Unfractionated Bone Marrow Mononuclear Cells Research Articles

Myeloperoxidase inhibition protects bone marrow mononuclear cells from DNA damage induced by the TOP2 poison anti-cancer drug etoposide.

Myeloperoxidase (MPO) is found almost exclusively in granulocytes and immature myeloid cells. It plays a key role in the innate immune system, catalysing the formation of reactive oxygen species that are important in anti-microbial action, but MPO also oxidatively transforms the topoisomerase II (TOP2) poison etoposide to chemical forms that have elevated DNA damaging properties. TOP2 poisons such as etoposide are widely used anti-cancer drugs, but they are linked to cases of secondary acute myeloid leukaemias through a mechanism that involves DNA damage and presumably erroneous repair leading to leukaemogenic chromosome translocations. This leads to the possibility that myeloperoxidase inhibitors could reduce the rate of therapy-related leukaemia by protecting haematopoietic cells from TOP2 poison-mediated genotoxic damage while preserving the anti-cancer efficacy of the treatment. We show here that myeloperoxidase inhibition reduces etoposide-induced TOP2B-DNA covalent complexes and resulting DNA double-strand break formation in primary ex vivo expanded CD34+ progenitor cells and unfractionated bone marrow mononuclear cells. Since MPO inhibitors are currently being developed as anti-inflammatory agents this raises the possibility that repurposing of these potential new drugs could provide a means of suppressing secondary acute myeloid leukaemias associated with therapies containing TOP2 poisons.

Functional and Molecular Heterogeneity of Aging Hematopoietic Stem Cells

Background: Aging hematopoiesis is characterized by increased numbers of hematopoietic stem cells (HSCs) that exhibit impaired self-renewal and long-term reconstitution potential. We previously demonstrated that differences in surface expression of c-Kit in young mouse HSCs could identify distinct functional HSC subsets, with c-Kithi HSCs exhibiting reduced self-renewal and megakaryocytic biased differentiation. We therefore sought to determine if similar functional and molecular differences may be observed in old HSCs. Methods: We evaluated c-Kithi and c-Kitlo subsets of HSCs (CD34-CD150+LSK) from young (8-12 wk), and old (18-20 mo) C57BL/6J mice. For competitive transplantation assays, 250 double sorted c-Kithi or c-Kitlo HSCs from aged CD45.2 mice were mixed with 5x105 unfractionated bone marrow mononuclear cells (BMMCs) from young CD45.1 mice. In parallel, we generated the chromatin accessibility and transcriptional profiles of c-Kithi and c-Kitlo HSCs from young and old mice. Results: We first performed competitive transplants of old c-Kithi and c-Kitlo HSCs into young recipients. These studies demonstrated age-related myeloid bias exhibited by both HSC subsets. In addition, similar to young HSCs, old c-Kithi HSCs exhibit significantly reduced long-term reconstitution capacity compared to old c-Kitlo HSCs. Next, to evaluate age-related functional differences, we performed equal competitive transplants in which equal numbers of c-Kithi or c-Kitlo old and young HSCs were transplanted into the same young recipient. These studies demonstrated that c-Kithi HSCs exhibit similar reconstituting capacity independent of age. In contrast, old c-Kitlo HSCs exhibited significantly reduced long-term reconstitution compared to their young counterparts, though with better-preserved self-renewal capacity, as reflected in their self-renewal quotients (SRQ). However, when we quantified the capacity of transplanted HSCs to give rise to downstream committed progenitors and mature hematopoietic cells, calculated as their differentiation quotient (DQ), old c-Kitlo HSCs exhibited markedly reduced DQ compared to their young counterparts. In contrast, c-kithi recipients showed that old c-Kithi HSCs gave rise to mature cell lineages more efficiently. To investigate molecular mechanisms driving heterogeneity in HSC aging, we evaluated the transcriptional profiles of young and old HSCs. Old c-Kitlo HSCs showed significant enrichment of genes associated with inflammation, interferon responses, and the quiescent state in comparison to old c-Kithi HSCs. On the other hand, old c-Kithi HSCs were enriched for cell cycle related and MYC target genes compared to old c-Kitlo HSCs and young HSCs. Old c-Kithi HSCs also exhibited increased expression of genes associated with high cell output state and high mitochondrial membrane potential, which is associated with a more active HSC cell state. To investigate the potential role of chromatin accessibility in determining the observed transcriptional changes, we performed ATAC-seq of young and old HSC subsets. Old HSCs exhibited more open chromatin peaks than young HSCs, with the number of peaks decreasing in the order of old c-Kitlo > old c-Kithi > young c-Kithi > young c-Kitlo HSCs. Evaluation of predicted transcription factor binding sites showed enrichment for CTCF and PU.1 sites in young c-Kitlo and c-Kithi HSCs, respectively, with PU.1 associated with high cell output states. In contrast, old c-Kitlo and c-Kithi HSCs were enriched for ERG and CTCF sites, respectively, with predicted binding sites enriched in genes associated with low and high cell output, respectively. These data suggest that differential ERG binding of quiescence and cellular output-related gene signatures regulate functional differences between old c-Kitlo and old c-Kithi HSCs. Conclusion: Overall, our studies demonstrate functional heterogeneity among old HSCs and identify a novel strategy to identify old HSCs with preserved self-renewal and long-term reconstitution capacity. Identifying and prospectively fractionating old HSCs offers a novel approach for investigating the molecular mechanisms underlying HSC aging.

Day +60 WT1 assessment on CD34 selected bone marrow better predicts relapse and mortality after allogeneic stem cell transplantation in acute myeloid leukemia patients.

The aim of this study was to evaluate the role of WT1 expression after allogeneic stem cell transplantation (alloHSCT) in patients with acute myeloid leukemia (AML). We studied WT1 expression in bone marrow cells from 50 patients in complete remission on day +60 after transplant. WT1 was assessed on unfractionated bone marrow mononuclear cells (MNC) and on CD34+ selected cells (CD34+). A ROC curve analysis identified 800 WT1 copies on CD34+ selected cells, as the best cut-off predicting relapse (AUC 0.842, p=0.0006, 85.7% sensitivity and 81.6% specificity) and 100 copies in MNC (AUC 0.819, p=0.007, 83.3% sensitivity and 88.2% specificity). Using the 800 WT1 copy cut off in CD34+ cells, the 2 year cumulative incidence of relapse was 12% vs 38% (p=0.005), and 2 year survival 88% vs 55% (p=0.02). Using the 100 WT1 copy cut off in unfractionated MNC, the 2 year cumulative incidence of relapse 13% vs 44% (p=0.01) and the 2 year survival 88% vs 55% (p=0.08). In a multivariate Cox analysis WT1 expression in CD34 cells proved to highly predictive of relapse (p=0.004); also WT1 expression on unfractionated cells predicted relapse (p=0.03). In conclusion, day-60 WT1 expression after allogeneic HSCT is a significant predictor of relapse, particularly when tested on CD34+ selected bone marrow cells.

Gene expression and risk of leukemic transformation in myelodysplasia

AbstractMyelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic disorders with a highly variable prognosis. To identify a gene expression–based classification of myelodysplasia with biological and clinical relevance, we performed a comprehensive transcriptomic analysis of myeloid neoplasms with dysplasia using transcriptome sequencing. Unsupervised clustering of gene expression data of bone marrow CD34+ cells from 100 patients identified 2 subgroups. The first subtype was characterized by increased expression of genes related to erythroid/megakaryocytic (EMK) lineages, whereas the second subtype showed upregulation of genes related to immature progenitor (IMP) cells. Compared with the first so-called EMK subtype, the IMP subtype showed upregulation of many signaling pathways and downregulation of several pathways related to metabolism and DNA repair. The IMP subgroup was associated with a significantly shorter survival in both univariate (hazard ratio [HR], 5.0; 95% confidence interval [CI], 1.8-14; P < .001) and multivariate analysis (HR, 4.9; 95% CI, 1.3-19; P = .02). Leukemic transformation was limited to the IMP subgroup. The prognostic significance of our classification was validated in an independent cohort of 183 patients. We also constructed a model to predict the subgroups using gene expression profiles of unfractionated bone marrow mononuclear cells (BMMNCs). The model successfully predicted clinical outcomes in a test set of 114 patients with BMMNC samples. The addition of our classification to the clinical model improved prediction of patient outcomes. These results indicated biological and clinical relevance of our gene expression–based classification, which will improve risk prediction and treatment stratification of MDS.

CXCR4(+)CD45(-) BMMNC subpopulation is superior to unfractionated BMMNCs for protection after ischemic stroke in mice.

Cell-based therapy is considered to be a promising therapeutic strategy for stroke treatment. Although unfractionated bone marrow mononuclear cells (BMMNCs) have been tried in both preclinical and clinical trials, the effective subpopulations need to be identified. In this study, we used fluorescence-activated cell sorting to harvest the CXCR4(+)CD45(+) and CXCR4(+)CD45(-) BMMNC subpopulations from transgenic mice that express enhanced green fluorescent protein. We then allogeneically grafted unfractionated BMMNCs or a subpopulation into mice subjected to transient middle cerebral artery occlusion (tMCAO) and compared the effects on stroke outcomes. We found that CXCR4(+)CD45(-) BMMNCs, but not CXCR4(+)CD45(+) BMMNCs, more effectively reduced infarction volume and neurologic deficits than did unfractionated BMMNCs. Brain tissue from the ischemic hemisphere of mice treated with CXCR4(+)CD45(-) BMMNCs had higher levels of vascular endothelial growth factor and lower levels of TNF-α than did tissue from mice treated with unfractionated BMMNCs. In contrast, CXCR4(+)CD45(+) BMMNCs showed an increase in TNF-α. Additionally, CXCR4(+)CD45(+) and CXCR4(+)CD45(-) populations exhibited more robust migration into the lesion areas and were better able to express cell-specific markers of different linages than were the unfractionated BMMNCs. Endothelial and astrocyte cell markers did not colocalize with eGFP(+) cells in the brains of tMCAO mice that received CXCR4(+)CD45(+) BMMNCs. In vitro, the CXCR4(+)CD45(-) BMMNCs expressed significantly more Oct-4 and Nanog mRNA than did the unfractionated BMMNCs. However, we did not detect gene expression of these two pluripotent markers in CXCR4(+)CD45(+) BMMNCs. Taken together, our study shows for the first time that the CXCR4(+)CD45(-) BMMNC subpopulation is superior to unfractionated BMMNCs in ameliorating cerebral damage in a mouse model of tMCAO and could represent a new therapeutic approach for stroke treatment.

Bone Marrow Mesenchymal Stem and Progenitor Cells Are Altered in Sipa1 Deficient Mice before Initiation of Myeloproliferative Neoplasm

Hematopoiesis is tightly regulated by bone marrow (BM) stromal cells through the production of soluble factors, matrix proteins, and intracellular signals initiated by cell-cell adhesive interactions. Despite the involvement of mesenchymal stem and progenitor cells in the maintenance of normal hematopoiesis and the development of hematological disorders have been reported (Mendez-Ferrer et al., 2010, Nature)(Raaijmakers et al., 2010, Nature), little is known how the different BM stromal cell fractions contribute to the processes.Signal-induced proliferation-associated gene 1 (Sipa1), a principal Rap1 GTPase activating protein, is expressed in hematopoietic cells (Ishida et al., 2003, Cancer Cell). Deletion of Sipa1 results in age-dependent development of myeloproliferative neoplasm (MPN) in mice, resembling human chronic myelogenous leukemia (Ishida et al., 2003, Cancer Cell). However, it is not known whether the deletion of Sipa1 affects BM cellular niche components, which could lead to the development of the MPN. Knowledge of this is important for better understanding the role of BM stromal cells in the initiation and progression of MPN.In the present study, we first analyzed Sipa1 expression by multicolor fluorescent activated cell sorting (FACS) using hematopoietic cell marker CD45 and TER119, mesenchymal stem cell (MSC) associated marker CD51 and SCA1, and endothelial cell marker CD31. We found that Sipa1 gene was also expressed in different BM stromal cell subsets including MSCs (CD45-TER119-CD31-CD44-CD51+SCA1+), more differentiated mesenchymal progenitor cells (CD45-TER119-CD31-CD44-CD51+SCA1-), and endothelial cells (CD45-TER119-CD31+). Among all the cell types, Sipa1 expression seemed to be higher in MSCs and endothelial cells than other cell types. We next studied whether the gene deficiency led to the alteration of BM stromal cell composition and function by phenotypically, functionally, and molecularly analyzing BM stromal cell subsets in young (<3 months) Sipa1-/- mice where hematopoiesis remained normal. Phenotypic multicolor FACS analysis showed significant increase in percentages and numbers of BM stromal cell (CD45-TER119-) subsets including endothelial cells, MSCs, and the mesenchymal progenitor cells in Sipa1-/- mice compared to age and sex-matched Sipa1+/+ mice (P<0.05). The alteration in the frequency of BM MSCs in the Sipa1-/- mice was further confirmed by colony-forming unit-fibroblast assay (CFU-F) on unfractionated BM mononuclear cells (P=0.0302) and FACS-sorted MSCs (P=0.0066). It is important to note that most (75% on average) of CFU-Fs generated from FACS-sorted Sipa1-/- stromal cells contained simultaneously differentiated adipocytes whereas around 57% of CFU-Fs from Sipa1+/+ stromal cells did (P=0.0184). In keeping with this, in vitro differentiation assays revealed greater adipocyte differentiation potential of Sipa1-/- MSCs. Similarly, an increase of adipocytes associated gene FABP4 expression was determined by Q-PCR. Our preliminary Q-PCR data indicated differential expression of Kit Ligand and Angipoietinlike 1 in the Sipa1-/- MSCs and the CD51+SCA1- cells compared to that in the Sipa1+/+ cells. Global gene expression analysis by RNA sequencing and more multilineage differentiation assays on FACS-sorted different stromal cell subsets of Sipa1-/- and Sipa1+/+ BM are currently ongoing.Taken together, our data suggested the BM stromal cell composition and function in the Sipa1-/- mice have already been altered before the initiation of MPN. Transplantations of normal hematopoietic stem cells into Sipa1 deficient BM have been initiated for examining if the Sipa1-/- BM niches play an instructive role in the initiation of MPN. DisclosuresNo relevant conflicts of interest to declare.

Stem Cells for Cardiac Repair: Problems and Possibilities

Ischemic heart disease is a major cause of death throughout the world. In order to limit myocardial damage and possibly generate new myocardium, stem cells are currently being injected into patients with ischemic heart disease. Three major patient investigations, The LateTIME, the TIME and the Swiss Myocardial Infarction trials, have recently addressed the questions of whether progenitor cells from unfractionated bone marrow mononuclear cells limit myocardial damage and what the optimal time to inject these cells after acute myocardial infarctions (AMIs) is. In each of these trials, there were no significant differences between treated and control patients when bone marrow cells were administered 5-7 days or 2-3 weeks after AMIs. Nevertheless, these investigations provide important information regarding clinical trial designs. Patients with AMIs in these trials were treated with percutaneous coronary intervention within a median of 4-5 h after the onset of chest pain. Thereafter, all patients received guideline-guided optimal medical therapy. Consequently, the sizes of AMIs were significantly limited. In patients with small AMIs and near-normal left ventricular ejection fractions, progenitor cells are least effective. However, these trials do question whether autologous bone marrow mononuclear cells are the optimal cells for myocardial repair owing to low numbers of progenitor cells in bone marrow aspirates and the significant variability in potency and efficacy of these cells in patients with chronic multisystem diseases. In contrast, the SCIPIO and the CAUDUCEUS trials examined cardiac progenitor cells in patients with ischemic cardiomyopathies. These trials reported over 1-2 years that cardiac progenitor cells produced significant improvements in left ventricular contractility due to 12-24 g decreases in myocardial scars and 18-23 g increases in viable myocardial muscle. However, caution must be exercised in the interpretation of these studies due to the small numbers of highly selected patients and intra- and inter-observer variability in infarct size measurements. Anatomical and histological examinations of large numbers of patients treated with these cells are necessary to confirm significant generation of myocytes and decreases in infarct size and fibrosis.

Extracellular High Mobility Group Box 1 Plays a Role in the Effect of Bone Marrow Mononuclear Cell Transplantation for Heart Failure

Transplantation of unfractionated bone marrow mononuclear cells (BMCs) repairs and/or regenerates the damaged myocardium allegedly due to secretion from surviving BMCs (paracrine effect). However, donor cell survival after transplantation is known to be markedly poor. This discrepancy led us to hypothesize that dead donor BMCs might also contribute to the therapeutic benefits from BMC transplantation. High mobility group box 1 (HMGB1) is a nuclear protein that stabilizes nucleosomes, and also acts as a multi-functional cytokine when released from damaged cells. We thus studied the role of extracellular HMGB1 in the effect of BMC transplantation for heart failure. Four weeks after coronary artery ligation in female rats, syngeneic male BMCs (or PBS only as control) were intramyocardially injected with/without anti-HMGB1 antibody or control IgG. One hour after injection, ELISA showed that circulating extracellular HMGB1 levels were elevated after BMC transplantation compared to the PBS injection. Quantitative donor cell survival assessed by PCR for male-specific sry gene at days 3 and 28 was similarly poor. Echocardiography and catheterization showed enhanced cardiac function after BMC transplantation compared to PBS injection at day 28, while this effect was abolished by antibody-neutralization of HMGB1. BMC transplantation reduced post-infarction fibrosis, improved neovascularization, and increased proliferation, while all these effects in repairing the failing myocardium were eliminated by HMGB1-inhibition. Furthermore, BMC transplantation drove the macrophage polarization towards alternatively-activated, anti-inflammatory M2 macrophages in the heart at day 3, while this was abolished by HMGB1-inhibition. Quantitative RT-PCR showed that BMC transplantation upregulated expression of an anti-inflammatory cytokine IL-10 in the heart at day 3 compared to PBS injection. In contrast, neutralizing HMGB1 by antibody-treatment suppressed this anti-inflammatory expression. These data suggest that extracellular HMGB1 contributes to the effect of BMC transplantation to recover the damaged myocardium by favorably modulating innate immunity in heart failure.

Removing the cells from adult bone marrow derived stem cell therapy does not eliminate cardioprotection†

The debate as to whether adult stem cell therapy is regenerative or not continues. The non-regenerative benefits of adult bone marrow-derived stem cell therapy were investigated by testing whether the supernatant derived from unfractionated bone marrow mononuclear cells might be cardioprotective in an animal model of myocardial ischaemia-reperfusion injury. Regional myocardial reperfusion injury was acquired by 25 min reversible left anterior descending coronary artery (LAD) occlusion followed by 2 h reperfusion, in anaesthetized Wistar male rats. Unfractionated bone marrow mononuclear cells (BMMNC) isolated from sibling Wistar male rat whole bone marrow were phenotyped by fluorescence activated cell sorting flowcytometry for the haematopoietic stem cell surface markers c-kit, CD34, CD45 and CD133. Animals subjected to regional myocardial reperfusion injury received either 10 million BMMNC or BMMNC supernatant (BMS); both were collected in 0.5 ml phosphate-buffered saline and delivered by intravenous bolus at the onset of reperfusion. The left ventricular region distal to the LAD occlusion point was excised for measurement of myocardial infarct size and proteomic analysis, which was used to identify whether there were any differences in myocardial proteins associated with intravenous injection of either BMMNC or BMS. BMMNC were phenotyped to be c-kit(+) (7 ± 1%), CD34(+) (7 ± 1%), CD45(+) (54 ± 6%), CD133(+) (15 ± 1%). The supernatant reduced myocardial infarct size (BMS 34 ± 2%, n = 15 vs control 57 ± 2%, n = 7, P < 0.0001), which was comparable to the reduction in infarct size afforded by the injection of cells (BMMNC 33 ± 3% vs control 57 ± 2%, n = 10, P < 0.0001). Proteomics of hearts treated with either BMS or BMMNC demonstrated higher expression of (i) anti-apoptotic signal transduction protein: 14-3-3-epsilon (1.5-fold); (ii) anti-oxidants: peroxiredoxin-6 (2.1-fold); (iii) heat shock proteins: alpha B-crystallin (1.7-fold), heat shock protein 72 (2.8-fold), tumour necrosis factor receptor-1 associated protein (2.3-fold), ischaemia responsive protein-94 (1.6-fold); (iv) glycolytic protein: glyceraldehyde-3-phosphate dehydrogenase (2.3-fold); (v) mitochondrial respiratory proteins: mitochondrial aconitase (4.7-fold), voltage-dependent anion-selective channel protein-1 (VDAC-1) (2.7-fold). Regional myocardial reperfusion injury can be attenuated by intravenous administration of either BMMNC or BMS at the onset of reperfusion, which suggests adult stem cells mediate non-regenerative cardioprotection.

Therapeutic effects of human STRO‐3‐selected mesenchymal precursor cells and their soluble factors in experimental myocardial ischemia

Stromal precursor antigen (STRO)-3 has previously been shown to identify a subset of adult human bone marrow (BM)-derived mesenchymal lineage precursors, which may have cardioprotective potential. We sought to characterize STRO-3-immunoselected and culture-expanded mesenchymal precursor cells (MPCs) with respect to their biology and therapeutic potential in myocardial ischemia. Immunoselection of STRO-3+ MPCs enriched for fibroblastic colony forming units from unfractionated BM mononuclear cells (MNCs). Compared to mesenchymal stem cells conventionally isolated by plastic adherence, MPCs demonstrated increased proliferative capacity during culture expansion, expressed higher levels of early ‘stem cell’ markers and various pro-angiogenic and cardioprotective cytokines, and exhibited greater trilineage developmental efficiency. Intramyocardial injection of MPCs into a rat model of myocardial infarction (MI) promoted left ventricular recovery and inhibited left ventricular dilatation. These beneficial effects were associated with cardioprotective and pro-angiogenic effects at the tissue level, despite poor engraftment of cells. Treatment of MI rats with MPC-conditioned medium (CM) preserved left ventricular function and dimensions, reduced myocyte apoptosis and fibrosis, and augmented neovascularization, involving both resident vascular cells and circulating endothelial progenitor cells (EPCs). Profiling of CM revealed various cardioprotective and pro-angiogenic factors, which had biological activity in cultures of myocytes, tissue-resident vascular cells and EPCs. Prospective immunoselection of STRO-3+ MPCs from BM MNCs conferred advantage in maintaining a population of immature MPCs during ex vivo expansion. Transplantation of culture-expanded MPCs into the post-MI heart resulted in therapeutic benefit, attributable at least in part to paracrine mechanisms of action. Thus, MPCs represent a promising therapy for myocardial ischemia.

Lentivirus-mediated Wnt11 Gene Transfer Enhances Cardiomyogenic Differentiation of Skeletal Muscle-derived Stem Cells

Wnt signaling plays a crucial role in regulating cell proliferation, differentiation and inducing cardiomyogenesis. Skeletal muscle-derived stem cells (MDSCs) have been shown to be multipotent; however, their potential to aid in the healing of the heart after myocardial infarction appears to be due to the paracrine effects they impart on the host environment. The goal of this study was to investigate whether Wnt11 could promote the differentiation of MDSCs into cardiomyocytes and enhance the repair of infarcted myocardium. MDSCs transduced with a lentivirus encoding for Wnt11 increased mRNA and protein expression of the early cardiac markers NK2 transcription factor related 5 (NKx2.5) and Connexin43 (Cx43) and also led to an increased expression of late-stage cardiac markers including: α, β-myosin heavy chain (MHC) and brain natriuretic protein (BNP) at the mRNA level, and MHC and Troponin I (TnI) at the protein level. We also observed that Wnt11 expression significantly enhanced c-jun N-terminal kinase activity in transduced MDSCs, and that some of the cells beat spontaneously but are not fully differentiated cardiomyocytes. Finally, lentivirus-Wnt11-transduced MDSCs showed greater survival and cardiac differentiation after being transplanted into acutely infarct-injured myocardium. These findings could one day lead to strategies that could be utilized in cardiomyoplasty treatments of myocardial infarction.

Absence of accelerated atherosclerotic disease progression after intracoronary infusion of bone marrow derived mononuclear cells in patients with acute myocardial infarction—Angiographic and intravascular ultrasound—Results from the TErapia Celular Aplicada al Miocardio Pilot study

We tried to evaluate a putative negative effect on coronary atherosclerosis in patients receiving intracoronary infusion of unfractionated bone marrow mononuclear cells (BMMC) following an acute ST-elevation myocardial infarction. Peripheral blood mononuclear cells or enriched CD133(+) BMMC have been associated with accelerated atherosclerosis of the distal segment of the infarct related artery (IRA). Thirty-seven patients with ST-elevation myocardial infarction from the TECAM pilot study underwent intracoronary infusion of autologous BMMC 9 +/- 3.1 days after onset of symptoms. We compared angiographic changes from baseline to 9 months of follow-up in the distal non-stented segment of the IRA, as well as in the contralateral coronary artery, with a matched control group. A subgroup of 15 treated patients underwent additional IVUS within the distal segment of the IRA. No difference between stem cell and control group were found regarding changes in minimum lumen diameter (0.006 +/- 0.42 vs 0.06 +/- 0.41 mm, P = ns) and the percentage of stenosis (-2.68 +/- 12.33% vs -1.78 +/- 8.75%, P = ns) at follow-up. Likewise, no differences were seen regarding changes in the contralateral artery (minimum lumen diameter -0.004 +/- 0.54 mm vs -0.06 +/- 0.35 mm, P = ns). In the intravascular ultrasound substudy, no changes were demonstrated comparing baseline versus follow-up in maximum area stenosis and plaque volume. In this pilot study, analysis of a subgroup of patients found that intracoronary injection of unfractionated BMMC in patients with acute ST-elevation myocardial infarction was not associated with accelerated atherosclerosis progression at mid term. Prospective, randomised studies in large cohorts with long-term angiographic and intravascular ultrasound follow-up are necessary to determine the safety of this therapy.

Bone-Marrow-Derived Side Population Cells for Myocardial Regeneration

Bone-marrow-derived stem cells have displayed the potential for myocardial regeneration in animal models as well as in clinical trials. Unfractionated bone marrow mononuclear cell (MNC) population is a heterogeneous group of cells known to include a number of stem cell populations. Cells in the side population (SP) fraction have a high capacity for differentiation into multiple lineages. In the current study, we investigated the role of murine and human bone-marrow-derived side population cells in myocardial regeneration. In these studies, we show that mouse bone-marrow-derived SP cells expressed the contractile protein, alpha-actinin, following culture with neonatal cardiomyocytes and after delivery into the myocardium following injury. Moreover, the number of green-fluorescent-protein-positive cells, of bone marrow side population origin, increased progressively within the injured myocardium over 90 days. Transcriptome analysis of these bone marrow cells reveals a pattern of expression consistent with immature cardiomyocytes. Additionally, the differentiation capacity of human granulocyte colony-stimulating factor stimulated peripheral blood stem cells were assessed following injection into injured rat myocardium. Bone marrow mononuclear cell and side population cells were both readily identified within the rat myocardium 1 month following injection. These human cells expressed human-specific cardiac troponin I as determined by immunohistochemistry as well as numerous cardiac transcripts as determined by polymerase chain reaction. Both human bone marrow mononuclear cells and human side population cells augmented cardiac systolic function following a modest drop in function as a result of cryoinjury. The augmentation of cardiac function following injection of side population cells occurred earlier than with bone marrow mononuclear cells despite the fact that the number of side population cells used was one tenth that of bone marrow mononuclear cells (9 x 10(5) cells per heart in the MNC group compared to 9 x 10(4) per heart in the SP group). These results support the hypotheses that rodent and human-bone-marrow derived side population cells are capable of acquiring a cardiac fate and that human bone-marrow-derived side population cells are superior to unfractionated bone marrow mononuclear cells in augmenting left ventricular systolic function following cryoinjury.

Noncanonical Wnt11 Signaling Is Sufficient to Induce Cardiomyogenic Differentiation in Unfractionated Bone Marrow Mononuclear Cells

Despite the frequent clinical use of adult unfractionated bone marrow mononuclear cells (BMMNCs) for cardiac repair, whether these cells are capable of undergoing cardiomyogenic differentiation in vitro remains uncertain. In addition, the role of Wnt signaling in cardiomyogenic differentiation of adult cells is unclear. Unfractionated BMMNCs were isolated from adult mice via Ficoll-Paque density-gradient centrifugation and cultured in the presence of Wnt3a or Wnt11. In control BMMNCs, Wnt11 was not expressed, whereas the expression of markers of pluripotency (Oct-4 and Nanog), as well as that of Wnt3a and beta-catenin, decreased progressively during culture. Exposure to Wnt3a rescued beta-catenin expression and markedly increased the expression of Oct-4 and Nanog, concomitant with increased cell proliferation and CD45 expression. In contrast, exposure to ectopically expressed noncanonical Wnt11 markedly decreased the expression of Oct-4 and Nanog and induced mRNA expression (quantitative real-time reverse-transcription polymerase chain reaction) of cardiac-specific genes (Nkx2.5, GATA-4, atrial natriuretic peptide, alpha- and beta-myosin heavy chain, and cardiac troponin T) by day 3 with subsequent progression to a pattern characteristic of the cardiac fetal gene program. After 21 days, 27.6+/-0.6% and 29.6+/-1.4% of BMMNCs expressed the cardiac-specific antigens cardiac myosin heavy chain and cardiac troponin T, respectively (immunocytochemistry), indicating cardiomyogenic lineage commitment. Wnt11-induced cardiac-specific expression was completely abolished by the protein kinase C inhibitor bisindolylmaleimide I, partially abolished by the c-Jun-N-terminal kinase inhibitor SP600125, and attenuated by the Wnt inhibitor Dickkopf-1. In adult density-gradient separated BMMNCs, canonical Wnt3a promotes stemness, proliferation, and hematopoietic commitment, whereas noncanonical signaling via Wnt11 induces robust cardiomyogenic differentiation in a protein kinase C- and c-Jun-N-terminal kinase-dependent manner.

Enhanced growth of myelodysplastic colonies in hypoxic conditions

To determine the response of bone marrow progenitor cells from patients with myelodysplastic syndromes (MDS) to culture in physiologic oxygen tension. Methylcellulose progenitor assays using both unfractionated bone marrow mononuclear cells (MNCs) and purified CD34(+) progenitors were performed in atmospheric oxygen (18.6% O(2)) or one of two levels of hypoxia (1% and 3% O(2)). Assays were performed using normal donor marrow, MDS patient marrow, acute myelogenous leukemia marrow or peripheral blood blasts, chronic phase chronic myelogenous leukemia (CML) marrow MNCs, and blast crisis CML peripheral blood. The majority of MDS samples showed decreased colony-forming units (CFU) in 18.6% O(2) compared to normal controls, as expected. However, in either 1% or 3% O(2), 9 of 13 MDS samples demonstrated augmentation of CFUs beyond that observed in normal controls, with 6 of 13 demonstrating a greater than ninefold augmentation. This effect is cell autonomous, as it persisted after purification of CD34(+) progenitor cells. Additionally, the augmented response to physiologic oxygen tension is specific to MDS, as it was not observed in either acute or chronic myelogenous leukemia samples. These results suggest that the reported decrease in MDS CFUs reflects greater sensitivity of MDS progenitors or their progeny to the nonphysiologic oxygen tensions routinely used in vitro, rather than a true decrease in progenitor frequency. Importantly, these experiments for the first time describe an experimental system that can be used to study the growth of primary cells from patients with MDS.

Differential promotion of angiogenesis by distinct bone marrow cell subpopulations

Abstract Introduction: Bone marrow mononuclear cells (BMMC) have the capacity to differentiate into endothelial cells and promote neovascularization in hindlimb ischemia. However, the specific bone marrow cell population that promotes revascularization is unknown. Therefore, we determined the ability of unfractionated BMMC, CD34+ and CD34− from bone marrow to promote angiogenesis in an in-vivo murine model. Methods: BMMC were obtained from total bone marrow harvested from C57Bl/6 mice. CD34+ and CD34− subpopulations were isolated using magnetic bead separation. 1cc Matrigel was admixed with either unfractionated BMMC or CD34+ or CD34− cells was injected subcutaneously. Matrigel +/− VEGF (500ng/cc) was injected as positive and negative controls. Matrigel implants were harvested 14 days later and were analyzed by immunohistochemistry with determination of CD31+ and smooth-muscle actin (SMA) positive areas by morphometry. Results: No angiogenesis was observed in plain Matrigel. All implants seeded with bone-marrow-derived cells promoted neovessel formation in a dose-dependent manner. Unfractionated BMMC promoted formation of neovessels containing both CD31+ and SMA + cells. CD31+ area was greatest in implants seeded with CD34+ cells. In contrast, SMA+ area was greatest in implants containing CD34− cells, which promoted development of larger SMA-containing vessels. Conclusions: BMMC are capable of promoting angiogenesis in an in-vivo Matrigel model in a dose-dependent manner. CD34+ bone marrow cells are most capable of promoting endothelial development. In contrast, the CD34− subpopulation promotes development of larger arteries containing vascular SMA. These results have important implications for the development of cell-based therapies for limb ischemia.

Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors

Fanconi anemia (FA) is an inherited cancer susceptibility syndrome caused by mutations in a DNA repair pathway including at least 6 genes (FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG). The clinical course of the disease is dominated by progressive, life-threatening bone marrow failure and high incidence of acute myelogenous leukemia and solid tumors. Allogeneic bone marrow transplantation (BMT) is a therapeutic option but requires HLA-matched donors. Gene therapy holds great promise for FA, but previous attempts to use retroviral vectors in humans have proven ineffective given the impaired proliferation potential of human FA hematopoietic progenitors (HPCs). In this work, we show that using lentiviral vectors efficient genetic correction can be achieved in quiescent hematopoietic progenitors from Fanca(-/-) and Fancc(-/-) mice. Long-term repopulating HPCs were transduced by a single exposure of unfractionated bone marrow mononuclear cells to lentivectors carrying the normal gene. Notably, no cell purification or cytokine prestimulation was necessary. Resistance to DNA- damaging agents was fully restored by lentiviral transduction, allowing for in vivo selection of the corrected cells with nonablative doses of cyclophosphamide. This study strongly supports the use of lentiviral vectors for FA gene therapy in humans.

Effects of stem cell factor on osteoclast-like cell formation in long-term human marrow cultures.

Stem cell factor (SCF) is a newly described hematopoietic growth factor that stimulates the growth of primitive hematopoietic progenitors and mast cells. Since the osteoclast precursor is hematopoietic in origin, we tested SCF for its capacity to stimulate the formation of osteoclast-like multinucleated cells (MNC) in long-term human marrow cultures. These MNC express an osteoclast phenotype and form resorption lacunae on calcified matrices. Addition of SCF alone (0.1 pg/ml to 100 ng/ml) to long-term marrow cultures did not increase MNC formation. However, treatment of these cultures sequentially with SCF for 1 week followed by 1,25-(OH)2D3 for the second and third weeks of culture significantly enhanced MNC formation. [3H]Thymidine incorporation studies showed that SCF increased the proliferation of MNC precursors. These data suggested that SCF was acting on early MNC precursors. We then tested the capacity of SCF to stimulate the formation of colonies of committed precursors for osteoclast-like MNC. SCF (20 pg/ml to 20 ng/ml) enhanced osteoclast precursor formation in unfractionated bone marrow mononuclear cells but was unable to increase osteoclast precursor formation when a highly purified population of hematopoietic precursors was used as the target cells for SCF. These data suggest that SCF works in concert with other factors produced by nonhematopoietic marrow cells to increase the precursor pool for osteoclasts and that other factors, such as 1,25-(OH)2D3, complete the differentiation process to the mature osteoclast.

Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer.

Moloney Murine Leukemia Virus (MoMuLV) causes T cell neoplasms in rodents but is not known to be a pathogen in primates. The core protein and enzyme genes of the MoMuLV genome together with an amphotropic envelope gene are utilized to engineer the cell lines that generate retroviral vectors for use in current human gene therapy applications. We developed a producer clone that generates a very high concentration of retroviral vector particles to optimize conditions for gene insertion into pluripotent hematopoietic stem cells. This producer cell line also generates a much lower concentration of replication-competent virus that arose through recombination. Stem cells from rhesus monkeys were purified by immunoselection with an anti-CD34 antibody, incubated in vitro for 80-86 h in the presence of retroviral vector particles with accompanying replication-competent virus and used to reconstitute recipients whose bone marrow had been ablated by total body irradiation. The retroviral vector genome was detected in circulating cells of five of eight transplant recipients of CD34+ cells and in the circulating cells of two recipients of infected, unfractionated bone marrow mononuclear cells. Three recipients of CD34+ cells had a productive infection with replication-competent virus. Six or seven mo after transplantation, each of these animals developed a rapidly progressive T cell neoplasm involving the thymus, lymph nodes, liver, spleen, and bone marrow. Lymphoma cells contained 10-50 copies of the replication-competent virus, but lacked the retroviral vector genome. We conclude that replication-competent viruses arising from producer cells making retroviral vectors can be pathogenic in primates, which underscores the importance of carefully screening retroviral producer clones used in human trials to exclude contamination with replication-competent virus.

An intrinsic progenitor defect in Diamond-Blackfan anaemia.

To determine whether the erythropoietin (epo) insensitivity of erythroid progenitor differentiation in congenital pure red cell aplasia or Diamond-Blackfan anaemia is intrinsic to the progenitor itself or is due to defective accessory cell function or active suppression, progenitors from normals and two patients (one steroid resistant and one spontaneously remitting), separated from all known accessory cells using sequential negative selection techniques (adherence, E-rosetting, and direct and indirect immune-panning), were studied. Initially, we evaluated three patients with DBA using unfractionated bone marrow mononuclear cells. Progenitors from two steroid non-responsive patients showed insensitivity to crude epo (c-epo) while one steroid responsive patient demonstrated normal in vitro sensitivity to c-epo. When recombinant epo (r-epo) was used in place of c-epo, the two steroid non-responders continued to demonstrate in vitro progenitor epo insensitivity. However, sensitivity of progenitors from the steroid responder, which was normal in the presence of c-epo, became abnormal when recombinant epo (r-epo) was substituted. Thus, using unfractionated bone marrow, the abnormal response to epo of progenitors from some patients with DBA appears to be obscured by stimulating factors termed erythroid burst-promoting activity (BPA) which are present in c-epo. Using fractionated highly enriched progenitors, from normals and a steroid responsive patient a final 3-10-fold enrichment of progenitors was achieved, but no such enrichment was seen when marrow from a steroid resistant patient was cultured. The epo sensitivities of normal and of patient erythroid progenitors were similar. However, at sub-optimal epo concentrations in both patients CFU-E responsiveness to crude BPA was abnormal compared to the three controls. We conclude from these studies that in DBA: (a) the failure of erythropoiesis is due to an intrinsic progenitor defect; (b) this defect involves progenitor insensitivity to factors in addition to erythropoietin: and (c) there exists a spectrum of disease reflected in the degree of the in vitro abnormality observed.