Osteoblastic adherence regulates hematopoietic stem cell self-renewal and differentiation: a conceptional in vitro and in vivo study

Abstract A tight control of hematopoietic stem cell (HSC) quiescence, self-renewal and differentiation is crucial for lifelong blood production. The mechanisms behind this control are still poorly understood. Here we show that mitochondrial activity determines HSC fate decisions. A low mitochondrial membrane potential (Δψm) predicts long-term multi-lineage blood reconstitution capability, as we show for freshly isolated and in vitro-cultured HSCs. However, as in vivo both quiescent and cycling HSCs have comparable Δψm distributions, a low Δψm is not per se related to quiescence but is also found in dividing cells. Indeed, using divisional tracking, we demonstrate that daughter HSCs with a low Δψm maintain stemness, whereas daughter cells with high Δψm have undergone differentiation. Strikingly, lowering the Δψm by chemical uncoupling of the electron transport chain leads to HSC self-renewal under culture conditions that normally induce rapid differentiation. Taken together, these data show that mitochondrial activity and fate choice are causally related in HSCs, and provides a novel method for identifying HSC potential after in vitro culture. Disclosures No relevant conflicts of interest to declare.

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Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801480116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1447-1456 ◽

Cited By ~ 29

Author(s):

Rong Lu ◽

Agnieszka Czechowicz ◽

Jun Seita ◽

Du Jiang ◽

Irving L. Weissman

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Lineage Commitment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Different Levels

While the aggregate differentiation of the hematopoietic stem cell (HSC) population has been extensively studied, little is known about the lineage commitment process of individual HSC clones. Here, we provide lineage commitment maps of HSC clones under homeostasis and after perturbations of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been perturbed by irradiation or by an antagonistic anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiate. Some of these clones dominantly expand and exhibit lineage bias. We identified the cellular origins of clonal dominance and lineage bias and uncovered the lineage commitment pathways that lead HSC clones to different levels of self-renewal and blood production under various transplantation conditions. This study reveals surprising alterations in HSC fate decisions directed by conditioning and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.

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New genetic tools for the in vivo study of hematopoietic stem cell function

Experimental Hematology ◽

10.1016/j.exphem.2018.02.004 ◽

2018 ◽

Vol 61 ◽

pp. 26-35 ◽

Cited By ~ 5

Author(s):

Samik Upadhaya ◽

Boris Reizis ◽

Catherine M. Sawai

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cell Function ◽

In Vivo Study ◽

Hematopoietic Stem ◽

Genetic Tools

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Making HSCs in vitro: don’t forget the hemogenic endothelium

Blood ◽

10.1182/blood-2018-04-784140 ◽

2018 ◽

Vol 132 (13) ◽

pp. 1372-1378 ◽

Cited By ~ 11

Author(s):

Bradley W. Blaser ◽

Leonard I. Zon

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Autologous Transplantation ◽

Cell Types ◽

Therapeutic Modality ◽

Hemogenic Endothelium ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Ontogeny

Generating a hematopoietic stem cell (HSC) in vitro from nonhematopoietic tissue has been a goal of experimental hematologists for decades. Until recently, no in vitro–derived cell has closely demonstrated the full lineage potential and self-renewal capacity of a true HSC. Studies revealing stem cell ontogeny from embryonic mesoderm to hemogenic endothelium to HSC provided the key to inducing HSC-like cells in vitro from a variety of cell types. Here we review the path to this discovery and discuss the future of autologous transplantation with in vitro–derived HSCs as a therapeutic modality.

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Growth Factor Receptor-Bound Protein 10 (Grb10) Regulates Hematopoietic Stem Cell (HSC) Self-Renewal Via Control of mTOR Signaling

Blood ◽

10.1182/blood.v126.23.1160.1160 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1160-1160

Author(s):

Xiao Yan ◽

Heather A Himburg ◽

Phuong L Doan ◽

Mamle Quarmyne ◽

Evelyn Tran ◽

...

Keyword(s):

Stem Cell ◽

Growth Factor ◽

Hematopoietic Stem Cell ◽

Growth Factor Receptor ◽

Post Transplantation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Donor Cells ◽

Duke University

Abstract Elucidation of the mechanisms governing HSC regeneration has been impeded by difficulty in isolating HSCs early following genotoxic injury, such as total body irradiation (TBI). Using multiparametric flow cytometric cell sorting of BM ckit+sca-1+lin- cells coupled with gene expression analysis, we identified growth factor receptor-bound protein 10 (Grb10), a co-receptor which regulates Insulin Receptor/IGF-1 signaling, to be significantly overexpressed by BM KSL cells at the earliest detectable point of regeneration (day +10) following TBI (3.3-fold, p<0.0001). Grb10 is a member of the imprinted gene family which is predominately expressed in the stem cells of a variety of tissues, including embryonic stem cells, bone marrow, skin and muscle. Viral shRNA-mediated knockdown of Grb10 in BM KSL cells caused a significant decrease in KSL cells and colony forming cells (CFCs) detected in 7-day culture (p=0.03 and p=0.002, respectively). Furthermore, mice competitively transplanted with Grb10-deficient HSCs displayed 10-fold lower donor, multilineage hematopoietic cell engraftment than mice transplanted with Grb10-expressing HSCs (p=0.007 for %CD45.1+ donor cells). Secondary competitive repopulation assays confirmed a greater than 10-fold deficit in long-term repopulating capacity in Grb10-deficient KSL cells compared to Grb10-expressing KSL cells (p=0.006 for %CD45.1+ donor cells). In order to determine if Grb10 was necessary for HSC maintenance and normal hematopoiesis in vivo, we generated maternally-derived Grb10-deficient mice. Heterozygous 8 week old Grb10m/+ (1 mutant allele, 1 wild type allele) had 10-fold decreased Grb10 expression in BM lin-cells. BM CFCs and SLAM+ KSL cells were significantly decreased in Grb10m/+ mice compared to Grb10+/+ mice (p=0.006 and p=0.04, respectively). Competitive repopulation assays demonstrated significantly decreased donor hematopoietic cell repopulation in recipient mice transplanted with Grb10m/+ BM cells versus mice transplanted with Grb10+/+ BM cells (p=0.003 for %CD45.1+ donor cells). Mice transplanted with BM cells from homozygous Grb10-/- mice showed a similar decrease in donor-derived hematopoietic repopulation compared to mice transplanted with BM cells from Grb10+/+ mice (p=0.02 at 20 weeks post-transplantation). These results confirmed that Grb10 regulates HSC self-renewal capacity in vivo. To determine whether Grb10 regulates HSC regeneration after myelotoxic injury, we irradiated Grb10m/+ mice with 550cGy TBI, and monitored hematopoietic recovery over time in comparison to Grb10+/+ controls. Interestingly, Grb10m/+ mice displayed accelerated hematopoietic regeneration early following TBI. At day+10 after 550cGy, Grb10m/+ mice contained significantly increased numbers of BM SLAM+ KSL cells (p=0.04) and CFCs (p=0.009), compared to Grb10+/+ littermates. Similarly, mice transplanted with BM cells from irradiated, Grb10m/+ mice displayed 5-fold increased donor hematopoietic repopulation at 20 weeks post-transplantation compared to mice transplanted with BM cells from irradiated, Grb10+/+ mice (p=0.006). These data suggest that Grb10 deficiency accelerates hematopoietic recovery in the early period following myelosuppressive radiation injury. Mechanistically, Grb10-deficiency caused an increase in the percentage of BM KSL cells in G1 and G2/S/M phase of cell cycle compared to Grb10+/+ KSL cells (p=0.003). We also observed significantly increased levels of mTOR activation in Grb10m/+ BM KSL cells compared to Grb10+/+ BM KSL cells (p=0.001 for pS6, p=0.001 for pS6k and p=0.02 for p4EBP1). Furthermore, mTOR inhibition via siRNA-mTOR targeting rescued the defect in BM hematopoietic progenitor content (colony forming cells) in Grb10-deficient BM cells (p<0.0001). Taken together, our results suggest that Grb10 is necessary for HSC maintenance in steady state, while, paradoxically, Grb10 inhibition accelerates HSC regeneration early following injury. Furthermore, our data suggest that Grb10 mediates these effects via regulation of mTOR signaling. Selective modulation of Grb10 signaling has the potential to augment HSC self-renewal in steady state and to accelerate HSC regeneration following myelotoxic injury. Disclosures Himburg: Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.

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Chromatin Remodeling Subunit Brm Regulates Hematopoietic Stem Cell Self-Renewal By Limiting Intracellular Valine Levels

Blood ◽

10.1182/blood-2019-128867 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3702-3702

Author(s):

Samisubbu R Naidu ◽

Maegan L. Capitano ◽

Scott Cooper ◽

Xinxin Huang ◽

Hal E. Broxmeyer

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Chromatin Remodeling ◽

Normal Mouse ◽

Self Renewal ◽

Atpase Subunit ◽

Hematopoietic Stem ◽

Cell Fate Decisions

Chromatin remodeling complexes facilitate gene expression and control cell fate decisions. The ATPase subunit of chromatin remodeling complex BRG1 is essential for stem cell function, but the role of its paralog Brm remains essentially unknown. To assess a role(s) for Brm in hematopoietic cell regulation in vivo, we studied hematopoietic stem (HSCs) and progenitor cells (HPCs) in bone marrow (BM) of Brm -/- vs. wildtype (WT) control mice. While BM from Brm -/- mice contain increased numbers of rigorously-defined phenotypic populations of long- and short-term repopulating HSCs and granulocyte macrophage progenitors (GMPs) and increased numbers and cycling status of functional HPC (assessed by CFU-GM, BFU-E, and CFU-GEMM colony assays), they were defective in self-renewal capacity upon in vivo serial transplantation using congenic mice (CD45.2+ donor cells, CD45.1+ competitor cells, and F1 (CD45.2+/CD45.1+) recipient mice). Increased numbers of HSCs from Brm-/- BM failed to show competitive advantage over wild type (WT) control BM cells in primary (1°) transplantation in lethally irradiated mice (based on month 4 donor cell chimerism and phenotypically defined HSC numbers). Moreover, 2° and 3° engraftment at 4 months post transplantation each, a measure of HSC self-renewal capacity, revealed much reduced engraftment of donor Brm -/- BM cell chimerism and HSC numbers compared to the extensive 2° and 3° engraftment of control WT BM. No significant differences in myeloid/lymphoid ratios were noted in 1° or 2° engrafted mice, suggesting no differentiation lineage bias of donor Brm -/- BM cells. This demonstrates a critical role for Brm in controlling in vivo self-renewal of mouse BM HSCs. Valine [(2S)-2 amino-3 methylbutanoic acid (C5H11N02)] is implicated in hematopoietic regulation, since depleting dietary valine permitted non-myeloablative mouse HSC transplantation (Taya et. al. Science 354:1152-1155, 2016). Metabolic analysis of lineage negative (lin-) cells demonstrated that valine, but not leucine, levels were very highly elevated in Brm -/- BM cells, thus linking intracellular valine levels with Brm expression. Exogenously added valine significantly increased basal oxygen consumption rates of both total WT BM and WT lin- cells, but not of total or lin-Brm -/- BM cells in vitro (via Seahorse machine analysis). To study effects of valine on HPCs, we assessed the addition of exogenously added valine on mouse BM and human cord blood (CB) cells cultured in the presence of cytokines with either non-dialyzed or dialyzed fetal bovine serum (FBS). Valine, but not leucine, dose-dependently enhanced HPC (CFU-GM, BFU-E, and CFU-GEMM) colony formation and secondary replating capacity of cytokine stimulated CFU-GM and CFU-GEMM derived colonies of normal mouse BM cells in vitro in presence of non-dialyzed FBS; additional enhanced valine effects were noted when dialyzed FBS (lacking valine and other amino acids) was used. Valine also enhanced mouse BM HPC survival in vitro in context of delayed addition of growth factors, and cytokine stimulated (SCF, FL, TPO) ex-vivo expansion of normal mouse BM HSCs and HPCs. Valine enhancement of the above noted functional mouse BM HPC assays in the presence of dialyzed FBS was also apparent with low density and CD34+ purified CB cells, demonstrating that valine effects are not species specific. Our results suggest that valine is an enhancing factor for HSC maintenance of self-renewal capacity and HPC proliferation, and that Brm gene expression limits intracellular valine levels, thereby controlling HSC self-renewal and HPC proliferation. This information is of potential use for future translation to modulate self-renewal of HSCs and survival and proliferation of HPCs for clinical advantage. Disclosures No relevant conflicts of interest to declare.

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A genetic disorder reveals a hematopoietic stem cell regulatory network co-opted in leukemia

10.1101/2021.12.09.471942 ◽

2021 ◽

Author(s):

Richard A. Voit ◽

Liming Tao ◽

Fulong Yu ◽

Liam D. Cato ◽

Blake Cohen ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Regulatory Network ◽

Genetic Disorder ◽

Transcriptional Network ◽

Self Renewal ◽

Hematopoietic Stem ◽

Functional Studies ◽

Experimental Systems

The molecular regulation of human hematopoietic stem cell (HSC) self-renewal is therapeutically important, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. Here, we have studied a rare genetic disorder due to MECOM haploinsufficiency, characterized by an early-onset absence of HSCs in vivo. By generating a faithful model of this disorder in primary human HSCs and coupling functional studies with integrative single-cell genomic analyses, we uncover a key transcriptional network involving hundreds of genes that is required for HSC self-renewal. Through our analyses, we nominate cooperating transcriptional regulators and identify how MECOM prevents the CTCF-dependent genome reorganization that occurs as HSCs exit quiescence. Strikingly, we show that this transcriptional network is co-opted in high-risk leukemias, thereby enabling these cancers to acquire stem cell properties. Collectively, we illuminate a regulatory network necessary for HSC self-renewal through the study of a rare experiment of nature.

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Identification of hematopoietic stem cell subsets on the basis of their primitiveness using antibody ER-MP12

Blood ◽

10.1182/blood.v85.4.952.bloodjournal854952 ◽

1995 ◽

Vol 85 (4) ◽

pp. 952-962 ◽

Cited By ~ 37

Author(s):

JC van der Loo ◽

WA Slieker ◽

D Kieboom ◽

RE Ploemacher

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Antigen Expression ◽

Chimeric Mouse ◽

Hematopoietic Stem ◽

Colony Forming Units

Monoclonal antibody ER-MP12 defines a novel antigen on murine hematopoietic stem cells. The antigen is differentially expressed by different subsets in the hematopoietic stem cell compartment and enables a physical separation of primitive long-term repopulating stem cells from more mature multilineage progenitors. When used in two-color immunofluorescence with ER-MP20 (anti-Ly-6C), six subpopulations of bone marrow (BM) cells could be identified. These subsets were isolated using magnetic and fluorescence-activated cell sorting, phenotypically analyzed, and tested in vitro for cobblestone area-forming cells (CAFC) and colony-forming units in culture (CFU-C; M/G/E/Meg/Mast). In addition, they were tested in vivo for day-12 spleen colony-forming units (CFU-S-12), and for cells with long-term repopulating ability using a recently developed alpha-thalassemic chimeric mouse model. Cells with long-term repopulation ability (LTRA) and day-12 spleen colony-forming ability appeared to be exclusively present in the two subpopulations that expressed the ER-MP12 cell surface antigen at either an intermediate or high level, but lacked the expression of Ly- 6C. The ER-MP12med20- subpopulation (comprising 30% of the BM cells, including all lymphocytes) contained 90% to 95% of the LTRA cells and immature day-28 CAFC (CAFC-28), 75% of the CFU-S-12, and very low numbers of CFU-C. In contrast, the ER-MP12hi20- population (comprising 1% to 2% of the BM cells, containing no mature cells) included 80% of the early and less primitive CAFC (CAFC-5), 25% of the CFU-S-12, and only 10% of the LTRA cells and immature CAFC-28. The ER-MP12hi cells, irrespective of the ER-MP20 antigen expression, included 80% to 90% of the CFU-C (day 4 through day 14), of which 70% were ER-MP20- and 10% to 20% ER-MP20med/hi. In addition, erythroblasts, granulocytes, lymphocytes, and monocytes could almost be fully separated on the basis of ER-MP12 and ER-MP20 antigen expression. Functionally, the presence of ER-MP12 in a long-term BM culture did not affect hematopoiesis, as was measured in the CAFC assay. Our data demonstrate that the ER-MP12 antigen is intermediately expressed on the long-term repopulating hematopoietic stem cell. Its level of expression increases on maturation towards CFU-C, to disappear from mature hematopoietic cells, except from B and T lymphocytes.

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Abstract 020: Angiotensin Ii-induced Hypertension Has Significant Effects On Proliferation, Differentiation And Engraftment Efficacy Of Hematopoietic Stem Cell.

Hypertension ◽

10.1161/hyp.64.suppl_1.020 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Seungbum Kim ◽

Christopher R Cogle ◽

Michael Zingler ◽

Edward W Scott ◽

Mohan K Raizada

Keyword(s):

Blood Pressure ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Inflammatory Cells ◽

Time Lapse ◽

Immunosuppressive Drugs ◽

Ang Ii ◽

Hematopoietic Stem

Cyclosporin and other immunosuppressive drugs are used in bone marrow (BM) transplantation to increase engraftment efficacy and reduce rejection. However, their chronic clinical use is closely associated with increase in blood pressure and development of hypertension (HTN). Despite these significant side effects, little is known about the influence of high blood pressure on hematopoietic stem cell (HSC) and BM activity. Thus, the objective of this study was to investigate if Ang II induced HTN exerts influence on HSC proliferation, differentiation and engraftment in the BM. Infusion of Ang II (1000ng/kg/min for 21 days) and establishment of HTN resulted in increased proliferation of HSCs as evidenced by 87% increase in Sca-1+, c-Kit+, Lin- (SKL) HSC and 254% increase in CD150+, CD48- SKL long-term HSC in the BM. Furthermore, this was associated with significant accumulation of monocytes in both BM (30% increase) and spleen (250% increase). These changes in HSC and inflammatory cells were blocked by co-infusion of Ang II and losartan (60mg/kg/day), In order to understand the effect of Ang II on HSC homing, GFP+ HSCs were injected into the lethally irradiated and saline or Ang II infused C57BL6 mice. FACS analysis of GFP+ donor derived cells showed that hypertensive animals has poor engraftment efficacy on both BM and peripheral blood (35-52% compared to saline controls). Time-lapse in vivo imaging of mouse tibia showed that HSC failed to engraft to the BM osteoblastic niche in hypertensive mice. HSCs pretreated with 100nM Ang II for 18 hours in vitro also showed significantly diminished ability (16% compared to control) to engraft in normal recipient mice. These observations demonstrate that 1) chronic Ang II induced HTN regulates HSC proliferation and impairs the homing ability and reconstitution potential of HSC in BM, 2) These effects are mediated by the AT1 receptor on HSC and 3) Ang II accelerates HSC differentiation leading the increase of inflammatory cells in BM and spleen. The results suggest that hypertensive status and BP control should be strictly taken into account in consideration for BM transplantation.

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Downregulation of Mpl Marks the Transition to Lymphoid-Primed Multipotent Progenitors with Gradual Loss of Granulocyte-Monocyte Potential.

Blood ◽

10.1182/blood.v110.11.2227.2227 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2227-2227

Author(s):

Sidinh Luc ◽

Kristina Anderson ◽

Ms Shabnam Kharazi ◽

Natalija Buza-Vidas ◽

Charlotta Boiers ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Gradual Loss ◽

Thrombopoietin Receptor ◽

Abrupt Changes ◽

Lineage Priming

Abstract Evidence for a novel route of adult hematopoietic stem cell (HSC) lineage commitment through Lin−Sca-1+Kit+Flt3hi (LSKFlt3hi) lymphoid-primed multipotent progenitors (LMPPs) with granulocyte/monocyte (GM) and lymphoid but little or no megakaryocyte/erythroid (MkE) potential was recently challenged, as LSKFlt3hi cells were reported to possess MkE potential. Herein residual MkE potential segregated almost entirely with LSKFlt3hi cells expressing the thrombopoietin receptor (Thpor), whereas LSKFlt3hiThpor− LMPPs lacked significant MkE potential in vitro and in vivo, but sustained combined GM and lymphoid potentials, and co-expressed GM and lymphoid but not MkE transcriptional lineage programs. Gradually increased transcriptional lymphoid priming in single LMPPs from Rag1GFP mice was shown to occur in the presence of maintained GM lineage priming, but gradually reduced GM lineage potential. These functional and molecular findings reinforce the existence of GM-lymphoid progenitors with dramatically downregulated probability for committing towards MkE fates, and support that lineage restriction occurs through gradual rather than abrupt changes in specific lineage potentials.

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