scholarly journals The Glucocorticoid Receptor-Dependent Stress Response in Human Erythropoiesis Is BCL11A-Dependent

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 939-939
Author(s):  
Lilian Varricchio ◽  
Eliza Geer ◽  
Fabrizio Martelli ◽  
Maria Mazzarini ◽  
Alister Funnell ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Progenitor Cells ◽  
Stem Cell Marker ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Stress Erythropoiesis ◽  
Restricted Form ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Cortisol Levels

Abstract Extrapolations from data in mouse models and from human erythroid cells expanded ex vivo with the glucocorticoid receptor (GR) agonist Dexamethasone (Dex), suggest that GR plays an important role in the regulation of stress erythropoiesis 1. However, the mechanistic details of stress erythropoiesis are still poorly understood. By exploring the effects of Dex on erythroid expansion of CD34+ cells from a large number of healthy adult donors (n=25), we documented that Dex expands a population of immature erythroid cells which express high levels of BCL11A. In addition, in these cells BCL11A is mostly in the nuclei, compared to cells grown without Dex. These results suggest that GR regulates the transcriptional activity of BCL11A. To validate the role of BCL11A in the Dex response, we made observations in cells from patients with BCL11A deficiencies 2. We found that BCL11A-deficient CD34+ cells generate lower numbers of maturing erythroid cells compared to controls with Dex addition, suggesting that they respond poorly to Dex (Fig 1). Of note, RNAseq analyses indicate that erythroid cells expanded from the patients express levels of BCL11A lower than that of their parent cells while the levels of expression of other genes known to mediate the response to Dex of erythroid cells (ZFP36L2, CDKNIC and PPARA 3-5) expressed by these cells is normal. Further we extended our observations to Cushing's patients with highier cortisol levels, before(V1) and after their treatment(V2) using concurrent age and weight matched healthy controls (MC). There is no significant difference in frequency of CD34+ cells among V1, V2 and MC (range 0.3-2% in all cases). CD34 pos cells from all groups express similar levels of cKIT, IL-3Rβ, CXCR4 and CALR. However, a greater proportion of CD34+ cells from V1 express CD36 and CD110 (the thrombospondin and thrombopoietin receptor) and CD133 (the hematopoietic stem cell marker prominin) than those from V2 and MC (60-80% vs 10-20%), suggesting that the circulating progenitor cells from active Cushing's patients are a unique population likely generated in response to their high cortisol levels given that the phenotype of CD34+ cells from the blood of V2 patients is similar to that found in MC. The phenotype of CD34+ cells from Cushing's patients in vivo is similar to that of CD34+ cells generated in vitro after 2-4 days of culture with Dex 3,6,7. V1 progenitor cells generate similarly large number of immature erythroid cells in culture with and without Dex. By contrast with normal cells, these active cells also express lower levels of the cytoplasm-restricted form of GRα (GRS203) and greater levels of GILZ and BCL11A than normal or V2 cells. In conclusion, the data presented here indicate that GR activation switches the erythroid differentiation program from the steady-state to the stress mode and that activation of BCL11A is part of the response of erythroid cells to Dex. References 1) Varricchio et al Am J Blood Res. 2014;4:53; 2) Funnell et al Blood 2015; 126:89; 3) Ashley et al JCI 2020;130:2097; 4) Zhang L et al Nature 2013;499:92; 5) Lee et al Nature 2015;522:474; 6) Heideveld et al Haematologica 2015; 100:1396; 7) Xiang et al Blood 2015;125:1803. Figure 1 Figure 1. Disclosures Migliaccio: Dompe farmaceutici Spa R&D: Other: received funding for reserach .

scholarly journals Wharton’s jelly mesenchymal stem cell-based or umbilical vein endothelial cell-based serum-free coculture with cytokines supports the ex vivo expansion/maintenance of cord blood hematopoietic stem/progenitor cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Qiuyang Li ◽  
Dewan Zhao ◽  
Qiang Chen ◽  
Maowen Luo ◽  
Jingcao Huang ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Feeder Layer ◽  
Hematopoietic Stem ◽  
Serum Free ◽  
Cd34 Cells ◽  
Significant Difference

Abstract Background The umbilical cord blood (UCB) has been widely accepted as an alternative source of hematopoietic stem/progenitor cells (HSPCs) for transplantation, and its use in adults is still restricted because of low absolute numbers. To overcome this obstacle, expansion of UCB-HSPCs under feeder cell-based coculture is a promising possibility. In this study, we explored UCB-CD34+ cells ex vivo expansion using Wharton’s jelly mesenchymal stem cells (WJ-MSCs) or umbilical vein endothelial cells (UVECs) as feeder layer-based serum-free coculture system with a cocktail of cytokines. Methods UCB-CD34+ cells were cultured in five different coculture conditions composed of umbilical cord stromal cells (WJ-MSCs or UVECs) with or without a cocktail of cytokines (SCF, FLT3L, and TPO). The cultured cells were harvested at day 10 and analyzed for phenotypes and functionalities, including total nuclear cells (TNCs), CD34+ cells, CD34+CD38− cells, colony-forming unit (CFU) for committed progenitors, and long-term culture initiating cells (LTC-ICs) for HSPCs. Results Our work showed the numbers of TNC cells, CD34+ cells, and CD34+CD38− cells were expanded under five coculture conditions, and the feeder layer-based cocultures further promoted the expansion. The numbers of colonies of CFU-GM, CFU-E/BFU-E, and CFU-GEMM in the cocultures with cytokines were significantly higher than their counterparts at day 0 (p < 0.05), while no significant difference (p > 0.05) in those without the addition of cytokines. The numbers of LTC-ICs were increased both under the WJ-MSCs and UVECs with cytokine cocultures, but only in the UVECs group showed a significant difference (p < 0.05), and were decreased under conditions without cytokine (p < 0.05). Conclusion Our data demonstrate that both WJ-MSCs and UVECs as feeder layer could efficiently support the expansion of UCB-CD34+ cells in synergy with SCF, FLT3L, and TPO under serum-free culture condition. The UVECs combined with the 3GF cytokine cocktail could maintain the growth of LTC-ICs derived from UCB-CD34+ cells and even expand to some extent.

scholarly journals Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2812-2821 ◽  
Author(s):  
Fabiana Perna ◽  
Nadia Gurvich ◽  
Ruben Hoya-Arias ◽  
Omar Abdel-Wahab ◽  
Ross L. Levine ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Myeloproliferative Neoplasms ◽  
Erythroid Differentiation ◽  
Polycomb Group ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Cd34 Cells

Abstract L3MBTL1, the human homolog of the Drosophila L(3)MBT polycomb group tumor suppressor gene, is located on chromosome 20q12, within the common deleted region identified in patients with 20q deletion-associated polycythemia vera, myelodysplastic syndrome, and acute myeloid leukemia. L3MBTL1 is expressed within hematopoietic CD34+ cells; thus, it may contribute to the pathogenesis of these disorders. To define its role in hematopoiesis, we knocked down L3MBTL1 expression in primary hematopoietic stem/progenitor (ie, CD34+) cells isolated from human cord blood (using short hairpin RNAs) and observed an enhanced commitment to and acceleration of erythroid differentiation. Consistent with this effect, overexpression of L3MBTL1 in primary hematopoietic CD34+ cells as well as in 20q− cell lines restricted erythroid differentiation. Furthermore, L3MBTL1 levels decrease during hemin-induced erythroid differentiation or erythropoietin exposure, suggesting a specific role for L3MBTL1 down-regulation in enforcing cell fate decisions toward the erythroid lineage. Indeed, L3MBTL1 knockdown enhanced the sensitivity of hematopoietic stem/progenitor cells to erythropoietin (Epo), with increased Epo-induced phosphorylation of STAT5, AKT, and MAPK as well as detectable phosphorylation in the absence of Epo. Our data suggest that haploinsufficiency of L3MBTL1 contributes to some (20q−) myeloproliferative neoplasms, especially polycythemia vera, by promoting erythroid differentiation.

scholarly journals Notch-Mediated Expansion of Human Hematopoietic Stem and Progenitor Cells By Culture Under Hypoxia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Daisuke Araki ◽  
Stefan Cordes ◽  
Fayaz Seifuddin ◽  
Luigi J. Alvarado ◽  
Mehdi Pirooznia ◽  
...  
Keyword(s):  
Er Stress ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Human Adult ◽  
Hypoxic Conditions ◽  
Cd34 Cells ◽  
Human Hscs ◽  
Ex Vivo Culture ◽  
Cells Cultured

Notch activation in human CD34+ hematopoietic stem/progenitor cells (HSPCs) by treatment with Delta1 ligand has enabled clinically relevant ex vivo expansion of short-term HSPCs. However, sustained engraftment of the expanded cells was not observed after transplantation, suggesting ineffective expansion of hematopoietic stem cells with long-term repopulating activity (LTR-HSCs). Recent studies have highlighted how increased proliferative demand in culture can trigger endoplasmic reticulum (ER) stress and impair HSC function. Here, we investigated whether ex vivo culture of HSPCs under hypoxia might limit cellular ER stress and thus offer a simple approach to preserve functional HSCs under high proliferative conditions, such as those promoted in culture with Delta1. Human adult mobilized CD34+ cells were cultured for 21 days under normoxia (21% O2) or hypoxia (2% O2) in vessels coated with optimized concentrations of Delta1. We observed enhanced progenitor cell activity within the CD34+ cell population treated with Delta1 in hypoxia, but the benefits provided by low-oxygen cultures were most notable in the primitive HSC compartment. At optimal coating densities of Delta1, the frequency of LTR-HSCs measured by limiting dilution analysis 16 weeks after transplantation into NSG mice was 4.9- and 4.2-fold higher in hypoxic cultures (1 in 1,586 CD34+ cells) compared with uncultured cells (1 in 7,706) and the normoxia group (1 in 5,090), respectively. Conversely, we observed no difference in expression of the homing CXCR4 receptor between cells cultured under normoxic and hypoxic conditions, indicating that hypoxia increased the absolute numbers of LTR-HSCs but not their homing potential after transplantation. To corroborate these findings molecularly, we performed transcriptomic analyses and found significant upregulation of a distinct HSC gene expression signature in cells cultured with Delta1 in hypoxia (Fig. A). Collectively, these data show that hypoxia supports a superior ex vivo expansion of human HSCs with LTR activity compared with normoxia at optimized densities of Delta1. To clarify how hypoxia improved Notch-mediated expansion of LTR-HSCs, we performed scRNA-seq of CD34+ cells treated with Delta1 under normoxic or hypoxic conditions. We identified 6 distinct clusters (clusters 0 to 5) in dimension-reduction (UMAP) analysis, with a comparable distribution of cells per cluster between normoxic and hypoxic cultures. Most clusters could be computationally assigned to a defined hematopoietic subpopulation, including progenitor cells (clusters 0 to 4) and a single transcriptionally defined HSC population (cluster 5). To assess the relative impact of normoxia and hypoxia on the HSC compartment, we performed gene set enrichment analysis (GSEA) of cells within HSC cluster 5 from each culture condition. A total of 32 genes were differentially expressed, and pathways indicative of cellular ER stress (unfolded protein response [UPR], heat shock protein [HSP] and chaperone) were significantly downregulated in hypoxia-treated cells relative to normoxic cultures (Fig. B). When examining expression of cluster 5 top differentially expressed genes across all cell clusters, we observed a more prominent upregulation of these genes within transcriptionally defined HSCs exposed to normoxia relative to more mature progenitors (Fig. C, red plots). Hypoxia lessened the cellular stress response in both progenitors and HSCs, but the mitigation was more apparent in the HSC population (Fig. C, grey plots), and decreased apoptosis was observed only within the HSC-enriched cluster 5 (Fig. D). These findings are consistent with several reports indicating that HSCs are more vulnerable to strong ER stress than downstream progenitors due to their lower protein folding capacity. In conclusion, we provide evidence that ex vivo culture of human adult CD34+ cells under hypoxic conditions enables a superior Delta1-mediated expansion of hematopoietic cells with LTR activity compared with normoxic cultures. Our data suggest a two-pronged mechanism by which optimal ectopic activation of Notch signaling in human HSCs promotes their self-renewal, and culture under hypoxia mitigates ER stress triggered by the increased proliferative demand, resulting in enhanced survival of expanding HSCs. This clinically feasible approach may be useful to improve outcomes of cellular therapeutics. Disclosures No relevant conflicts of interest to declare.

scholarly journals Very Small Embryonic-Like Stem Cells, Endothelial Progenitor Cells, and Different Monocyte Subsets Are Effectively Mobilized in Acute Lymphoblastic Leukemia Patients after G-CSF Treatment

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Andrzej Eljaszewicz ◽  
Lukasz Bolkun ◽  
Kamil Grubczak ◽  
Malgorzata Rusak ◽  
Tomasz Wasiluk ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Lymphoblastic Leukemia ◽  
Monocyte Subsets ◽  
Endothelial Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Background. Acute lymphoblastic leukemia (ALL) is a malignant disease of lymphoid progenitor cells. ALL chemotherapy is associated with numerous side effects including neutropenia that is routinely prevented by the administration of growth factors such as granulocyte colony-stimulating factor (G-CSF). To date, the effects of G-CSF treatment on the level of mobilization of different stem and progenitor cells in ALL patients subjected to clinically effective chemotherapy have not been fully elucidated. Therefore, in this study we aimed to assess the effect of administration of G-CSF to ALL patients on mobilization of other than hematopoietic stem cell (HSCs) subsets, namely, very small embryonic-like stem cells (VSELs), endothelial progenitor cells (EPCs), and different monocyte subsets. Methods. We used multicolor flow cytometry to quantitate numbers of CD34+ cells, hematopoietic stem cells (HSCs), VSELs, EPCs, and different monocyte subsets in the peripheral blood of ALL patients and normal age-matched blood donors. Results. We showed that ALL patients following chemotherapy, when compared to healthy donors, presented with significantly lower numbers of CD34+ cells, HSCs, VSELs, and CD14+ monocytes, but not EPCs. Moreover, we found that G-CSF administration induced effective mobilization of all the abovementioned progenitor and stem cell subsets with high regenerative and proangiogenic potential. Conclusion. These findings contribute to better understanding the beneficial clinical effect of G-CSF administration in ALL patients following successful chemotherapy.

scholarly journals Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- progenitor cells

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1218-1227 ◽  
Author(s):  
LW Terstappen ◽  
S Huang ◽  
M Safford ◽  
PM Lansdorp ◽  
MR Loken
Keyword(s):  
Progenitor Cells ◽  
Lineage Commitment ◽  
Multiparameter Flow Cytometry ◽  
Differentiation Markers ◽  
Single Class ◽  
Hematopoietic Stem ◽  
Cd34 Antigen ◽  
Cd34 Cells ◽  
Cell Fractions ◽  
Cd38 Antigen

Abstract Multiparameter flow cytometry was applied on normal human bone marrow (BM) cells to study the lineage commitment of progenitor cells ie, CD34+ cells. Lineage commitment of the CD34+ cells into the erythroid lineage was assessed by the coexpression of high levels of the CD71 antigen, the myeloid lineage by coexpression of the CD33 antigen and the B-lymphoid lineage by the CD10 antigen. Three color immunofluorescence experiments showed that all CD34+ BM cells that expressed the CD71, CD33, and CD10 antigens, concurrently stained brightly with anti-CD38 monoclonal antibodies (MoAbs). In addition, the CD38 antigen was brightly expressed on early T lymphocytes in human thymus, characterized by CD34, CD5, and CD7 expression. Only 1% of the CD34+ cells, 0.01% of nucleated cells in normal BM, did not express the CD38 antigen. The CD34+, CD38- cell population lacked differentiation markers and were homogeneous primitive blast cells by morphology. In contrast the CD34+, CD38 bright cell populations were heterogeneous in morphology and contained myeloblasts and erythroblasts, as well as lymphoblasts. These features are in agreement with properties expected from putative pluripotent hematopoietic stem cells; indeed, the CD34 antigen density decreased concurrently with increasing CD38 antigen density suggesting an upregulation of the CD38 antigen on differentiation of the CD34+ cells. Further evidence for a strong enrichment of early hematopoietic precursors in the CD34+, CD38- cell fraction was obtained from culture experiments in which CD34+ cell fractions with increasing density of the CD38 antigen were sorted singularly and assayed for blast colony formation. On day 14 of incubation, interleukin-3 (IL-3), IL-6, and GM-CSF, G-CSF, and erythropoietin (Epo) were added in each well. Twenty-five percent of the single sorted cells that expressed CD34 but lacked CD38 antigen gave rise to primitive colonies 28 to 34 days after cell sorting. The ability to form primitive colonies decreased rapidly with increasing density of the CD38 antigen. During 120 days of culture, up to five sequential generations of colonies were obtained after replating of the first-generation primitive colonies. This study provides direct evidence for the existence of a single class of progenitors with extensive proliferative capacity in human BM and provides an experimental approach for their purification, manipulation, and further characterization.

scholarly journals Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis

Blood ◽  
1994 ◽  
Vol 83 (10) ◽  
pp. 2844-2850 ◽  
Author(s):  
N Yanai ◽  
C Sekine ◽  
H Yagita ◽  
M Obinata
Keyword(s):  
Progenitor Cells ◽  
Adhesion Molecules ◽  
Stromal Cells ◽  
Ligand Molecule ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Erythroid Progenitor Cells ◽  
Stem And Progenitor Cells ◽  
Activation Antigen

Abstract Adhesion molecules are required for development of hematopoietic stem and progenitor cells in the respective hematopoietic microenvironments. We previously showed that development of the erythroid progenitor cells is dependent on their direct adhesion to the stroma cells established from the erythropoietic organs. In this stroma-dependent erythropoiesis, we examined the role of adhesion molecules in erythropoiesis by blocking antibodies. The development of the erythroid cells on stroma cells was inhibited by anti-very late activation antigen-4 (VLA-4 integrin) antibody, but not by anti-VLA-5 antibody, although the erythroid cells express both VLA-4 and VLA-5. Whereas high levels of expression of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin, ligands for VLA-4, were detected in the stroma cells, the adhesion and development of the erythroid progenitor cells were partly inhibited by the blocking antibody against VCAM-1. VLA-5 and fibronectin could mediate adhesion of the erythroid progenitor cells to the stromal cells, but the adhesion itself may not be sufficient for the stroma-supported erythropoiesis. The stromal cells may support erythroid development by the adhesion through a new ligand molecule(s) for VLA-4 in addition to VCAM-1, and such collaborative interaction may provide adequate signaling for the erythroid progenitor cells in the erythropoietic microenvironment.

scholarly journals Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 811
Author(s):  
Pranav Oberoi ◽  
Kathrina Kamenjarin ◽  
Jose Francisco Villena Ossa ◽  
Barbara Uherek ◽  
Halvard Bönig ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 952-958 ◽  
Author(s):  
Mario Peichev ◽  
Afzal J. Naiyer ◽  
Daniel Pereira ◽  
Zhenping Zhu ◽  
William J. Lane ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Left Ventricular ◽  
Plating Efficiency ◽  
Stem Cell Marker ◽  
Ventricular Assist Devices ◽  
Human Model ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Endothelial Precursors

Emerging data suggest that a subset of circulating human CD34+ cells have phenotypic features of endothelial cells. Whether these cells are sloughed mature endothelial cells or functional circulating endothelial precursors (CEPs) is not known. Using monoclonal antibodies (MoAbs) to the extracellular domain of the human vascular endothelial receptor-2 (VEGFR-2), we have shown that 1.2 ± 0.3% of CD34+ cells isolated from fetal liver (FL), 2 ± 0.5% from mobilized peripheral blood, and 1.4 ± 0.5% from cord blood were VEGFR-2+. In addition, most CD34+VEGFR-2+ cells express hematopoietic stem cell marker AC133. Because mature endothelial cells do not express AC133, coexpression of VEGFR-2 and AC133 on CD34+ cells phenotypically identifies a unique population of CEPs. CD34+VEGFR-2+ cells express endothelial-specific markers, including VE-cadherin and E-selectin. Also, virtually all CD34+VEGFR-2+ cells express the chemokine receptor CXCR4 and migrate in response to stromal-derived factor (SDF)-1 or VEGF. To quantitate the plating efficiency of CD34+ cells that give rise to endothelial colonies, CD34+ cells derived from FL were incubated with VEGF and fibroblast growth factor (FGF)-2. Subsequent isolation and plating of nonadherent FL-derived VEGFR-2+ cells with VEGF and FGF-2 resulted in differentiation of AC133+VEGFR-2+ cells into adherent AC133−VEGFR-2+Ac-LDL+(acetylated low-density lipoprotein) colonies (plating efficiency of 3%). In an in vivo human model, we have found that the neo-intima formed on the surface of left ventricular assist devices is colonized with AC133+VEGFR-2+ cells. These data suggest that circulating CD34+ cells expressing VEGFR-2 and AC133 constitute a phenotypically and functionally distinct population of circulating endothelial cells that may play a role in neo-angiogenesis.

scholarly journals Granzyme B and perforin lytic proteins are expressed in CD34+ peripheral blood progenitor cells mobilized by chemotherapy and granulocyte colony-stimulating factor

Blood ◽  
1995 ◽  
Vol 86 (9) ◽  
pp. 3500-3506 ◽  
Author(s):  
C Berthou ◽  
JP Marolleau ◽  
C Lafaurie ◽  
A Soulie ◽  
L Dal Cortivo ◽  
...  
Keyword(s):  
Cell Line ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Granzyme B ◽  
Cellular Adhesion ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stimulating Factor

Granzyme B and perforin are cytoplasmic granule-associated proteins used by cytotoxic T lymphocytes and natural killer (NK) cells to kill their targets. However, granzyme B gene expression has also been detected in a non-cytotoxic hematopoietic murine multipotent stem cell line, FDCP-Mix. The objective of the present study was to investigate whether granzyme B and perforin could be expressed in human hematopoietic CD34+ cells and if present, discover what their physiologic relevance could be. The primitive CD34+ human cell line KG1a was investigated first and was found to express granzyme B and perforin. Highly purified hematopoietic stem/progenitor cells were then selected using the CD34 surface antigen as marker. Steady-state bone marrow (BM) CD34+ cells did not contain these proteins. Peripheral blood (PB) CD34+ cells, which had been induced to circulate, were also analyzed. After chemotherapy (CT) and granulocyte colony-stimulating factor (G-CSF) treatment, CD34+ cells strongly expressed mRNAs and proteins of granzyme B and perforin. In contrast, CD34+ cells mobilized by G-CSF alone were negative. Western blot analysis further showed that granzyme B and perforin proteins were identical in CD34+ cells and activated PBLs. Such proteins might be implicated in the highly efficient migration of CD34+ stem/progenitor cells from BM to PB after CT and G-CSF treatment. The cellular adhesion mechanisms involved in the BM homing of CD34+ cells are disrupted at least temporarily after CT. The Asp-ase proteolytic activity of granzyme B on extracellular matrix proteins could be used by progenitor cells for their rapid detachment from BM stromal cells and perforin might facilitate their migration across the endothelial cell barrier.

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