Cobblestone Area-forming Cell Assay Research Articles

The cross-talk between ROS and p38MAPKα in the Ex Vivo expanded human umbilical cord blood CD133+ cells

This study investigated the correlation between and compared the effects of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase α (p38MAPKα) in the ex vivo expanded umbilical cord blood (hUCB) CD133(+) cells. hUCB CD133(+) cells were cultured in the hematopoietic stem cells (HSCs) culture medium with N-acetylcysteine (NAC, an anti-oxidant), p38MAPKα-specific inhibitor (SB203580) or their combination. The levels of ROS and expression of phosphorylated p38MAPKα (p-p38) in CD133(+) cells were flow cytometrically detected. The efficacy of ex vivo expansion was evaluated by the density of CD133(+) cell sub-group colony-forming cells (CFC) and cobblestone area-forming cells (CAFC) assay. Our results showed decreased ROS levels in NAC, SB203580, and their combination treatment groups were almost 37%, 48%, and 85%, respectively. Furthermore, SB203580 abrogated the activation of p38MAPKα more obviously than NAC. Moreover, the CD133(+) cells in SB203580 treatment group had a 21.93±1.36-fold increase, and 14.50±1.19-fold increase in NAC treatment group, but only 10.13±0.57-fold increase in control group. In addition, SB203580 treatment led a higher level increase in the number of CFU and CAFC than NAC did. These findings suggested that, in expanded CD133(+) cells, ROS activates p38MAPKα, which, in turn, induces ROS production, and p38MAPKα might be the most suitable regulator in ROS-p38MAPKα pathway for the promotion of HSCs ex vivo expansion.

Endurance exercise training promotes medullary hematopoiesis

Endurance exercise is a poorly defined yet powerful mediator of hematopoiesis. The purpose of this study was to directly investigate the effects of endurance exercise training on hematopoiesis and to identify potential mechanisms responsible for any observed changes. Four-week-old male C57Bl/6 mice were trained on a treadmill at progressive speeds over a 10-wk period. Tissues were harvested 2 d following the final training session. Flow cytometry, the cobblestone area-forming cell assay, and the methycellulose colony-forming unit assay were used to assess medullary and mobilized hematopoietic stem and progenitor cells. Quantitative real-time PCR and Western blots were used to measure hematopoietic cytokine production. Histochemistry was also used to assess adaptations to exercise in the bone marrow niche. Depending on the cell type, endurance training increased medullary and mobilized hematopoietic stem and progenitor cell content from 50 to 800%. Training also reduced marrow cavity fat by 78%. Skeletal muscle hematopoietic cytokine expression was also increased at least 60% by training. Sedentary mice served as controls for the above experiments. In conclusion, endurance exercise training greatly promotes hematopoiesis and does so through improvements in medullary niche architecture as well as increased skeletal muscle hematopoietic cytokine production.

Inhibition of p38 Mitogen-Activated Protein Kinase Promotes Ex Vivo Hematopoietic Stem Cell Expansion

Hematopoietic stem cell (HSC) self-renewal is tightly regulated by a complex crosstalk between many cell-intrinsic regulators and a variety of extrinsic signals from the stem cell niche. In this study, we examined whether the p38 mitogen-activated protein kinase (p38) is one of the intrinsic regulators that can negatively regulate HSC self-renewal in vitro and whether inhibition of p38 activity with a small molecule inhibitor can promote HSC expansion ex vivo. The results from this study showed that sorted mouse bone marrow Lin(-)Sca1(+)c-kit(+) cells (LSK(+) cells) exhibited selective activation of p38 after culture in a serum-free medium supplemented with 100 ng/mL stem cell factor, thrombopoietin, and Flt3 ligand. The activation of p38 was associated with a significant reduction in HSCs and induction of apoptosis and cellular senescence in LSK(+) cells and their progeny. Addition of the specific p38 inhibitor SB203580 (SB, 5 μM) to the culture inhibited the activation of p38 in LSK(+) cells, which led to increase in HSC self-renewal and ex vivo expansion as shown by the cobblestone area forming cell assay, competitive repopulation, and serial transplantation. The increase in HSC expansion is likely attributable to SB-mediated inhibition of HSC apoptosis and senescence and upregulation of HoxB4 and CXCR4. These findings suggest that p38 plays an important role in the regulation of HSC self-renewal in vitro and inhibition of p38 activation with a small molecule inhibitor may represent a novel approach to promote ex vivo expansion of HSCs.

Mitigation of Ionizing Radiation-induced Bone Marrow Suppression by p38 Inhibition and G-CSF Administration

p38 mitogen-activated protein kinases (p38) has been shown to be activated in hematopoietic stem and progenitors cells after exposure to ionizing radiation (IR) and its activation has been implicated in bone marrow (BM) suppression under various pathological conditions. Therefore, in the present study we investigated whether inhibition of p38 activity alone with SB203580 (SB, a specific p38 inhibitor) or in combination with granulocyte colony-stimulating factor (G-CSF) can mitigate total body irradiation (TBI)-induced BM damage and lethality. Our results showed that p38 inhibition with SB had no significant effect on the 30-day survival rates of the mice exposed to 7.2 Gy TBI when it was used alone but increased the survival of the mice when it was combined with G-CSF. This combined effect may be attributable to a better preservation or stimulation of hematopoietic stem and progenitor cells, because BM cells from SB and G-CSF-treated mice produced more colony forming units-granulocyte-macrophage (CFU-GM) and 4-week cobblestone area forming cells (CAFCs) than the cells from either SB or G-CSF-treated mice after TBI in a colony forming cell assay and a CAFC assay, respectively. These findings suggest that the combined therapy with SB and G-GSF is more effective in mitigating TBI-induced acute BM injury than either agent alone.

The Acetylation of AML1-ETO Is Required for Leukemogenesis.

AbstractAbstract 1588Transcription factors and histones are similarly modified through acetylation, phosphorylation, ubiquitination and methylation, which impact on the transcriptional regulation of gene expression and various biological processes in normal and malignant hematopoiesis. The t(8;21) associated AML1-ETO fusion protein is found in 40% of the FAB M2 subtype of acute myeloid leukemia, but how the post-translational modification of AML1-ETO affects its leukemogenicity is largely unknown. Here we show that AML1-ETO directly interacts with the lysine acetyltransferase, p300, via the region containing NHR1 domain and that p300 can acetylate two lysine residues in AML1-ETO and AML1-ETO (exon 9a) in human and mouse leukemia cells. To understand the biological effects of AML1-ETO acetylation, we used human CD34+ cord blood cells as a preleukemia model. The maintenance of CD34+ cells by the acetylation defective form of AML1-ETO was 5 fold less than with AML1-ETO (p<0.01) in the liquid culture assay, and unlike the effect of AML1-ETO, the number of the cobble stone area forming cells (CAFC) was not increased by the mutant AML1-ETO in CAFC assay. However, the block in erythroid and myeloid differentiation conferred by AML1-ETO was still seen in the AML1-ETO acetylation mutant transduced human CD34+ cells. We then approved the impact of acetylation on leukemogenicity using the AML1-ETO9a (AE9a) mouse leukemia model. Mice receiving AE9a acetylation mutant transduced fetal liver cells have not developed leukemia by Day 250, whereas all the mice receiving AE9a transduced cells died due to leukemia before Day 160, with a mean survival time of 109 days (p<0.001). These results suggest that the acetylation of AML1-ETO is required not only for its self-renewal promoting effects and but also for the development of acute leukemia. To gain insight into the mechanisms of AML1-ETO acetylation, we performed luciferase assays and found that the AML1-ETO acetylation mutant lost the ability to activate an M-CSFR promoter driven reporter construct. Furthermore, the expression levels of AML1-ETO activated target genes related to self-renewal were not upregulated in AML1-ETO acetylation mutant transduced human CD34+ cells. These results indicated that the acetylation is crucial to AML1-ETO induced transcription activation. We have also been studying the role of the region containing NHR1 domain (245 to 430 aa) in AML1-ETO: deletion of this region abrogated the binding of p300 to AML1-ETO and led to loss of AML1-ETO lysine acetylation. Furthermore, loss of the region containing NHR1 domain abrogated the self-renewal properties of AML1-ETO and the activation of AML1-ETO target genes in human CD34+ cord blood cells, without affecting its differentiation-blocking activity or its ability to repress gene expression. Given the importance of the acetylation of AML1-ETO in its biological effects, we inhibited p300 function, chemically and using RNA interference; this blocked the transcriptional activation of AML1-ETO target genes, and inhibited the growth of AML1-ETO expressing AML cells in both pre-leukemic and leukemia models. All together, we have found that the acetylation of AML1-ETO via p300 is indispensable for its leukemia-promoting activity and for its ability to activate gene expression. Our work suggests that inhibition of p300 function may represent an important new anti-leukemia strategy that targets self-renewing, leukemia-initiating cells. Disclosures:No relevant conflicts of interest to declare.

Abstract C8: Putative tumor-initiating cell populations in mantle cell lymphoma exhibit microenvironmentally influenced differences in stem cell-like and tumorigenic properties

Abstract The presence of subpopulations of cells possessing enhanced tumor formation capabilities has been described in several tissues. These cells have been termed tumor-initiating cells (TICs) or cancer stem cells and have been isolated using various techniques, including the side population (SP), the use of cell surface markers including CD133 and CD44 and aldehyde dehydrogenase activity. We have elected to evaluate the stem cell-like and tumor forming capabilities of SP and CD133+/CD44+ cells in Mantle Cell Lymphoma (MCL), an aggressive and relatively therapy unresponsive form of Non-Hodgkin's Lymphoma. All analyses were conducted in the presence of micro-environmental niche cells (murine stromal 5 cells) or extracellular matrix components (Matrigel) as these factors are known to play an integral role in processes associated with stem cell self-renewal and differentiation. SP cells and CD133+/CD44+ cells were present in Granta 519, an aggressive MCL cell line. The in-vitro technique of the Cobblestone Area Forming Cell (CAFC) assay was utilized to assess the self-renewal capabilities of SP and CD133+/CD44+ cells when the two cell types were cultured in the presence and absence of Matrigel, a mixture of extracellular matrix proteins derived from the Englebreth-Holm murine sarcoma. CAFC assays revealed enhanced self-renewal capabilities for Granta SP cells over non-SP cells, with the addition of Matrigel increasing the self-renewal frequency of SP and non-SP cells by 5.8 and 8.6 fold respectively. Conversely, Granta CD133+/CD44+ and CD133−/CD44− cells possessed similar self-renewal capabilities, with the addition of Matrigel having a negligible effect. When evaluated for chemotherapy resistance, the murine stromal 5 (MS5)-adherent CD133+/CD44+ cells and the non-adherent SP cells were more resistant to hydroxyurea while all MS5-adherent Granta cells were resistant to this agent. In-vivo analyses demonstrated tumor formation of Matrigel mixed SP and non-SP populations but not for Matrigel mixed CD133+/CD44+ or CD133−/CD44− cells. Although micro-metastatic tumors were not detected for SP and non-SP injected mice, flow cytometric analyses identified Granta cells, characterized by CD20 expression, in all murine tissues examined. IL-8, a proangiogenic cytokine implicated in tumor metastasis, may be involved in the metastatic spread of Granta cells as it was significantly present in the supernatants of Granta SP MS5 co-cultures, but was negligible or undetected when non-SP, CD133+/CD44+ and CD133−/CD44− cells were co-cultured with MS5 cells. These studies suggest that the SP phenotype may be a better marker for the isolation of stem-like cells in MCL. Moreover, the micro-environmental components found in Matrigel, rather than cell phenotype differences, appear to play a vital role in the initiation of tumor development by SP and non-SP cells. The tumor micro-environmental niche may therefore be an important target for development of novel therapeutic agents. Citation Information: Cancer Res 2009;69(23 Suppl):C8.

Inhibition of p38 MAPK Promotes Hematopoietic Stem Cells Self-Renewal and Ex Vivo Expansion.

Abstract 34Activation of the p38 mitogen-activated protein kinase (p38 MAPK) is implicated in the inhibitory effects of TNF-α, TGF-β, interferons and reactive oxygen species (ROS) on hematopoiesis and self-renewal of hematopoietic stem cells (HSCs). Clinically, overactivated p38 MAPK contributes to the pathogenesis of myelodysplastic syndromes (MDS) and Fanconi anemia. Inhibition of p38 MAPK with pharmacological agents improves hematopoietic progenitors' function in MDS. However, it has yet to be determined if p38 MAPK plays a role in regulation of normal HSC self-renewal and whether inhibition of p38 MAPK can improve HSC ex vivo expansion. In the present study, we found that sorted mouse bone marrow HSCs (Lin− Sca1+ c-kit+ cells or LSK+ cells) exhibited specific activation of p38 MAPK after seven days culture in serum-free medium supplemented with stem cell growth factors (SCF, Tpo and Flt3 ligand). The activation of p38 MAPK was associated with rapid differentiation of HSCs and induction of cellular senescence. Addition of SB203580 (SB, a specific p38 MAPK inhibitor) to the culture abrogated the activation of p38 MAPK, inhibited the induction of cellular senescence and increased the expression of several HSC self-renewing genes (such as CXCR4, HoxB4 and Gfi1). Moreover, HSCs cultured with SB resulted in a significantly greater HSC expansion than HSCs cultured without SB as assessed by flow cytometry and cobblestone area-forming cell (CAFC) assay. Finally, competitive repopulation assays revealed that HSCs expanded with SB produced a dramatic increase in donor-derived engraftments after transplantation to irradiated recipients. These findings suggest that p38 MAPK plays an important role in the regulation of HSC self-renewal and its inhibitors (e.g. SB203580) may be clinically useful in the ex vivo expansion of HSCs. Disclosures:No relevant conflicts of interest to declare.

Glycosaminoglycan mimetics–induced mobilization of hematopoietic progenitors and stem cells into mouse peripheral blood: Structure/function insights

Glycosaminoglycans (GAG) are major components of bone marrow extracellular matrix because they have the property to interact with cells and growth factors in hematopoietic niches. In this study, we investigated the effect of two different chemically defined GAG mimetics on mobilization of hematopoietic stem and progenitor cells (HSPCs) in mice peripheral blood. Mobilization was achieved by intraperitoneal injection of GAG mimetics. Mobilized cells were characterized phenotypically by reverse transcription polymerase chain reaction and fluorescence-activated cell sorting analysis and functionally by colony-forming cell, cobblestone area-forming cell and long-term culture-initiating cell assays in vitro. Radioprotection assays were performed to confirm the functionality of primitive hematopoietic cells in vivo. Involvement of stromal-derived factor-1 (SDF-1) and matrix metalloproteinase-9 (MMP-9) were investigated. GAG mimetics treatment induces hyperleukocytosis and mobilization of HSPC. They synergize with the effects of granulocyte colony-stimulating factor or AMD3100 on hematopoietic progenitors mobilization. Reconstitution of lethally irradiated recipient mice with peripheral blood mononuclear cells from GAG mimetic-treated donor mice improves engraftment and survival. BiAcore studies indicate that the mimetics interact directly with SDF-1. In addition, GAG mimetics-induced mobilization is associated with increased levels of pro- and active MMP-9 from bone marrow cells and increased level of SDF-1 in peripheral blood. Finally, mobilization is partially inhibited by co-injection with anti-SDF-1 antibody. This study demonstrates that GAG mimetics induce efficient mobilization of HSPCs, associated with an activation of pro-MMP-9 and a modification in the SDF-1 concentration gradient between bone marrow and peripheral blood. We suggest that structural features of GAGs can modify the nature of mobilized cells.

A Cell-Autonomous Defect of Bone Marrow Hematopoietic Stem/Progenitor Cells and Impaired Stromal Function in FA-a Patients

Fanconi anemia (FA) is a devastating blood disease associated with bone marrow (BM) failure. Currently the effective treatment for FA is BM transplantation. We and others showed that FA murine hematopoietic stem and progenitor cells (HSC/Ps) have defective hematopoietic reconstitution after BM transplantation. In addition, several clinical gene therapy trials in FA patients have failed to show sustained engraftment of FA HSC/Ps complemented with a functional FA gene. The mechanism underlying this engraftment defect remains unknown. To determine whether the poor engraftment of FA HSC/Ps observed in mouse knockout models and clinical gene therapy trials in FA patients results from an intrinsic defect of the FA HSC/Ps or an impaired microenvironment in the FA bone marrow, we have assessed the self-renewal ability of HSC/Ps and hematopoietic supportive capacity of BM stromal cells from FA-A patients. First, we performed quantitative cobblestone area-forming cell (CAFC) assays using MS-5 stromal layer. CAFCs were enumerated at 2 and 5 weeks of cocultures between BM mononuclear cells from a normal donor and three FA-A patients. Early-appearing CAFCs (week 2) represent transient repopulating cells equivalent to progenitor colony-forming units (CFUs), whereas late-appearing CAFCs (week 5) are representative of long-term repopulating stem cells. The frequency of FA-A CAFCs was 15–20 folds decreased compared with that of the normal donor at both week 2 and week 5. Second, we carried out long-term culture-initiating cell (LTC-IC) assays using BM mononuclear cells and BM-derived stromals from normal donors or FA-A patients. Compared to coculture with normal stromal cells, the recovery of colony forming cells (CFCs) that grew on FA-A stromal layer after 5–6 weeks incubation was significantly decreased for either normal or FA-A HSC/Ps (41.4% ± 3.1 CFC in normal HSC/Ps - normal stroma compared to 14.3% ± 1.6 CFC in normal HSC/Ps – FA-A stroma and 2.4 ± 0.6 CFC in FA-A HSC/Ps- FA-A stroma). Finally, we conducted CFC and cell-cell adhesion assays using BM-derived stromal cells from normal donors and FA-A patients. We observed that FA-A BM stroma failed to support HSC/P cells from either normal donors or FA-A patients. Specifically, severe adhesion defect was found in adhesion of normal and FA-A HSC/Ps with FA-A stromal layer (5.5% ± 0.6 CFC adhesion in normal HSC/Ps - normal stroma compared to 2.4% ± 0.5 CFC adhesion in normal HSC/Ps – FA-A stroma and 1.0 ± 0.7 CFC adhesion in FA-A HSC/Ps- FA-A stroma). These results suggest that both a cell-autonomous defect of FA HSC/Ps and an impaired FA BM microenvironment may contribute to the abnormal hematopoiesis in FA.

CD97 is differentially expressed on murine hematopoietic stem-and progenitor-cells

CD97 is a member of the epidermal growth factor-seven transmembrane (EGF-TM7) family of adhesion receptors and is broadly expressed on hematopoietic cells. The aim of this study was to investigate the expression of CD97 on hematopoietic stem- and progenitor cells (HSC/HPC). CD97 expression on hematopoietic stem- and progenitor cells was studied in BALB/c, C57BL/6 and DBA/1 mice using flow cytometry. Functional hematopoietic stem- and progenitor cell characteristics were investigated in vitro and in vivo by progenitor cell assays, cobblestone area forming cell assays and bone marrow cell transplantation. Analysis of CD97 expression on murine bone marrow cells showed three major populations i.e. CD97(HI), CD97(INT) and CD97(NEG) cells. Functional studies revealed that radioprotective capacity and cobblestone area forming cell day 28-35 activity resides in the CD97(INT) bone marrow cell fraction while CFU-GM colony-forming capacity mainly resides in the CD97(NEG) population in all strains. In C57BL/6 and DBA/1 mice CD97(NEG) and CD97(HI) bone marrow cells show hematopoietic stem cell characteristics as well. Further functional analysis of BALB/c CD97(INT) bone marrow cells revealed that c-Kit(HI)CD97(INT) bone marrow cells exhibit HSC activity and are 1.5-fold enriched for cobblestone area forming cell-day 35 activity compared to c-Kit(HI) bone marrow cells. Moreover, phenotypical analysis showed that BALB/c and C57BL/6 HSC are CD97(INT), while DBA/1 HSC are CD97(HI). CD97 is differentially expressed on hematopoietic stem cells and hematopoietic progenitor cells. Committed progenitor cell activity is largely comprised in the CD97(NEG) fraction, while the CD97(INT) population contains hematopoietic stem cell activity. In BALB/c mice, CD97 expression can be applied to almost completely separate colony-forming cells and cells exhibiting radioprotective capacity. In addition we propose that the CD97(INT)c-Kit(HI) phenotype allows simple and rapid purification of murine hematopoietic stem cells.

Genome-wide identification of functional polymorphisms modulating individual risk of lung cancer

Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AX (γH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34 + Lin-) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure.

Cobblestone Area-Forming Cell (CAFC) Content of Cord Blood Does Not Predict Engraftment in Double Cord Blood Transplantation.

INTRODUCTION: Delayed engraftment remains a serious problem following cord blood (CB) transplantation. It may be due, at least in part, to the limited dose of CB hematopoietic stem and progenitor cells (HSPC) transplanted. Limitations associated with HSPC dose may be reduced by the transplantation of 2 CB units. In an accompanying abstract, de Lima et al. report in detail on an ongoing MDACC randomized study in which patients received double CB units as either 2 unmanipulated units (2×UN), or 1 unmanipulated and 1 ex vivo expanded CB unit (UN+EX). This study has revealed that one CB unit ultimately predominates as the source of long-term, sustained hematopoiesis. We hypothesized that the number of primitive HSPC in the CB unit might ultimately predict which CB unit would ultimately prevail. The in vitro cobblestone area-forming cell (CAFC) assay was used to provide a measure of primitive components of the CB HSPC in each unit. A photomicrograph of a typical cobblestone area (CA) derived from a single CB-derived CAFC is shown. (Figure) CA persisting in in vitro culture for ≥6 weeks (derived from CAFCwk6) represent relatively primitive HSPC and their numbers may provide a qualitative profile for CB units. We hypothesized that of the two CB units transplanted, the one with the greater number of CAFCwk6 would ultimately persist long-term in the patient.METHODS: Clinical samples of 2×UN or UN+EX CB cells were plated in the in vitro CAFC assay.1 The frequency of CAFCwk6 was estimated and total CAFCwk6 numbers transplanted for each CB unit calculated. CAFCwk6 data for each CB unit and engraftment data from patients were analyzed to determine whether the number of CAFCwk6 transplanted was predictive of which CB unit would ultimately be responsible for long-term, sustained engraftment.RESULTS: Preliminary data was accrued from 10 patients.(Table) Six patients received 2×UN and 4 patients received UN+EX (EX indicated by #). CAFCwk6 content at transplant was predictive of which CB unit would ultimately be responsible for long-term, sustained engraftment in only 6 of the 10 cases (60%).CONCLUSION: CAFCwk6 represent a relatively primitive component of the HSPC compartment, however, these data suggest that CAFCwk6 numbers, although possibly a qualitative measure, are not a predictor of long-term sustained engraftment. [Display omitted]

Microencapsulated feeder cells as a source of soluble factors for expansion of CD34 + hematopoietic stem cells

Expansion of hematopoietic stem cells (HSCs) from cord blood is highly desired for treatment and transplantation of adult patients for hematologic diseases. For efficient proliferation of HSCs, CD34 + cells from cord blood were co-cultured with microencapsulated murine stromal cells (HESS-5) or immortalized human mesenchymal stem cells (MSCs) in their conditioned media (CM). Bioactive substances for HSC proliferation in CM at the onset of culture are likely consumed by HSCs with time, and co-culturing with microencapsulated feeder cells ensures a continuous supply. The cell number of CD34 + cell progeny efficiently increased under these culture conditions, and progeny were analyzed by flow cytometry, the colony assay and the cobblestone area-forming cell (CAFC) assay. Total nucleated cells and CD34 + cell number increased 194- and 7.4-fold, respectively, in the presence of microencapsulated HESS-5 in CM. Colony forming cells and CAFCs were well maintained. The effective expansion of total cells and maintenance of primitive progenitor cells suggest that transfusion of the progeny obtained from CD34 + cell culture with microencapsulated HESS-5 in CM could shorten the time to engraftment by bridging the pancytopenic period and support functional hematopoietic repopulation.

Enforced Expression of NUP98-HOXA9 in Human CD34+ Cells Enhances Stem Cell Proliferation

The t(7;11)(p15;p15) translocation, observed in acute myelogenous leukemia and myelodysplastic syndrome, generates a chimeric gene where the 5' portion of the sequence encoding the human nucleoporin NUP98 protein is fused to the 3' region of HOXA9. Here, we show that retroviral-mediated enforced expression of the NUP98-HOXA9 fusion protein in cord blood-derived CD34(+) cells confers a proliferative advantage in both cytokine-stimulated suspension cultures and stromal coculture. This advantage is reflected in the selective expansion of hematopoietic stem cells as measured in vitro by cobblestone area-forming cell assays and in vivo by competitive repopulation of nonobese diabetic/severe combined immunodeficient mice. NUP98-HOXA9 expression inhibited erythroid progenitor differentiation and delayed neutrophil maturation in transduced progenitors but strongly enhanced their serial replating efficiency. Analysis of the transcriptosome of transduced cells revealed up-regulation of several homeobox genes of the A and B cluster as well as of Meis1 and Pim-1 and down-modulation of globin genes and of CAAT/enhancer binding protein alpha. The latter gene, when coexpressed with NUP98-HOXA9, reversed the enhanced proliferation of transduced CD34(+) cells. Unlike HOXA9, the NUP98-HOXA9 fusion was protected from ubiquitination mediated by Cullin-4A and subsequent proteasome-dependent degradation. The resulting protein stabilization may contribute to the leukemogenic activity of the fusion protein.

Fresh and Cryopreserved Umbilical Cord Blood Side Population Cells Have Comparable Immunophenotype and Function but Are Not Characterised by ABCG2 Expression.

The side population (SP) phenotype resulting from efflux of the fluorescent dye Hoechst 33342 has been associated with high quality stem cells from both hematopoietic and non-hematopoietic organs. While expression of the transporter proteins MDR1 and ABCG2 has been strongly implicated in the SP phenotype of murine bone marrow stem cells, relatively little is known about the SP in human umbilical cord blood. The aim of this study was to investigate the relationship between the expression of common stem cell markers, the SP phenotype and the expression of the MDR1 and ABCG2 transporters in both fresh and cryopreserved umbilical cord blood. Assays of long-term Cobblestone Area Forming Cell (CAFC) activity at week 13 confirmed a 22 fold enrichment (p=0,002) of CAFC potential in the sorted SP (0,11% +/−0,06%, n=7) compared to the control living lymphocyte population subjected to Hoechst-free preparation and sorting (0,005% +/−0,0023%, n=6). SP cells from fresh (n=3) and cryopreserved (n=4) blood samples performed equally well in CAFC assays (p=0,28). A multiparameter FACS analysis involving a combination of four colors plus SP controlled with verapimil revealed the CD133+ and CD34+ cells to be distributed towards the upper part of the SP plot and to extend into the verapimil-sensitive SP. However, only 20,5% +/−15,6% (n=9) of the SP cells expressed either CD34 or CD133. ABCG2+ cells were found to be distributed throughout the main population but to be specifically absent from the side population. Fresh and cryopreserved blood samples were similar in all of these respects. In contrast to the SP cells, the MACS-purified ABCG2+ population failed to grow in cytokine-supplemented media and yielded no colonies in the CAFC assay. Semi-quantitative RT-PCR assays comparing cDNA prepared from the SP to that from the entire mononuclear population revealed a moderate but consistent enrichment of MDR1 mRNA in the SP, but no enrichment of ABCG2 mRNA. A similar comparison of the MACS-separated CD133+ (n=4) and CD133− (n=3) cells revealed the CD133+ population to be enriched (4 fold) for MDR1 but depleted (19-fold) for ABCG2 mRNA. We conclude that ABC transporters other than ABCG2 are responsible for the SP phenotype in human UCB; that MDR1 remains a potential candidate in this respect and that the frequency, immunophenotype and CAFC function of SP cells from fresh and cryopreserved blood are indistinguishable.

A Critical Role for the Retinoblastoma Tumor Suppressor Gene in Hematopoietic Stem Cells.

The retinoblastoma tumor suppressor protein (RB) plays important roles in the control of the cell cycle, DNA-damage checkpoint, differentiation and apoptosis. It is estimated that RB is dysfunctional/inactivated in up to 40% of human leukemias. Positive as well as inhibitory signals are integrated into the phosphorylation of the RB protein to regulate the G1 to S-phase progression of the cell cycle. Despite the importance of RB in leukemia, the consequences of loss of RB on hematopoietic stem and progenitor cell (HSPC) function in vivo are still not clear and have been controversially discussed. Using Cre-enzyme expression driven by the hematopoietic specific Vav1-promotor, we generated mice that are constitutively deficient in RB (hemRb−/− animals) in HSPCs. HemRb−/− mice showed anemia with an increased number of reticulocytes in PB, consistent with a published role of RB in erythroid differentiation. In addition, the frequency of Mac-1 positive cells in BM was increased to 67% compared to 47% in control animals, whereas the frequency of B220 positive B-lymphoid cells was almost 10-fold reduced, without affecting the T-lymphoid compartment. HemRb−/− mice possessed a 3-fold enlarged spleen with a 5-fold increased number of colony-forming cells (CFCs) and severe extramedullary hematopoiesis, a phenotype also reported for animals transplanted with Rb−/− fetal liver cells.BM of hemRb−/− mice showed an almost 3-fold reduction of HSC frequency, measured by the cobblestone-area forming cell assay (CAFC) assay, but not a decrease in the number of HSCs determined by cell surface staining and flow cytometry. Upon transplantation into NOD/SCID animals or upon competitive transplantation into C57BL/6. CD45.1 animals, HSPCs from hemRb−/− mice contributed 4 to 6-fold less to hematopoiesis. HSPCs from hemRb−/− animals were neither impaired in their ability to home to the BM, nor did they show increased apoptosis. Finally, we detected a significant 4-fold decrease in stem cell function/numbers upon stress caused by 5-FU treatment in hemRB−/− mice compared to control animals. We conclude that upon transplantation/stress, HSPCs from hemRb−/− animals are impaired in their self-renewal function. HemRb−/− animals also showed a 2-fold increase in the frequency of CFCs in peripheral blood. As we detected no increased leukemia incidence in the hemRb−/− animals (now up to 1 year of age), loss of the tumor suppressor RB in hematopoietic cells might be regarded as necessary, but not sufficient for causing early onset leukemia. In summary, loss of RB results in context/localization dependent phenotypes in the hematopoietic hierarchy, influencing stem and progenitor cells in function, localization and differentiation ability.

The transcription factor MEF/ELF4 regulates the quiescence of primitive hematopoietic cells

The transcriptional circuitry that regulates the quiescence of hematopoietic stem cells is largely unknown. We report that the transcription factor known as MEF (or ELF4), which is targeted by the t(X;21)(q26;q22) in acute myelogenous leukemia, regulates the proliferation of primitive hematopoietic progenitor cells at steady state, controlling their quiescence. Mef null HSCs display increased residence in G0 with reduced 5-bromodeoxyuridine incorporation in vivo and impaired cytokine-driven proliferation in vitro. Due to their increased HSC quiescence, Mef null mice are relatively resistant to the myelosuppressive effects of chemotherapy and radiation. Thus, MEF plays an important role in the decision of stem/primitive progenitor cells to divide or remain quiescent by regulating their entry to the cell cycle.

Murine Side Population Cells Contain Cobblestone Area-forming Cell Activity in Mobilized Blood

Primitive hematopoietic stem cells (HSCs) can be purified from murine bone marrow by sorting Hoechst 33342-effluxing side population (SP) cells. The aim of this study was to establish whether SP cells from peripheral blood contain primitive HSCs and whether this is altered in mice following mobilization. SP cells were analyzed and isolated from bone marrow and blood of mice after mobilization; the HSC content of isolated SP cells was determined through surrogate cobblestone area-forming cell (CAFC) assays. SP cells in normal blood were not found in the high Hoechst dye effluxing portion of the SP tail, did not express the stem cell markers c-Kit and CD34, and did not have measurable CAFC activity. In contrast, SP cells in mobilized blood expressed both stem cell markers, contained cells in the high dye efflux portion of the SP tail, and displayed significant day- 28 to day-35 CAFC activity with 165- to 334-fold enrichment. In comparison to mobilized blood SP cells, normal marrow SP cells contained a higher proportion of cells expressing c-Kit and CD34 and had a greater percentage of cells in the high Hoechst dye-effluxing portion of the SP tail. Analysis of SP cells in the bone marrow after mobilization revealed a decrease in the frequency of SP cells, in expression of c-Kit and Sca+ CD34(+)/CD34(-), and in day-7 to day-35 CAFC activity, consistent with mobilization into blood. We conclude that murine SP cells mobilized into blood contain primitive hematopoietic stem cell activity (day-28 to day-35 CAFC activity). This model offers a means to study the mechanisms of mobilization of primitive stem cells directly in a murine model.

In vitro functional defects of bone marrow-derived CD34 + progenitors from newly diagnosed mature B-cell malignancies with bone marrow tumor involvement

We hypothesized that the presence of tumor cells in bone marrow (BM) could alter hematopoietic progenitor cell functions. Therefore, we evaluated phenotypic and in vitro functional properties of BM-derived CD34+ progenitors issued from untreated and newly diagnosed patients presenting a mature B-lymphoproliferative disorder (LPD) involving the BM (Inv+). In vitro proliferation and differentiation capacities of primitive and committed progenitors were evaluated by cobblestone area-forming cell (CAFC) and colony-forming cell (CFC) assays, and ex vivo cell expansion. Migratory capacities of CD34+ cells were explored by chemotaxis assays using a CXCL12alpha gradient. Our results showed that CD34+ cells from Inv+ patients overexpressed CD117 and had a significant decrease of week-3 and -6 CAFC, and CFC frequencies, compared to cells obtained from healthy volunteers and LPD patients without BM involvement (Inv-). In addition, progenitors from Inv+ patients maintained a significantly decreased CFC capacity after ex vivo cell expansion, compared to healthy volunteers. However, the former cells held their migratory capacity in response to CXCL12alpha. Functional defects of primitive and committed CD34+ progenitors detected among LPD patients with BM tumor involvement suggest either that tumor cells may induced bystander effects on progenitors or that "unusual" CD34+ cells may exist in the BM that could belong to the proliferating tumor tissue.

Evidence That More Primitive Hematopoietic Progenitor Cells Are Concentrated within the CD133+ Cell Compartment of Cord Blood.

We report the use of the in vitro cobblestone area-forming cell (CAFC) assay to investigate the hematopoietic progenitor cell (HPC) profile of pre-selection, post-selection positive (CD133+) and post-selection negative (CD133−) fractions of cord blood (CB). The CAFC assay provides a qualitative measure of the HPC contained within a given cell population at a given time-point. Briefly, CAFC give rise to cobblestone areas (CA) beneath a stromal cell layer that can be visualized by light microscopy at weekly intervals and their frequencies determined by the use of a limiting dilution strategy. CA that persist in culture for a relatively long period of time are considered to be derived from more primitive, potentially higher quality HPC than those that persist in culture for a shorter period of time. Using this approach, changes within the HPC compartment within a specific cell population can be quantified.Methods: Three frozen CB units were thawed, washed and pre-selection samples removed for CAFC and flow cytometric (CD133+) analysis. Samples were then subjected to CD133+ selection using the Miltenyi MidiMACS device and positive and negative fractions collected for CAFC and CD133+ analysis. Limiting dilution within the CAFC assay was performed in flat-bottomed 96-well plates, over 12, 2.5-fold serial dilutions with 10 wells per dilution. The murine FBMD-1 cell line provided the stromal component of the assay. Culture was performed at 33°C in Iscove's Modified Dulbecco's Medium containing 10% each of fetal bovine and horse serum and supplemented with 10ng/ml recombinant human interleukin 3 (R & D Systems) and 20ng/ml recombinant human granulocyte colony-stimulating factor (Neupogen, Amgen). Cultures were assayed and medium refreshed at weekly intervals.Results: Purity of CD133+ cells in pre-selection CB samples ranged from 1-3% and reached a maximum of 98% following positive selection. Analysis of negative fractions revealed a CD133+ cell purity of 1–2%. As shown in the figure, the frequency of CAFC in the pre-selection fraction reached a maximum at week 5 (~400/106 cells) and by week 10 of culture was reducing (~40/106 cells). The frequency of CAFC in the positive fraction also reached a maximum at week 5 (~6500/106 cells) however, by week 10 of culture the frequency of CAFC was still ~2000/106 cells. The pattern of CAFC in the negative fraction closely mirrored that observed in the pre-selection sample although with reduced frequencies of CAFC at each time-point.Conclusion: CAFC activity present in CB is concentrated in the CD133+ fraction rather than the CD133− fraction and, in particular, CAFC that persist longer in culture (potentially higher quality HPC) are also concentrated in the CD133+ fraction. The CAFC assay is currently being used to track changes that occur within the HPC compartment with ex vivo expansion of isolated CD133+ CB cells to determine whether any one population is preferentially expanded at the expense of any other. This will address concerns that ex vivo expansion techniques expand more mature HPC at the expense of more primitive HPC thereby potentially compromising stem cell reserve. [Display omitted]