Differential Localization of RAC1 and RAC2 Reflects Their Specific Functions in Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells.

Abstract Abstract 2302 Hematopoietic stem cells (HSCs) depend on the bone marrow niche to provide signals for their survival, quiescence and differentiation. Many of these microenvironmental signals converge on RAC GTPases. In the hematopoietic system, two members of the RAC family are expressed, RAC1 and RAC2. Although RAC1 and RAC2 share a very high sequence homology, specific functions of these proteins have been suggested. However, little has been revealed about the downstream effectors and molecular mechanisms. In this study, we used multiple approaches to gain insight into the molecular biology of RAC1 and RAC2 in normal and leukemic human HSCs. Firstly, GFP-tagged constructs of RAC1 and RAC2 were used to study localization of these proteins in CD34+/CD38−/Lin− HSCs. Time-lapse confocal imaging of living cells plated on stroma revealed that RAC1 was strongly enriched in the plasma membrane. In contrast, RAC2 localized predominantly in the cytoplasm of both resting and dividing HSCs, whereby localization changed dramatically when cells progressed from S to the G2 phase of the cell cycle. This very distinct localization pattern implied different functions of RAC1 and RAC2. Therefore, we specifically downregulated RAC1 and/or RAC2 to study the effects of their depletion in normal and BCR-ABL-transduced leukemic HSCs. In normal HSCs, simultaneous downregulation of RAC1 and RAC2 resulted in a modest but significant decrease in proliferation and progenitor frequencies in the long term stromal co-cultures. However, in BCR-ABL-transduced HSCs depletion of RAC2 alone, but not RAC1, was sufficient to induce a marked proliferative disadvantage, decreased progenitor frequency, reduced leukemic cobblestone formation and diminished replating capacity. To elucidate the mechanisms involved in the observed phenotypes, we employed an in vivo biotin labeling strategy of Avi-tagged RAC1 and RAC2 followed by pull down and mass-spectrometry to identify specific interaction partners of RAC1 and RAC2 in BCR-ABL-expressing hematopoietic cells. Several of the RAC1-specific interaction partners were annotated as plasma membrane proteins, involved in cell adhesion, cytoskeleton assembly and regulation of endocytosis. In contrast, RAC2-interacting proteins were cytoplasmic and involved in processes such as cell cycle progression, mitosis and regulation of apoptosis. Consistently with these findings, confocal time-lapse imaging of living hematopoietic cells revealed that pharmacological inhibition of RAC2 activity resulted in greatly decreased frequency of cell division. Moreover, the average division time was significantly extended upon RAC2 inhibition. Further functional characterization of RAC1 and RAC2-specific interactions is currently ongoing and will be discussed, but our data clearly indicate that distinct subcellular localization of RAC1 and RAC2 dictates their interaction with specific sets of proteins and consequently their specific functions in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Differential Localization Of RAC1 and RAC2 Reflects Their Specific Functions In Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v122.21.2892.2892 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2892-2892

Author(s):

Marta Ewa Capala ◽

Francesco Bonardi ◽

Henny Maat ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Cell Cycle ◽

Plasma Membrane ◽

Molecular Mechanisms ◽

Specific Interaction ◽

Hematopoietic Cells ◽

Confocal Imaging ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Interaction Partners

Abstract Hematopoietic stem cells (HSCs) depend on the bone marrow niche to provide signals for their survival, quiescence and differentiation. Many of these microenvironmental signals converge on RAC GTPases. In the hematopoietic system, two members of the RAC family are expressed, RAC1 and RAC2. Although RAC1 and RAC2 share a very high sequence homology, specific functions of these proteins have been suggested. However, little has been revealed about the downstream effectors and molecular mechanisms. In this study, we used multiple approaches to gain insight into the molecular biology of RAC1 and RAC2 in normal and leukemic human HSCs. Firstly, GFP-tagged constructs of RAC1 and RAC2 were used to study localization of these proteins in CD34+/CD38-/Lin- HSCs. Time-lapse confocal imaging of living cells plated on stroma revealed that RAC1 was strongly enriched in the plasma membrane. In contrast, RAC2 localized predominantly in the cytoplasm of both resting and dividing HSCs, whereby localization changed dramatically when cells progressed from S to the G2 phase of the cell cycle. This very distinct localization pattern implied different functions of RAC1 and RAC2. Therefore, we specifically downregulated RAC1 and/or RAC2 to study the effects of their depletion in normal and BCR-ABL-transduced leukemic HSCs. In normal HSCs, simultaneous downregulation of RAC1 and RAC2 resulted in a modest but significant decrease in proliferation and progenitor frequencies in the long term stromal co-cultures. However, in BCR-ABL-transduced HSCs depletion of RAC2 alone, but not RAC1, was sufficient to induce a marked proliferative disadvantage, decreased progenitor frequency, reduced leukemic cobblestone formation and diminished replating capacity, indicative for reduced self-renewal. Consistently, the frequency of long-term culture initiating leukemic cells was markedly reduced upon RAC2 downregulation. To elucidate the mechanisms involved in the observed phenotypes, we employed an in vivo biotin labeling strategy of Avi-tagged RAC1 and RAC2 followed by pull down and mass-spectrometry to identify specific interaction partners of RAC1 and RAC2 in BCR-ABL-expressing hematopoietic cells. Several of the RAC1-specific interaction partners were annotated as plasma membrane proteins, involved in cell adhesion, cytoskeleton assembly and regulation of endocytosis. In contrast, RAC2-interacting proteins were cytoplasmic or mitochondria-associated, and involved in processes such as cell cycle progression and regulation of apoptosis. Consistently, the proportion of dividing cells was decreased in RAC2-depleted BCR-ABL leukemic cobblestones coinciding with an increased apoptosis. Finally, a marked decrease in mitochondrial membrane potential was observed upon RAC2 but not RAC1 downregulation pointing to mitochondrial dysfunction as the initiating event of the apoptotic response. Moreover, preliminary electron microscopy data suggest that this functional change may be paralleled by structural aberrations of mitochondria. Further functional characterization of RAC1 and RAC2-specific interactions is currently ongoing and will be discussed, but our data clearly indicate that distinct subcellular localization of RAC1 and RAC2 dictates their interaction with specific sets of proteins and consequently their specific functions in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients

Blood ◽

10.1182/blood.v99.5.1585 ◽

2002 ◽

Vol 99 (5) ◽

pp. 1585-1593 ◽

Cited By ~ 83

Author(s):

Anna Jetmore ◽

P. Artur Plett ◽

Xia Tong ◽

Frances M. Wolber ◽

Robert Breese ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Hematopoietic Cells ◽

Cycle Progression ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Nonobese Diabetic ◽

Cd34 Cells ◽

G1 Cells ◽

Active Proliferation

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34+ cells, or their G0(G0CD34+) or G1(G1CD34+) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34+ cells harbored similar numbers of CD34+ and CD34− cells, indicating that CD34+ cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G2+M than in recipients of G0/G1 cells (34.6% ± 5.9% and 17.1% ± 6.3%, respectively; P < .01). These results suggest that active proliferation inhibition in the BM of irradiated recipients maintains mitotic quiescence of transplanted HSCs early AT and may trigger apoptosis of cycling cells. These data also illustrate that trafficking of transplanted cells to the BM is not selective, but lodgment of BM-homed cells may be specific.

Download Full-text

Transcriptional signature of prion-induced neurotoxicity in a Drosophila model of transmissible mammalian prion disease

Biochemical Journal ◽

10.1042/bcj20190872 ◽

2020 ◽

Vol 477 (4) ◽

pp. 833-852

Author(s):

Alana M. Thackray ◽

Brian Lam ◽

Anisa Shahira Binti Ab Razak ◽

Giles Yeo ◽

Raymond Bujdoso

Keyword(s):

Cell Cycle ◽

Plasma Membrane ◽

Prion Disease ◽

Molecular Mechanisms ◽

Control Strategies ◽

Prion Diseases ◽

Transcriptional Signature ◽

Natural Hosts ◽

Regulation Of Cell Cycle ◽

Transgenic Drosophila

Prion diseases are fatal transmissible neurodegenerative conditions of humans and animals that arise through neurotoxicity induced by PrP misfolding. The cellular and molecular mechanisms of prion-induced neurotoxicity remain undefined. Understanding these processes will underpin therapeutic and control strategies for human and animal prion diseases, respectively. Prion diseases are difficult to study in their natural hosts and require the use of tractable animal models. Here we used RNA-Seq-based transcriptome analysis of prion-exposed Drosophila to probe the mechanism of prion-induced neurotoxicity. Adult Drosophila transgenic for pan neuronal expression of ovine PrP targeted to the plasma membrane exhibit a neurotoxic phenotype evidenced by decreased locomotor activity after exposure to ovine prions at the larval stage. Pathway analysis and quantitative PCR of genes differentially expressed in prion-infected Drosophila revealed up-regulation of cell cycle activity and DNA damage response, followed by down-regulation of eIF2 and mTOR signalling. Mitochondrial dysfunction was identified as the principal toxicity pathway in prion-exposed PrP transgenic Drosophila. The transcriptomic changes we observed were specific to PrP targeted to the plasma membrane since these prion-induced gene expression changes were not evident in similarly treated Drosophila transgenic for cytosolic pan neuronal PrP expression, or in non-transgenic control flies. Collectively, our data indicate that aberrant cell cycle activity, repression of protein synthesis and altered mitochondrial function are key events involved in prion-induced neurotoxicity, and correlate with those identified in mammalian hosts undergoing prion disease. These studies highlight the use of PrP transgenic Drosophila as a genetically well-defined tractable host to study mammalian prion biology.

Download Full-text

CXCR4 is required for the quiescence of primitive hematopoietic cells

Journal of Experimental Medicine ◽

10.1084/jem.20072513 ◽

2008 ◽

Vol 205 (4) ◽

pp. 777-783 ◽

Cited By ~ 243

Author(s):

Yuchun Nie ◽

Yoon-Chi Han ◽

Yong-Rui Zou

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Stromal Cells ◽

Hematopoietic Cells ◽

Regulatory Factor ◽

Cell Compartment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Stem Cell Compartment

The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is thought that specialized niches in which HSCs reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. We report that CXCL12 produced by bone marrow (BM) stromal cells is not only the major chemoattractant for HSCs but also a regulatory factor that controls the quiescence of primitive hematopoietic cells. Addition of CXCL12 into the culture inhibits entry of primitive hematopoietic cells into the cell cycle, and inactivation of its receptor CXCR4 in HSCs causes excessive HSC proliferation. Notably, the hyperproliferative Cxcr4−/− HSCs are able to maintain a stable stem cell compartment and sustain hematopoiesis. Thus, we propose that CXCR4/CXCL12 signaling is essential to confine HSCs in the proper niche and controls their proliferation.

Download Full-text

C/EBPβ Isoforms Regulate Proliferation and Differentiation of Regenerating Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood-2019-128026 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3713-3713

Author(s):

Atsushi Sato ◽

Asumi Yokota ◽

Yoshihiro Hayashi ◽

Naoka Kamio ◽

Satoshi Sagai ◽

...

Keyword(s):

Cell Cycle ◽

Molecular Mechanisms ◽

Expression Patterns ◽

Flow Cytometric Analysis ◽

Myeloid Cells ◽

Stress Conditions ◽

Hematopoietic Stem ◽

N Terminus ◽

Factor C ◽

The Impact

Under stress or regenerative conditions, HSCs rapidly enter into cell cycle and are reprogrammed toward myeloid-biased hematopoiesis to meet the increasing demand of myeloid cells. We have previously shown that the transcription factor C/EBPβ plays critical roles at the level of HSPCs under stress conditions (Nat Immunol 2006, J Immunol 2012, Leukemia 2013 and Blood Adv 2019). However, underlying molecular mechanisms of action remain largely unknown. In this study, we have investigated the detailed function of C/EBPβ in regulation of HSPCs. We first evaluated the impact of C/EBPβ on the cell cycle status of LT-HSCs. To exclude the cell-extrinsic contribution of C/EBPβ, CD45.2+ BM cells from WT or Cebpb-/- mice were transplanted into lethally irradiated CD45.1+ WT mice, and these "BM-replaced" recipients were subjected to the following experiments. At steady state, the cell cycle statuses and the numbers of HSPCs did not significantly differ between the recipients of WT cells and those of Cebpb-/- cells. Immediately after 5-FU treatment, WT LT-HSCs entered the cell cycle, as revealed by the decreased percentage of cells in G0 phase and the increased percentage of cells in S/G2M phase. All these parameters of cell cycle acceleration were observed prior to the nadir of LT-HSCs induced by 5-FU and were significantly attenuated in Cebpb-/- LT-HSCs. Next, we assessed the numbers of LT-HSCs, KSL cells, and KL cells after 5-FU treatment. Following the nadir, the recovery of LT-HSCs preceded that of KSL and KL cells, suggesting the differentiation of LT-HSCs to KSL and KL cells. In the recipients of Cebpb-/- cells, the recovery of KSL and KL cells was delayed significantly. Collectively, cell cycle acceleration and subsequent differentiation of LT-HSCs under stress conditions were impaired in the absence of Cebpb. The Cebpb is a single exon gene, and three isoforms, namely, LAP*, LAP and LIP which lacks N-terminus, are translated from its unique mRNA. Due to their structural difference, they should have distinct functions. Here, we focused on expression and functions of these isoforms in regenerating HSPCs. To monitor expression of these isoforms in small numbers of HSCs, we devised a novel intracellular double staining method for flow cytometric analysis using two distinct anti-C/EBPβ antibodies. An antibody against the C-terminus of C/EBPβ recognized all three isoforms, while an antibody against the N-terminus of C/EBPβ only recognized LAP* and LAP. Thus, simultaneous staining with both antibodies should enable us to distinguish cells that dominantly expressed LIP (C-term+ N-term-) from those that expressed all three isoforms (C-term+ N-term+). Using this method, we monitored the expression patterns of these isoforms in LT-HSCs after 5-FU treatment. LT-HSCs initially became C-term single positive in response to 5-FU and subsequently changed to C- and N-term double positive, suggesting that LIP was upregulated prior to LAP/LAP* under stress conditions. These results suggest that phase-specific upregulation of LIP and LAP/LAP* is strongly associated with phase-specific functions of C/EBPβ in cell cycle activation and differentiation, respectively. Indeed, when EML cells, a mouse HSC line, were retrovirally transduced with LIP, the transduced cells were more proliferative and actively cycling than those transduced with the control vector, whereas proliferation and cell cycle were markedly suppressed in LAP*- and LAP-expressing EML cells. LIP-expressing cells remained undifferentiated, while LAP*- and LAP-expressing cells rapidly differentiated into CD11b+ myeloid cells and eventually stopped proliferating. In summary, our findings clearly suggest that sequential upregulation of C/EBPβ isoforms is critical for the regulation of HSCs under stress conditions. LIP amplifies the "reservoir" of HSPCs by accelerating the proliferation of HSCs during the early phase of regeneration, while LAP*/LAP induce their myeloid differentiation at a later phase. These findings should facilitate our understanding of the pathophysiology of infection, inflammation, and regenerating hematopoiesis in response to myeloablative chemotherapies or hematopoietic stem cell transplantation, all of which increase the hematopoietic demands. Disclosures Hirai: Kyowa Kirin: Research Funding.

Download Full-text

Kruppel-Like Factor 4 Upregulates p21 and Downregulates Proliferation of Human and Mouse HSPCs, but Is Not Essential for Mouse HSPC Repopulation.

Blood ◽

10.1182/blood.v108.11.1317.1317 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1317-1317

Author(s):

Jonathan K. Alder ◽

Robert W. Georgantas ◽

Richard L. Hildreth ◽

Xiaobing Yu ◽

Curt I. Civin

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Fetal Liver ◽

Family Members ◽

Hematopoietic Cells ◽

Normal Numbers ◽

Hematopoietic Stem ◽

Cycle Arrest ◽

Klf4 Expression ◽

And Function

Abstract Several Kruppel-like factor family members, including KLF1, KLF2, KLF3, and KLF6 have pivotal roles in hematopoiesis. Experiments in zebrafish have suggested that KLF4 may play a similar role. Here we found that enforced expression of KLF4 in hematopoietic cells induced cell cycle arrest without triggering apoptosis. Based on the high levels of expression of KLF4 in mouse and human hematopoietic stem-progenitor cells (HSPCs), we hypothesized and demonstrated that KLF4 regulates proliferation of these cells through regulation of p21cip1/waf1 (p21). Nevertheless, KLF4−/− mouse fetal liver cells had normal numbers of all mature lineages and provided radioprotection, similar to wild type (wt) controls. Furthermore, in long-term competitive repopulation assays, KLF4−/− mouse HSPCs demonstrated hematopoietic potency equivalent to wt. We found that KLF2 is expressed at higher levels than KLF4 in mouse HSPCs and is a more potent activator of p21, suggesting that KLF2 (and/or other KLF family members) may play a compensatory role in KLF4−/− HSPCs. Thus, although is not essential for their normal development and function, KLF4 expression is sufficient to induce p21-mediated cell cycle arrest in hematopoietic cells.

Download Full-text

Limited Regeneration but Functional Preservation of Hematopoietic Stem Cells in the Mice Developing Leukemia via Constitutive Expression of Notch1.

Blood ◽

10.1182/blood.v110.11.204.204 ◽

2007 ◽

Vol 110 (11) ◽

pp. 204-204 ◽

Cited By ~ 1

Author(s):

Xiaoxia Hu ◽

Hongmei Shen ◽

Hui Yu ◽

Feng Xu ◽

Jianmin Wang ◽

...

Keyword(s):

Bone Marrow ◽

Molecular Mechanisms ◽

Lymphoblastic Leukemia ◽

Hematopoietic Cells ◽

Constitutive Expression ◽

Hematopoietic Progenitor Cell ◽

Control Group ◽

Extrinsic Factors ◽

Hematopoietic Stem ◽

The Impact

Abstract Leukemia development is a complex process involving both intrinsic and extrinsic factors. While many environmental factors have been studied, the impact of leukemic environment on normal hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) has not been definitively investigated. In this study, we have formally addressed this important issue by examining the potential functional alterations of HSC and HPC in the mice bearing Notch1-induced T acute lymphoblastic leukemia (T-ALL). The MSCV retrovirus vector containing cDNA encoding oncogenic intracellular domain of Notch1 (ICN1) pseudotyped with VSV-G was used to infect Lin−Sca-1+ cells in order to induce leukemic development. Normal hematopoietic cells from the B6.SJL strain (CD45.1+) were co-transplanted with Notch1 transduced Lin−Sca-1+ cells (CD45.2+) into lethally irradiated recipients. In this robust leukemia model with 100% penetrance, the normal hematopoietic cell compartment marked by CD45.1 in the leukemic marrow was sorted for phenotypic analyses and functional assays at different time points. Same numbers of the normal hematopoietic cells without Notch1-transduced cells were transplanted into the irradiated recipients as controls. As expected, progressive hematopoietic suppression was observed at both HSC and HPC levels in the leukemic mice. The frequency of HSC enriched population (Lin−c-Kit+Sca-1+, LKS) in the leukemic group was 7 times lower than that in the control at the 4th week of leukemogensis. When normalized to the bone marrow cellularity, the absolute yield of each population was 246 times lower in the leukemic group than that in the control group. These data were highly consistent with significantly lower yields of colony forming unit (CFU) and cobblestone area forming cell (CAFC). To measure the long-term engraftment of HSCs from leukemic environment, we performed the competitive bone marrow transplantation (cBMT), in which equal numbers of CD45.1+ cells isolated from leukemic or control mice and competitor cells (CD45.1/.2) at the 2nd week of leukemogenesis were co-transplanted into lethally irradiated C57BL/6J recipients. Unexpectedly, the multilineage engraftment of the hematopoietic cells isolated from the leukemic mice was 3 times more than that of the control group. Moreover, HSCs from the leukemic environment remained functional in serial transplant recipients. Finally, to explore the underlying molecular mechanisms for the enhanced function of normal HSC in the cBMT model, we examined a number of cell cycle and self-renewal regulators in HSC and HPC from leukemic marrow and control group at the time of harvest prior to transplantation by qRT-PCR. There was a significant decrease in p18 expression when compared with the control, whereas p21 expression was significantly increased. Notch1, Gfi1 and c-myc signalings were also elevated in the HSCs from leukemic environment. In summary, our current work provides the first definitive evidence for the reversible inhibition of normal HSC growth by the leukemic environment, thereby having important implications for HSC transplantation as well as leukemogenesis.

Download Full-text

Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.2504.2504 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2504-2504

Author(s):

Russell Garrett ◽

Gerd Bungartz ◽

Alevtina Domashenko ◽

Stephen G. Emerson

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Poly I:C ◽

Hematopoietic Stem ◽

Marrow Cells ◽

Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Investigating The Role Of ARAP3 In The Regulation Of Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v122.21.2409.2409 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2409-2409

Author(s):

Yiwen Song ◽

Sonja Vermeren ◽

Wei Tong

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Progenitor Cells ◽

Hematopoietic Cells ◽

Conditional Knockout ◽

Ph Domain ◽

Hematopoietic Stem ◽

Normal Peripheral Blood ◽

Stem And Progenitor Cells

Abstract ARAP3 is a member of the dual Arf-and-Rho GTPase-activating proteins (GAP) family, functioning specifically to inactivate its substrates Arf6 and RhoA GTPases. ARAP3 is translocated to the plasma membrane after PIP3 binding to the first two of its five PH domains, facilitating its GAP activity in a PI3K-mediated manner. Rho family GTPases are found to play critical roles in many aspects of hematopoietic stem and progenitor cells (HSPCs), such as engraftment and migration, while a role for Arf family GTPases in hematopoiesis is less defined. Previous studies found that either exogenous ARAP3 expression in epithelial cells or RNAi-mediated ARAP3 depletion in endothelial cells disrupts F-actin or lamellipodia formation, respectively, resulting in a cell rounding phenotype and failure to spread. This implies that ARAP3 control of Arf6 and RhoA is tightly regulated, and maintaining precise regulation of ARAP3 levels is crucial to actin organization in the cell. Although ARAP3 was first identified in porcine leukocytes, its function in the hematopoietic system is incompletely understood. Germline deletion of Arap3 results in embryonic lethality due to angiogenic defects. Since endothelial cells are important for the emergence of HSCs during embryonic development, early lethality precludes further studying the role of ARAP3 in definitive hematopoiesis. Therefore, we generated several transgenic mouse models to manipulate ARAP3 in the hematopoietic compartment: (1) Arap3fl/fl;Vav-Cretg conditional knockout mice (CKO) deletes ARAP3 specifically in hematopoietic cells, (2) Arap3fl/fl;VE-Cadherin -Cretg CKO mice selectively deletes ARAP3 in embryonic endothelial cells and thereby hematopoietic cells, and (3) Arap3R302,3A/R302,3A germline knock-in mice (KI/KI) mutates the first PH domain to ablate PI3K-mediated ARAP3 activity in all tissues. We found an almost 100% and 90% excision efficiency in the Vav-Cretg- and VEC-Cretg- mediated deletion of ARAP3 in the bone marrow (BM), respectively. However, the CKO mice appear normal in steady-state hematopoiesis, showing normal peripheral blood (PB) counts and normal distributions of all lineages in the BM. Interestingly, we observed an expansion of the Lin-Scal+cKit+ (LSK) stem and progenitor compartment in the CKO mice. This is due to an increase in the multi-potent progenitor (MPP) fraction, but not the long-term or short-term HSC (LT- or ST-HSC) fractions. Although loss of ARAP3 does not alter the frequency of phenotypically-characterized HSCs, we performed competitive BM transplantation (BMT) studies to investigate the functional impact of ARAP3 deficiency. 500 LSK cells from Arap3 CKO (Arap3fl/fl;Vav-Cretg and Arap3fl/fl;VEC-Cretg) or Arap3fl/fl control littermate donors were transplanted with competitor BM cells into irradiated recipients. We observed similar donor-derived reconstitution and lineage repopulation in the mice transplanted with Arap3fl/fl and Arap3 CKO HSCs. Moreover, Arap3 CKO HSCs show normal reconstitution in secondary transplants. Arap3 KI/KI mice are also grossly normal and exhibit an expanded MPP compartment. Importantly, Arap3KI/KI LSKs show impaired reconstitution compared to controls in the competitive BMT assays. Upon secondary and tertiary transplantation, reconstitution in both PB and BM diminished in the Arap3KI/KI groups, in contrast to sustained reconstitution in the control group. Additionally, we observed a marked skewing towards the myeloid lineage in Arap3KI/KI transplanted secondary and tertiary recipients. These data suggest a defect in HSC function in Arap3KI/KI mice. Myeloid-skewed reconstitution also points to the possibility of selection for “myeloid-primed” HSCs and against “balanced” HSCs, as HSCs exhaust during aging or upon serial transplantation. Taken together, our data suggest that ARAP3 plays a non-cell-autonomous role in HSCs by regulating HSC niche cells. Alternatively, the ARAP3 PH domain mutant that is incapable of locating to the plasma membrane in response to PI3K may exert a novel dominant negative function in HSCs. We are investigating mechanistically how ARAP3 controls HSC engraftment and self-renewal to elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in HSCs. In summary, our studies identify a previously unappreciated role of ARAP3 as a regulator of hematopoiesis and hematopoietic stem and progenitor cell function. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Importance of Matrix-dimensionality in Regulating the Bone Marrow Hematopoietic Cells Pool

10.1101/720359 ◽

2019 ◽

Author(s):

P. Zhang ◽

C. Zhang ◽

J. Han ◽

J. Gao ◽

W. Zhao ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Ex Vivo ◽

Hematopoietic Cells ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Factors Affecting ◽

Lymphoid Lineage ◽

3D Matrix

AbstractIn bone marrow, hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor cells (HPCs) cooperate to differentiate and replenish blood and immune cells. It has long been recognized bone marrow niche parameters interact with hematopoietic stem and progenitor cells (HSPCs) and additional work is required to study niche physical signals controlling cell behavior. Here we presented that important biophysical signals, stiffness and dimensionality, regulating expansion of bone marrow HSPCs. Mice bone marrow derived progenitor cells were cultured in collagen I hydrogel in vitro. We found stiffer 3D matrix promoted the expansion of lineage negative (Lin−) progenitor cells and Lin−Sca-1+c-kit+ (LSK) HSPCs compared to softer hydrogel. Compared with cells cultured in 2D environment, 3D embedded construct had significant advantage on HSPCs expansion, accompanied by increases on myeloid and lymphoid lineage fractions. Bright changes on gene expression were subsequently discovered. According to these data, we concluded that culture matrix dimensionality is an important factor to regulate the behavior of subpopulations in hematopoietic cell pool, which should be considered in attempts to illuminate HSCs fate decision in vitro.Statement of SignificanceWe would like to submit the enclosed manuscript entitled "Importance of Niche-dimensionality in Regulating the Bone Marrow Hematopoietic Cells Pool", which we wish to be considered for publication in Biophysical Journal. Studies about the interaction between HSCs and factors provided by their microenvironment is largely focus on pure perspective of biology. But biophysical factors affecting HSC fate and behavior needs to be further explore. Herein we found ex vivo culture dimensionality affected HSPC expansion. Cell surface marker detection and mRNA expression analysis predicted the changes on myeloid and lymphoid lineage fractions. We hope niche physical signals which we identified will be considered to design HSC biomimetic niches in clinical applications. And we believe that our study will make it interesting to general readers. We deeply appreciate your consideration of our manuscript, and we look forward to receiving comments from the reviewers.

Download Full-text