Relationship between selectin-mediated rolling of hematopoietic stem and progenitor cells and progression in hematopoietic development

Current understanding of the adhesion molecules and mechanisms regulating hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow is limited. In contrast, the process by which mature leukocytes are able to home to and extravasate out of blood vessels at sites of inflammation has been well characterized and invites comparison. We studied the interaction of human HSPC from adult bone marrow (ABM) and fetal liver (FL) with E-, P-, and L-selectin immobilized in a flow chamber. CD34+ HSPC from both ABM and FL rolled avidly on E-, P-, and L-selectin across a range of physiologic shear rates, indicating the presence of ligands for all three selectins on HSPC. Results indicate that CD34+ ABM and FL cells roll more efficiently (to a greater extent and more slowly) than more differentiated CD34− cells, especially on P- and L-selectin. In a similar fashion, increased rolling efficiency was seen with CD34+CD38− ABM cells when compared with committed progenitor cells of the CD34+CD38+ phenotype. Rolling of CD34+ ABM cells on P-selectin could be partially inhibited by monoclonal antibody (mAb) against PSGL-1, and was not inhibited by a mAb against CD34, suggesting that HSPC have unique carbohydrate repertoires that facilitate selectin-mediated rolling. Our results provide direct evidence of selectin ligands on HSPC under physiologic flow conditions and are the first to show a correlation between the maturity of HSPC during development and rolling efficiency on selectins, suggesting a mechanism by which HSPC subsets may differentially home to the extravascular spaces of the bone marrow.

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Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽

10.1182/blood.v106.11.1387.1387 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1387-1387

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Fetal Liver ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

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AC133, a Novel Marker for Human Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v90.12.5002 ◽

1997 ◽

Vol 90 (12) ◽

pp. 5002-5012 ◽

Cited By ~ 1209

Author(s):

Amy H. Yin ◽

Sheri Miraglia ◽

Esmail D. Zanjani ◽

Graca Almeida-Porada ◽

Makio Ogawa ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Fetal Liver ◽

Umbilical Vein ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Fetal Sheep ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract AC133 is one of a new panel of murine hybridoma lines producing monoclonal IgG antibodies (mAbs) to a novel stem cell glycoprotein antigen with a molecular weight of 120 kD. AC133 antigen is selectively expressed on CD34bright hematopoietic stem and progenitor cells (progenitors) derived from human fetal liver and bone marrow, and blood. It is not detectable on other blood cells, cultured human umbilical vein endothelial cells (HUVECs), fibroblast cell lines, or the myeloid leukemia cell line KG1a by standard flow cytometric procedures. All of the noncommitted CD34+ cell population, as well as the majority of CD34+ cells committed to the granulocytic/monocytic pathway, are stained with AC133 antibody. In vitro clonogenicity assays have demonstrated that the CD34+AC133+ double-positive population from adult bone marrow contains the majority of the CFU-GM, a proportion of the CFU-Mix, and a minor population of BFU-E. The CD34dim and AC133− population has been shown to contain the remaining progenitor cells. AC133-selected cells engraft successfully in a fetal sheep transplantation model, and human cells harvested from chimeric fetal sheep bone marrow have been shown to successfully engraft secondary recipients, providing evidence for the long-term repopulating potential of AC133+ cells. A cDNA coding for AC133 antigen has been isolated, which codes for a polypeptide consisting of 865 amino acids (aa) with a predicted size of 97 kD. This antigen is modeled as a 5-transmembrane molecule, a structure that is novel among known cell surface structures. AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for both short- and long-term engraftment, in transplant situations, for studies of ex vivo expansion strategies, and for gene therapy.

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

Blood ◽

10.1182/blood.v114.22.4593.4593 ◽

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.

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The Combination of AMD3100 + G-CSF Restores the Ineffective Hematopoietic Stem Cell Mobilization with G-CSF Alone in a Thalassemic Mouse Model.

Blood ◽

10.1182/blood.v114.22.3234.3234 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3234-3234

Author(s):

Evangelia Yannaki ◽

Nikoleta Psatha ◽

Maria Demertzi ◽

Evangelia Athanasiou ◽

Eleni Sgouramali ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Steady State ◽

Mouse Model ◽

Progenitor Cells ◽

State Condition ◽

Hematopoietic Stem ◽

Steady State Condition ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract Abstract 3234 Poster Board III-171 Gene therapy has been recently postulated as a realistic therapeutic potential for thalassemia and the mobilized autologous hematopoietic stem cells (HSCs) may represent the preferable source of stem cells for genetic modification due to the higher yield of HSCs compared to conventional bone marrow (bm) harvest. We have previously shown (manuscript under revision) that G-CSF mobilization in the HBBth-3 thalassemic mouse model is less efficient compared to normal C57Bl6 strain, mainly due to increased trapping of hematopoietic stem (Lin-sca-1+ckit+–LSK) and progenitor cells (CFU-GM) in the enlarged thalassemic spleen. The novel mobilizer, AMD3100 (plerixafor, mozobil), has been shown to reversibly bind to CXCR4 and inhibit the interaction between SDF-1 and CXCR4 within the bm microenvironment, resulting in the egress of CD34+ cells into the circulation of healthy donors and cancer patients. The addition of AMD to G-CSF results in even greater increases in circulating CD34+cells. We explored in the current study whether AMD alone or in combination with G-CSF improves the mobilization efficiency of thalassemic mice. C57 and HBBth-3 mice received G-CSF-alone at 250microgr/kgX7 days, AMD-alone at 5mg/kgX3 days or the combination of two with AMD administered in the evening of days 5-7 of G-CSF administration. Hematopoietic tissues (blood, bm, spleen) were collected and the absolute LSK and CFU-GM numbers were calculated based on their frequency within tissues (by FCM and clonogenic assays) in relation to the individual cell count per tissue. AMD-alone didn't significantly affect the HSC yield as compared to G-CSF mobilization in thal mice (LSK/μl blood: 103±85 vs 69±26 p=ns), although it significantly increased the circulating Colony Forming Cells (CFU-GM/ml blood: 1205±533 vs 330±87, p=0,05). In contrast, the AMD+G-CSF combination significantly improved the mobilization efficiency of HBBth-3 mice over the G-CSF-treated group (LSK cells/μl blood: 224±104 vs 69±26 p=0,04, CFU-GM/ml blood: 1671±984 vs 330±87 p=0,05, respectively) at levels comparable to normal mice treated with G-CSF (LSK cells/ μl blood: 241±167, CFU-GM/ml blood: 1235±1140, respectively). AMD induced a “detachment” of stem cells from the bm because reduced numbers of bm LSK cells were counted in the AMD-alone group as compared to the untreated group (LSK/2 femurs×103: 692±429 vs 1687±1016, respectively, p=0,05). This was in contrast to the marrow hyperplasia caused by G-CSF over the steady-state condition (LSK/2 femurs×103: 2684±1743 vs 1687±1016 p=0,02 / CFU-GM/2femurs:111.841±15.391 vs 76.774±31.728 p=0,01). Consequently, the combination of AMD+G-CSF resulted in increased numbers of circulating stem and progenitor cells without inducing marrow hyperplasia as compared to steady-state condition (LSK/2femurs×103: 1681±862 vs 1686±1017, p=ns / CFU-GM/2femurs: 76.774±31.728 vs 82.905±26.277, p=ns). AMD, also in contrast to G-CSF, did not cause increased trapping of stem and progenitor cells in the spleen compared to the untreated condition (LSK cells/spleen×103: 4738±2970 vs 8303±4166 p=ns / CFU-GM/spleen:146.269±93.174 vs 98.518±25.549, p=ns). However, the combination of AMD+G-CSF still resulted in splenic sequestration of progenitor cells (CFU-GM/spleen: 412.176±157.417 vs 98.518±25.549, p=0,0003) but not of LSK cells (LSK cells/spleen×103: 10.200±7.260 vs 8.300±4.166 p=ns). Overall, the combination of AMD3100+G-CSF seems to restore the less efficient mobilization in a thalassemic mouse model. This combination may prove beneficial in a GT setting for obtaining the high numbers of HSCs needed for genetic correction. In addition, the combination of AMD3100+G-CSF, by avoiding the marrow hyperplasia induced by G-CSF alone, indicates a better safety profile because it will not further burden the hyperplastic –due to the increased erythroid demand and the intramarrow destruction of erythroblasts-thalassemic bone marrow. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v128.22.5048.5048 ◽

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.

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P-Selectin–Coated Microtube for Enrichment of CD34+ Hematopoietic Stem and Progenitor Cells from Human Bone Marrow

Clinical Chemistry ◽

10.1373/clinchem.2007.089896 ◽

2008 ◽

Vol 54 (1) ◽

pp. 77-85 ◽

Cited By ~ 37

Author(s):

Srinivas D Narasipura ◽

Joel C Wojciechowski ◽

Nichola Charles ◽

Jane L Liesveld ◽

Michael R King

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Adhesion Molecule ◽

Mononuclear Cells ◽

Air Embolism ◽

Cell Capture ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Coated Surfaces

Abstract Background: Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematologic disorders and in basic stem cell research. Primitive CD34+ HSPCs have demonstrated stronger rolling adhesion on selectins than mature CD34− mononuclear cells (MNCs). We have exploited this differential rolling behavior to capture and purify HSPCs from bone marrow by perfusing MNCs through selectin-coated microtubes. Methods: Bone marrow MNCs were perfused through the cell-capture microtubes coated with adhesion molecules. We washed the device lumen and visualized and estimated captured cells by video microscopy. Adherent cells were eluted by high shear, calcium-free buffer, and air embolism. We used immunofluorescence staining followed by flow cytometry to analyze CD34+ HSPCs. Results: CD34+ HSPC purity of cells captured in adhesion molecule–coated devices was significantly higher than the fraction of CD34+ cells found in bone marrow MNCs [mean (SE) 2.5% (0.8%)]. P-selectin–coated surfaces yielded 16% to 20% CD34+ cell purity, whereas antibody-coated surfaces yielded 12% to 18%. Although CD34+ cell purity was comparable between selectin and antibody surfaces, the total number of CD34+ HSPCs captured was significantly higher in P-selectin devices (approximately 5.7 × 104 to 7.1 × 104) than antibody devices (approximately 1.74 × 104 to 2.61 × 104). Conclusions: P-selectin can be used in a compact flow device to capture HSPCs. Selectin-mediated capture of CD34+ HSPCs resulted in enrichment approximately 8-fold higher than the CD34+ cell population from bone marrow MNCs. This study supports the hypothesis that flow-based, adhesion molecule–mediated capture may be a viable alternative approach to the capture and purification of HSPCs.

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

Blood ◽

10.1182/blood.v99.6.2037 ◽

2002 ◽

Vol 99 (6) ◽

pp. 2037-2044 ◽

Cited By ~ 113

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.

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AC133, a Novel Marker for Human Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v90.12.5002.5002_5002_5012 ◽

1997 ◽

Vol 90 (12) ◽

pp. 5002-5012 ◽

Cited By ~ 27

Author(s):

Amy H. Yin ◽

Sheri Miraglia ◽

Esmail D. Zanjani ◽

Graca Almeida-Porada ◽

Makio Ogawa ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Fetal Liver ◽

Umbilical Vein ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Fetal Sheep ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

AC133 is one of a new panel of murine hybridoma lines producing monoclonal IgG antibodies (mAbs) to a novel stem cell glycoprotein antigen with a molecular weight of 120 kD. AC133 antigen is selectively expressed on CD34bright hematopoietic stem and progenitor cells (progenitors) derived from human fetal liver and bone marrow, and blood. It is not detectable on other blood cells, cultured human umbilical vein endothelial cells (HUVECs), fibroblast cell lines, or the myeloid leukemia cell line KG1a by standard flow cytometric procedures. All of the noncommitted CD34+ cell population, as well as the majority of CD34+ cells committed to the granulocytic/monocytic pathway, are stained with AC133 antibody. In vitro clonogenicity assays have demonstrated that the CD34+AC133+ double-positive population from adult bone marrow contains the majority of the CFU-GM, a proportion of the CFU-Mix, and a minor population of BFU-E. The CD34dim and AC133− population has been shown to contain the remaining progenitor cells. AC133-selected cells engraft successfully in a fetal sheep transplantation model, and human cells harvested from chimeric fetal sheep bone marrow have been shown to successfully engraft secondary recipients, providing evidence for the long-term repopulating potential of AC133+ cells. A cDNA coding for AC133 antigen has been isolated, which codes for a polypeptide consisting of 865 amino acids (aa) with a predicted size of 97 kD. This antigen is modeled as a 5-transmembrane molecule, a structure that is novel among known cell surface structures. AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for both short- and long-term engraftment, in transplant situations, for studies of ex vivo expansion strategies, and for gene therapy.

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Ex Vivo Expansion of Cord Blood Hematopoietic Stem and Progenitor Cells in a Wharton’s Jelly and Bone Marrow Stromal Cell Based Co-Culture System

Blood ◽

10.1182/blood.v124.21.1129.1129 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1129-1129

Author(s):

Dandan Li ◽

Omar S. Aljitawi ◽

Richard A. Hopkins

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Progenitor Cells ◽

Stromal Cell ◽

Bone Marrow Stromal Cell ◽

Hematopoietic Stem ◽

Marrow Stromal Cell ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Cord Blood Cd34

Abstract Umbilical cord blood (UCB) is of great value by providing transplantable hematopoietic stem and progenitor cells (HSPCs). Compared with HSPCs from adult bone marrow and periferial blood, UCB cells are more primitive with higher proliferation ability, and UCB HSPC transplantation requires less HLA matching. The major problems in UCB transplantation is the limited number of transplantable cells in each unit which are often insufficient for transplantation in adults In order to elucidate the effects of the main components of bone marrow on cord blood CD34+ expansion, freshly enriched cord blood CD34+ cells were cultured in contact with bone marrow stromal cell (BM-MSC) monolayer, BM-MSCs pre-seeded in decellularized Wharton's jelly matrix (DWJM), and in DWJM alone, as well as separated by a transwell preventing the physical contact between CD34+ cells and BM-MSCs in a medium supplemented with a cytokine cocktail including Flt-3 igand, stem cell factor, and thrombopoietin. Expansion patterns were analyzed by CD34+ and CD45+ expansion, as well as colony-forming unit (CFU) assay. We found that the number of CFU increases significantly in cord blood CD34+ cells co-cultured with BM-MSCs in DWJM compared to that of control. Particularly, the increase in CFU-GEMM, BFU-E and CFU-GM number is more significant compared to other colonies. Importantly, DWJM alone is not able to increase CFU numbers compared to that of the control indicating there is a synergistic effect between DWJM and BM-MSCs on cell stemness. Surprisingly, BM-MSCs in DWJM also increses CD34+ cell expansion by 2 to 3 fold after one week culture, presumably from enhanced ablilty of self-renewal from CD34+ cells. Therefore, our data suggest DWJM synergizes with BM-MSCs to increase CD34 cell stemness, which can potentially be used in the clinic therapy. Disclosures No relevant conflicts of interest to declare.

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