scholarly journals Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting Angiopoietin-1

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Bo O Zhou ◽  
Lei Ding ◽  
Sean J Morrison
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Leptin Receptor ◽  
Hematopoietic Cells ◽  
Perivascular Niche ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
And Function ◽  
Angiopoietin 1

Hematopoietic stem cells (HSCs) are maintained by a perivascular niche in bone marrow but it is unclear whether the niche is reciprocally regulated by HSCs. Here, we systematically assessed the expression and function of Angiopoietin-1 (Angpt1) in bone marrow. Angpt1 was not expressed by osteoblasts. Angpt1 was most highly expressed by HSCs, and at lower levels by c-kit+ hematopoietic progenitors, megakaryocytes, and Leptin Receptor+ (LepR+) stromal cells. Global conditional deletion of Angpt1, or deletion from osteoblasts, LepR+ cells, Nes-cre-expressing cells, megakaryocytes, endothelial cells or hematopoietic cells in normal mice did not affect hematopoiesis, HSC maintenance, or HSC quiescence. Deletion of Angpt1 from hematopoietic cells and LepR+ cells had little effect on vasculature or HSC frequency under steady-state conditions but accelerated vascular and hematopoietic recovery after irradiation while increasing vascular leakiness. Hematopoietic stem/progenitor cells and LepR+ stromal cells regulate niche regeneration by secreting Angpt1, reducing vascular leakiness but slowing niche recovery.

Mesenchymal Stem and Progenitor Cells In the Mouse Bone Marrow Lack Expression of CD44.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2581-2581
Author(s):  
Hong Qian ◽  
Mikael Sigvardsson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Colony Forming Unit ◽  
Stem And Progenitor Cells ◽  
Cell Fractions

Abstract Abstract 2581 The bone marrow (BM) microenvironment consists of a heterogeneous population including mesenchymal stem cells and as well as more differentiated cells like osteoblast and adipocytes. These cells are believed to be crucial regulators of hematopoetic cell development, however, so far, their identity and specific functions has not been well defined. We have by using Ebf2 reporter transgenic Tg(Ebf2-Gfp) mice found that CD45−TER119−EBF2+ cells are selectively expressed in non-hematopoietic cells in mouse BM and highly enriched with MSCs whereas the EBF2− stromal cells are very heterogenous (Qian, et al., manuscript, 2010). In the present study, we have subfractionated the EBF2− stromal cells by fluorescent activated cell sorter (FACS) using CD44. On contrary to previous findings on cultured MSCs, we found that the freshly isolated CD45−TER119−EBF2+ MSCs were absent for CD44 whereas around 40% of the CD45−TER119−EBF2− cells express CD44. Colony forming unit-fibroblast (CFU-F) assay revealed that among the CD45−LIN−EBF2− cells, CD44− cells contained generated 20-fold more CFU-Fs (1/140) than the CD44+ cells. The EBF2−CD44− cells could be grown sustainably in vitro while the CD44+ cells could not, suggesting that Cd44− cells represents a more primitive cell population. In agreement with this, global gene expression analysis revealed that the CD44− cells, but not in the CD44+ cells expressed a set of genes including connective tissue growth factor (Ctgf), collagen type I (Col1a1), NOV and Runx2 and Necdin(Ndn) known to mark MSCs (Djouad et al., 2007) (Tanabe et al., 2008). Furthermore, microarray data and Q-PCR analysis from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca7,-8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the EBF2−CD44− cells, compared to the CD44+ cells indicating a relatively quiescent state of the CD44− cells ex vivo. This was confirmed by FACS analysis of KI67 staining. Furthermore, our microarray analysis suggested high expression of a set of hematopoietic growth factors and cytokines genes including Angiopoietin like 1, Kit ligand, Cxcl12 and Jag-1 in the EBF2−CD44− stromal cells in comparison with that in the EBF2+ or EBF2−CD44+ cell fractions, indicating a potential role of the EBF2− cells in hematopoiesis. The hematopoiesis supporting activity of the different stromal cell fractions were tested by in vitro hematopoietic stem and progenitor assays- cobblestone area forming cells (CAFC) and colony forming unit in culture (CFU-C). We found an increased numbers of CAFCs and CFU-Cs from hematopoietic stem and progenitor cells (Lineage−SCA1+KIT+) in culture with feeder layer of the EBF2−CD44− cells, compared to that in culture with previously defined EBF2+ MSCs (Qian, et al., manuscript, 2010), confirming a high capacity of the EBF2−CD44− cells to support hematopoietic stem and progenitor cell activities. Since the EBF2+ cells display a much higher CFU-F cloning frequency (1/6) than the CD44−EBF2− cells, this would also indicate that MSCs might not be the most critical regulators of HSC activity. Taken together, we have identified three functionally and molecularly distinct cell populations by using CD44 and transgenic EBF2 expression and provided clear evidence of that primary mesenchymal stem and progenitor cells reside in the CD44− cell fraction in mouse BM. The findings provide new evidence for biological and molecular features of primary stromal cell subsets and important basis for future identification of stage-specific cellular and molecular interaction pathways between hematopoietic cells and their cellular niche components. Disclosures: No relevant conflicts of interest to declare.

Characterization of Hematopoietic Stem Cell Frequency and Function In Unexplained Anemia of the Elderly

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2047-2047
Author(s):  
Wendy Pang ◽  
Elizabeth Price ◽  
Irving L. Weissman ◽  
Stanley L. Schrier
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
In Vitro Culture ◽  
Progenitor Cells ◽  
The Elderly ◽  
Elderly Subjects ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
And Function

Abstract Abstract 2047 Anemia is both a highly prevalent and clinically important condition that causes significant morbidity and mortality in the elderly population. While anemia in the elderly can be attributed to a number of causes, approximately 30% of elderly subjects with anemia have no overt etiology and fall under the category of unexplained anemia of the elderly (UA). There is increasing evidence to suggest that changes in the frequency and/or function of hematopoietic stem and progenitor cells may contribute to the onset and pathophysiology of age-associated hematological conditions, such as UA. Hematopoietic stem cells (HSC) reside at the top of the hematopoietic hierarchy and can differentiate, via increasingly committed downstream progenitors, into all the mature cells of the hematopoietic system. Human myelo-erythroid development proceeds through a set of oligopotent progenitors: HSC give rise to multipotent progenitors (MPP), which give rise to common myeloid progenitors (CMP), which in turn give rise to granulocyte-macrophage progenitors (GMP) and megakaryocyte-erythrocyte progenitors (MEP). We use flow cytometry and in vitro culture of sorted human HSC (Lin-CD34+CD38-CD90+CD45RA-), MPP (Lin-CD34+CD38-CD90-CD45RA-), CMP (Lin-CD34+CD38+CD123+CD45RA-), GMP (Lin-CD34+CD38+CD123+CD45RA+), and MEP (Lin-CD34+CD38+CD123-CD45RA-) from hematologically normal young (23 samples; age 20–35) and elderly (11 samples; age 65+) and UA (5 samples; age 65+) bone marrow samples in order to characterize the changes in the distribution and function of hematopoietic stem and progenitor populations during the aging process and, in particular, in the development of UA. We found that UA patients contain higher frequencies of HSC compared to both elderly normal (1.5-fold; p<0.03) and young normal samples (2.8-fold; p<10-5). We also found increased frequencies of MPP from UA patients compared to MPP from elderly normal (2.6-fold; p<0.002) and young normal samples (5.8-fold; p<0.04). While we observed similar frequencies of CMP among the three groups, we found a notable trend suggesting decreased frequencies of GMP and corresponding increased frequencies of MEP in UA patients. Functionally, HSC from the three groups exhibit statistically insignificant differences in the efficiency of colony formation under the myeloid differentiation-promoting methylcellulose-based in vitro culture conditions; however, on average, HSC from elderly bone marrow samples, regardless of the presence or absence of anemia, tend to form fewer colonies in methylcellulose. Interestingly, HSC from UA patients produce more granulocyte-monocyte (CFU-GM) colonies and fewer erythroid (CFU-E and BFU-E) colonies, compared to HSC from normal samples (p<0.001). Similarly, CMP from UA patients, compared to normal CMP, yield skewed distributions of myeloid-erythroid colonies when plated in methylcellulose, significantly favoring production of CFU-GM colonies over CFU-E and BFU-E colonies (p<0.003). Additionally, MEP from UA patients form both CFU-E and BFU-E colonies in methylcellulose albeit at a significantly lower efficiency than MEP from normal bone marrow samples (p<0.01). This is the first study to examine the changes in hematopoietic stem and progenitor populations in UA patients. The changes in the distribution of hematopoietic stem and progenitor cells in UA patients indicate that the HSC and MPP populations, and possibly also the MEP population, expand in the context of anemia, potentially in response to homeostatic feedback mechanisms. Nevertheless, these expanded populations are functionally impaired in their ability to differentiate towards the erythroid lineage. Our data suggest that there are intrinsic defects in the HSC population of UA patients that lead to poor erythroid differentiation, which can be readily observed even in the earliest committed myelo-erythroid progenitors. We have generated gene expression profiling data from these purified hematopoietic stem and progenitor populations from UA patients to try to identify biological pathways and markers relevant to disease pathogenesis and potential therapeutic targets. Disclosures: Weissman: Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Schrier:Celgene: Research Funding.

scholarly journals Deletion of proapoptotic Puma selectively protects hematopoietic stem and progenitor cells against high-dose radiation

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4707-4714 ◽  
Author(s):  
Lijian Shao ◽  
Yan Sun ◽  
Zhonghui Zhang ◽  
Wei Feng ◽  
Yongxing Gao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Cells ◽  
High Dose ◽  
Adverse Side Effect ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Novel Strategy ◽  
Induced Apoptosis ◽  
Dose Radiation

Abstract Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted, but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here, we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell–autonomous manner. Unexpectedly, loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly, null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation but selectively protects HSCs and HPCs against high-dose radiation, thereby accelerating hematopoietic regeneration. Consistent with these findings, Puma is required for radiation-induced apoptosis in HSCs and HPCs, and Puma is selectively induced by irradiation in primitive hematopoietic cells, and this induction is impaired in Puma-heterozygous cells. Together, our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy.

scholarly journals Hematopoietic Stem and Progenitor Cells as Effectors in Innate Immunity

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Jennifer L. Granick ◽  
Scott I. Simon ◽  
Dori L. Borjesson
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
Progenitor Cells ◽  
Innate Immune Response ◽  
Innate Immune ◽  
Phenotypic Characterization ◽  
Hematopoietic Stem ◽  
Effector Cells ◽  
Stem And Progenitor Cells ◽  
And Function

Recent research has shed light on novel functions of hematopoietic stem and progenitor cells (HSPC). While they are critical for maintenance and replenishment of blood cells in the bone marrow, these cells are not limited to the bone marrow compartment and function beyond their role in hematopoiesis. HSPC can leave bone marrow and circulate in peripheral blood and lymph, a process often manipulated therapeutically for the purpose of transplantation. Additionally, these cells preferentially home to extramedullary sites of inflammation where they can differentiate to more mature effector cells. HSPC are susceptible to various pathogens, though they may participate in the innate immune response without being directly infected. They express pattern recognition receptors for detection of endogenous and exogenous danger-associated molecular patterns and respond not only by the formation of daughter cells but can themselves secrete powerful cytokines. This paper summarizes the functional and phenotypic characterization of HSPC, their niche within and outside of the bone marrow, and what is known regarding their role in the innate immune response.

Cell Therapy in a Mouse Model of Immune-Mediated Bone Marrow Failure.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1689-1689
Author(s):  
Jichun Chen ◽  
Neal S. Young
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Adherent Cells ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Immune Mediated ◽  
Stem And Progenitor Cells

Abstract Immune-mediated bone marrow (BM) failure has been modeled in the mouse by infusion of lymph node cells from allogeneic C57BL/6 (B6) donors into major or minor histocompatibility antigen-mismatched recipients (Chen et al., Blood 2004; Bloom et al., Exp Hematol 2004, Chen et al., J Immunol 2007). Co-infusion of limited numbers of CD4+CD25+ regulatory T lymphocytes (Tregs) can alleviate clinical manifestations by suppressing the expansion of pathogenic T cells (Chen et al., J Immunol 2007). In the current study, we investigated the effectiveness of Tregs and suppressor cells contained in BM stroma in this fatal disease. Infusion of fewer than 3 × 103 Tregs to each recipient mouse had only a minor effect in preserving BM cells and did not prevent pancytopenia. Fifteen-50 × 103 thymic Tregs was moderately protective: blood WBC, RBC, platelet and BM cell counts at three weeks after cell infusion were 197%, 116%, 155% and 158% of those of control animals that did not receive Treg infusion; 5–10 × 103 B6 splenic Tregs produced the largest effect as WBC, RBC, platelet and BM cell counts were 275%, 143%, 276%, and 198% of controls. Overall, Treg therapy was helpful but its effectiveness was limited and variable among individual recipients as no antigen-specific Tregs can be identified for the treatment of BM failure. Learned about the immunosuppressive effects of mesenchymal stem cells (MSCs), we went on to test the effectiveness of stromal cells as another therapeutic modality for BM failure, since stromal cells contain MSCs. These cells were derived from B6 BM by culture in α-modified Eagle medium at 33°C with 5% CO2 for two weeks. After separating the non-adherent cells, we detached the adherent stromal cells and infused them into TBI + B6 LN-infused C.B10 mice. Injection of 106 stromal cells at the time of LN cell infusion effectively preserved WBCs (3.09 ± 0.51 vs 0.61 ± 0.18), RBCs (8.72 ± 0.14 vs 3.52 ± 0.46), platelets (924 ± 93 vs 147 ± 25) and BM cells (186.6 ± 8.7 vs 52.7 ± 7.8) when compared to LN-cell-infused mice without stromal cell addition. Delayed stromal cell injection at day 9 after LN cell infusion had only a mild effect on the preservation of RBCs (147%), platelets (276%) and BM cells (223%) and no effect on WBCs (64%), and infusion of non-adherent cells from the same stromal cell culture had no therapeutic effect. Stromal cell-infused mice had higher proportion of FoxP3+CD4+ cells in the peripheral blood (59.7 ± 10.7% vs 29.8 ± 5.4%) and more Lin−CD117+CD34− hematopoietic stem and progenitor cells in the BM (591 ± 95 vs 60 ± 43, thousand) in comparison to LN cell infused mice without stromal cell treatment. Mitigation of pathogenic T cells, including both CD4 and CD8 T lymphocytes, is the potential mechanism for the effectiveness of Treg and stromal cell therapies that helped to protect hematopoietic stem and progenitor cells in the BM of affected animals. Figure Figure

Bone Marrow Derived Mesenchymal Cells Secrete Granulopoietic Cytokines upon Danger Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4115-4115
Author(s):  
Stefan Wirths ◽  
Stefanie Bugl ◽  
Markus P. Radsak ◽  
Melanie Märklin ◽  
Martin R. Müller ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Feedback Loop ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Caspase 1 ◽  
Stem And Progenitor Cells ◽  
Myd88 Signaling

Abstract Granulopoietic homeostasis is regulated at steady-state to supply sufficient numbers of pooled and circulating neutrophils to maintain barrier function against commensal flora. In addition, upon pathogenic microbial challenge, an increased formation of neutrophils is induced, termed ‘emergency granulopoiesis’. Antibody-mediated reduction of neutrophil numbers in steady-state induces a feedback loop leading to an increase of bone marrow granulopoiesis with expansion of hematopoetic stem and progenitor cells. This feedback loop was demonstrated to depend on TLR4 and TRIF, but not MyD88 signaling (Bugl et al. Blood 2013). In contrast, emergency granulopoiesis was shown to be dependent on MyD88 signaling in endothelial cells (Boettcher et al. Blood 2014). Bone marrow mesenchymal stromal cells (MSC) are niche-forming cells, harboring and regulating hematopoiesis. Upon steady-state neutropenia an increase of niche size was observed. Here we investigated, whether niche-forming MSC act as sensors of pathogen-associated molecular patterns (PAMPs) and induce granulopoietic cytokines to stimulate expansion of adjacent hematopoietic stem and progenitor cells. MSC of C57BL/6 and TLR4-KO mice were cultured in vitro and treated with LPS for 24 hours. Cells were harvested and qRT-PCR for G-CSF, TLR4, MyD88, TRIF, GM-CSF, IL-1β, IL-18 and Casp-1 was performed After treatment with LPS, RNA of granulopoietic cytokines G-CSF and GM-CSF were massively up regulated in MSC of WT mice. Upstream regulating, inflammasome components IL-1ß and caspase-1 RNA levels increased as well, with little changes in IL-18, TLR4, MyD88 and TRIF. Unexpectedly, TLR4-KO MSC up regulated transcription of IL-1β and G-CSF upon LPS stimulation as well, and caspase-1 was found to be strongly up-regulated in unstimulated TLR4-KO compared to WT MSC. In summary, bone marrow stromal cells are found to be PAMP-sensing and secrete cytokines that regulate granulopoiesis. TLR4-independent sensing of LPS by MSC might correspond to the alternative noncanonical inflammasome pathway recently described (Kayagaki et al. Science 2013). Disclosures No relevant conflicts of interest to declare.

scholarly journals Acute exercise mobilizes hematopoietic stem and progenitor cells and alters the mesenchymal stromal cell secretome

2016 ◽  
Vol 120 (6) ◽  
pp. 624-632 ◽  
Author(s):  
Russell Emmons ◽  
Grace M. Niemiro ◽  
Olatomide Owolabi ◽  
Michael De Lisio
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Acute Exercise ◽  
Marrow Cell ◽  
Exercise Bout ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Transplantation of hematopoietic stem and progenitor cells (HSPC), collected from peripheral blood, is the primary treatment for many hematological malignancies; however, variable collection efficacy with current protocols merits further examination into factors responsible for HSPC mobilization. HSPCs primarily reside within the bone marrow and are regulated by mesenchymal stromal cells (MSC). Exercise potently and transiently mobilizes HSPCs from the bone marrow into peripheral circulation. Thus the purpose of the present study was to evaluate potential factors in the bone marrow responsible for HSPC mobilization, investigate potential sites of HSPC homing, and assess changes in bone marrow cell populations following exercise. An acute exercise bout increased circulating HSPCs at 15 min (88%, P < 0.001) that returned to baseline at 60 min. Gene expression for HSPC homing factors (CXCL12, vascular endothelial growth factor-a, and angiopoietin-1) were increased at 15 min in skeletal muscle and HSPC content was increased in the spleen 48 h postexercise (45%, P < 0.01). Acute exercise did not alter HSPCs or MSCs quantity in the bone marrow; however, proliferation of HSPCs (40%, P < 0.001), multipotent progenitors (40%, P < 0.001), short-term hematopoietic stem cells (61%, P < 0.001), long-term hematopoietic stem cells (55%, P = 0.002), and MSCs (20%, P = 0.01) increased postexercise. Acute exercise increased the content of the mobilization agent granulocyte-colony stimulating factor, as well as stem cell factor, interleukin-3, and thrombopoeitin in conditioned media collected from bone marrow stromal cells 15 min postexercise. These findings suggest that the MSC secretome is responsible for HSPC mobilization and proliferation; concurrently, HSPCs are homing to extramedullary sites following exercise.

scholarly journals EBF1 As a Novel Regulator of Bone Marrow Mesenchymal Stromal Progenitors

Blood ◽  
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.

In Vitro Hematopoietic Lineage Interconversion from Human Bone Marrow Stem and Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4160-4160
Author(s):  
Ling Chen ◽  
Stephanie Jean-Noel ◽  
Kevin Hall ◽  
Ying Shi ◽  
Griffin P. Rodgers
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Surface ◽  
Progenitor Cells ◽  
Hematopoietic Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Two Populations

Abstract The cell surface antigen, CD133, marks a fraction of hematopoietic stem and progenitor cells and has been successfully used to study their differential biology. To evaluate the differentiating capacity of stem/progenitor cells, we cultivated purified normal human bone marrow CD133 selected cells for 2 weeks with erythropoietin (EPO) or granulocyte colony-stimulating factor (G-CSF) to induce erythroid or myeloid differentiation, respectively. After the second week of cultivation, we reversed the seeding environment of the two populations by placing EPO treated cells into a G-CSF environment and G-CSF treated cells into an EPO environment for an additional 2-week culture. The cells produced in the culture were phenotypically defined by morphology and flow cytometry, and genotypically by RNA and proteomic analyses. Three-color flow cytometry was used for identifying CD133+ progenitors, CD36+ erythroid and CD13+ myeloid cells, as shown in Table 1. The morphology of the cultured cells, assessed by Wright-Giemsa staining, is consistent with the conversion of cellular specific markers. Rapid analysis of gene expression demonstrated co-expression of 76% of 266 genes analyzed among the erythroid and myeloid lineages. Furthermore, proteomic analysis exhibited the sharing of 33% of 9518 expressed protein spots assayed in the two populations after the first 2-week culture, and 32% after 2 weeks of the switch culture. Our data clearly demonstrate that the committed erythroid and myeloid precursors are able to change their fate and can switch into the opposite cell type by a conversion pathway under a specifically defined condition. We termed this switch as interconversion, considering conversion of hematopoietic cells to non-hematopoietic cells. Furthermore, the observations presented in this study show that cytokines used can improve the conversion. We are developing a mathematical model describing the kinetics of hematopoietic stem/progenitor cell transitions into specific lineages, along with the conversion of committed cells based on multiple potential energy wells corresponding to different cell states and cytokines. Table 1. Expression of cell surface markers after 4-week culture D0 1 week 2 weeks 4 weeks CD expression (%) E G E G E2w →G2w → G2w E2w Data are presented as a mean of at least 2 experiments. E: EPO; G: G-CSF; E2w or G2w: EPO or G-CSF treatment for two weeks. CD133+ 96.19 15.74 13.6 0.24 0.36 0.01 0.63 CD36+ 0 60.37 27.39 96.37 25.87 45.41 68.54 CD13+ 0.43 35.41 57.29 24.41 92.1 85.87 37.76 CD133+ / CD36+ 0.44 22.24 15.97 0.12 0.18 1.55 7.65 CD133+ / CD13+ 1.24 19.43 13.36 0.36 1.09 13.31 14.92 CD36+ / CD13+ 0.09 41.25 17.80 23.69 54.1 46.60 79.41

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