CD166+CD34+ Cells Exhibit Marked Functional Differences During Fetal and Adult Life

Abstract ALCAM/CD166 is expressed from the onset of hematopoiesis in the yolk sac and in a variety of hematopoietic tissues throughout ontogeny. Both hematopoietic and stromal cells in the AGM region, fetal liver, and fetal and adult marrow express this molecule. CD166 double knockout mice are viable and fertile, without any major blood defects, but their microenvironment and hematopoietic stem cells (HSC) exhibit deficiencies in their ability to support and engraft long-term, respectively. In order to further study the role of CD166 in hematopoiesis, we characterized, during ontogeny, the origin, function, and sub-populations of CD166+ cells in different hematopoietic organs. To this end, we used flow cytometry, confocal microscopy, and colony-forming assays to analyze human fetal liver (FL) at 18 and 20 gestational weeks (gw), bone marrow (BM) from 10 to 20gw, and adult BM. Flow cytometric analysis of FL at 18 and 20gw demonstrated that although 3±1% of liver cells at this age were CD166+, less than 1% were endothelial CD166+CD34+ cells, and no hematopoietic CD166+CD34+CD45+ cells were detected. In fetal BM, CD166+ cells emerged after 15gw, expressed Flk-1 and CD34, and their percentage increased progressively with gestational age. Flow cytometric analysis at 20gw showed that 1.5±1% of cells were CD166+CD34+, of which 93±0.5% were CD45+. Human adult BM contained 2±0.5% of CD166+CD34+ cells, of which only 66±0.6% were CD45+. In order to functionally characterize CD166+CD34+ cells from adult and fetal BM (20gw), we plated these cells in mesenchymal cell growth medium (MSCGM), endothelial growth medium (EGM-2), and complete methylcellulose (MC). MSCGM and EGM-2 did not support growth and expansion of fetal or adult CD166+CD34+ cells. Quantification of the hematopoietic colony-forming potential of these cells demonstrated that fetal CD166+CD34+ generated/1000 cells: 8±1 Blast; 27±2 CFU-Mix; 51±10 CFU-GM; and 0 BFU-E, while adult CD166+CD34+ gave rise to 7 Blast; 17 CFU-Mix; 36 CFU-GM; and 10 BFU, demonstrating differences in the hematopoietic potential of these cells. Furthermore, at day 12 of MC culture, adherent stromal cells were detected underneath MC, but only in cultures from fetal BM. Characterization of these cells by flow cytometry showed that more than 90±2% of these cells were CD166+CD9+, and 30±5% were CD146+. Furthermore, these stromal CD166+ cells did not express CD34, CD45, CD31, CD209, or CD6. Immunostaining demonstrated that the CD166+CD146+ cells expressed osteopontin and Stro-1. A CD41+CD68+ population of cells was also found. In conclusion, we found that, during ontogeny, expression of CD166 in FL is not associated with hematopoietic cells. In the BM, expression of CD166 is associated with CD34 and Flk2, and its expression on HSC commences later in gestation, suggesting that these cells either arise in the BM, or that CD166 expression is triggered at a certain time point in gestation, probably associated with rapid proliferation of HSC during this time period. Furthermore, we demonstrated that CD34+CD166+ cells from 20gw fetal BM contain hematopoietic and stromal cell populations, while adult BM-derived CD34+ CD166+ cells are exclusively hematopoietic. Disclosures: No relevant conflicts of interest to declare.

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High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo

Blood ◽

10.1182/blood.v95.3.855.003k41_855_862 ◽

2000 ◽

Vol 95 (3) ◽

pp. 855-862 ◽

Cited By ~ 82

Author(s):

Robert A. J. Oostendorp ◽

Julie Audet ◽

Connie J. Eaves

Keyword(s):

Stem Cells ◽

Fetal Liver ◽

Flow Cytometric Analysis ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Flow Cytometric ◽

Differentiation Status ◽

Kinetics Of

The kinetics of proliferation of primitive murine bone marrow (BM) cells stimulated either in vitro with growth factors (fetal liver tyrosine kinase ligand 3 [FL], Steel factor [SF], and interleukin-11 [IL-11], or hyper–IL-6) or in vivo by factors active in myeloablated recipients were examined. Cells were first labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and then incubated overnight prior to isolating CFSE+ cells. After 2 more days in culture, more than 90% of the in vivo lymphomyeloid repopulating activity was associated with the most fluorescent CFSE+ cells (ie, cells that had not yet divided), although this accounted for only 25% of the repopulating stem cells measured in the CFSE+ “start” population. After a total of 4 days in culture (1 day later), 15-fold more stem cells were detected (ie, 4-fold more than the day 1 input number), and these had become (and thereafter remained) exclusively associated with cells that had divided at least once in vitro. Flow cytometric analysis of CFSE+ cells recovered from the BM of transplanted mice indicated that these cells proliferated slightly faster (up to 5 divisions completed within 2 days and up to 8 divisions completed within 3 days in vivo versus 5 and 7 divisions, respectively, in vitro). FL, SF, and ligands which activate gp130 are thus efficient stimulators of transplantable stem cell self-renewal divisions in vitro. The accompanying failure of these cells to accumulate rapidly indicates important changes in their engraftment potential independent of accompanying changes in their differentiation status.

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MT1-MMP Associates with Membrane Lipid Rafts and Is Required for G-CSF-Induced Hematopoietic Stem/Progenitor Cell Mobilization.

Blood ◽

10.1182/blood.v114.22.3536.3536 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3536-3536

Author(s):

Neeta Shirvaikar ◽

Leah A. Marquez-Curtis ◽

Andrew Shaw ◽

A. Robert Turner ◽

Anna Janowska-Wieczorek

Keyword(s):

Flow Cytometry ◽

Steady State ◽

Western Blot ◽

Lipid Rafts ◽

Stromal Cells ◽

Hematopoietic Cells ◽

Membrane Lipid ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 3536 Poster Board III-473 Hematopoietic stem/progenitor cells (HSPC) that have been mobilized from bone marrow (BM) to peripheral blood (PB) by granulocyte-colony stimulating factor (G-CSF) are being used for autologous and allogeneic transplantation. However, the molecular mechanisms of HSPC mobilization are not completely understood. The key molecules and interactions that regulate HSPC mobilization include various adhesion molecules, chemokine stromal cell-derived factor (SDF)-1 and its receptor CXCR4, and proteases including the soluble matrix metalloproteinase (MMP)-9. Membrane type (MT)-1 MMP, which is localized on the leading edge of migrating cells, has strong pericellular proteolytic activity, activates the latent MMPs especially proMMP-2, and has been implicated in mediating migration of tumor cells, monocytes, endothelial as well as CD34+ HSPC. MT1-MMP not only degrades several extracellular matrix molecules in the pericellular space, but also cleaves cell surface molecules such as CXCR4 and CD44, cytokines, and chemokines including SDF-1. In this study we focused on characterizing the role of MT1-MMP during G-CSF-induced migration, its regulation and subcellular localization in HSPC and mature cells. We found that MT1-MMP mRNA and protein expression (as determined by RT-PCR and flow cytometry) in G-CSF-mobilized mature hematopoietic cells (monocytes and neutrophils) as well as immature CD34+ cells was significantly higher than in their steady-state BM counterparts. Moreover, G-CSF stimulation (i) upregulated MT1-MMP transcription (RT-PCR) and protein synthesis (flow cytometry, Western blot, and confocal microscopy) in BM MNC and CD34+ cells but not in BM stromal cells; and (ii) increased their trans-Matrigel chemoinvasion towards an SDF-1 gradient which was inhibited by the MT1-MMP inhibitor epigallocatechin 3-gallate, by anti-MT1-MMP mAb, and by siRNA silencing of MT1-MMP. To determine the effect of high MT1-MMP expression in hematopoietic cells on the BM microenvironment we co-cultured steady-state BM CD34+ cells with BM fibroblasts. Zymographic analysis of the cell-conditioned media revealed that activation of proMMP-2 occurs only when the co-cultures were stimulated with G-CSF indicating that upregulation of MT1-MMP in CD34+ cells is necessary for proMMP-2 activation as media conditioned by CD34+ cells (silenced with MT1-MMP siRNA) co-cultured with stromal cells did not show proMMP-2 activation. We next focused on determining the signaling pathways that regulate MT1-MMP expression and localization in hematopoietic cells including HSPC during G-CSF-induced migration. We found that although G-CSF activated both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways (Western blot), upregulation of MT1-MMP by G-CSF, and proMMP-2 activation were PI3K-dependent. Moreover, we demonstrated for the first time that G-CSF incorporated MT1-MMP to membrane lipid rafts of hematopoietic cells in a PI3K-dependent manner since inhibition of this axis by PI3K inhibitor LY290042 reduced MT1-MMP incorporation, an effect not observed with the MAPK inhibitor PD98059. We further demonstrated that by disrupting raft formation using the cholesterol sequestering agent methyl-beta-cyclodextrin, PI3K phosphorylation was inhibited. Subsequently MT1-MMP incorporation into lipid rafts was abrogated resulting in reduced both proMMP-2 activation and HSPC trans-Matrigel migration. We conclude that G-CSF-induced upregulation of MT1-MMP and its incorporation into membrane lipid rafts of hematopoietic cells contributes to the activation of proMMP-2 and to the generation of a highly proteolytic microenvironment in BM, which facilitates egress of HSPC into circulation. Our results suggest that manipulating MT1-MMP expression could become a new strategy to enhance mobilization of HSPC and improve the outcome of transplantation. Disclosures: No relevant conflicts of interest to declare.

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Characterization of the Hematopoietic Supporting Activity of Osteoblasts Derived from Bone Marrow Mesenchymal Stromal Cells

Blood ◽

10.1182/blood.v124.21.4358.4358 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4358-4358

Author(s):

Manal Alsheikh ◽

Roya Pasha ◽

Nicolas Pineault

Keyword(s):

Bone Marrow ◽

Osteogenic Differentiation ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Soluble Factors ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

The Impact ◽

Myeloid Progenitors

Abstract Osteoblasts (OST) found within the endosteal niche are important regulators of Hematopoietic Stem and Progenitor Cells (HSPC) under steady state and during hematopoietic reconstitution. OST are derived from mesenchymal stromal cells (MSC) following osteogenic differentiation. MSC and OST secrete a wide array of soluble factors that sustain hematopoiesis. Recently, we showed that media conditioned with OST derived from MSC (referred as M-OST) after 6 days of osteogenic differentiation were superior to MSC conditioned media (CM) for the expansion of cord blood (CB) progenitors, and CB cells expanded with M-OST CM supported a more robust engraftment of platelets in NSG mice after transplantation. These findings raised the possibility that M-OST could be superior to MSC for the ex vivoexpansion HSPC. In this study, we set out to test the hypothesis that the growth modulatory activity of M-OST would vary as a function of their maturation status. The objectives were to first monitor the impact of M-OST differentiation and maturation status on the expression of soluble factors that promote HSPC expansion and in second, to investigate the capacity of M-OST CMs prepared from M-OST at distinct stages of differentiation to support the expansion and differentiation of HSPCs in culture. M-OST at distinct stages of differentiation were derived by culturing bone marrow MSC in osteogenic medium for various length of time (3 to 21 days). All CB CD34+ enriched (92±7% purity) cell cultures were done with serum free media conditioned or not with MSC or M-OST and supplemented with cytokines SCF, TPO and FL. We first confirmed the progressive differentiation and maturation of M-OST as a function of osteogenic culture length, which was evident by the induction of the osteogenic transcription factors Osterix, Msx2 and Runx2 mRNAs, the gradual increase in osteopontin and alkaline phosphatase positive cells and quantitative increases in calcium deposit. Next, we investigated the expression in MSC and M-OSTs of genes known to collaborate for the expansion of HSPCs by Q-PCR. Transcript copy numbers for IGFBP-2 increased swiftly during osteogenic differentiation, peaking at day-3 (˃100-fold vs MSC, n=2) and returning below MSC level by day-21. In contrast, ANGPTL members (ANGPTL-1, -2, -3 and -5) remained superior in M-OSTs throughout osteogenic differentiation with expression levels peaking around day 6 (n=2). Next, we tested the capacity of media conditioned with primitive (day-3, -6), semi-mature (day-10, -14) and mature M-OST (day-21) to support the growth of CB cells. All M-OST CMs increased (p˂0.03) the growth of total nucleated cells (TNC) after 6 days of culture compared to non-conditioned medium used as control (mean 2.0-fold, n=4). Moreover, there was a positive correlation between cell growth and M-OST maturation status though differences between the different M-OST CMs tested were not significant. The capacity of M-OST CMs to increase (mean 2-fold, n=4) the expansion of CD34+ cells was also shared by all M-OST CMs (p˂0.05), as supported by significant increases with immature day-3 (mean ± SD of 18 ± 6, p˂0.02) and mature day-21 M-OST CMs (14 ± 5, p˂0.05) vs. control (8 ± 3, n=4). Conversely, expansions of TNC and CD34+ cells in MSC CM cultures were in-between that of control and M-OST CMs cultures. Interestingly, M-OST CMs also modulated the expansion of the HSPC compartment. Indeed, while the expansion of multipotent progenitors defined as CD34+CD45RA+ was promoted in control culture (ratio of 4.5 for CD34+CD45RA+/CD34+CD45RA- cells), M-OST CMs supported greater expansion of the more primitive CD34+CD45RA- HSPC subpopulation reducing the ratio to 3.3±0.4 for M-OST cultures (cumulative mean of 10 cultures, n=2). Moreover, the expansions of CD34+CD38- cells and of the long term HSC-enriched subpopulation (CD34+CD38-CD45RA-Thy1+) in M-OST CM cultures were respectively 2.7- and 2.8-fold greater than those measured in control cultures (n=2-4). Finally, the impact of M-OST CMs on the expansion of myeloid progenitors was investigated using a colony forming assay; expansion of myeloid progenitors were superior in all M-OST CM cultures (1.6±0.2 fold, n=2). In conclusion, our results demonstrate that M-OST rapidly acquire the expression of growth factors known to promote HSPC expansion. Moreover, the capacity of M-OST CMs to support the expansion of HSPCs appears to be a property shared by M-OST at various stages of maturation. Disclosures No relevant conflicts of interest to declare.

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Myelodysplastic Syndrome-Derived Stromal Cells Support Leukemia-Initiating Cells and Contradirectly Eliminate Normal CD34+ Clonogenic Cells

Blood ◽

10.1182/blood.v122.21.1219.1219 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1219-1219

Author(s):

Hiroto Horiguchi ◽

Masayoshi Kobune ◽

Shohei Kikuchi ◽

Satoshi Iyama ◽

Kohichi Takada ◽

...

Keyword(s):

Stem Cell ◽

Myelodysplastic Syndrome ◽

Progenitor Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Methylation Status ◽

Gene Expressions ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Clonogenic Cells

Abstract Introduction The failure of normal hematopoiesis in myeloid neoplasm could be induced by a variety of mechanism. Regarding myelodysplastic syndrome (MDS)/acute leukemia (AML), aberrant hematopoietic stem/progenitor cells with exhibiting ineffective hematopoiesis and impaired differentiation ability gradually substitute it for normal hematopoietic stem/progenitor cells during a long term as a consequent of replacement of stem cell niche. However, it has not yet been clarified precise mechanism how MDS stem/progenitor cells could replace normal hematopoietic stem/progenitor cells. Methods In an attempt to analyze the supporting activity of bone marrow (BM) stromal cells, we first established the MDS/AML-derived stromal cells and healthy volunteer (HV)-derived-stromal cells. Next, MDS/AML-derived CD34+ cells or normal CD34+ cells were cocultured with established stromal cells using cytokines including stem cell factor, thrombopoietin, flt3-ligand in the presence of notch ligand (for normal CD34+ cells) or IL-3 (for AML/MDS derived cells). Subsequently, we analyzed clonogenic cells after 2 weeks coculture, 5 week cobblestone area-forming cells (CAFC) and repopulating cells in immunedeficient mice (NSG mice). Results The support of clonogenic cells after 2 weeks coculture and 5 weeks CAFCs was observed after coculture with normal CD34+ cells and HV-derived stromal cells. Furthermore, these cocultured cells engrafted into immunedeficient mice. Interestingly, the number of colony-forming units (CFU) mixed cells (MIXs) and CAFC derived from CD34+ cells was drastically reduced after coculture with MDS/AML-derived stromal cells. Nevertheless, MDS/AML-derived stromal cells support the proliferation of leukemia-initiating cells (L-ICs) and L-ICs were detected after third replating. These results indicate that MDS/AML-derived stromal cells preferentially support leukemia stem/progenitor cells, but not normal CD34+ cells. We compared the mRNA expression between (HV)-derived-stromal cells, MDS/AML-derived stromal cells and 5-aza-dC-treated stromal cells. The expression of several factors including hedgehog-interacting protein (HHIP) was reduced in MDS/AML-derived stromal cells. 5-aza-dC treatment restored the expression in some of genes and the stromal supporting activity for normal CD34+ cells partially recovered. Conclusion These results suggest that reduction of several gene expressions was detected in MDS/AML stromal cells by changes of methylation status. The epigenetic alteration of stromal genome may be involved in the progression of myeloid disorders. Disclosures: No relevant conflicts of interest to declare.

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Erythroid Lineage Cells Are Found In Close Association With Bone In The Marrow Microenvironment

Blood ◽

10.1182/blood.v122.21.945.945 ◽

2013 ◽

Vol 122 (21) ◽

pp. 945-945

Author(s):

Rialnat Adebisi Lawal ◽

Kathleen E. McGrath ◽

Laura M. Calvi

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Erythroid Differentiation ◽

Enzymatic Digestion ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Osteolineage Cells

Abstract Osteolineage cells within the bone marrow microenvironment have been implicated in support and regulation of hematopoietic stem cells (HSCs). Recently, augmented hypoxia-inducible factor (HIF) signaling in osteoprogenitors has been shown to expand the HSC niche, and surprisingly these cells have also been demonstrated to express erythropoietin, the critical cytokine stimulating erythropoiesis. We therefore hypothesize that endosteal cells may represent an additional regulatory site for erythropoiesis. To further delineate the role of the osteolineage cells in the support of erythropoiesis, we isolated bone associated cells (BACs) with enzymatic digestion of adult C57bl/6 mice hind limbs after bone marrow flushing and depleted the BACs of CD45+ cells to enrich for osteogenic cells. We suspected some contribution of erythroid cells to CD45- BACs, however we were surprised to find that ter119+ cells represented a large percentage of BACs after enzymatic digestion. After CD45 depletion, ter119+ cells constituted about 30% percent compared to approximately 0.85% of CD45+ cells (33 ± 4.4vs. 0.85 ± 0.26, p= 0.0018) by flow cytometric analysis. Additionally, CD45 depleted BACs had approximately 46 fold higher osteocalcin expression than CD45+ cells (1300 ± 120 vs. 28 ± 9.5, p < 0.0001), while CD45/Ter119/CD31 depleted BACs had approximately 2000 fold higher osteocalcin expression than CD45/Ter119/CD31 (+) cells (2000 ± 520 vs. 0.98 ± 0.02, p= 0.0044) by qRT-PCR, confirming enrichment of the osteoblastic lineage by this immunophenotypic panel. These data suggest that there are a large number of erythroid lineage cells associated with the BACs along the endosteum. In the bone marrow of adult mice, ter119 + cells represented approximately 85% in the CD45- pool as compared to 5% in the CD45+ cell pool. To determine if the endosteum is an active site of erythropoiesis, we quantified erythroid progenitors and precursors in the BAC pool compared to whole bone marrow (wbm) and peripheral blood (pb) by both flow cytometric analysis and colony forming assays. Flow cytometric analysis demonstrated the presence of every phase of erythroid differentiation in the BAC pool, including the presence of phenotypic MEPs (wbm vs bac vs pb: 250 ± 30 vs 84 ± 22 vs 0), BFU-E (wbm vs bac vs pb: 300 ± 14 vs 110 ± 36 vs 0 ), CFU-E (wbm vs bac vs pb: 2900 ± 2 vs 430 ± 23 vs 1 ± 0.8) and proerythroblasts (wbm vs bac vs pb: 11000 ± 2500 vs 7600 ± 1600 vs 2300 ± 920) per million cells. The phenotypic frequency of CFU-E was particularly remarkable in the BACs (430 ± 23) as compared to peripheral blood (1 ± 0.8) , demonstrating that all stages of erythroid differentiation are found in tight association with the endosteum and are not due to contamination from circulating erythroid progenitors. Colony assays were performed for CFU-E (wbm vs. bac 108 ± 16 vs 6.3 ± 2 colonies per 20,000cells plated), BFU-E (wbm vs. bac 55 ±1.0 vs 2 ±1.0; colonies per 40,000 cells plated) and myeloid progenitors (wbm vs. bac 66 ± 28 vs 11 ± 2.5 ; colonies per 10,000 cells plated) also confirmed the presence of erythroid progenitors at endosteal sites. Together these results identify the endosteal surface as a site for erythroid differentiation. The presence of all phases of erythroid lineage differentiation in the BACs suggests a potential role for osteolineage cells for maintenance and regulation of erythropoiesis. Whether osteolineage cells contribute to erythroid lineage homeostasis and/or stress response, and whether activation or damage to osteolineage cells alters local erythroid differentiation remains to be demonstrated. However our data suggest further study of the endosteum and osteolineage cells as a potential and unexpected site of erythroid regulation, which could potentially be targeted to accelerate erythropoiesis and treat anemia. Disclosures: No relevant conflicts of interest to declare.

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TIMP–1 Plays a Functional Role in CD34. + Hematopoietic Stem and Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.1487.1487 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1487-1487

Author(s):

C. Matthias Wilk ◽

Akos G. Czibere ◽

Ron-Patrick Cadeddu ◽

Sebastian Buest ◽

Frank A. Schildberg ◽

...

Keyword(s):

Stem Cell ◽

Flow Cytometric Analysis ◽

Integrin Signaling ◽

Receptor Complex ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Beta 1 Integrin

Abstract Abstract 1487 Poster Board I-510 TIMP–1 protein (Tissue Inhibitor of Metalloproteinases) is a recently identified tetraspanin interacting cell surface protein in the immortalized human breast epithelial cell line MCF10A. Tetraspanins like CD63 are proteins that consist of four transmembrane domains and are known to interact with integrins. Integrins play a crucial role in hematopoietic stem cell homing and mobilization. We first screened gene array data sets of CD34+ human hematopoietic stem and progenitor cells (HSPCs) and found TIMP–1 mRNA expression. In this study we show that TIMP–1 co-localizes with the tetraspanin CD63 and Beta-1-Integrin. Furthermore, we found a functional interaction of TIMP-1 with its receptor complex on G-CSF mobilized HSPCs. All experiments were carried out using highly enriched CD34+ cells. Co-immunoprecipitation shows that TIMP–1 binds to CD63. Using high resolution Stimulated Emission Depletion (STED) microscopy we could confirm co-localization of TIMP-1 and CD63 as well as Beta-1 Integrin and CD63. To further characterize the role of TIMP-1 in the Beta-1-Integrin signaling, we used an antibody specific to the active form of Beta-1- Integrin. Flow cytometric analysis revealed a significantly higher number of active Beta-1-Integrin in TIMP-1 stimulated cells suggesting TIMP-1 to activate the receptor complex on CD34+ cells. For functional analysis of the receptor complex formation, transwell migration assays were performed showing significantly increased migratory capacities of TIMP–1 treated cells. Additionally, TIMP–1 stimulation leads to a significantly increased adhesion rate of CD34+ cells to the fibronectin-coated dish. To assess a potential role of TIMP–1 in apoptosis, CD34+ HSPCs were co-incubated with thapsigargin and TIMP–1 or DMSO as a control. Subsequent flow cytometric analysis of cleaved Caspase-3 revealed a decrease of apoptotic cells in the TIMP-1 treated samples. In summary, we can show that TIMP-1, CD63 and Beta-1-Integrin form a complex on CD34+ HSPCs. TIMP-1 activates the Beta-1-Integrin signaling in HSPCs and alters the adhesive as well as the migratory behavior of CD34+ HSPCs. Furthermore, TIMP-1 induces an antiapoptotic effect in CD34+ cells. The functional effects of TIMP-1 in HSPCs might be of relevance in clinical hematopoietic stem cell transplantation so that we are currently about to verify these effects in an in vivo model. Disclosures: No relevant conflicts of interest to declare.

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Timing of Mesenchymal Stem Cell Co-Transplantation and Site of Bone Engraftment Determine Levels of Hematopoietic Engraftment and Contribution of HSC to the Bone Niche

Blood ◽

10.1182/blood.v116.21.940.940 ◽

2010 ◽

Vol 116 (21) ◽

pp. 940-940

Author(s):

Evan Colletti ◽

Saloomeh Mokhtari ◽

Chad Sanada ◽

Esmail D Zanjani ◽

Christopher D Porada ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Flow Cytometric Analysis ◽

Microscopic Analysis ◽

Donor Cell ◽

Osteoblastic Cell ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Hsc Transplantation

Abstract Abstract 940 We and others have previously demonstrated that the co-transplantation of marrow-derived stromal cells (MSC) with hematopoietic stem cells (HSC) results in higher levels of engraftment and acceleration of the rate of appearance of donor derived hematopoietic cells in the peripheral blood (PB) following transplantation. Possible explanations for this effect are MSC immunomodulatory properties and/or the ability of MSC to produce factors which support the transplanted HSC or prevent them from undergoing apoptosis. Here, we hypothesized that transplanted MSC are able to engraft within the recipient's bone marrow and integrate into vascular and/or osteoblastic niches to selectively create HSC donor optimized sites, and thereby enhance the rate and level of donor-derived hematopoietic reconstitution. Using an allogeneic sheep-to-sheep in-utero transplantation model, we administered intra-peritoneally 1.4×10^5 CD34+ cells transduced with a lentiviral vector encoding eGFP, (eGFP-CD34+) in combination with 2.5×10^5 same donor MSC transduced with a lentiviral vector encoding mKate (mKateMSC) (n=4). Another group of animals (n=4) received 2.5×10^5 mKateMSC, 3 days prior to transplantation of same donor 1.4×10^5 eGFP-CD34+. At 60 days post-transplant we performed flow cytometric analysis and Colony Forming Unit assay (CFU) of PB and BM to assess the levels of donor cell engraftment. Confocal microscopic analysis of bone sections was also performed in order to identify the localization and interaction between the transplanted HSC and MSC. Animals receiving mKateMSC 3 days prior to HSC transplantation displayed a 1.6 and a 1.1 fold increase in circulating donor GFP+cells and donor GFP+BM cells, respectively than animals receiving MSC+HSC simultaneously. However the latter had significantly higher levels of CD34 engraftment in BM (4.5-fold) than animals receiving mKateMSC 3 days prior to HSC transplantation. This also corresponded to higher levels of GFP+CFU in animals transplanted with MSC+HSC simultaneously. Confocal microscopy revealed that regardless of whether animals received mKateMSC 3 days prior to HSC or MSC+HSC simultaneously, HSC and MSC engrafted in clusters; however, there was no preferential interaction of the transplanted HSC with autologous MSC over the recipient's own cells. Nevertheless, animals receiving mKateMSC 3 days prior to HSC had higher levels of MSC in their BM than animals receiving MSC+HSC simultaneously. Furthermore, independent of the regimen of cells transplanted, depending on the site of bone engraftment, i.e., metaphysis or diaphysis, transplanted HSC localized preferentially in perivascular areas in the diaphysis, while in the metaphysic they appeared to contribute to the osteoblastic cell layer coating the ossifying bone. Also, HSC contributed to the osteoblastic layer more consistently and robustly than the transplanted MSC. These results show that the delivery of MSC prior to HSC results in higher levels of MSC engraftment in the bone marrow and higher levels of donor derived blood cells in circulation. However, the presence of MSC in the transplanted graft is necessary for optimal engraftment of CD34+ cells. Furthermore, CD34+ cells, and not MSC, migrated efficiently to the metaphysis where they were able to integrate into the developing bone and contribute to the osteoblastic layer. Disclosures: No relevant conflicts of interest to declare.

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The ETS family transcription factor PU.1 is necessary for the maintenance of fetal liver hematopoietic stem cells

Blood ◽

10.1182/blood-2002-08-2425 ◽

2004 ◽

Vol 104 (13) ◽

pp. 3894-3900 ◽

Cited By ~ 42

Author(s):

Hyung-Gyoon Kim ◽

Cristina G. de Guzman ◽

C. Scott Swindle ◽

Claudiu V. Cotta ◽

Larry Gartland ◽

...

Keyword(s):

Stem Cells ◽

Fetal Liver ◽

Flow Cytometric Analysis ◽

Absolute Number ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Fold Reduction ◽

Ets Family

Abstract PU.1 is a member of the ETS family of transcription factors and is required for the development of multiple hematopoietic lineages. PU.1-/- mice die from hematopoietic failure at about embryonic day 18.5 (e18.5) and show a complete absence of B cells, mature T cells, and macrophages. This phenotype suggests that PU.1 may function at the level of the hematopoietic stem cell (HSC) or a multilineage progenitor. To investigate the role of PU.1 in the regulation of HSCs, PU.1-/- embryos were analyzed at various stages of embryonic development. The absolute number and frequency of HSCs were determined by flow cytometric analysis of c-Kit+Thy-1.1loLin-Sca-1+ (KTLS) cells. We found that KTLS cells were absent or severely reduced in PU.1-/- fetal liver from e12.5 to e15.5. Progenitor cells with a c-Kit+Lin-AA4.1+ and c-Kit+Lin-CD34+ phenotype were also severely reduced. In addition, PU.1-/- fetal liver at e14.5 lacked common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) but retained megakaryocyteerythroid progenitors (MEPs). Consistent with the loss of HSC activity, a 10-fold reduction in erythroid progenitors (mature erythroid burst-forming units [BFUEs]) was observed between e14.5 and e16.5. These data suggest that PU.1 plays an important role in the maintenance or expansion of HSC number in murine fetal liver. (Blood. 2004;104:3894-3900)

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βig-h3 Regulates Differentiation and Survival of Human Hematopoietic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v116.21.2591.2591 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2591-2591

Author(s):

Sofieke Klamer ◽

Paula van Hennik ◽

Daphne C Thijssen-Timmer ◽

Ellen van der Schoot ◽

Carlijn Voermans

Keyword(s):

Progenitor Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Annexin V ◽

Hematopoietic Stem ◽

Knock Down ◽

Colony Forming Unit ◽

Human Cd34 ◽

Cd34 Cells

Abstract Abstract 2591 Adult hematopoietic stem cells (HSC) reside in dedicated niches in the bone marrow (BM). Within this specialized microenvironment, the various interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are critical for the maintenance of the HSC population and the concomitant development of the distinct blood cell lineages. Comparative gene-expression profiling of purified HSC identified ECM proteins that are differentially expressed in homeostatic and regenerative conditions. The ECM protein βig-h3 was one of the proteins upregulated in regenerative conditions. Therefore, we characterized the role of βig-h3 in the regulation of HSC self-renewal and differentiation. A comparison between human CD34+ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB), revealed the highest βig-h3 expression in BM-HSPC (3.9-fold increased compared to MPB, 1.7-fold increased compared to CB), which may implie a role for βig-h3 in retaining HSC in the BM. To examine the functional relevance of βig-h3 in HSC, we first increased βig-h3 expression by transducing human HSPC with a lentiviral βig-h3-SIN-GFP expression vector or a control SIN-GFP vector. Over-expression of βig-h3 (80-fold) in HSPC decreased colony-forming-unit-granulocyte-monocyte (CFU-GM) formation from 130 (SEM=47) to 73 (SEM=19, n=3) CFU-GM per 500 plated CD34+ cells, while megakaryopoiesis was accelerated and the number of mature megakaryocytic cells increased from 16% (SEM=6%) to 30% (SEM=7%, n=4) at day 14 of culture. Ectopic expression of βig-h3 did not affect differentiation along the erythroid or granulopoietic lineage. The development of megakaryocytes at the cost of pluripotent CFU-GM suggests that βig-h3 drives differentiation. In addition, βig-h3 expression in HSPC was reduced by two different short-hairpin-RNAs (shRNA) expressed from lentiviral vectors, which resulted in decreased proliferation (from 19.6- to 5.8-fold per input cell at day 13) and increased apoptosis (from 13.5% to 25.3% at day 13) in liquid HSPC cultures, as analyzed by Annexin V staining. Similarly, knock-down of βig-h3 in various cell lines also resulted in a decreased proliferation and increased apoptosis. Knock-down of βig-h3 in primary HSPC dramatically reduced CFU-GM from 73 (SEM=8.7) to 31 (SEM=14.4, n=6) CFU-GM per 500 CD34+ cells plated, and reduced colony-forming-unit-erythrocyte (CFU-E) formation from 30 (SEM=6.5) to 9 (SEM=1.6, n=4) CFU-E per 500 CD34+ cells plated. This can be explained by increased apoptosis of βig-h3 knock-down cells. Notably, co-culture of βig-h3 knock-down HSPC with stromal feeder cells, known to express high levels of βig-h3, showed no difference compared to control HSPC in cobblestone area formation within two weeks, indicating that stromal cells can counteract apoptosis in βig-h3 knock-down cells. Remarkably, long-term-culture CFU-GM (LTC-CFU) formation of HSPC that were co-cultured with stromal cells during two weeks, was even significantly increased (1.9-fold, n=2) in βig-h3 knock-down cells, indicating that decreased endogenous levels of βig-h3 stimulates the maintenance or expansion of HSPC on stroma. In conclusion, ectopic expression of βig-h3 decreased CFU-GM in HSPC and accelerated differentiation towards megakaryocytes, suggesting that βig-h3 might drive lineage commitment of HSC. Conversely, knock-down of βig-h3 in HSPC stimulated LTC-CFU formation, indicating that decreased βig-h3 levels in HSPC maintain their undifferentiated state. In absence of stroma, however, knock-down of βig-h3 induces apoptosis, indicating βig-h3 as an essential survival factor, which expression levels regulate differentiation and maintenance of HSC. Disclosures: No relevant conflicts of interest to declare.

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Vascular Pericytes Sustain Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.2394.2394 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2394-2394 ◽

Cited By ~ 2

Author(s):

Mirko Corselli ◽

Chintan Parekh ◽

Elisa Giovanna Angela Montelatici ◽

Arineh Sahghian ◽

Wenyuan Wang ◽

...

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Stem Cell ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Cell Interactions ◽

Lymphoid Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 2394 Mesenchymal stromal (or stem-) cells (MSC) are culture-selected, heterogeneous supporting cells that can differentially regulate hematopoietic stem cell (HSC) behavior in vitro. The elusive identity of native MSC has obscured the contribution, if any, of these cells to HSC support in vivo. Having previously demonstrated that vascular pericytes (ubiquitous cells encircling endothelial cells in capillaries and microvessels) are ancestors of human MSC, we now hypothesize that pericytes are a critical component of the HSC “niche”. Consequently, pericyte isolation from total stroma would allow to develop co-culture systems for human HSC maintenance. In the present study, human cord blood CD34+ cells were cultured onto confluent human pericytes isolated from adipose tissue as CD146+CD34-CD45-CD56- cells. Co-culture of CD34+ cells on pericytes, for up to 6 weeks in the absence of any added growth factor, produced significantly i) higher numbers of CD45+ and CD34+ cells (p<0.05), ii) higher percentages of primitive CD34+CD33-CD10-CD19- progenitors (p<0.05), iii) higher percentages of single- and multi-lineage CFU (p<0.05) and iv) lower percentages of mature myeloid and lymphoid cells (p<0.05), compared to control co-cultures on unfractionated adipose stromal cells (ASC) (n=10 individual experiments, n=4 biological replicates). Most importantly, only pericytes could maintain HSC with self-renewal and long-term repopulating potential, as demonstrated by transplantation into primary and secondary NOD/SCID/IL2Rg−/− mouse recipients (n=3 individual experiments). In the latter setting, none of the mice receiving CD34+ cells co-cultured with ASC engrafted (n=10), whereas all recipients of CD34+ cells cultured in the presence of pericytes developed lympho-myeloid hematopoietic human cells (n=10). Altogether, these results support the hypothesis that pericytes maintain hematopoietic cell stemness. Conversely, unfractionated stromal cell cultures may promote HSC differentiation at the expense of self-renewal. Both tentative scenarios were explored. Co-cultures with pericytes in a transwell system revealed that cell-to-cell contact is required for HSC survival. Since Notch signaling regulates stem cell maintenance by inhibiting cell differentiation through cell-cell interactions, we hypothesized that pericytes purified from total stroma express specific Notch ligands. As shown by qPCR, the expression of Jagged-1 is 2 fold higher in pericytes compared to unfractionated ASC. Addition of a Notch inhibitor (DAPT) to pericyte/HSC co-cultures resulted in the significant reduction of CFU numbers (p<0.05) and increase in B-cell development. Furthermore, increased myeloid differentiation was observed when ASC conditioned medium was added to pericytes/HSC co-cultures. In conclusion, we demonstrate that vascular pericytes sustain HSC by promoting survival and preventing differentiation via cell-to-cell interactions involving Notch activation, whereas unfractionated stroma promotes HSC differentiation through a paracrine mechanism. We thus infer that HSC-supporting stromal cells are not confined within blood-forming organs (similar observations, not reported here, have been made on skeletal muscle pericytes). This novel concept is not easy to reconcile with normal hematopoiesis, but may be highly relevant in the context of the dissemination of malignant hematopoietic cells. Of important note, adipose tissue used in this study represents a convenient, safe and often abundant source of autologous therapeutic cells. Therefore, human fat-derived pericytes emerge as a candidate cell product for HSC ex vivo manipulation in the clinic. Disclosures: No relevant conflicts of interest to declare.

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