The Tetraspanin CD82 Defines Erythroid Committment, Regulates Stem- and Progenitor Cell Survival and Mediates Adhesion.

Abstract CD82 belongs to the tetraspanin superfamily, members of which regulate multiple aspects of cell biology such as T-cell activation, proliferation, differentiation, and adhesion.We have previously shown expression of CD82 on hematopoietic progenitor cells. Here we were seeking to shed light on the functional role of CD82 in hematopoiesis. First, we found that CD82 expression is strongly associated with an erythroid committment as exclusively the CD82bright fraction but not the CD82medium or CD82dim fraction of CD34+ cells gave rise to erythroid colony forming units (CFU). In order to manipulate CD82 activity, we used as surrogate ligand, a previously described, CD82-activating monoclonal antibody (moAb), clone 50F11, to activate CD82 on hematopoietic precursors. We found that 50F11, but not another CD82 specific antibody clone, BL-2, specifically induced tyrosine phosphorylation of CD34+ cells. In Dexter-type long-term cultures (D-LTC) 50F11, and not IgG1 isotype control moAbs significantly inhibited myelopoiesis and the number of CD34+ clonogenic progenitors. Moreover, in long term culture initiating cell (LTC-IC) assays, 50F11as compared to isotype control antibodies substantially inhibited, but not entirely abolished, the number of 5 week-LTC-IC′s, indicating that CD82 activation inhibits progenitor - and stem cell proliferation or self renewal. Finally, plastic immobilized 50F11-antibodies caused a time-, and concentration dependent induction of adhesion of CD34+ cells, which was associated with the formation of F-actin and development of multipolar extensions. Finally, CD82 ligation by 50F11 caused a statistically significant down-regulation of the integrin CD49d (p=0,036) and CD62L (p=0,010). Together, it is shown that CD34+/CD82high cells characterize an erythroid committment implying a role for this tetraspanin in erythroid hematopoiesis. Activation of CD82 induces adhesion and negatively regulates proliferation of adult stem- and progenitor cells. This implicates a so far unknown role for CD82 in the regulation of early hematopoiesis.

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HIV-specific T-cell Responses and Generalized Activation in HIV-1 Infected Long-term Non-progressors and Progressors from South India

Current HIV Research ◽

10.2174/1570162x17666181212122607 ◽

2019 ◽

Vol 16 (4) ◽

pp. 302-314

Author(s):

Chinnambedu Ravichandran Swathirajan ◽

Ramachandran Vignesh ◽

Greer Waldrop ◽

Uma Shanmugasundaram ◽

Pannerselvam Nandagopal ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Cd4 T Cell ◽

Cell Responses ◽

Activation Rates ◽

Hiv 1

Background:Anti-viral cytokine expressions by cytotoxic T-cells and lower activation rates have been reported to correlate with suppressed HIV replication in long-term non-progressors (LTNP). Immune mechanisms underlying disease non-progression in LTNP might vary with HIV-1 subtype and geographical locations.Objective:This study evaluates cytokine expression and T-cells activation in relation to disease non-progression in LTNP.Methods:HIV-1 Subtype C infected LTNP (n=20) and progressors (n=15) were enrolled and flowcytometry assays were performed to study HIV-specific CD8 T-cells expressing IL-2, IFN-γ, TNF-α and MIP-1β against gag and env peptides. CD4+ T-cell activation was evaluated by surface expression of HLADR and CD38.Results:Proportions of cytokines studied did not differ significantly between LTNP and progressors, while contrasting correlations with disease progression markers were observed in LTNP. CD4+ T-cell activation rates were significantly lower in LTNP compared to progressors which indicate the potential role of T-cell activation rates in disease non-progression in LTNP.Conclusion:LTNP and progressors showed similar CD8+ T-cell responses, but final conclusions can be drawn only by comparing multiple immune factors in larger LTNP cohort with HIV-1 infected individuals at various levels of disease progression. A possible role of HIV-1 subtype variation and ethnic differences in addition to host-genetic and viral factors cannot be ruled out.

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STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells

Blood ◽

10.1182/blood-2006-08-039073 ◽

2007 ◽

Vol 110 (8) ◽

pp. 2880-2888 ◽

Cited By ~ 68

Author(s):

Hein Schepers ◽

Djoke van Gosliga ◽

Albertus T. J. Wierenga ◽

Bart J. L. Eggen ◽

Jan Jacob Schuringa ◽

...

Keyword(s):

Progenitor Cells ◽

Myeloid Leukemia ◽

Cell Number ◽

Hematopoietic Stem ◽

Fold Reduction ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Precise Role ◽

Term Maintenance

Abstract The transcription factor STAT5 fulfills a distinct role in the hematopoietic system, but its precise role in primitive human hematopoietic cells remains to be elucidated. Therefore, we performed STAT5 RNAi in sorted cord blood (CB) and acute myeloid leukemia (AML) CD34+ cells by lentiviral transduction and investigated effects of STAT5 downmodulation on the normal stem/progenitor cell compartment and the leukemic counterpart. STAT5 RNAi cells displayed growth impairment, without affecting their differentiation in CB and AML cultures on MS5 stroma. In CB, limiting-dilution assays demonstrated a 3.9-fold reduction in progenitor numbers. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays, and the average LTC-IC frequency was 3.25-fold reduced from 0.13% to 0.04% by STAT5 down-regulation. Single-cell sorting experiments of CB CD34+/CD38− cells demonstrated a 2-fold reduced cytokine-driven expansion, with a subsequent 2.3-fold reduction of progenitors. In sorted CD34+ AML cells with constitutive STAT5 phosphorylation (5/8), STAT5 RNAi demonstrated a reduction in cell number (72% ± 17%) and a decreased expansion (17 ± 15 vs 80 ± 58 in control cultures) at week 6 on MS5 stroma. Together, our data indicate that STAT5 expression is required for the maintenance and expansion of primitive hematopoietic stem and progenitor cells, both in normal as well as leukemic hematopoiesis.

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Long-Term Hydroxyurea Use Is Associated with Lower Levels of Hematopoietic Stem and Progenitor Cells in Patients with Sickle Cell Disease

Blood ◽

10.1182/blood-2019-131648 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 985-985

Author(s):

Seda S. Tolu ◽

Kai Wang ◽

Zi Yan ◽

Andrew Crouch ◽

Gracy Sebastian ◽

...

Keyword(s):

Gene Therapy ◽

Progenitor Cells ◽

Research Funding ◽

Cell Disease ◽

Transfusion Therapy ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Background Sickle cell disease (SCD) is curable by transplantation and potentially by gene therapy, and is generally treated by a combination of blood transfusion and Hydroxyurea (HU). Characterizing the hematopoietic system in SCD patients is important because the long-term effect of HU treatment are not known, and because of lower than expected efficacy of transduction and transplantation in Hematopoietic Stem and Progenitor Cells (HSPCs) in recent gene therapy trials. Previous studies have shown that the number of bone marrow (BM) CD34+ cells is elevated in SCD patients and that HU treatment is associated with decreased level of CD34+ cells in the peripheral blood (PB) and BM relative to steady state patients. However, hematopoiesis in SCD patients naive or treated with HU or transfusion remains poorly understood. Here, we report on the characterization of the HSPC compartment in patients with SCD by prospective isolation of 49f+ long-term Hematopoietic Stem Cells (49f+LT-HSCs), Multipotent Progenitors (MPPs), Common Myeloid Progenitors (CMPs), Megakaryocyte-Erythroid Progenitors (MEPs), and Granulocyte-Monocyte Progenitors (GMPs). Methods After obtaining consent, PB and/or BM were collected from 69 patients with HBSS/SB0, aged 12 to 45years, and 25 healthy adult African American controls. Patients were divided into chronic transfusion therapy (n=19), HU (n=31) and naïve (n=19) groups. Frozen mono-nuclear cells were analyzed by flow cytometry on a BD LSRII using CD 49f, 90 45Ra, 123, 235a, 38, 34, 33 and lineage antibodies. Results FACS analysis revealed that the number PB CD34+ cells was 2.5 CD34+/uL of blood in the HU group as compared to 19 CD34+/uL in the exchange and naive groups, and 7.3 CD34+/uL in the control group (q-value <0.05 in all cases). Analysis with additional markers revealed that the decrease in circulating HSPCs in the HU group affected the entire hematopoietic system since the number of 49f+LT-HSCs, MPPs, CMPs, MEPs were all significantly lower in the HU group. The decrease in cell number in the HU group, however, was not homogeneous. The proportion of LT-HSCs was higher in the HU and transfusion groups when compared to the naive and control groups. The HU group also had the lowest proportion of MPPs and GMPs, as well as the highest proportion of MEPs. We then investigated hematopoiesis as a function of the length of HU treatment to elucidate the long-term treatment effect of this cytotoxic agent. Patients > 18 years of age that had been treated on HU for at least three years exhibited a strong statistically significant negative correlation between years on HU and CD34+/uL (R2 = 0.41), LT-HSC/uL (R2 = 0.35), MPP/uL (R2 =0.43), CMP/uL (R2 = 0.37), MEP/uL (R2 = 0.25) and GMP/uL (R2 0.39, p<0.01 in all cases). Importantly there was no correlation between WBC counts, age, HU dose, or serum erythropoietin level versus the numbers of any HSPC/uL. Lastly, we compared the number of HSPCs in paired PB and BM samples 10 controls and 4 SCD patients. This revealed that the numbers of CD34+, HSCs, MPPs, CMPs, GMPs and MEPs in the PB and BM were well correlated (r2 in 0.6-0.8 range) suggesting that in first approximation, results obtained in the PB reflect changes in the BM rather than changes in egress of HSPCs from the BM. Discussion We have observed lower numbers of circulating CD34+,49f+LT- HSCs, MPPs, CMP, GMP and MEPs in individuals with HBSS on HU therapy when compared to naive, chronic transfusion and, to a lesser extent, controls. Furthermore we observed subtle differences in the proportion of various circulating stem and progenitor cells (HSPCs) suggesting that the various treatments affects hematopoiesis in complex ways. The strong negative correlations between the length of HU treatment and the numbers of HSPCs can be explained either by decreased cell mobilization to the periphery, or by a depletion of the HSPC numbers in the BM overtime. Most patients undergoing gene therapy trials are currently taken off HU and placed on transfusion therapy for several months to increase CD34+ cell collection and LT-HSC transduction efficiency. We observed a greater number of circulating 49f+LT-HSC/uL of blood in the transfusion group than in the HU group, but the proportion of 49f+LT-HSC relative to the number of CD34+ were similar in both groups. Functional studies may help determine whether 49f+LT-HSCs from the transfusion group are qualitatively different from of the HU group and more amenable to gene therapy. Figure Disclosures Manwani: Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding. Minniti:Doris Duke Foundation: Research Funding.

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STROME-DEPENDENT EXPANSION OF HEMOPOIETIC PRECURSORS DURING 7-DAY MODELING OF THE EFFECTS OF MICROGRAVITY

Aerospace and Environmental Medicine ◽

10.21687/0233-528x-2021-55-3-28-35 ◽

2021 ◽

Vol 55 (3) ◽

pp. 28-35

Author(s):

Е.А. Tyrina (Golikova) ◽

◽

P.I. Bobyleva ◽

E.R. Andreeva ◽

L.B. Buravkova ◽

...

Keyword(s):

Progenitor Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Space Missions ◽

Hemopoietic Precursors ◽

Modeled Microgravity ◽

Colony Forming Units ◽

Stem And Progenitor Cells

It is well known that long-term space missions influence various myeloid shoots of hemopoiesis in humans and animals. In this investigation, we used several optimized models of co-culturing mesenchymal stromal cells (MSCs) and hemopoietic stem and progenitor cells (HSPCs) to analyze the HSPCs suspension fractions following 7-day modeling of the effects of microgravity. We determined the number of developing HSPCs, presence of colony-forming units (CFU) of various hemopoietic shoots and phenotype spectrum of cells in colonies. We observed an increased number of HSPCs, decreased number of CFUs, and changes in the structure of CFU population after the exposure to modeled microgravity.

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CITED2 Modulates Differentiation and Proliferation of Normal and Leukemic Hematopoietic Stem and Progenitor Cells Downstream of PU.1

Blood ◽

10.1182/blood.v118.21.922.922 ◽

2011 ◽

Vol 118 (21) ◽

pp. 922-922

Author(s):

Hein Schepers ◽

Patrick M. Korthuis ◽

Jan J. Schuringa ◽

Gerald de Haan ◽

Edo Vellenga

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Binding Sites ◽

Myeloid Differentiation ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Cell Numbers ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract Abstract 922 Recently, it was demonstrated that the transcriptional co-activator CITED2 has a conserved role in the maintenance of normal adult hematopoiesis. Little is known regarding the regulation of CITED2, its expression levels in leukemic stem cells (LSCs) and whether CITED2 expression contributes to leukemogenesis. Using microarray data, we found variable CITED2 expression in ∼90% of sorted CD34+ AML cells (n=46). Q-PCR on 12 of these samples indicated that 50% of the leukemic samples displayed higher expression of CITED2 as compared to mobilized peripheral blood (PB) or cord blood (CB)-derived CD34+ hematopoietic stem and progenitor cells (HSCPs). To verify whether this expression of CITED2 in leukemic cells is functional, we performed RNAinterference on primary CD34+ cells from acute myeloid leukemia (AML) patients (n=9). AML samples with high CITED2 expression were susceptible to lentiviral downregulation of CITED2 as evidenced by the loss of transduced cells from long-term leukemic cultures. To investigate a potential role of CITED2 in leukemogenesis, lentiviral gain-of-function experiments were performed with CB-derived CD34+ HSCPs. These experiments indicate that CITED2 expression increases cell numbers up to 5-fold in long-term culture-initiating cell (LTC-iC) experiments. This cell expansion on MS5-stromal cultures was paralleled by a short-term maintenance of progenitors. Colony-forming-cell (CFC) assays demonstrated a 3.5 fold higher output of CFC colonies compared to control cultures up to 2 weeks of MS-5 co-culture. CITED2-expressing colonies were larger than control colonies, verifying the increased cell numbers in LTC-iC experiments. To further dissect the effects of CITED2, transduced CD34+CD38− HSCs and CD34+CD38+ progenitors were sorted for single-cell liquid cultures and cell-divisions were followed for 6 days. Analysis of 360 single CD34+CD38− HSCs indicated that CITED2 overexpression leads to an enhanced quiescence (cells with no division) and decreased proliferation (cells that divide). However, tracking the divisions of 360 single CD34+CD38+ progenitor cells demonstrated the opposite: Cells overexpressing CITED2 divided more than control cells. These data are consistent with a role for CITED2 in leukemic stem cells (LSCs), where LSCs are thought to be more quiescent than leukemic progenitors. Since leukemias are also characterized by a differentiation block, we subsequently analyzed the differentiation of CB-derived CD34+ HSCPs upon overexpression of CITED2. In LTC-iC experiments, a shift in myelo-monocytic differentiation was observed. Enhanced CITED2 expression led to an increased percentage of CD15-positive cells (64%) at the expense of CD14-positive cells (9%) compared to control cultures (35% and 32% respectively). This bias towards granulocytic differentiation was also observed on May-Grunwald-Giemsa (MGG) stains and was furthermore confirmed in CFC assays. Furthermore, when analyzing erythroid differentiation, a clear reduction in CD71bright GPA+ cells could be observed in CITED2 expressing cells, compared to control cells (2% vs. 12% respectively), which was confirmed by CFC assays and MGG stains. Towards clarifying the regulation of CITED2, we scanned the CITED2 promoter for transcription factor binding sites and identified several PU.1 binding sites. Gene expression comparison between PU.1 and CITED2 in a panel of primary AML samples indicated that, apart from FAB M2 AMLs, PU.1 expression is inversely correlated with CITED2 expression. To functionally investigate this inverse correlation, we performed chromatin immunoprecipitations (ChIP) and demonstrated that PU.1 is indeed able to bind to the PU.1 binding sites in the CITED2 promoter of CB CD34+ cells. Subsequent overexpression of PU.1 in CB CD34+ HSPCs led to a 2-fold reduction in CITED2 expression. Taken together, we propose a model in which PU.1 tightly regulates CITED2 expression during normal myeloid differentiation. In certain AML subsets, this model would predict that low PU.1 expression results in failure to lower CITED2 expression below a certain threshold, which subsequently results in maintenance of LSC quiescence. Furthermore, the enhanced CITED2 levels result in an increased proliferation of downstream leukemic progenitors, where together with low PU.1 levels the normal myeloid differentiation program is perturbed contributing to leukemic development. Disclosures: No relevant conflicts of interest to declare.

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The Scribble Complex PDZ Proteins in Immune Cell Polarities

Journal of Immunology Research ◽

10.1155/2020/5649790 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Dante Barreda ◽

Luis H. Gutiérrez-González ◽

Erasmo Martínez-Cordero ◽

Carlos Cabello-Gutiérrez ◽

Rommel Chacón-Salinas ◽

...

Keyword(s):

Immune System ◽

T Cell Activation ◽

Cell Biology ◽

Cell Activation ◽

Immune Cell ◽

Immunological Synapse ◽

Cell Polarization ◽

Cell Functions ◽

Wide Range

hScrib and hDlg belong to the PDZ family of proteins. Since the identification of these highly phylogenetically conserved scaffolds, an increasing amount of experiments has elucidated the roles of hScrib and hDlg in a variety of cell functions. Remarkably, their participation during the establishment of polarity in epithelial cells is well documented. Although the role of both proteins in the immune system is scantly known, it has become a growing field of investigation. Here, we summarize the interactions and functions of hScrib and hDlg1, which participate in diverse functions involving cell polarization in immune cells, and discuss their relevance in the immune cell biology. The fundamental role of hScrib and hDlg1 during the establishment of the immunological synapse, hence T cell activation, and the recently described role of hScrib in reactive oxygen species production in macrophages and of hDlg1 in cytokine production by dendritic cells highlight the importance of both proteins in immune cell biology. The expression of these proteins in other leukocytes can be anticipated and needs to be confirmed. Due to their multiple interaction domains, there is a wide range of possible interactions of hScrib and hDlg1 that remains to be explored in the immune system.

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BSTM-03. INCREASING ANTI-TUMOR T CELL ACTIVATION, VELOCITY, AND MIGRATION TO BRAIN STEM GLIOMA USING HEMATOPOIETIC STEM AND PROGENITOR CELLS

Neuro-Oncology ◽

10.1093/neuonc/noz036.020 ◽

2019 ◽

Vol 21 (Supplement_2) ◽

pp. ii67-ii67

Author(s):

Catherine Flores ◽

Cameron Morley ◽

Ginger Moore ◽

Thomas Angelini ◽

Duane Mitchell

Keyword(s):

T Cell ◽

Brain Stem ◽

Progenitor Cells ◽

T Cell Activation ◽

Cell Activation ◽

Brain Stem Glioma ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

And Migration

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Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis

Blood ◽

10.1182/blood-2009-03-209734 ◽

2009 ◽

Vol 114 (8) ◽

pp. 1498-1505 ◽

Cited By ~ 93

Author(s):

Aleksandra Rizo ◽

Sandra Olthof ◽

Lina Han ◽

Edo Vellenga ◽

Gerald de Haan ◽

...

Keyword(s):

Progenitor Cells ◽

Myeloid Leukemia ◽

Oxygen Species ◽

Self Renewal ◽

Human Cd34 ◽

Bmi1 Expression ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Cord Blood Cd34

Abstract High expression of BMI1 in acute myeloid leukemia (AML) cells is associated with an unfavorable prognosis. Therefore, the effects of down-modulation of BMI1 in normal and leukemic CD34+ AML cells were studied using a lentiviral RNA interference approach. We demonstrate that down-modulation of BMI1 in cord blood CD34+ cells impaired long-term expansion and progenitor-forming capacity, both in cytokine-driven liquid cultures as well as in bone marrow stromal cocultures. In addition, long-term culture-initiating cell frequencies were dramatically decreased upon knockdown of BMI1, indicating an impaired maintenance of stem and progenitor cells. The reduced progenitor and stem cell frequencies were associated with increased expression of p14ARF and p16INK4A and enhanced apoptosis, which coincided with increased levels of intracellular reactive oxygen species and reduced FOXO3A expression. In AML CD34+ cells, down-modulation of BMI1 impaired long-term expansion, whereby self-renewal capacity was lost, as determined by the loss of replating capacity of the cultures. These phenotypes were also associated with increased expression levels of p14ARF and p16INK4A. Together our data indicate that BMI1 expression is required for maintenance and self-renewal of normal and leukemic stem and progenitor cells, and that expression of BMI1 protects cells against oxidative stress.

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Alloantigen presenting function of normal human CD34+ hematopoietic cells

Blood ◽

10.1182/blood.v88.7.2619.bloodjournal8872619 ◽

1996 ◽

Vol 88 (7) ◽

pp. 2619-2675 ◽

Cited By ~ 32

Author(s):

D Rondelli ◽

RG Andrews ◽

JA Hansen ◽

R Ryncarz ◽

MA Faerber ◽

...

Keyword(s):

T Cells ◽

T Cell Activation ◽

Cell Activation ◽

Autologous Transplantation ◽

Allogeneic Transplantation ◽

Hematopoietic Stem ◽

Hla Dr ◽

Cd34 Cells ◽

Marrow Cells

The identification of the CD34 molecule, expressed almost exclusively on human hematopoietic stem cells and committed progenitors, and the development of CD34-specific monoclonal antibodies have made procurement of relatively pure populations of CD34+ marrow cells for autologous transplantation feasible. Characterization of the immunogenicity of CD34+ marrow cells may facilitate the design of successful strategies to use these cells for allogeneic transplantation. CD34+ marrow cells from normal volunteers were enriched to greater than 98% purity by immunoaffinity chromatography on column followed by fluorescence-activated cell sorting. Purified CD34+ cells were tested for expression of HLA-DR and other accessory molecules, and function in hematopoietic colony growth and mixed leukocyte culture (MLC) assays. Greater than 95% CD34+ cells were positive for HLA-DR and 74% +/- 10% were highly positive for CD18, the common beta-chain of a leukointegrin family. CD34+/CD18- cells were small, agranular lymphocytes which contained the majority of precursors for colony-forming cells detected in long-term cultures. They produced almost no stimulation of purified T cells from HLA-DR-incompatible individuals in bulk MLC or in limiting dilution assay. In contrast, CD34+/CD18+ cells were large, were enriched for cells forming mixed colonies in short- but not long-term assays, and were capable of stimulating allogeneic T cells. CD86, a natural ligand for the T-cell activation molecule CD28, was coexpressed with CD18 in 6% +/- 3% of CD34+ cells. CD34+/CD86+ cells, but not CD34+/CD86- cells, exhibited strong alloantigen presenting function. Thus, pluripotent hematopoietic activity and alloantigen presenting function are attributes of distinct subsets of CD34+ marrow cells. CD34+/CD18- or CD34+/CD86- cells may be more effective than either the whole CD34+ population or unseparated marrow in engrafting allogeneic recipients and may also facilitate induction of tolerance.

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Multicolor Flowcytometry Analysis of Hematopoietic Stem and Progenitor Cells Subsets Among Basal and Mobilized Peripheral CD34+ Cells

Blood ◽

10.1182/blood.v124.21.5117.5117 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5117-5117

Author(s):

Valentina Giai ◽

Elona Saraci ◽

Eleonora Marzanati ◽

Christian Scharenberg ◽

Monica De Stefanis ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Healthy Volunteers ◽

Progenitor Cells ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Progenitor Compartment

Abstract BACKGROUND: In the recent years, numerous studies based on multicolor flowcytometry have analyzed the different subpopulations of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) (Manz MG et al, PNAS 2002; Majeti R et al, Cell Stem Cell 2007): the common myeloid progenitors (CMPs: Lin-CD34+CD38+CD45RA-CD123+), the granulocyte-macrophage progenitors (GMPs: Lin-CD34+CD38+CD45RA+CD123+) and the megakaryocyte-erythroid progenitors (MEPs: Lin-CD34+CD38+CD45RA-CD123-) constitute the progenitor compartment, while the hematopoietic stem cells (HSCs: Lin-CD34+CD38- CD45RA-CD90+), the multipotent progenitors (MPPs: Lin-CD34+CD38- CD45RA-CD90-) and the lymphoid-myeloid multipotent progenitors (LMPPs: Lin-CD34+CD38- CD45RA+CD90-) represent the more immature HSPCs. In animal models, the progenitor compartment includes short-term repopulating cells, leading to the hematological recovery in the first 5 weeks after transplantation, whereas the stem cell compartment comprehends the long-term repopulation cells, responsible for the long-term hematological recovery. However, very little is known about the different subpopulations of HSPCs among peripheral blood (PB) CD34+ in basal state and after mobilization for harvest and transplantation. Our study was conducted to analyze PB CD34+ cells from healthy volunteers and from hematological patients during CD34+ cells mobilization. Our main aim was to understand if the proportions of different HSPCs among PB CD34+ cells were similar to those found in BM and whether the mobilizing regimens employed in chemo treated patients differently affected CD34+ cells subfractions in PB. METHODS: multicolor flowcytometry was used to analyze CD34+ cells from 4 BM samples and 9 PB samples from healthy volunteers and 32 PB samples from hematological patients prior CD34+ cells harvesting. RESULTS: Percentages of CD34+ cells subpopulations were different in basal PB compared to the BM: indeed, CMPs, GMPs and MEPs constituted respectively 27.6% ± 9.5, 23.8% ± 7.2 and 27.6% ± 16.2 of BM CD34+ cells and 47.8% ± 9.5, 10.3% ± 6.9 and 16.1% ± 7.6 of the total PB CD34+ cells. HSCs constituted 2.1% of BM and 1.5% of PB CD34+ cells. The differences between BM and circulating CMPs and GMPs were significant (p<0.005 and p<0.01). No differences in subpopulations proportions were shown comparing G-CSF mobilized and basal PB CD34+ cells. Interestingly, the 2 patients mobilized with AMD3100 (the inhibitory molecule for CXCR4) showed a higher percentage of GMPs (33.8% and 37.8% versus the average 16.3% ± 9.8 in G-CSF mobilized samples) and a lower fraction of CMPs (29.5% and 41.6% versus the average 58% ± 12 in G-CSF mobilized samples). In order to understand this result, we looked then at the CXCR4 mean fluorescence intensity among the progenitor subsets: GMPs showed significantly higher levels of this molecule compared to CMPs and MEPs. Regarding the mobilizing chemotherapy regimens, CMPs percentages were higher (61.1% versus 49.1%, p: 0.038) and GMPs’ were significantly lower (11.1% versus 27.6%, p<0.0001) in cyclophosphamide treated patients, compared to patients mobilized with other chemotherapy regimens. The percentage of HSCs did not significantly differ among bone marrow, unmobilized and mobilized PB CD34+ cells. Therefore, since an average collection of mobilized PB cells contains approximately one log more CD34+ cells than a BM harvest, a similarly higher amount of HSC are infused with mobilized CD34+ cell transplantation. A linear positive correlation between the number of mobilized CD34+ cells and the number of mobilized CMPs, GMPs, and MEPs was observed indicating that the proportions of different HSPCs did not significantly change among high- and low-mobilizers. There were no correlations between the number of mobilized subpopulations and leucocytes, hemoglobin and platelets levels. CONCLUSIONS: Our data displayed the heterogeneity of HSPC compartment between PB and BM. Many factors could contribute to this variegated scenario. These mechanisms comprehension can help us to choose the most suitable chemotherapy and cytokine administrations in order to improve clinical outcomes as infections complications, length of aplasia and transfusion requirements during an hematopoietic stem cell transplantation. Disclosures Palumbo: Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Boccadoro:Celgene: Honoraria; Janssen: Honoraria; Onyx: Honoraria.

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