scholarly journals Colony formation of clone-sorted human hematopoietic progenitors

Blood ◽  
1990 ◽  
Vol 75 (10) ◽  
pp. 1941-1946 ◽  
Author(s):  
H Ema ◽  
T Suda ◽  
Y Miura ◽  
H Nakauchi
Keyword(s):  
Single Cell ◽  
Cell Cultures ◽  
Mononuclear Cells ◽  
Direct Action ◽  
Single Cells ◽  
Accurate Estimation ◽  
Hematopoietic Progenitors ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Sorting System

Abstract To characterize human hematopoietic progenitors, we performed methylcellulose cultures of single cells isolated from a population of CD34+ cells by fluorescence-activated cell-sorting (FACS) clone-sorting system. CD34+ cells were detected in bone marrow (BM) and peripheral blood (PB) cells at incidences of 1.0% and 0.01% of total mononuclear cells, respectively. Single cell cultures revealed that approximately 37% of BM CD34+ cells formed colonies in the presence of phytohemagglutinin-leukocyte conditioned medium and erythropoietin. Erythroid bursts-, granulocyte-macrophage (GM) colony-, and pure macrophage (Mac) colony-forming cells were 10% each in CD34+ cells. Approximately 15% of PB CD34+ cells formed colonies in which erythroid bursts were predominant. CD34+ cells were heterogeneous and fractionated by several antibodies in FACS multicolor analysis. In these fractionated cells, CD34+, CD33+ cells formed GM and Mac colonies 7 to 10 times as often as CD34+, CD33- cells. Most of the erythroid bursts and colonies were observed in the fraction of CD34+, CD13- cells or CD34+, CD33- cells. The expression of HLA-DR on CD34+ cells was not related to the incidence, size, or type of colonies. There was no difference in the phenotypical heterogeneity of CD34+ cells between BM and PB. About 10% of CD34+ cells were able to form G colonies in response to granulocyte colony-stimulating factor (G-CSF) and to form Mac colonies in GM-CSF or interleukin-3 (IL-3). Progenitors capable of generating colonies by stimulation of G-CSF were more enriched in CD34+, CD33+ fraction than in CD34+, CD33- fraction. Thus, single cell cultures using the FACS clone-sorting system provide an accurate estimation of hematopoietic progenitors and an assay system for direct action of colony-stimulating factors.

scholarly journals Colony formation of clone-sorted human hematopoietic progenitors

Blood ◽  
1990 ◽  
Vol 75 (10) ◽  
pp. 1941-1946 ◽  
Author(s):  
H Ema ◽  
T Suda ◽  
Y Miura ◽  
H Nakauchi
Keyword(s):  
Single Cell ◽  
Cell Cultures ◽  
Mononuclear Cells ◽  
Direct Action ◽  
Single Cells ◽  
Accurate Estimation ◽  
Hematopoietic Progenitors ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Sorting System

To characterize human hematopoietic progenitors, we performed methylcellulose cultures of single cells isolated from a population of CD34+ cells by fluorescence-activated cell-sorting (FACS) clone-sorting system. CD34+ cells were detected in bone marrow (BM) and peripheral blood (PB) cells at incidences of 1.0% and 0.01% of total mononuclear cells, respectively. Single cell cultures revealed that approximately 37% of BM CD34+ cells formed colonies in the presence of phytohemagglutinin-leukocyte conditioned medium and erythropoietin. Erythroid bursts-, granulocyte-macrophage (GM) colony-, and pure macrophage (Mac) colony-forming cells were 10% each in CD34+ cells. Approximately 15% of PB CD34+ cells formed colonies in which erythroid bursts were predominant. CD34+ cells were heterogeneous and fractionated by several antibodies in FACS multicolor analysis. In these fractionated cells, CD34+, CD33+ cells formed GM and Mac colonies 7 to 10 times as often as CD34+, CD33- cells. Most of the erythroid bursts and colonies were observed in the fraction of CD34+, CD13- cells or CD34+, CD33- cells. The expression of HLA-DR on CD34+ cells was not related to the incidence, size, or type of colonies. There was no difference in the phenotypical heterogeneity of CD34+ cells between BM and PB. About 10% of CD34+ cells were able to form G colonies in response to granulocyte colony-stimulating factor (G-CSF) and to form Mac colonies in GM-CSF or interleukin-3 (IL-3). Progenitors capable of generating colonies by stimulation of G-CSF were more enriched in CD34+, CD33+ fraction than in CD34+, CD33- fraction. Thus, single cell cultures using the FACS clone-sorting system provide an accurate estimation of hematopoietic progenitors and an assay system for direct action of colony-stimulating factors.

scholarly journals The Single-Cell Transcriptional Landscape of Human Hematopoietic Progenitors

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1266-1266
Author(s):  
Mariana Loperfido ◽  
Danilo Pellin ◽  
Cristina Baricordi ◽  
Samuel Louis Wolock ◽  
Allon Moshe Klein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cell ◽  
Mast Cells ◽  
Single Cell ◽  
Cell Fate ◽  
Blood Cells ◽  
Single Cells ◽  
Hematopoietic Cell ◽  
Hematopoietic Progenitors ◽  
Cd34 Cells

Abstract In humans, hematopoietic stem/progenitor cells (HSPCs) constitute a heterogeneous population in the bone marrow (BM) endowed with the potential of generating and maintaining an adequate and diverse pool of blood cells lifelong. However, the nature of hematopoietic cell fate choice remains unresolved and is still highly contentious. In fact, despite the recent efforts and the advent of single-cell technologies, conflicting classical, early-split or continuous models for the generation of human blood cells still coexist in the literature as of today. We here used single-cell RNA-Seq (scRNA-Seq) to provide a detailed map of the transcriptional states of hematopoietic progenitors in the human BM. We believe that this work provides unprecedented insights into the structure of early cell-fate choices of the human hematopoietic system. Single-cell analysis of human HSPCs is a well-worn path, but all previous studies aimed at investigating the shape of early cell fate choices human hematopoiesis have been based on the use of immune-selected CD34+ cells. We too started by separating CD34+ cells purified by magnetic beads selection into seven subpopulations, marking cells of differing fate potential and we successfully tagged and sequenced the transcriptome of 6,011 single cells. Our scRNA-Seq map of CD34+ subpopulations shows that HSPCs do not undergo a single-step transition from HSPCs to unilineage states. Instead, they form a clear hierarchy. The earliest fate split separates erythroid-megakaryocyte progenitors from lymphoid-myeloid progenitors, which separate further into lymphoid, dendritic cell and granulocytic progenitors. We noted, however, that branches towards basophils/eosinophils/mast cells and monocytes commitments were missing. Indeed, many cells negative for mature lineage markers in human BM are CD34low/- and could account for additional transitional states at which the CD34 expression is rapidly downregulated, therefore greatly reducing their probability of capture. Thus, differently from previous works, we also extended our analysis to the whole bone marrow fraction lacking the main markers of terminal differentiation (Lin- cells). In doing so we used a novel graded FACS-sorting strategy that corrects for expansion of cells as they differentiated, and thus allows examining early states alongside later ones that comprise the vast majority of Lin- progenitors. In fractionating the cells by maturity, we made use of a cell surface marker, GeneA, which we identified from the initial data set as capturing cells that are multipotent until just beyond the first branch-point.The result of this analysis was a high resolution landscape from the transcriptome of 15,401 single cells, showing multiple branch points, and a topology that remarkably similar to what we reported in mice (Tusi et al. Nature 2018). This map defines the early branching of dendritic cell vs monocyte precursors and supports the novel concept that basophils/eosinophils/mast cells do not derive from common granulocytes/monocytes progenitors but originate instead from what are currently defined as megakaryo/erythroid progenitors.By the in depth analysis of our single-cell maps we generated a number of information of value for the broader hematology community: * We put the "classical" CD34+ FACS-based cell subpopulations into the context of the newly generated BM Lin- population structure (valuable for transplantation research) * We investigated the expression of transcription factors along differentiation trajectories and branching points (valuable for in vitro reprogramming efforts) * We analyzed the expression of several blood cancer genes over the main differentiation hubs and hematopoietic branches (valuable for the study, diagnosis and therapy of hematological tumors) * We provided a detailed comparison of the expression of gene orthologues in human vs mouse hematopoiesis (valuable for the modeling of blood cell disorders in the mouse) * We identified and validated GeneA as a reliable marker for the earliest branches of HSPCs specification in transplantation cell products (valuable for transplantation research and gene therapy) Overall, our study provides a detailed view of the early hematopoietic cell state hierarchy in humans and a valuable resource for HSPC biology. Disclosures No relevant conflicts of interest to declare.

Immunomodulatory Effects of CC-4047 on Hematopoietic Progenitors Are Associated with a Shift in the Cytokine Secretion Profile Favoring Myeloid Differentiation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2917-2917
Author(s):  
Margarethe Gries ◽  
Markus Y. Mapara ◽  
David Stirling ◽  
Martin Janz ◽  
Suzanne Lentzsch
Keyword(s):  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Cytokine Secretion ◽  
Cytokine Gene Expression ◽  
Lineage Commitment ◽  
Red Cell ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
Cd34 Cells

Abstract CC-4047 is an immunomodulatory analog of thalidomide with stronger anti-myeloma and anti-angiogenic activity than thalidomide. We could show that CC-4047 directly influences lineage commitment and differentiation of hematopoietic stem cells (Koh et al., Blood 2004 in press). We found that CC-4047 effectively inhibits erythroid cell colony formation from CD34+ cells and increases the frequency of myeloid colonies. We also demonstrated that development of both erythropoietin-independent and erythropoietin-dependent red cell progenitors was strongly inhibited by CC-4047, while terminal red cell differentiation was unaffected. However, there is little information regarding the mechanism by which CC-4047 affect hematopoiesis. Due to the fact that CC-4047 has been shown to influence secretion of proinflammatory cytokines of peripheral mononuclear cells after LPS stimulation we investigated the cytokine profile of hematopoietic progenitors treated with this drug. CD34+ cells were cultured with SCF, IL-3 and IL-6 in the presence of thalidomide (100μM) or CC-4047 (100μM) for 1, 3 or 6 days and cytokine gene expression was studied in these hematopoietic progenitor cells using gene array analyses. Furthermore, supernatants were collected and examined for IL-1b, IL-2, IL-4, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF-a, IFN-g, GM-CSF, G-CSF, MCP-1, and MIP-1b. Our analysis revealed that cytokines supporting myelopoiesis increased very early after treatment with CC-4047. After CC-4047 stimulation, secretion of G-CSF increased within 24 hours 10-fold in comparison to control cells. MCP-1, which is known to support predominantly the granulocytic lineage and to augment the clonal expansion of hematopoietic progenitor cells, increased also up to 5-fold on day 1 under CC-4047 treatment in compared to control. Secretion of IL-10, a pro-inflammatory cytokine known to inhibit erythropoiesis, was also up regulated. In addition, IL-13, which favors the development of erythroid progenitors, decreased 3-fold by CC-4047 on day 1 compared to control. In contrast, thalidomide induced much weaker changes in cytokine secretion. This is in line with our observation that thalidomide has only weak effects on lineage commitment. Cytokine analysis after 24 hours G-CSF MCP-1 IL-10 IL-5 IL-13 pg/ml Control 162 3543 7.9 6191 2806 Thal 455 7653 10.8 3425 2563 CC-4047 1514 17734 28 968 1748 In contrast to the previous findings that CC-4047 inhibits TNF-a, IL-12 and IL-1b synthesis in activated mononuclear cells are our results showing that secretion of TNF-a, IL-12, IL-1b and also of IL-2, IL-4, IL-7, IL-8, IL-17, IFN-g, GM-CSF and MIP-1b is not significantly affected by CC-4047 and thalidomide. Analyses of cytokine gene expression confirmed our results. In conclusion, these data indicate that CC-4047 might directly influence lineage commitment of hematopoietic cells by modulation of cytokine secretion increasing the propensity of stem and/or progenitor cells to undergo myeloid cell development and concomitantly inhibiting red cell development. The influence on cytokine secretion is an early event since these changes can observed within the first 24 hours of CC-4047 treatment and depends strictly on cell type and differentiation level. CC-4047 provides a valuable tool to study the mechanisms underlying lineage commitment.

scholarly journals CD14+ monocytes repress gamma globin expression at early stages of erythropoiesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Steven Heshusius ◽  
Esther Heideveld ◽  
Marieke von Lindern ◽  
Emile van den Akker
Keyword(s):  
Mononuclear Cells ◽  
Therapeutic Option ◽  
Globin Genes ◽  
Beta Globin ◽  
Hematopoietic Progenitors ◽  
Peripheral Blood Mononuclear ◽  
Temporal Regulation ◽  
Gamma Globin ◽  
Cd34 Cells ◽  
Adult Hemoglobin

AbstractIn β-hemoglobinopathies, reactivation of gamma- at the expense of beta-globin is a prominent therapeutic option. Expression of the globin genes is not strictly intrinsically regulated during erythropoiesis, supported by the observation that fetal erythroid cells switch to adult hemoglobin expression when injected in mice. We show cultured erythroblasts are a mix of HbA restrictive and HbA/HbF expressing cells and that the proportion of cells in the latter population depends on the starting material. Cultures started from CD34+ cells contain more HbA/HbF expressing cells compared to erythroblasts cultured from total peripheral blood mononuclear cells (PBMC). Depletion of CD14+ cells from PBMC resulted in higher HbF/HbA percentages. Conversely, CD34+ co-culture with CD14+ cells reduced the HbF/HbA population through cell–cell proximity, indicating that CD14+ actively repressed HbF expression in adult erythroid cultures. RNA-sequencing showed that HbA and HbA/HbF populations contain a limited number of differentially expressed genes, aside from HBG1/2. Co-culture of CD14+ cells with sorted uncommitted hematopoietic progenitors and CD34-CD36+ erythroblasts showed that hematopoietic progenitors prior to the hemoglobinized erythroid stages are more readily influenced by CD14+ cells to downregulate expression of HBG1/2, suggesting temporal regulation of these genes. This possibly provides a novel therapeutic avenue to develop β-hemoglobinopathies treatments.

scholarly journals Transcriptome Based Projection of Single Cells to Uncover Development and Heterogeneity of Abnormal Hematopoietic Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2520-2520
Author(s):  
Parashar Dhapola ◽  
Mikael Sommarin ◽  
Mohamed Eldeeb ◽  
Amol Ugale ◽  
David Bryder ◽  
...  
Keyword(s):  
Single Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Target Cells ◽  
Cell Identity ◽  
Large Patient ◽  
Low Dimensional

Single-cell transcriptomics (scRNA-Seq) has accelerated the investigation of hematopoietic differentiation. Based on scRNA-Seq data, more refined models of lineage determination in stem- and progenitor cells are now available. Despite such advances, characterizing leukemic cells using single-cell approaches remains challenging. The conventional strategies of scRNA-Seq analysis map all cells on the same low dimensional space using approaches like tSNE and UMAP. However, when used for comparing normal and leukemic cells, such methods are often inadequate as the transcriptome of the leukemic cells has systematically diverged, resulting in irrelevant separation of leukemic subpopulations from their healthy counterpart. Here, we have developed a new computational approach bundled into a tool called Nabo (nabo.readthedocs.io) that has the capacity to directly compare cells that are otherwise unalignable. First, Nabo creates a shared nearest neighbor graph of the reference population, and the heterogeneity of this population is subsequently defined by performing clustering on the graph and calculating a low dimensional representation using t-SNE or UMAP. Nabo then calculates the similarity of incoming cells from a target population to each cell in the reference graph using a modified Canberra metric. The reference cells with higher similarity to the target cells obtain higher mapping scores. The built-in classifier is used to assign each target cell a reference cluster identity. We tested Nabo's accuracy on control datasets and found that Nabo's performance in terms of accuracy and robustness of projection is comparable to state-of-art methods. Moreover, Nabo is a generalized domain adaptation algorithm and hence can perform classification of target cells that are arbitrarily dissimilar to reference cells. Nabo could identify the cell-identity of sorted CD19+ B cells, CD14+ monocytes and CD56+ by projecting these unlabeled cells onto labelled peripheral blood mononuclear cells with an average specificity higher than 0.98. The general applicability of Nabo was demonstrated by successfully integrating pancreatic cells, sequenced in three different studies using different sequencing chemistries with comparable or better accuracy than existing methods. Also, it was conclusively demonstrated that Nabo can predict the identity of human HSPC subpopulations to the same accuracy as can be achieved by established cell-surface markers. Having Nabo at hand, we aimed to uncover the heterogeneity of hematopoietic cells from different stages of AML. Nabo showed that AML cells lacked the heterogeneity of normal CD34+ cells and were devoid of cells with HSC gene signature. A large patient-to-patient variability was found where leukemic cells mapped to distinct stages of myeloid progenitors. To ask whether this variability could reflect differences in leukemia-initiating cell identity, we induced leukemia in murine granulocyte-monocyte-lymphoid progenitors (GMLPs) using an inducible model for MLL-ENL-driven AML. On projection, more than 70% of MLL-ENL-activated cells mapped to a distinct Flt3+ subpopulation present within healthy GMLPs. Statistical validity of this projection was verified using two novel null models for testing cell projections: 1) ablated node model, wherein the mapping strength of target cells are evaluated after removal of high mapping score source nodes, and 2) high entropy features model, which rules out the background noise effect. By separating Flt3+ and Flt3- cells prior to activation of the fusion gene and performing in vitro replating assays, we could demonstrate that Flt3+ GMLPs contained 3-4 fold more leukemia-initiating cells (1/1.34 cells) than Flt3- GMLPs (1/4.89 cells), indicating that leukemia-initiating cells within GMLPs express Flt3. Taken together, Nabo represents a robust cell projection strategy for relevant analysis of scRNA-Seq data that permits an interpretable inference of cross-population relationships. Nabo is designed to compare disparate cellular populations by using the heterogeneity of one population as a point of reference allowing for cell-type specification even following perturbations that have resulted in large molecular changes to the cells of interest. As such, Nabo has critical implementation for delineation of leukemia heterogeneity and identification of leukemia-initiating cell population. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hepatocyte Growth Factor Is Constitutively Produced by Human Bone Marrow Stromal Cells and Indirectly Promotes Hematopoiesis

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1560-1565 ◽  
Author(s):  
Kenji Takai ◽  
Junichi Hara ◽  
Kunio Matsumoto ◽  
Gaku Hosoi ◽  
Yuko Osugi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Gm Csf ◽  
Tumor Growth Factor ◽  
Cd34 Cells ◽  
Factor Α ◽  
Hepatocyte Growth

Bone marrow (BM) stromal cells are required for normal hematopoiesis. A number of soluble factors secreted by these cells that mediate hematopoiesis have been characterized. However, the mechanism of hematopoiesis cannot be explained solely by these known factors, and the existence of other, still unknown stromal factors has been postulated. We showed that hepatocyte growth factor (HGF ) is one such cytokine produced by human BM stromal cells. BM stromal cells were shown to constitutively produce HGF and also to express the c-MET/HGF receptor. The production of HGF was enhanced by addition of heparin and phorbol ester. Dexamethasone and tumor growth factor-β (TGF-β) inhibited the production of HGF. Interleukin-1α (IL-1α) tumor necrosis factor-α (TNF-α), and N6,2′-o-dibutyryl-adenosine-3′:5′-cyclic monophosphate (dbc-AMP) showed no obvious influence on HGF production. Western blot analysis of HGF derived from BM stromal cells showed two bands at 85 and 28 kD corresponding to native and variant HGF, respectively. Addition of recombinant HGF significantly promoted the formation of burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte erythroid macrophage (CFU-GEM) by BM mononuclear cells in the presence of erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF ), but the formation of CFU-GM was not modified. However, HGF had no effects on colony formation by purified CD34+ cells. Within BM mononuclear cells, c-MET was expressed on a proportion of cells (CD34−, CD33+, CD13+, CD14+, and CD15+), but was not found on CD34+ cells. We conclude that HGF is constitutively produced by BM stromal cells and that it enhances hematopoiesis. In addition, expression of c-MET on the stromal cells suggests the presence of an autocrine mechanism, operating through HGF, among stromal cells.

A new approach reveals syncytia within the visceral musculature ofDrosophila melanogaster

Development ◽  
2001 ◽  
Vol 128 (13) ◽  
pp. 2517-2524 ◽  
Author(s):  
Robert Klapper ◽  
Sandra Heuser ◽  
Thomas Strasser ◽  
Wilfried Janning
Keyword(s):  
Drosophila Melanogaster ◽  
Single Cell ◽  
Cell Transplantation ◽  
Reporter Gene ◽  
Mononuclear Cells ◽  
Single Cells ◽  
New Approach ◽  
Transplantation Technique ◽  
Somatic Musculature ◽  
Somatic Muscles

In order to reveal syncytia within the visceral musculature of Drosophila melanogaster, we have combined the GAL4/UAS system with the single-cell transplantation technique. After transplantation of single cells from UAS-GFP donor embryos into ubiquitously GAL4-expressing recipients, the expression of the reporter gene was exclusively activated in syncytia containing both donor- and recipient-derived nuclei. In the first trial, we tested the system in the larval somatic musculature, which is already known to consist of syncytia. By this means we could show that most of the larval somatic muscles are generated by clonally non-related cells. Moreover, using this approach we were able to detect syncytia within the visceral musculature – a tissue that has previously been described as consisting of mononuclear cells. Both the longitudinal visceral musculature of the midgut and the circular musculature of the hindgut consist of syncytia and persist through metamorphosis. This novel application of the transplantation technique might be a powerful tool to trace syncytia in any organism using the GAL4/UAS system.

scholarly journals Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART

2017 ◽  
Vol 114 (18) ◽  
pp. E3659-E3668 ◽  
Author(s):  
Ann Wiegand ◽  
Jonathan Spindler ◽  
Feiyu F. Hong ◽  
Wei Shao ◽  
Joshua C. Cyktor ◽  
...  
Keyword(s):  
Single Cell ◽  
Mononuclear Cells ◽  
Single Cells ◽  
Constitutive Expression ◽  
Multiple Time ◽  
Hiv Rna ◽  
Infected Cells ◽  
The Impact ◽  
Hiv 1

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2–18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

scholarly journals The action of bryostatin on normal human hematopoietic progenitors is mediated by accessory cell release of growth factors

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 716-720 ◽  
Author(s):  
SJ Sharkis ◽  
RJ Jones ◽  
ML Bellis ◽  
GD Demetri ◽  
JD Griffin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Single Cell ◽  
Cell Cultures ◽  
Bone Marrow Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Accessory Cell ◽  
Bryostatin 1 ◽  
Gm Csf

Abstract Since enrichment of human bone-marrow hematopoietic progenitors is becoming more feasible and since purified growth factors are now available, we sought to study the action of growth factors on CD34- positive enriched cultures of human bone-marrow cells. We tested the effect of recombinant human (rh) granulocyte-macrophage colony- stimulating factor (GM-CSF), rh interleukin-3 (IL-3), or a unique biologic response modifier, bryostatin 1, on the growth of purified CD34 cells obtained by limiting dilution in single-cell cultures. We have shown previously that bryostatin 1 stimulates both myeloid and erythroid progenitors of human origin in vitro. In this study both IL-3 and GM-CSF supported colony formation from 500, 100, or single-cell cultures at equivalent plating efficiences, suggesting a direct action of these factors on hematopoietic cell growth. Conversely, bryostatin 1 did not support the growth of CD34 cells in single-cell cultures, and the cloning efficiency increased with increasing the number of cells in the culture. To test whether the indirect action of bryostatin 1 might be mediated through the production of growth factors by accessory cells, studies were performed using antibodies directed against human IL-3 and GM-CSF in culture with bryostatin 1 and normal human bone- marrow cells. Results are consistent with the hypothesis that bryostatin 1 could have a stimulatory effect on the accessory cell populations to produce either IL-3 or GM-CSF. Further support for this notion was obtained by demonstrating that T cells, which are cells known to be able to produce IL-3 and GM-CSF, are stimulated by bryostatin 1 to express messenger RNA (mRNA) for specific growth factors, including GM-CSF. These results provide further support that bryostatin 1 may be a useful clinical agent to stimulate hematopoiesis in vivo.

FLT3 Mediated MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1532-1532
Author(s):  
Christophe Desterke ◽  
Hans Hasselbalch ◽  
Dominique Bordessoule ◽  
Heinz Gisslinger ◽  
Alessandro Vannucchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cultures ◽  
Mononuclear Cells ◽  
Mapk Pathway ◽  
Primary Myelofibrosis ◽  
Mapk Activation ◽  
Specific Chemical ◽  
Mutational Status ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Myeloproliferation, myelofibrosis, osteosclerosis and neo-angiogenesis are the major intrinsic pathophysiological features of Primary Myelofibrosis (PMF). The myeloproliferation is characterized by an increased number of circulating CD34+ cells with the prominent amplification of “dystrophic” megakaryocytes (MK) through to be responsible for myelofibrosis thought fibrogenic factor release. Comparison of CD34+ and MK cell gene expression profiling between PMF patients and healthy donors revealed a global deregulation of the MAPK pathway genes. This alteration is associated with a modulation of the FLT3 tyrosine kinase gene expression in CD34+ and MK cells from patients, independently of the JAK2V617F mutation presence. Quantification of the FLT3 transcript in mononuclear cells from patients with Polycythemia Vera and Essential Thrombocythemia showed that this over expression is mainly observed in JAK2WT PMF patients. This is associated with a higher proportion of FLT3+CD34+CD41+ cells in the blood of patients. Analysis of FLT3 membrane expression in MK-derived CD34+ cultures revealed that its expression was maintained all along MK differentiation in patients in contrast to healthy donors. Such a higher expression of FLT3 is associated with an increased concentration of its ligand in the platelet rich plasma from patients, independently of their JAK2 mutational status. The role of FLT3 in the regulation of hematopoiesis incited us to analyse whether its alteration could take part in the myeloproliferation and dysmegakaryopoiesis that characterizes PMF. A flow cytometry analysis of FLT3-downstream MAPK activation in PMF CD34+ cells showed a hyperphosphorylation of p38 and JNK as compared to CD34+ cells from normal blood. This phosphorylation was maintained in PMF MK-derived CD34+ cells at day 10. Addition of PD98059, a MAPK inhibitor, induced a dose dependent restoration of the in vitro megakaryopoiesis in PMF as shown by an increase in MK ploidy with apparition of 32N cells associated with a mature cytological aspect and an increase in CD41, CD42a and CD9 MK differentiation marker expression. PD98059 also increased the MK clonogenicity of CD34+ cells from all patients tested (5/5) as compared to healthy donors. Preliminary results using a specific chemical inhibitor of FLT3 in MK-derived CD34+ cell cultures reinforced the involvement of FLT3 in PMF MK differentiation. In presence of FLT3 ligand, the FLT3 mediated MAPK hyperphosphorylation in PMF MK cultures (D6) is reversed by either PD98059 or UO126, another ERK inhibitor and is accompanied by a slight increase in proliferative MK. This effect is not observed in MK cultures from normal CD34+ cells. Surprisingly, ligation of FLT3 by a monoclonal anti-FLT3 antibody in CD34+ cell cultures resulted in an increase MK proliferation. In conclusion, this work shows a deregulation of FLT3 and MAPK pathway in the PMF CD34+ cells and suggests that the persistence of the FLT3 mediated MAPK activation participates in the dysmegakaryopoiesis of PMF patients.

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