the molecular profile of their secretome, which could eventually be used as a cell-free biologic in clinical settings.

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Regulation of β1-Integrin by Mir-134 in Mesenchymal Stromal Cells – Implications for Mesenchymal Stromal Cell Adherence and Hematopoietic Stem Cell Interaction

Blood ◽

10.1182/blood.v120.21.3459.3459 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3459-3459

Author(s):

Friedrich Stölzel ◽

David M. Poitz ◽

Laleh S. Arabanian ◽

Jens Friedrichs ◽

Denitsa Docheva ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mesenchymal Stromal Cell ◽

Stromal Cell ◽

Luciferase Activity ◽

Cell Types ◽

Bone Marrow Niche ◽

Β1 Integrin ◽

Hematopoietic Stem ◽

Transfected Cells

Abstract Abstract 3459 The different intra- and extracellular constituents of the hematopoietic stem cell (HSC) niche in the human bone marrow are tightly regulated and of momentous importance for various properties of HSCs. Some of these are regulated through β1-Integrins (CD29) which therefore dramatically influence HSC and mesenchymal stromal cell (MSC) interaction in the niche. Important regulators within these cells are microRNAs (miRNAs). These small, non-coding RNAs control the expression of around two-thirds of the human protein-coding genes. One of these miRNAs, miR-134, previously referred to be a “brain-specific” miRNA was shown to be highly expressed in MSCs in tissue-studies conducted by our group. Since the central nervous system was recently shown to be closely connected to the regulation of HSCs and MSCs, we asked whether miR-134 which has several conserved binding seed-match sequences within the 3'UTR of β1-Integrin, regulates MSC mediated properties in the bone marrow niche. Screening of human MSC cell lines (n=4) by western blotting revealed highest β1-Integrin expression in SCP-1 cells. Transfection of SCP-1 with either siRNA directed against β1-Integrin (siCD29) or pre-miRNA-134 (pre134) revealed a downregulation of β1-Integrin at the mRNA level only in siRNA transfected cells, p=0.01. In contrast, at the protein level, as measured by western blot and FACS analysis, p=0.002, β1-Integrin was downregulated by siCD29 as well as by pre134, indicating a miRNA-specific action of repression. Confirmatory, the 3'UTR of β1-Integrin, which contains several putative binding sites for miR-134, was cloned into a pMiRReporter vector and luciferase activity was measured after cotransfection with pre134. The luciferase activity was significantly reduced in pre134 transfected cells [1.80 ± 0.46 (preCo) vs. 0.99 ± 0.49 (pre134); p<0.001]. To evaluate whether pre134 mediated reduction of β1-Integrin can modulate the adhesion potential of SCP-1, atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) was performed. Indeed, transfection of SCP-1 with siCD29 or pre134 resulted in a significantly reduced adherence as compared to their respective controls, p<0.001 and p<0.01. Furthermore, using AFM-based SCFS we investigated the interaction between 32D-cells, which have a high surface expression of the natural interaction partner of β1-Integrin VCAM-1, and SCP-1 cells. Here again, we were able to show, that 32D show a significantly lower adhesion potential to siCD29 and pre134-transfected SCP-1, p<0.001 and p<0.001, respectively. In a translational approach MSCs from healthy bone marrow donors (n=30) and from MDS patients (n=17) were screened for miRNA-expression. This analysis revealed 50% higher miR-134 transcript levels in MSCs from MDS patients [0.0057 ± 0.0021 (healthy) vs. 0.0127 ± 0.0045 (MDS); p<0.001], suggesting a potential role of this miRNA in regulating its MSC adhesion. Regulation of adhesion of MSCs and to MSCs is important for various components of the bone marrow niche. Here, we demonstrate for the first time that β1-Integrin mediated adhesion of MSCs themselves and other cell types onto MSCs via β1-Integrin receptors can be inhibited via miR-134 overexpression. Furthermore, this newly characterized mechanism provides evidence for a potential anti-adhesive influence of miR-134. While this might not only influence adhesion, other mechanisms such as homing of HSCs as well as other cell types, might be affected by modification of miR-134 expression in the stromal niche. Disclosures: Platzbecker: Amgen: Consultancy; GlaxoSmithKline: Consultancy; Celgene: Consultancy; Novartis: Consultancy.

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Selective Deletion of ATG5 in Bone Marrow Leptin-Receptor-Expressing Perivascular Stromal Cells Causes Leukopenia

Blood ◽

10.1182/blood.v126.23.2395.2395 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2395-2395

Author(s):

Shabnam Arsiwala ◽

Wei Ding ◽

Hong-Gang Wang

Keyword(s):

Bone Marrow ◽

T Cells ◽

Cell Count ◽

Stromal Cells ◽

Peripheral Blood ◽

Leptin Receptor ◽

Critical Role ◽

Bone Marrow Niche ◽

Male Mice ◽

Hematopoietic Stem

Abstract Bone marrow niche cells, specifically the Leptin-receptor-expressing perivascular stromal cells (LepR+), CXCL12-abundant reticular cells (CAR), and endothelial cells play an important role in the maintenance and self-renewal functions of hematopoietic stem cells (HSCs). This maintenance function provided by the niche cells is mediated by a number of secretory-related molecules such as stem cell factor (SCF), CXCL12 and Angiopoietin. In addition, functional autophagy within HSCs is known to play a critical role for maintaining HSC homeostasis, as the loss of ATG7 in HSCs leads to loss of normal HSC functions and severe myeloproliferation. However, it remains unclear as to how hematopoiesis will be affected if autophagy is selectively blocked in bone marrow niche cells. To investigate the effect of autophagy deficiency within bone marrow niche cells on hematopoiesis, we generated a knockout mouse model allowing for a selective ablation of an autophagy-essential protein, ATG5, in LepR+ perivascular stromal cells. We have confirmed that the deletion of Atg5 is indeed restricted to LepR+ cells, and that the loss of ATG5 is sufficient to block autophagic function, as evident through the accumulation of p62 protein. We found that the white blood cell count (WBC) is significantly reduced in 16-week old male ATG5 KO mice compare to wild type (wt) littermate control mice (p<0.05). Further, the myeloid population (CD11b+ Ly6G+) in both bone marrow and peripheral blood is significantly decreased in ATG5 KO mice (p value < 0.05, ATG KO vs. wt control). WBC count continues to decline in 24-week old ATG5 KO male mice (p=0.02) but not in female mice (p=0.58). Both LSK (Lin- Sca-1+ c-Kit+) and long term HSC (LT-HSCs, CD150+ CD48- LSK) populations are decreased in both 16-week and 24-week old male mice; however, this trend did not reach statistical significance (p>0.05). Although the absolute cell count of lymphocytes, including B and T cells (CD19+ and CD3+, respectively) in peripheral blood, is significantly dropped in ATG5 KO mice compared to wt controls, there is no significant change of mature B and T cells in bone marrow. These preliminary results suggest that deletion of a key autophagy regulator in LepR+ bone marrow niche cells will cause leukopenia. The underlying mechanism is currently under investigation and it will help us to better understand the role of autophagy in hematopoiesis within the bone marrow microenvironment. Disclosures No relevant conflicts of interest to declare.

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Niche-mediated depletion of the normal hematopoietic stem cell reservoir by Flt3-ITD–induced myeloproliferation

Journal of Experimental Medicine ◽

10.1084/jem.20161418 ◽

2017 ◽

Vol 214 (7) ◽

pp. 2005-2021 ◽

Cited By ~ 26

Author(s):

Adam J. Mead ◽

Wen Hao Neo ◽

Nikolaos Barkas ◽

Sahoko Matsuoka ◽

Alice Giustacchini ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Stromal Cells ◽

Negative Regulator ◽

Bone Marrow Niche ◽

Strong Negative Correlation ◽

Hematopoietic Stem ◽

A Cell ◽

Tandem Duplications

Although previous studies suggested that the expression of FMS-like tyrosine kinase 3 (Flt3) initiates downstream of mouse hematopoietic stem cells (HSCs), FLT3 internal tandem duplications (FLT3 ITDs) have recently been suggested to intrinsically suppress HSCs. Herein, single-cell interrogation found Flt3 mRNA expression to be absent in the large majority of phenotypic HSCs, with a strong negative correlation between Flt3 and HSC-associated gene expression. Flt3-ITD knock-in mice showed reduced numbers of phenotypic HSCs, with an even more severe loss of long-term repopulating HSCs, likely reflecting the presence of non-HSCs within the phenotypic HSC compartment. Competitive transplantation experiments established that Flt3-ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSC-supporting bone marrow stromal cells, with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. Tumor necrosis factor (TNF), a cell-extrinsic potent negative regulator of HSCs, was overexpressed in bone marrow niche cells from FLT3-ITD mice, and anti-TNF treatment partially rescued the HSC phenotype. These findings, which establish that Flt3-ITD–driven myeloproliferation results in cell-extrinsic suppression of the normal HSC reservoir, are of relevance for several aspects of acute myeloid leukemia biology.

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EBF1 As a Novel Regulator of Bone Marrow Mesenchymal Stromal Progenitors

Blood ◽

10.1182/blood.v130.suppl_1.96.96 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 96-96

Author(s):

Marta Derecka ◽

Senthilkumar Ramamoorthy ◽

Pierre Cauchy ◽

Josip Herman ◽

Dominic Grun ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Myeloid Cells ◽

Bone Marrow Niche ◽

Wild Type ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract Hematopoietic stem and progenitor cells (HSPC) are in daily demand worldwide because of their ability to replenish entire blood system. However, the in vitro expansion of HSPC is still a major challenge since the cues from bone marrow microenvironment remain largely elusive. Signals coming from the bone marrow niche, and specifically mesenchymal stem and progenitor cells (MSPC), orchestrate maintenance, trafficking and stage specific differentiation of HSPCs. Although, it is generally accepted that MSPCs are essential for hematopoietic homeostasis and generating multiple types of stromal cells, the exact transcriptional networks regulating MSPCs are not well established. Early B-cell factor 1 (Ebf1) has been discovered as lineage-specific transcription factor governing B lymphopoiesis. Additionally, it has been shown to play important role in differentiation of adipocytes, which are a niche component supporting hematopoietic regeneration. Thus, in this study we seek to examine if Ebf1 has an alternative function in non-hematopoietic compartment of bone marrow, specifically in mesenchymal stromal cells that maintain proper hematopoiesis. Here, we identified Ebf1 as new transcription regulator of MSPCs activity. Mesenchymal progenitors isolated from Ebf1-/- mice show diminished capacity to form fibroblasticcolonies (CFU-F) indicating reduced self-renewal. Moreover, cells expanded from these colonies display impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. In order to test how this defective MSPCs influence maintenance of HSPCs, we performed long-term culture-initiating cell assay (LTC-IC). After 5 weeks of co-culture of Ebf1-deficient stromal cells with wild type HSPCs we could observe significantly decreased number of cobblestone and CFU colonies formed by primitive HSPCs, in comparison to co-cultures with control stromal cells. Furthermore, in vivo adoptive transfers of wild type HSPCs to Ebf1+/- recipient mice showed a decrease in the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Additionally, Prx1-Cre-mediated deletion of Ebf1 specifically in MSPCs of mice leads to reduced frequency and numbers of HSPCs and myeloid cells in the bone marrow. These results confirm that mesenchymal stromal cells lacking Ebf1 render insufficient support for HSPCs to sustain proper hematopoiesis. Interestingly, we also observed a reduced ability of HSPCs sorted from Prx1CreEbf1fl/fl mice to form colonies in methylcellulose, suggesting not only impaired maintenance but also hindered function of these cells. Moreover, HSPCs exposed to Ebf1-deficient niche exhibit changes in chromatin accessibility with reduced occupancy of AP-1, ETS, Runx and IRF motifs, which is consistent with decreased myeloid output seen in Prx1CreEbf1fl/fl mice. These results support the hypothesis that defective niche can cause epigenetic reprograming of HSPCs. Finally, single cell and bulk transcriptome analysis of MSPCs lacking Ebf1 revealed differences in the niche composition and decreased expression of lineage-instructive signals for myeloid cells. Thus, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs. Disclosures No relevant conflicts of interest to declare.

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