Marrow-derived Mononuclear Cells Research Articles

Human Cytomegalovirus Selectively Suppresses the Megakaryo/Thrombopoiesis of PDGFR+ and αvβ3+ Megakaryocytes Via the TPO/c-Mpl Pathway after Allo-HSCT

Introduction Virus-induced thrombocytopenia is a severe complication in immunocompromised hosts. Among patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT), human cytomegalovirus (HCMV) infection contributes to a variety of end-organ diseases and hematological complications, leading to increased mortality. Even with antiviral treatment, HCMV remains a potentially lethal infection due to the lack of understanding of the underlying mechanisms of host-virus interactions. The key to solving this problem is to identify the factors that predispose patients to HCMV infection and carry out targeted therapy. Here, we investigated the megakaryo/thrombopoiesis process, including the thrombopoietin (TPO)/c-Mpl pathway, after HCMV infection in vivo and in vitro, screened for susceptible subsets of megakaryocytes (MKs) and explored novel therapeutic targets for HCMV infection. Methods To test whether thrombocytopenia induced by HCMV results from an impaired megakaryo/thrombopoiesis process, we studied the impact of HCMV in an in vivo model of HCMV DNAemia patients following allo-HSCT and an in vitro model of bone marrow CD34+-derived MKs infected with serum from HCMV DNAemia patients. Forty patients who had received allo-HSCT were enrolled in this study, among whom 18 recipients had HCMV DNAemia and 22 were HCMV negative, and bone marrow-derived mononuclear cells (MNCs) from patients were tested for CD41, vWF, pp65, c-Mpl, PDGFR, αvβ3 and TLR2 using flow cytometry (FCM). Transmission electron microscopy (TEM) was used to detect HCMV capsids inside MKs. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. Finally, inhibitors of PDGFR (IMC-3G3 and Gleevec), αvβ3 and TLR2 were cocultured with MKs. Results Our data showed that pp65+ cells accounted for 40.59±6.12% of total CD41+vWF+ MKs from HCMV DNAemia patients, and there was a significant increase in the expression of αvβ3, PDGFR and TLR2 in pp65+ MKs compared with that in control patients. Furthermore, the percentage of PDGFR+αvβ3+ MKs emerged as an independent factor associated with HCMV infection in multivariate analysis (p = 0.008). MKs in HCMV-infected patients showed increased apoptosis and necrosis and different patterns of MK ploidy distribution compared with those in HCMV-negative patients, with a decreased proportion from 16N to 64N and a peak at 8N. Meanwhile, the expression of TPO receptor c-Mpl was lower in pp65+ MKs from HCMV DNAemia patients (0.77±0.38% in pp65+ MKs from HCMV DNAemia patients, 1.75±0.40% in pp65- MKs from HCMV DNAemia patients, 1.97±0.67% in MKs from HCMV-negative patients, and 2.06±0.29% in MKs from healthy controls, p<0.01) while the TPO level in serum was increased compared with that in controls. Next, we established an in vitro HCMV infection model of CD34+-derived MKs with serum from HCMV DNAemia patients, and the laboratory HCMV strain Towne was used as a positive control. After 9 days of coculturing, the viral capsids of HCMV were observed in the nuclei of MKs (Figure 1A), and HCMV infection increased the apoptosis of MKs and shifted them to low ploidy, with a significant decrease in platelet release. As with the in vivo results, c-Mpl was downregulated in HCMV-infected MKs. The expression levels of PDGFR, TLR2 and αvβ3 on MKs were increased in coculture with HCMV DNAemia serum, and pp65-positive MKs were decreased compared with the control after treatment with inhibitors of PDGFR and αvβ3 (Figure 1B). However, neither Gleevec nor anti-TLR2 altered the HCMV infection rate. Conclusions Our study showed that HCMV could impair megakaryopoiesis throughout maturation, apoptosis, and platelet generation via the TPO/c-Mpl pathway both in vivo and in vitro. MKs with PDGFR+ and αvβ3+ phenotypes are susceptible to HCMV infection and we proposed PDGFR and αvβ3 inhibitors as potential therapeutic alternatives for allo-HSCT patients with HCMV infection. Disclosures No relevant conflicts of interest to declare.

Enhancing the Therapeutic Efficacy of Bone Marrow-Derived Mononuclear Cells with Growth Factor-Expressing Mesenchymal Stem Cells for ALS in Mice.

SummarySeveral treatments have been attempted in amyotrophic lateral sclerosis (ALS) animal models and patients. Recently, transplantation of bone marrow-derived mononuclear cells (MNCs) was investigated as a regenerative therapy for ALS, but satisfactory treatments remain to be established. To develop an effective treatment, we focused on mesenchymal stem cells (MSCs) expressing hepatocyte growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor using human artificial chromosome vector (HAC-MSCs). Here, we demonstrated the transplantation of MNCs with HAC-MSCs in ALS mice. As per our results, the progression of motor dysfunction was significantly delayed, and their survival was prolonged dramatically. Additional analysis revealed preservation of motor neurons, suppression of gliosis, engraftment of numerous MNCs, and elevated chemotaxis-related cytokines in the spinal cord of treated mice. Therefore, growth factor-expressing MSCs enhance the therapeutic effects of bone marrow-derived MNCs for ALS and have a high potential as a novel cell therapy for patients with ALS.

PARP Inhibition Sensitize BCR-ABL1 Positive Cel

Introduction: BCR-ABL1 play a key role in the development of chronic myelogenous leukemia and a part of Ph1 positive acute lymphoblastic leukemia (ALL). BCR-ABL1 functions as a tyrosine kinase. Whereas, BCR-ABL1 induces genomic instability by downregulation of BRCA1. An innate error of BRCA1, a molecule involved in the homologous recombination repair pathway, causes hereditary breast and ovarian cancer. PARP inhibitor (PARPi) induces synthetic lethality in BRCA defective cell. Therefore, PARP inhibitor is expected to induce efficient cell death with BCR-ABL1 positive cell. In addition, in some previous reports, reduction of PARP1 activity leads to the upregulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and BCR-ABL1 tyrosine kinase activates PI3K/AKT pathway. These findings suggest activation of the PI3K/AKT pathway leading to PARPi resistance in BCR-ABL1 positive leukemic cells. Here, we demonstrate that PARP inhibition attenuates BCR-ABL1 mediated leukemogenesis and aberration of factors associated with PARP inhibitor resistance induces cell death to fully transformed leukemic cells. Method: Bone marrow-derived mononuclear cells (MNC) from wild type mice and BCR-ABL1 transgenic (Tg) mice were exposed to PARPi in vivo, and cell death was analyzed Annexin-V positivity. PARPi sensitivity to BCR-ABL1 expressed cell was also investigated in vivo bone marrow transplantation model using mouse hematopoietic stem cell (HCS) infected with BCR-ABL1 expressing retrovirus. To evaluate more precisely the results obtained in vitro and in vivo transplantation model, the genetical approach was also performed. The Parp1 knockout (KO) mice were crossed with BCR-ABL1 Tg mice. Then, Leukemia development and subsequent mouse death were observed. In vitro, HR activity was examined using DR-GFP assay. Genomic instability was investigated using the breakage-fusion-bridge (BFB) generation.Maintenance of HSC as a progenitor of the leukemic cell was analyzed by repopulation activity using colony assay. The growth-inhibitory effect was assessed using BCR-ABL positive cell lines with PARPi and PI3K inhibitor. Results: BCR-ABL1 Tg mice derived MNC showed more hypersensitivity to PARPi. Mouse HCS was infected with BCR-ABL1 expressing retrovirus and transplanted lethally Olaparib or vehicle was administrated intraperitoneal injection one day after transplantation. BCR-ABL1 mediated leukemic death was observed 1 month after transplantation in sham-treated mouse, whereas, Olaparib treated mouse did not develop BCR-ABL1 mediated leukemia. Parp1 KO BCR-ABL1 Tg mice attenuated leukemia development and extended their survival compared with BCR-ABL1 Tg mice. In vitro experiment revealed HR activity was down-regulated by BCR-ABL1 expression in DR-GFP assay. The number of BFB generation was increased in BCR-ABL1 Tg with Parp1 KO background. The colony-forming activity of BCR-ABL1 positive HSC was totally abolished by PARP inhibition after 3 times serial replating, whereas sham-treated HSC retained repopulation activity. However, the effect of PARPi on BCR-ABL positive leukemic cell lines was controversial. Therefore, leukemic cell lines were treated with the PARPi and inhibitors toward the molecules associated with PARPi resistance. As a result, a combination of PARPi with PI3K inhibitor effectively induce cell death in PARPi resistant BCR-ABL1 positive leukemic cell lines. Conclusion and discussion: Tyrosine kinase inhibitor (TKI) is the gold standard of the therapeutic option of BCR-ABL1 positive leukemia. However, TKI monotherapy is not sufficient for complete eradication of leukemic cells. It is highly expected that molecules effectively induce cell death to leukemic cells combined with TKI. PARPi would be one of these candidates. However, PARPi could not induces efficient death in all of the cancer cells that carry the mutation of molecules associated with the HR defect. Comprehensive genetic analysis to reveal PARPi resistance is important for HRR defective cancer cells. Combination therapy of PARPi and inhibitorstoward the molecules associated with PARPi resistance would be a good therapeutic option for Ph1 positive leukemia. Disclosures No relevant conflicts of interest to declare.

Serial Cerebral Metabolic Changes in Patients With Ischemic Stroke Treated With Autologous Bone Marrow Derived Mononuclear Cells.

Purpose: Cell-based therapy offers new opportunities for the development of novel treatments to promote tissue repair, functional restoration, and cerebral metabolic balance. N-acetylasperate (NAA), Choline (Cho), and Creatine (Cr) are three major metabolites seen on proton magnetic resonance spectroscopy (MRS) that play a vital role in balancing the biochemical processes and are suggested as markers of recovery. In this preliminary study, we serially monitored changes in these metabolites in ischemic stroke patients who were treated with autologous bone marrow-derived mononuclear cells (MNCs) using non-invasive MRS.Materials and Methods: A sub-group of nine patients (3 male, 6 female) participated in a serial MRS study, as part of a clinical trial on autologous bone marrow cell therapy in acute ischemic stroke. Seven to ten million mononuclear cells were isolated from the patient's bone marrow and administered intravenously within 72 h of onset of injury. MRS data were obtained at 1, 3, and 6 months using a whole-body 3.0T MRI. Single voxel point-resolved spectroscopy (PRESS) was obtained within the lesion and contralesional gray matter. Spectral analysis was done using TARQUIN software and absolute concentration of NAA, Cho, and Cr was determined. National Institute of Health Stroke Scale (NIHSS) was serially recoreded. Two-way analysis of variance was performed and p < 0.05 considered statistically significant.Results: All metabolites showed statistically significant or clear trends toward lower ipsilesional concentrations compared to the contralesional side at all time points. Statistically significant reductions were found in ipsilesional NAA at 1M and 3M, Cho at 6M, and Cr at 1M and 6M (p < 0.03), compared to the contralesional side. Temporally, ipsilesional NAA increased between 3M and 6M (p < 0.01). On the other hand, ipsilesional Cho showed continued decline till 6M (p < 0.01). Ipsilesional Cr was stable over time. Contralesional metabolites were relatively stable over time, with only Cr showing a reduction 3M (p < 0.02). There was a significant (p < 0.03) correlation between ipsilesional NAA and NIHSS at 3M follow-up.Conclusion: Serial changes in metabolites suggest that MRS can be applied to monitor therapeutic changes. Post-treatment increasing trends of NAA concentration and significant correlation with NIHSS support a potential therapeutic effect.

COMPARE CPM-RMI Trial: Intramyocardial Transplantation of Autologous Bone Marrow-Derived CD133+ Cells and MNCs during CABG in Patients with Recent MI: A Phase II/III, Multicenter, Placebo-Controlled, Randomized, Double-Blind Clinical Trial.

Objective:The regenerative potential of bone marrow-derived mononuclear cells (MNCs) and CD133+ stem cells in the heart varies in terms of their pro-angiogenic effects. This phase II/III, multicenter and double-blind trial is designed to compare the functional effects of intramyocardial autologous transplantation of both cell types and placebo in patients with recent myocardial infarction (RMI) post-coronary artery bypass graft.Materials and Methods:This was a phase II/III, randomized, double-blind, placebo-controlled trial COMPARE CPM-RMI (CD133, Placebo, MNCs - recent myocardial infarction) conducted in accordance with the Declaration of Helsinki that assessed the safety and efficacy of CD133 and MNCs compared to placebo in patients with RMI. We randomly assigned 77 eligible RMI patients selected from 5 hospitals to receive CD133+ cells, MNC, or a placebo. Patients underwent gated single photon emission computed tomography assessments at 6 and 18 months post-intramyocardial transplantation. We tested the normally distributed efficacy outcomes with a mixed analysis of variance model that used the entire data set of baseline and between-group comparisons as well as within subject (time) and group×time interaction terms.Results:There were no related serious adverse events reported. The intramyocardial transplantation of both cell types increased left ventricular ejection fraction by 9% [95% confidence intervals (CI): 2.14% to 15.78%, P=0.01] and improved decreased systolic wall thickening by -3.7 (95% CI: -7.07 to -0.42, P=0.03). The CD133 group showed significantly decreased non-viable segments by 75% (P=0.001) compared to the placebo and 60% (P=0.01) compared to the MNC group. We observed this improvement at both the 6- and 18-month time points.Conclusion:Intramyocardial injections of CD133+ cells or MNCs appeared to be safe and efficient with superiority of CD133+ cells for patients with RMI. Although the sample size precluded a definitive statement about clinical outcomes, these results have provided the basis for larger studies to confirm definitive evidence about the efficacy of these cell types (Registration Number: NCT01167751).

The use of stem cells in ischemic heart disease treatment.

Ischemic heart disease is a major cause of death and disabilities worldwide. Unfortunately, not all patients are suitable for direct revascularization. Cell-based therapies may be alternative options because of their potential to promote neovascularisation and endothelial repair, improving myocardial perfusion. The success of cell-based therapies depends on the type of implanted stem cells, delivery method and underlying disease. Several different cell populations including bone marrow-derived mononuclear cells (MNCs), mesenchymal stromal cells (MSCs), CD34+, CD133+, endothelial progenitor cells, adipose-derived mesenchymal stromal cells (ASCs) and stem cells from placenta and umbilical cord have been investigated. Presently, no consensus exists about the best cell type for clinical regenerative therapy. Because the system of coronary arteries in the ischemic area is poor and most of the coronary artery is significantly narrowed or closed, direct implantation of stem cells in the ischemic area of the heart muscle appears an attractive method.

Functional genomic fabrics are remodeled in a mouse model of Chagasic cardiomyopathy and restored following cell therapy

We previously found that, in a mouse model of Chagas cardiomyopathy, 18% of the 9390 quantified unigenes were significantly regulated by Trypanosoma cruzi infection. However, treatment with bone marrow-derived mononuclear cells (MNCs) resulted in 84% transcriptomic recovery. We have applied new algorithms to reanalyze these datasets with respect to specific pathways [Chagas disease (CHAGAS), cardiac muscle contraction (CMC) and chemokine signaling (CCS)]. In addition to the levels of expression of individual genes we also calculated gene expression variability and coordination of expression of each gene with all others. These additional measures revealed changes in the control of transcript abundances and gene networking in CHAGAS and restoration following MNC treatment, not accessible using the conventional approach limited to the average expression levels. Moreover, our weighted pathway regulation analysis incorporated the contributions of all affected genes, eliminating the arbitrary cut-off criteria of fold-change and/or p-value for significantly regulated genes. The new analyses revealed that T. cruzi infection had large transcriptomic consequences for the CMC pathway and triggered a huge cytokine signaling. Remarkably, MNC therapy not only restored normal expression levels of numerous genes, but it also recovered most of the CHAGAS, CMC and CCS fabrics that were altered by the infection.

Intraportal Infusion of Bone Marrow Mononuclear or CD133+ Cells in Patients With Decompensated Cirrhosis: A Double-Blind Randomized Controlled Trial.

The present study assessed the effects of intraportal infusions of autologous bone marrow-derived mononuclear cells (MNCs) and/or CD133+ cells on liver function in patients with decompensated cirrhosis. We randomly assigned 27 eligible patients to a placebo, MNCs, and/or CD133+ cells. Cell infusions were performed at baseline and month 3. We considered the absolute changes in the Model for End-Stage Liver Disease (MELD) scores at months 3 and 6 after infusion as the primary outcome. The participants and those who assessed the outcomes were unaware of the treatment intervention assignments. After 6 months, 9 patients were excluded because of liver transplantation (n=3), hepatocellular carcinoma (n=1), loss to follow-up (n=3), and death (n=2). The final analysis included 4 patients from the CD133+ group, 8 from the MNC group, and 6 from the placebo group. No improvement was seen in the MELD score at month 6 using either CD133+ cells or MNC infusions compared with placebo. However, at month 3 after infusion, a trend was seen toward a higher mean absolute change in the MELD score in patients who had received CD133+ cells compared with placebo (-2.00±1.87 vs. -0.13±1.46; p=.08). No significant adverse events occurred in the present study. A transient improvement in the MELD score was observed in subjects treated with CD133+ cells but not in the MNC or placebo group. Although the study was not powered to make definitive conclusions, the data justify further study of CD133+ therapy in cirrhotic patients. Cell therapy is a new approach in liver disease. Several clinical experiments have been reported on the safety of bone marrow-derived stem cells to treat liver disorders. However, the effectiveness of these approaches in the long-term follow-ups of patients initiated controversial discussions among the scientific community. A double-blind randomized controlled trial was designed to address this concern scientifically. A transient improvement in the patients' signs occurred; however, for a sustainable result, more work is needed. The results of multiple administrations of cells reported in the present study can be compared with the results from other single-injection studies.

Autologous Bone Marrow Mononuclear Cells Exert Broad Effects on Short- and Long-Term Biological and Functional Outcomes in Rodents with Intracerebral Hemorrhage.

Autologous bone marrow-derived mononuclear cells (MNCs) are a potential therapy for ischemic stroke. However, the effect of MNCs in intracerebral hemorrhage (ICH) has not been fully studied. In this study, we investigated the effects of autologous MNCs in experimental ICH. ICH was induced by infusion of autologous blood into the left striatum in young and aged male Long Evans rats. Twenty-four hours after ICH, rats were randomized to receive an intravenous administration of autologous MNCs (1 × 10(7) cells/kg) or saline. We examined brain water content, various markers related to the integrity of the neurovascular unit and inflammation, neurological deficit, neuroregeneration, and brain atrophy. We found that MNC-treated young rats showed a reduction in the neurotrophil infiltration, the number of inducible nitric oxide synthase-positive cells, and the expression of inflammatory-related signalings such as the high-mobility group protein box-1, S100 calcium binding protein B, matrix metalloproteinase-9, and aquaporin 4. Ultimately, MNCs reduced brain edema in the perihematomal area compared with saline-treated animals at 3 days after ICH. Moreover, MNCs increased vessel density and migration of doublecortin-positive cells, improved motor functional recovery, spatial learning, and memory impairment, and reduced brain atrophy compared with saline-treated animals at 28 days after ICH. We also found that MNCs reduced brain edema and brain atrophy and improved spatial learning and memory in aged rats after ICH. We conclude that autologous MNCs can be safely harvested and intravenously reinfused in rodent ICH and may improve long-term structural and functional recovery after ICH. The results of this study may be applicable when considering future clinical trials testing MNCs for ICH.

Repositioning Potential of PAK4 to Osteoclastic Bone Resorption.

Drug repositioning is a rational approach for expanding the use of existing drugs or candidate drugs to treat additional disorders. Here we investigated the possibility of using the anticancer p21-activated kinase 4 (PAK4)-targeted inhibitor PF-3758309 to treat osteoclast-mediated disorders. PAK4 was highly expressed in bone marrow cells and was phosphorylated during their differentiation into osteoclasts, and osteoclast differentiation was significantly inhibited by the dominant negative form of PAK4 and by PF-3758309. Specifically, PF-3758309 significantly inhibited the fusion of preosteoclasts, the podosome formation, and the migration of preosteoclasts. PF-3758309 also had in vivo antiresorptive activity in a lipopolysaccharide-induced bone erosion model and in vitro antiosteoclastogenic activity in the differentiation of human bone marrow-derived cells and peripheral blood mononuclear cells into osteoclasts. These data demonstrate the relevance of PAK4 in osteoclast differentiation and the potential of PAK4 inhibitors for treating osteoclast-related disorders.

Paracrine Effects of Bone Marrow–Derived Endothelial Progenitor Cells: Cyclooxygenase-2/Prostacyclin Pathway in Pulmonary Arterial Hypertension

BackgroundEndothelial dysfunction is the pathophysiological characteristic of pulmonary arterial hypertension (PAH). Some paracrine factors secreted by bone marrow–derived endothelial progenitor cells (BMEPCs) have the potential to strengthen endothelial integrity and function. This study investigated whether BMEPCs have the therapeutic potential to improve monocrotaline (MCT)-induced PAH via producing vasoprotective substances in a paracrine fashion.Methods and ResultsBone marrow-derived mononuclear cells were cultured for 7 days to yield BMEPCs. 24 hours or 3 weeks after exposure to BMEPCs in vitro or in vivo, the vascular reactivity, cyclooxygenase-2 (COX-2) expression, prostacyclin (PGI2) and cAMP release in isolated pulmonary arteries were examined respectively. Treatment with BMEPCs could improve the relaxation of pulmonary arteries in MCT-induced PAH and BMEPCs were grafted into the pulmonary bed. The COX-2/prostacyclin synthase (PGIS) and its progenies PGI2/cAMP were found to be significantly increased in BMEPCs treated pulmonary arteries, and this action was reversed by a selective COX-2 inhibitor, NS398. Moreover, the same effect was also observed in conditioned medium obtained from BMEPCs culture.ConclusionsImplantation of BMEPCs effectively ameliorates MCT-induced PAH. Factors secreted in a paracrine fashion from BMEPCs promote vasoprotection by increasing the release of PGI2 and level of cAMP.

Circulating Endothelial Progenitor Cells is a predictor in Atherosclerosis: Is it really a promising candle?

Circulating endothelial progenitor cells (CEPC) are supposed to be a subset of bone marrow-derived peripheral blood mononuclear cells (PBMC), revealing immature surface markers common to hematopoietic stem cells, such as CD 34 and CD 133 and endothelial lineage markers. These cells can be isolated from peripheral, umbilical cord, and bone marrow blood. CD 34 represents a marker of immature stem cells that is commonly used to characterize CEPC together with other surface antigens. Though, as CD 34 is also expressed at lower levels on mature endothelial cells, most recent studies used CD 133, a marker of more immature hematopoietic stem cells that is now considered the best surface marker to define, identify and isolate the CEPC1. CD 133 (also known as AC 133 or prominin) is highly conserved antigen with unknown biological activity. It would be expressed on hematopoietic stem cells, but not on mature endothelial cell and monocytes. In order to reflect the endothelial cells, there is general agreement for the use of at least one additional marker, such as vascular endothelial growth factor receptor-2 (VEGFR-2 or KDR), while others are platelet-endothelial cells adhesion molecules-1 (PECAM-1), von Willebrand factor, c-kit, Tie-2, vascular endothelial-cadherin and VEGFR-12.

Enhanced Growth of Endothelial Precursor Cells on PCG-Matrix Facilitates Accelerated, Fibrosis-Free, Wound Healing: A Diabetic Mouse Model

Diabetes mellitus (DM)-induced endothelial progenitor cell (EPC) dysfunction causes impaired wound healing, which can be rescued by delivery of large numbers of ‘normal’ EPCs onto such wounds. The principal challenges herein are (a) the high number of EPCs required and (b) their sustained delivery onto the wounds. Most of the currently available scaffolds either serve as passive devices for cellular delivery or allow adherence and proliferation, but not both. This clearly indicates that matrices possessing both attributes are ‘the need of the day’ for efficient healing of diabetic wounds. Therefore, we developed a system that not only allows selective enrichment and expansion of EPCs, but also efficiently delivers them onto the wounds. Murine bone marrow-derived mononuclear cells (MNCs) were seeded onto a PolyCaprolactone-Gelatin (PCG) nano-fiber matrix that offers a combined advantage of strength, biocompatibility wettability; and cultured them in EGM2 to allow EPC growth. The efficacy of the PCG matrix in supporting the EPC growth and delivery was assessed by various in vitro parameters. Its efficacy in diabetic wound healing was assessed by a topical application of the PCG-EPCs onto diabetic wounds. The PCG matrix promoted a high-level attachment of EPCs and enhanced their growth, colony formation, and proliferation without compromising their viability as compared to Poly L-lactic acid (PLLA) and Vitronectin (VN), the matrix and non-matrix controls respectively. The PCG-matrix also allowed a sustained chemotactic migration of EPCs in vitro. The matrix-effected sustained delivery of EPCs onto the diabetic wounds resulted in an enhanced fibrosis-free wound healing as compared to the controls. Our data, thus, highlight the novel therapeutic potential of PCG-EPCs as a combined ‘growth and delivery system’ to achieve an accelerated fibrosis-free healing of dermal lesions, including diabetic wounds.

Bone Marrow–Derived Cell Therapy After Myocardial Infarction

We would like to clarify several points in the editorial (1) accompanying our publication of the TIME report (2). First, the automated system for extracting bone marrow-derived mononuclear cells (MNCs) used for Cardiovascular Cell Therapy Research Network (CCTRN) clinical trials (2,3,4) employs Ficoll gradient centrifugation, similar to the manual method used for REPAIR AMI (5). Thus, the difference lies in improved reproducibility and standardization by the automated system, not some other fundamental characteristic. Second, our original publication erroneously listed the heparin concentration at 0.1 units/ml. In fact, no heparin was added to the automated system, and cells used in all CCTRN studies contained only residual heparin from bone marrow aspiration, estimated to be not more than 0.01 units per ml (Data Correction 6). This is five times below the lowest concentration evaluated (7) in a stromal cell-derived factor-1- (SDF-1)-stimulated invasion capacity assay, and 200-2000 times lower than heparin concentrations used in other functional assays reported by this group. Accordingly, the statement: “… the cell product used in TIME contained heparin at a dose that has been shown to impair the migratory capacity of MNCs” is no longer supported by the facts. We regret the confusion caused by our error. Third, the statement, “MNCs isolated by the automated system used in this study have not been tested in an animal model of MI, and cell bioactivity has not been verified in any human studies of heart disease” should be considered in view of the following: (a) MNCs from automated and manual preparations were found to have similar reperfusion activity in a murine hind-limb ischemia model (2); (b) A Cochrane review of stem cell studies (8) reported ten clinical trials cells were administered in a solution of “standard saline containing heparin.” Of these ten studies, we note that seven disclosed positive results (e.g., increased left ventricular ejection fraction, LVEF);(c) in the CCTRN FOCUS trial we found that LVEF increased 2.7% in patients receiving MNCs versus placebo, and that this increase was associated with higher percentages of CD34+ and CD133+ MNCs (3). Additionally, pre-clinical studies by CCTRN investigators demonstrated retention of SDF-1 migration and CFU-activity when MNCs isolated by the automated system were compared with MNCs from the manual method (unpublished observations). Thus, cell bioactivity was observed not only in one CCTRN trial (with miniscule residual heparin) but also in seven other trials in which cells were re-suspended in heparin. One explanation for this retention of bioactivity is that “…the effects of heparin can be ameliorated by excessive serum in vitro…” (7). Summarizing, the available evidence does not support the contention that bioactivity of cells administered in the TIME study was impaired by processing techniques or exposure to heparin. We believe that the TIME results are consistent with a limited effect of bone marrow cells on recovery of LV function, and support previous findings (9) demonstrating that MNCs have reduced cytokine and growth factor production compared to other bone marrow-derived stem and progenitor cells.

Challenges enrolling patients with acute ischemic stroke into cell therapy trials.

Infusion of autologous bone marrow-derived mononuclear cells (MNCs) is a promising investigational therapeutic approach for patients with acute ischemic stroke. Preclinical models indicate that MNCs can reduce neurological deficits and enhance recovery. We recently concluded a phase I clinical trial to determine the safety and feasibility of these cells in patients with acute ischemic stroke. In this article, we discuss practical barriers and challenges encountered during the trial and provide lessons learned for the design and planning of future clinical trials testing novel cell therapies for acute ischemic stroke.

Ischemic Stroke May Activate Bone Marrow Mononuclear Cells to Enhance Recovery After Stroke

Bone marrow-derived mononuclear cells (MNCs) enhance recovery in rodent stroke models. Since stroke activates the bone marrow, there may be biological differences of autologous MNCs derived poststroke compared with the prestroke setting. We analyzed MNCs harvested from the same Long Evans rats 1 day before and 1 day after ischemic stroke or sham stroke. Stroke was induced by suture occlusion of the middle cerebral artery for 90 min. MNCs were characterized by flow cytometry to identify differences in the percentages of different cell subpopulations. MNCs were also placed in culture and cytokines were measured in the media. In separate experiments, Long Evans rats received 24 h after stroke an intracarotid injection of saline or autologous MNCs, prepared from the same animal, either 1 day before or 1 day after stroke. The rats were then followed on the cylinder and corner tests for 28 days. In poststroke MNCs compared with prestroke MNCs, there was a significant reduction in T and mesenchymal stem cells and a significant increase in CD34+ and natural killer cells. Postsham MNCs showed an elevation in CD11b and CD45R cells compared with presham MNCs. The concentrations of IL-10, IL-6, MCP-1, vascular endothelial growth factor (VEGF), and tumor necrosis factor-α were significantly increased in poststroke MNCs compared with prestroke MNCs. Postsham MNCs showed a decrease in VEGF. Poststroke MNCs in comparison with prestroke MNCs led to a greater recovery on neurological testing and reduced lesion size. Autologous MNCs exert different biological responses when derived from the poststroke setting compared with normal animals.

Processing of equine bone marrow using the automated MarrowXpress System: RBC depletion, volume reduction, and mononuclear cell recovery

The therapeutic use of bone marrow-derived mononuclear cells (MNCs) and mesenchymal stem cells for the treatment of soft tissue and orthopedic injuries in equine patients is expanding. After collection, bone marrow must be reduced in volume and depleted of RBCs for immediate therapeutic use or to prepare cells for culture or cryopreservation and storage. The MarrowXpress (MXP) System is an automated, closed, sterile system designed to process human bone marrow samples. The purpose of this study was to evaluate the capacity of the MXP System to process equine bone marrow to reduce volume, deplete RBCs, and enhance recovery of MNCs. Bone marrow was collected from 47 horses into 2 60-mL syringes containing heparin and processed using the MXP System. HCT, total nucleated cell (TNC) count, and MNC count were obtained for each sample before and after processing using an Advia 120 hematology analyzer. Volume reduction, RBC depletion, and recovery of TNCs and MNCs were calculated. For equine bone marrow samples, mean values were 73.2% for RBC depletion and 78.0% for volume reduction. TNC count before processing was 2.5 ± 1.2 × 10(7) and after processing was significantly higher at 7.8 ± 3.3 × 10(7) (P < .0001), with a recovery of 68.5 ± 24.5% (mean ± SD). MNC count before processing was 1.1 ± 0.9 × 10(7) and after processing was significantly higher at 3.8 ± 1.9 × 10(7) (P < .0001), with a recovery 73.0 ± 31.5%. The MXP System can reliably reduce volume and deplete RBCs from aspirates of equine bone marrow aspirates. MNCs can be recovered in a reproducible and sterile manner. Further studies evaluating the effects of the MXP System on cell viability, identification of mesenchymal stem cells (MSCs), and the efficacy of MSC expansion are warranted.

Optimization of cryopreservation procedures for porcine endothelial progenitor cells

Endothelial progenitor cells (EPCs) provide a powerful option for therapeutic use in ischemic diseases. The cell therapy-induced vasculogenesis requires sufficient homogeneous cells, and cryopreservation is a prerequisite for long-term storage and quality assurance of EPCs. The aim of this study was to optimize cryopreservation protocols of EPCs derived from porcine bone marrow. Bone marrow-derived mononuclear cells (MNCs) were isolated by density centrifugation and differentiated into EPCs. The first passage EPCs were frozen by using different methodologies, and after cryopreservation the thawed cells were cultured to the fourth passage. The recovery efficiency and functions of these cells were evaluated by determination of cell viability, proliferation and migration. We found the optimal conditions for cryopreservation of EPCs as follows: (i) a cryopreservation medium consisting of 10% dimethylsulphoxide (DMSO) in combination with 50% fetal bovine serum (FBS); (ii) using a controlled freezing rate at 5°C/min; (iii) at an optimal density of 5×10⁶/ml for cryopreserved EPCs; (iv) a storage temperature of -156°C. Under these conditions we demonstrated that EPCs could be stored in mechanical freezer for up to 18 months after cryopreservation without losing their phenotypic characteristics and biological functions.

A Novel Model of Hind Limb Ischemia to Test Human Therapeutic Angiogenesis

Clinical trials injecting bone marrow-derived mononuclear cells (MNC) for therapeutic angiogenesis in patients with critical limb ischemia are currently underway. However, there are limited animal models available that adequately model human disease to allow direction of the human studies.

Treatment of murine fulminant hepatitis with genetically engineered endothelial progenitor cells

Cell therapy has been used to attenuate liver injury. Here we evaluated whether genetic engineering of either bone marrow-derived mononuclear cells (MNC) or endothelial progenitor cells (EPC) many enhance their hepatoprotective properties. Mice with ConA-induced hepatitis or with lethal fulminant hepatitis resulting from administration of an adenovirus encoding CD40L (AdCD40L) received an intra-splenic injection of saline or 2 × 10(6) unmodified MNC or EPC or the same cells transduced ex vivo with an adenovirus expressing luciferase (MNCLUC and EPCLUC) or encoding the hepatoprotective cytokine cardiotrophin-1 (CT-1) (MNCCT-1 and EPCCT-1). We analyzed the extent of liver damage, the intensity of inflammatory reaction, and animal survival. Luciferase immunohistochemistry showed that after injection into the spleen, the engineered cells migrated efficiently to the damaged liver. In mice with ConA hepatitis EPCCT-1, but not other forms of cell therapy, significantly decreased serum transaminases and induced more intense histological improvement than other treatments. This superior therapeutic effect was associated with upregulation of cytoprotective molecules including IGF-I and EGF, lower expression of proinflammatory cytokines, IL-1b and TNFα, and decreased granzyme B levels. In AdCD40L-induced lethal fulminant hepatitis, EPCCT-1 also exceeded other cell therapies in attenuating the expression of proinflammatory mediators and hepatic injury enabling 35.7% survival while mortality was 100% in the other treatment groups. Genetic engineering of EPC to overexpress CT-1 enhances the hepatoprotective properties of EPC and constitutes a therapy that deserves consideration for acute liver failure.