R4 Rgs Subfamily Proteins Negatively Regulates SDF-1/CXCR4 Signaling in CD34+ Hematopoietic Stem and Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5048-5048
Author(s):  
Kam Tong Leung ◽  
Yorky Tsin Sik Wong ◽  
Karen Li ◽  
Kathy Yuen Yee Chan ◽  
Xiao-Bing Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Rgs Proteins ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract RGS family proteins are known to negatively regulate G-protein-coupled receptor signaling through their GTPase-accelerating activity. In several types of hematopoietic cells (e.g., B lymphocytes and megakaryocytes), responses to stromal cell-derived factor-1 (SDF-1) are subjected to regulation by R4 subfamily RGS proteins. However, their expression patterns and functional roles in hematopoietic stem and progenitor cells (HSC) are poorly characterized. Here, we showed that human CD34+ HSC derived from cord blood (CB, n = 10) expressed 7 out of 10 R4 RGS proteins at mRNA level (RGS1-3, 5, 13, 16 and 18), whereas expressions of RGS4, 8 and 21 were undetectable. Exposure of CB CD34+ cells to SDF-1 significantly increased RGS1, 2, 13 and 16 expressions and decreased RGS3 and 18 expressions (P ≤ 0.0402, n = 5). Expressions of RGS1, 13 and 16 were significantly higher in bone marrow (BM, n = 10) CD34+ cells when compared to mobilized peripheral blood (MPB, n = 5) CD34+ cells (P ≤ 0.0160), while RGS3 and 18 expressions were lower in BM CD34+ cells (P ≤ 0.0471), suggesting a SDF-1- and niche-dependent regulation of RGS expressions. To investigate the potential involvement of RGS proteins in SDF-1-mediated homing-related functions, we introduced RGS overexpression constructs into CB CD34+ cells by lentiviral transduction. With >80% transduction efficiency, we showed that overexpression of RGS1, 13 and 16 but not RGS2 significantly inhibited migration of CD34+ cells to a SDF-1 gradient (P ≤ 0.0391, n = 4-5). Similarly, RGS1, 13 and 16 overexpression suppressed SDF-1-induced Akt phosphorylation (n = 2), but none of them affected SDF-1-mediated actin polymerization (n = 3). In the NOD/SCID mouse xenotransplantation model, preliminary results showed that bone marrow homing was impaired in RGS1- (16.3% reduction), RGS13- (12.7% reduction) or RGS16-overexpressing CD34+ cells (33.7% reduction). Taken together, we provided the first evidence that expressions of R4 RGS proteins are regulated by the SDF-1/CXCR4 axis in human CD34+ HSC. We also presented evidence that specific R4 RGS proteins (RGS1, 13 and 16) negatively regulate in vitro SDF-1-mediated responses and in vivo homing of CD34+ cells, suggesting that RGS proteins may serve as a feedback mechanism to regulate SDF-1/CXCR4 signaling. Strategies to inhibit RGS signaling could thus be a potential method for enhancing efficiency of HSC homing and long-term engraftment, which is particularly important in the setting of CB transplantation. Disclosures No relevant conflicts of interest to declare.

Neuropeptides Orexin A and B Are Funktionally Aktive in CD34+ Hematopoietic Stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4593-4593
Author(s):  
Ron-Patrick Cadeddu ◽  
Akos G. Czibere ◽  
Sebastian Büst ◽  
Johannes C Fischer ◽  
Ulrich Steidl ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Calcium Influx ◽  
Receptor Stimulation ◽  
Orexin A ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Orexin Receptors ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Abstract 4593 Orexin receptors are involved in the regulation of sleep-wake-rhythm, food intake and energy homeostasis and it was still recently believed that their expression is restricted to the nervous system. But, during the last years orexin receptors have been detected in an increasing number of peripheral tissues. We have earlier found orexin receptor 1 and 2 expression on human CD34+ hematopoietic stem and progenitor cells. Still, the sources of their physiological ligands, the peptides orexin A and B, seemed so far to be restricted to the central nerve system. Ca2+-dependent signaling and activation of mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (ERK1/2) pathways are considered as main downstream signaling pathways of the orexin receptors. In this study, we investigated the signaling and functional role of orexin receptors in CD34+ hematopoietic stem and progenitor cells. Using confocal fluorescence microscopy and flow cytometry we found that stimulation of purified CD34+ cells with orexin A and B led to an increase of the intracellular calcium concentration due to both calcium influx and calcium release from intracellular stores. Of interest, incubation with orexin reduces the SDF-1β-induced calcium influx. Furthermore orexin receptor stimulation led to a decrease of the intracellular cAMP concentration. Following orexin receptor stimulation with orexin A and B, we observed an initial increase of ERK1/2 phosphorylation up to 30 minutes upon incubation with orexin followed by a decrease at several time points up to 8 hours in comparison to the unstimulated control. To investigate a potential impact on the functional properties of human CD34+ cells we performed proliferation and apoptosis assays, migration and adhesion assays as well as colony forming and long-term culture assays. Remarkably, stimulation with orexin A and B led to a significant higher proportion of early pluripotent hematopoietic progenitor (CFU-GEMM) colonies and a significant reduction of erythroid precursors. A more immature phenotype of orexin-stimulated CD34+ cells is also reflected by array-based gene expression profiling. Long-term culture assays revealed a significant higher frequency of LTC-IC indicating also a more immature phenotype of orexin-stimulated cells. In line, orexin receptor stimulation led to a significant increase of the proportion of Lin-, CD34+, CD38- HSC in the G0-phase of the cell cycle. Furthermore, stimulation with orexin A and B increased the number of apoptotic cells in the Lin-, CD34+, CD38- HSC fraction and the total hematopoietic stem and progenitor population determined by flowcytometric analysis of intracellular cleaved caspase 3 content. The adhesive capacity of CD34+ cells to fibronectin and collagen coated dishes and the migratory capacity was significantly decreased upon orexin receptor stimulation. Concurrent incubation with the selective Gi-protein inhibitor pertussis toxin abrogated these effects. Given the functional impact of the orexin system on CD34+ cells, we asked if orexins are secreted locally in the bone marrow or autocrine by CD34+ cells or if they are humorally transported to the bone marrow cavity. Using FACS analysis, immunfluorescent staining and western blotting we could detect prepro-Orexin in CD34+ cells and using ELISA orexin was found in the serum obtained by bone marrow biopsies and peripheral blood. Taken together, the phenotype of orexin-stimulated hematopoietic stem and progenitor cells suggest a mobilizing effect of the orexin receptor stimulation as well as an increased repopulation capacity which might be of relevance in clinical stem cell mobilization and transplantation and is currently verified in murine models. Disclosures: No relevant conflicts of interest to declare.

scholarly journals R4 Rgs Subfamily Proteins Suppress Engraftment and Regulate SDF-1/CXCR4 Signaling in Human CD34+ Hematopoietic Stem/Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1927-1927
Author(s):  
Kathy Chan ◽  
Tsin Sik Wong ◽  
Karen Li ◽  
Xiao-Bing Zhang ◽  
Ronald Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Actin Polymerization ◽  
Conflicts Of Interest ◽  
Calcium Flux ◽  
Rgs Proteins ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Stat3 Phosphorylation ◽  
Cd34 Cells

Members of the Regulators of G-protein Signaling (RGS) are GTPase-accelerating proteins and have been implicated in SDF-1-directed trafficking of mature hematopoietic cells. However, their roles in hematopoietic stem and progenitor cells (HSPC) remain largely unknown. In this study, we investigated the expression, functions and mechanism of R4 RGS subfamily members on migration and engraftment of human HSPC. Our results demonstrated that cord blood (CB), bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells expressed specific RGS mRNAs, of which RGS1, RGS2, RGS13 and RGS16 were significantly upregulated by SDF-1 (1.6-1.9 fold, n=5, P<0.05). In the presence of AMD3100, a CXCR4 inhibitor, the stimulating effects of SDF-1 on RGS expression were completely abolished (n=6). SDF-1-directed functions (chemotaxis, trans-matrigel migration and calcium flux) and signaling (phosphorylation of Akt, ERK and Stat3) were significantly inhibited in RGS1, RGS13 and RGS16-overexpressing CD34+ cells (n=4-6, P<0.05) but not in RGS2-overexpressing cells, whereas actin polymerization, adhesion and colony formation were unaffected by these RGS members. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1, RGS13 or RGS16 in CD34+ cells did not impact their short-term homing but substantially compromised their long-term engraftment efficiency in bone marrow and spleens of recipient mice by 91.4%, 83.7% and 71.2%, respectively (n= 8-9; P<0.05). Genome-wide expression microarray and qPCR validation identified 32 common differentially expressed genes (1 upregulated and 31 downregulated) in RGS1, RGS13 or RGS16-overexpressing CD34+ cells. Network analysis revealed the potential mechanisms of RGS1, RGS13 and RGS16 downstream of SDF1/CXCR4 and Gαi protein, leading to compromised Akt, ERK and Stat3 phosphorylation and negative regulation of stem cell functions (CCNA1, SPP1, LPAR5, IL1RL1, HPSE), complement activation (C3AR1, C5AR2, C5AR1), proteolysis (TIMP3, MMP14) and cell migration (THBS1, F2RL2, PROS1, CCL1). Our results highlight the unprecedented functions of R4 RGS proteins in HSPC migration and engraftment, and provide the foundation of future design of RGS-targeting strategies to enhance the efficiency of clinical HSPC transplantation. Disclosures No relevant conflicts of interest to declare.

Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow–derived and circulating human CD34+ hematopoietic stem cells

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2037-2044 ◽  
Author(s):  
Ulrich Steidl ◽  
Ralf Kronenwett ◽  
Ulrich-Peter Rohr ◽  
Roland Fenk ◽  
Slawomir Kliszewski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Dna Synthesis ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract CD34+ hematopoietic stem cells are used clinically to support cytotoxic therapy, and recent studies raised hope that they could even serve as a cellular source for nonhematopoietic tissue engineering. Here, we examined in 18 volunteers the gene expressions of 1185 genes in highly enriched bone marrow CD34+(BM-CD34+) or granulocyte–colony-stimulating factor–mobilized peripheral blood CD34+(PB-CD34+) cells by means of cDNA array technology to identify molecular causes underlying the functional differences between circulating and sedentary hematopoietic stem and progenitor cells. In total, 65 genes were significantly differentially expressed. Greater cell cycle and DNA synthesis activity of BM-CD34+ than PB-CD34+ cells were reflected by the 2- to 5-fold higher expression of 9 genes involved in cell cycle progression, 11 genes regulating DNA synthesis, and cell cycle–initiating transcription factor E2F-1. Conversely, 9 other transcription factors, including the differentiation blocking GATA2 and N-myc, were expressed 2 to 3 times higher in PB-CD34+ cells than in BM-CD34+cells. Expression of 5 apoptosis driving genes was also 2 to 3 times greater in PB-CD34+ cells, reflecting a higher apoptotic activity. In summary, our study provides a gene expression profile of primary human CD34+ hematopoietic cells of the blood and marrow. Our data molecularly confirm and explain the finding that CD34+ cells residing in the bone marrow cycle more rapidly, whereas circulating CD34+ cells consist of a higher number of quiescent stem and progenitor cells. Moreover, our data provide novel molecular insight into stem cell physiology.

Multicolor Flow Cytometry Allows Quantitative Analysis of Signal Transduction in Murine and Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5393-5393
Author(s):  
Tamara Riedt ◽  
Claudia Lengerke ◽  
Lothar Kanz ◽  
Viktor Janzen
Keyword(s):  
Signal Transduction ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Intracellular Signaling ◽  
Hematopoietic Stem ◽  
Specific Antibodies ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract The regulation of cell cycle activity, differentiation and self-renewal of stem cells are dependent on accurate processing of intrinsic and extrinsic signals. Traditionally, signaling pathway activation has been detected by immunobloting using phospho-specific antibodies. However, detection of signal transduction in rare cells within heterogeneous populations, such as hematopoietic stem and progenitor cells (HSC) has been difficult to achieve. In a recently reported approach to visualize signaling in selected single c-Kit+ Sca-1+ Lin− (KSL) bone marrow cells, cells were sorted onto glas slides by flow cytometry and signaling was detected by confocal fluorescence microscopy, a very time consuming method that thus restricts the number of cells that can be analysed simultaneously. Moreover it permits only qualitative, but not quantitative signaling evaluation (Yamazaki et al., EMBO J. 2006). Here, we report a new protocol allowing quantitative measurement of signaling activity in large numbers of defined murine and human hematopoietic cells. The cells are stained with established surface markers and then phospho-specific antibodies are used to detect the levels of active intracellular signaling molecules. Signals are quantified by flow cytometry fluorescence measurement. Importantly, the protocol developed in our laboratory enables preservation of surface marker staining identifying the cells of interest inspite the fixation and permeabilization procedures necessary for intracellular signaling detection. This applies also for antigens previously reported to be particularly vulnerable to standard fixation and permeabilization approaches (e.g. the murine stem cell markers c-Kit and Sca1). Thus, our protocol provides an easy and reliable method for quantifying the activation degree of several intracellular signaling pathways on single cell level in defined hematopoietic (stem) cells within the heterogeous bone marrow (BM) compartment. Using cytokines known to exert a biological effect on HSCs, we have examined the susceptibility of KSL murine BM cells and human BM CD34+ cells to cytokine-induced signaling. We have performed extensive dosage titration and time course analysis for multiple cytokines (SCF, TPO, Flt-3, IL-3, IL-6, Ang-1, SDF-1α, TGF-β, and BMP-4) and signaling pathways (ERK, Akt, p38MAPK, Jak-Stat, TGF-β/BMP-Smad) in murine KSL BM cells. The activation intensity and the duration of signal activity as measured by the expression of corresponding phosphorylated proteins were cytokine specific. The obtained results can be used as a platform to explore signaling alterations in distinct compartments of the hematopoietic system, and may provide mechanistical insights for observed bone marrow defects (e.g impaired ERK signaling pathway has been detected as a possible cause of hematopoietic defects in Caspase-3 mutant murine HSCs, Janzen et al, Cell Stem Cell 2008). Furthermore, we could show that the technique is also applicable to human BM cells and that the human hematopoietic stem cell marker CD34 is also preserved by our fixation and permeabilization protocol. Preliminary results suggest that cytokines induce similar signaling activation in human CD34+BM cells collected from healthy donors. As observed in mouse KSL BM cells, stimulation of human CD34+cells with human stem cell factor (hSCF) induced activation of the ERK but not the Akt pathway. Ongoing experiments analyse the stimulatory effects of other cytokines such as thrombopoietin (TPO) and fms-related tyrosine kinase 3 (Flt-3) and their corresponding pathways. Moreover, comparative studies are underway analyzing cross-reactivity between mouse and human cytokines, aiming to provide insights into cytokine-induced biases in commonly used xenotransplantation models.

Sociology of Normal Stem and Progenitor Cells in CML Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1234-1234
Author(s):  
Robert S Welner ◽  
Giovanni Amabile ◽  
Deepak Bararia ◽  
Philipp B. Staber ◽  
Akos G. Czibere ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitor Cells ◽  
Quiescent State ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.

Blood Cell Replenishment and Bone Marrow Stem Cell Pool Renewal Are Regulated By Different Circadian Peaks Via Norepinephrine and TNFα/S1P Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 217-217
Author(s):  
Karin Golan ◽  
Aya Ludin ◽  
Tomer Itkin ◽  
Shiri Cohen-Gur ◽  
Orit Kollet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Daily Basis ◽  
Hematopoietic Stem ◽  
Activated Macrophage ◽  
Sphingosine 1 Phosphate ◽  
Stem And Progenitor Cells ◽  
Primitive State

Abstract Hematopoietic stem and progenitor cells (HSPC) are mostly retained in a quiescent, non-motile mode in the bone marrow (BM), shifting to a cycling, differentiating and migratory state on demand. How HSC replenish the blood with new mature leukocytes on a daily basis while maintaining a constant pool of primitive cells in the BM throughout life is not clear. Recently, we reported that the bioactive lipid Sphingosine 1-Phosphate (S1P) regulates HSPC mobilization via ROS signaling and CXCL12 secretion (Golan et al, Blood 2012). We hypothesize that S1P influences the daily circadian egress of HSPC and their proliferation. We report that S1P levels in the blood are increased following initiation of light at the peak of HSPC egress and are reduced towards the termination of light when circulating HSPC reach a nadir. Interestingly, mice with constitutively low S1P plasma levels due to lack of one of the enzymes that generates S1P (Sphingosine kinase 1), do not exhibit fluctuations of HSPC levels in the blood between day and night. We report that HSPC numbers in the BM are also regulated in a circadian manner. Unexpectedly, we found two different daily peaks: one in the morning, following initiation of light, which is accompanied by increased HSPC egress and the other at night after darkness, which is associated with reduced HSPC egress. In both peaks HSPC begin to cycle and differentiate via up-regulation of reactive oxygen species (ROS) however, the night peak had lower ROS levels. Concomitant with the peak of primitive stem and progenitor cells, we also observed (to a larger extent in the night peak), expansion of a rare activated macrophage/monocyte αSMA/Mac-1 population. This population maintains HSPC in a primitive state via COX2/PGE2 signaling that reduces ROS levels and increases BM stromal CXCL12 surface expression (Ludin et al, Nat. Imm. 2012). We identified two different BM peaks in HSPC levels that are regulated by the nervous system via circadian changes in ROS levels. Augmented ROS levels induce HSPC proliferation, differentiation and motility, which take place in the morning peak; however, they need to be restored to normal levels in order to prevent BM HSPC exhaustion. In the night peak, HSPC proliferate with less differentiation and egress, and activated macrophage/monocyte αSMA/Mac-1 cells are increased to restore ROS levels and activate CXCL12/CXCR4 interactions to maintain a HSPC primitive phenotype. Additionally, S1P also regulates HSPC proliferation, thus mice with low S1P levels share reduced hematopoietic progenitor cells in the BM. Interestingly S1P is required more for the HSPC night peak since in mice with low S1P levels, HSPC peak normally during day time but not at darkness. We suggest that the first peak is initiated via elevation of ROS by norepinephrine that is augmented in the BM following light-driven cues from the brain (Mendez-Ferrer at al, Nature 2008). The morning elevated ROS signal induces a decrease in BM CXCL12 levels and up-regulated MMP-9 activity, leading to HSC proliferation, as well as their detachment from their BM microenvironment, resulting in enhanced egress. Importantly, ROS inhibition by N-acetyl cysteine (NAC) reduced the morning HSPC peak. Since norepinephrine is an inhibitor of TNFα, upon light termination norepinephrine levels decrease and TNFα levels are up-regulated. TNFα induces activation of S1P in the BM, leading to the darkness peak in HSPC levels. S1P was previously shown also to induce PGE2 signaling, essential for HSPC maintenance by the rare activated αSMA/Mac-1 population. Indeed, in mice with low S1P levels, we could not detect a peak in COX2 levels in these BM cells during darkness. We conclude that S1P not only induces HSPC proliferation via augmentation of ROS levels, but also activates PGE2/COX2 signaling in αSMA/Mac-1 population to restore ROS levels and prevent HSPC differentiation and egress during the night peak. We hypothesize that the morning HSPC peak, involves proliferation, differentiation and egress, to allow HSPC to replenish the blood circulation with new cells. In contrast, the second HSPC night peak induces proliferation with reduced differentiation and egress, allowing the renewal of the BM HSPC pool. In summary, we identified two daily circadian peaks in HSPC BM levels that are regulated via light/dark cues and concomitantly allow HSPC replenishment of the blood and immune system, as well as maintenance of the HSPC constant pool in the BM. Disclosures: No relevant conflicts of interest to declare.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

scholarly journals The Neurotransmitter Receptor Gabbr1 Regulates Proliferation and Function of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-33
Author(s):  
Adedamola Elujoba-Bridenstine ◽  
Lijian Shao ◽  
Katherine Zink ◽  
Laura Sanchez ◽  
Kostandin V. Pajcini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Receptor Expression ◽  
Bone Marrow Niche ◽  
Transplant Recipients ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Null Mutants

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells which differentiate to maintain and replenish blood lineages throughout life. Due to these characteristics, HSPC transplants represent a cure for patients with a variety of hematological disorders. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. The nervous system has been shown to indirectly influence hematopoiesis by innervating the niche; however, we present a direct route of HSPC regulation via expression of neurotransmitter receptors on HSPC surface. We have identified Gamma Aminobutyric acid (GABA) receptor B subunit 1 (Gabbr1), a hitherto unknown hematopoietic player, as a regulator of HSPC function. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation remains unknown. Based on published RNA-seq data (Nestorowa et al., Blood 2016), we discovered that Gabbr1 is expressed on a subset of HSPCs. We confirmed this expression using RT-qPCR to assay hematopoietic populations in the bone marrow (BM). Surface receptor expression analysis showed that Gabbr1 protein is expressed on a subset of BM HSPCs. To detect GABA, the ligand for Gabbr1 in the BM microenvironment, we utilized imaging mass spectrometry (IMS). We detected regionally specific GABA signal in the endosteal region of the BM. We further identified B cells as a cellular source of GABA in the BM. To understand the role of Gabbr1 in hematopoiesis, we generated CRISPR-Cas9 Gabbr1 null mutants on a C57/BL6 background suitable for hematopoietic studies and studied their hematopoietic phenotype. We discovered a decrease in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but the long-term hematopoietic stem cells (LT-HSCs) remain unaffected. Further analysis of peripheral blood of Gabbr1 null mutants showed decreased white blood cells due to reduced B220+ cells. This differentiation defect was confirmed in an in vitro differentiation assay where Gabbr1 null HSPCs displayed an impaired ability to produce B cells. We show that Gabbr1 null HSCs show diminished reconstitution ability when transplanted in a competitive setting. Reduced Gabbr1 null HSC reconstitution persisted in secondary transplant recipients indicating a cell autonomous role for Gabbr1 in regulating reconstitution of HSCs in transplant recipients. Our results show a crucial role for Gabbr1 in HSPC regulation and may translate to human health as a rare human SNP within the GABBR1 locus that correlates with altered leukocyte counts has been reported (Astle et al., Cell 2016). Our studies indicate an important role for Gabbr1 in HSPC reconstitution and differentiation into B cell lineages. Disclosures No relevant conflicts of interest to declare.

scholarly journals Adult Donor-Derived Human CD34+ Cell Engraftment and Hemato-Lymphoid System Development in 3rd Generation Humanized Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4378-4378
Author(s):  
Yasuyuki Saito ◽  
Jana M. Ellegast ◽  
Rouven Müller ◽  
Richard A. Flavell ◽  
Markus G. Manz
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Lymphoid Organs ◽  
Humanized Mice ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Lymphoid System ◽  
Cell Engraftment ◽  
Cd34 Cells

Abstract Transplantation of human CD34+ hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) in vivo (Rongvaux et al. Ann. Rev. Immunol. 2013). While fetal liver- or cord blood- derived CD34+ cells lead to high levels of engraftment, adult donor-derived CD34+ cell transplantation usually led to low levels of engraftment in existing humanized mice models. We recently generated novel mouse strains called 3rd generation humanized mice (3rd gen. huMice) in which human versions of cytokines (M-CSF and TPO with or without IL-3/GM-CSF) are knocked into Balb/c Rag2-/-γC-/- strains (MISTRG or MSTRG, respectively). In addition, human Sirpα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene in both strains (Rongvaux et al., Nat. Biotechnol. 2014). To evaluate human adult CD34+ cell engraftment in 3rd gen. huMice, CD34+ cells obtained from peripheral blood after G-CSF administration (3.0 – 5.5 x105 cells) were i.h. injected into sub-lethally irradiated newborn MISTRG or MSTRG and NOD/scid/γC-/- (NSG) mice or Rag2-/-γC-/-hSirpαTg (RGS) mice as controls. Seventeen of 18 (94%) MISTRG/MSTRG mice showed human CD45+ cell engraftment (>1% of total CD45+ cells in BM) 10-16 weeks after injection, whereas 4 of 11 (36%) NSG/RGS mice supported human cell engraftment. Percentages of human cells in the BM of the engrafted MISTRG/MSTRG were 7- to 8 fold higher than in the BM of engrafted NSG/RGS mice (30.2% ± 6.9 vs 4.1% ± 0.9, respectively). MISTRG/MSTRG mice supported significantly increased numbers of non-classical monocytes and NKp46+ cells in BM compared with NSG/RGS mice. Moreover, we observed significantly increased numbers of CD34+ and CD34+CD38- cells, a population enriched for human early progenitor cells and HSCs, in the BM of MISTRG/MSTRG mice. In addition, MISTRG/MSTRG mice supported higher level of human thymocyte development compared to NSG/RGS mice. Besides lymphoid organs, we further observed increased human CD45+ cells, mostly myeloid lineage cells, in the liver and lung of MISTRG/MSTRG mice compared to NSG/RGS mice. Taken together, this study demonstrates that our 3rd gen. huMice models support adult donor-derived HSC engraftment and development of myeloid as well as lymphoid lineage cells at high levels in primary lymphoid and non-lymphoid organs. These models thus have the potential for personalized studies of healthy hematopoiesis as well as hemato-immune system diseases from adult individuals. Disclosures No relevant conflicts of interest to declare.

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