Mesenchymal Stem Cells For Repair Research Articles

Abstract 19437: Concomitant Overexpression of FGF2 and TGFbeta1 in Mesenchymal Stem Cells Improves Both Autocrine and Paracrine Properties Through Activation of Akt, ERK1/2 and SMAD2 Pathways and Upregulation of Soluble Factors

Background: mesenchymal stem cells (MSC) repair infarcted hearts mainly through paracrine mechanisms. However, donor age negatively influences paracrine properties of bone marrow MSC (BM-MSC). We have shown that in BM-MSC from elderly patients both TGFβ1 (T) and FGF2 (F) expression is decreased. We hypothesized that overexpression of these two factors involved in cytoprotection and angiogenesis may improve MSC repair capacity. Methods: rat BM-MSC were transduced with control (GFP-MSC) or with a bicistronic (TF-MSC) lentivirus. Cytoprotection was evaluated in MSC (autocrine) or in H9c2 (paracrine) cells exposed to 24 hrs of hypoxia in the presence of either unconditioned (CTRL-M) or conditioned medium (GFP-CM or TF-CM). Viability was measured by MTS assay and apoptosis by caspase-3 activation. Angiogenesis was assessed by evaluating HUVEC tube formation on Matrigel. Transcriptional levels of the pro-survival gene Bcl2 and genes encoding soluble factors were measured by RT-PCR. Activation of the three main TF-related pathways (ERK1/2, Akt and SMAD2) was tested by western blot. Results: TF-MSC showed a marked reduction of apoptosis rate (-27% p<0.001) compared with GFP-MSC after 24 hrs of hypoxia. Furthermore, TF-CM increased H9c2 viability compared with CTRL-M (+46.3% p<0.001). Caspase-3 activation in H9C2 was reduced by 60.3% in the presence of TF-CM vs CTRL-M (p<0.001) and by 44.7% vs GFP-CM (p<0.05). HUVEC tube formation was significantly enhanced by TF-CM compared to both CTRL-M (+57.5%; p<0.001) and GFP-CM (+45.2%; p<0.001). Both in TF-MSC and TF-CM treated-H9c2 we observed a strong activation of Akt, ERK1/2 and SMAD2 pathways and enhanced expression of Bcl2. Finally, in TF-MSC the transcriptional levels of soluble factors such as PDGF-β, VEGF, Ang1, IGF1, BMP2, and IL11 was upregulated. Conclusions: TF overexpression increases both autocrine and paracrine properties of MSC through upregulation of several soluble factors and activation of pathways known to be involved in cytoprotection and angiogenesis. These results pave the way for further research on modification of MSC as effective strategy to improve efficacy of stem cell therapy.

Novel vascular endothelial growth factor gene delivery system-manipulated mesenchymal stem cells repair infarcted myocardium

Transplantation of vascular endothelial growth factor (VEGF) gene-manipulated mesenchymal stem cells (MSCs) has been proposed as a promising therapy strategy for cardiac repair after myocardium infarction. However, the gene delivery system, including targeted VEGF gene and delivery vehicle, still needs to be optimized. In this study, a novel, hyperbranched poly(amidoamine) (hPAMAM), polymer-based, hypoxia-regulated VEGF(165) plasmid (pHRE-VEGF(165)) delivery system was constructed for effective, biocompatible and controllable gene expression. The hPAMAM demonstrated high transfection efficiency (38.98 ± 1.95%) with minor cytotoxicity (cell viability = 92.38 ± 1.09%) in primary MSCs under optimal conditions. Under hypoxia, hPAMAM-pHRE-hVEGF(165)-transfected MSCs could over-express hVEGF(165) stably for 14 days, with a peak expression at day 2, which promoted endothelial cell proliferation in vitro. The transplantation of hPAMAM-pHRE-hVEGF(165) gene delivery system-manipulated MSCs could enhance ischemic myocardium VEGF concentration obviously, which improved the graft MSC survival, increased neovascularization, and ultimately preserved cardiac function to a significantly greater degree than untreated MSC transplantation. This work demonstrated that hPAMAM-based pHRE-hVEGF(165) gene delivery combined with MSC transplantation is an economical, feasible and biocompatible strategy for cardiac repair.

Mesenchymal Stem Cells Stably Transduced with a Dominant-Negative Inhibitor of CCL2 Greatly Attenuate Bleomycin-Induced Lung Damage

Acute respiratory distress syndrome (ARDS) is a crippling disease with no effective therapy characterized by progressive dyspnea. Mesenchymal stem cells (MSCs) have emerged as a new therapeutic modality for ARDS because MSCs can attenuate inflammation and repair the damaged tissue by differentiating into several cell types. Macrophages participate in the development of ARDS; however, MSCs only weakly modulate macrophage function. The chemokine CCL2 is a potent inducer of macrophage recruitment and activation, and its expression is elevated in patients with ARDS. We established MSCs that are stably transduced by a lentiviral vector expressing 7ND, a dominant-negative inhibitor of CCL2, to enhance the therapeutic function of MSCs. 7ND-MSCs retained the innate properties of MSCs and produced a large amount of 7ND. Many 7ND-MSCs were detected in bleomycin-treated lungs (immunostaining 24 hours after injection), suggesting that MSCs could work as a drug delivery tool. Mice treated with 7ND-MSCs showed significantly milder weight loss, lung injury, collagen content, accumulation of inflammatory cells and inflammatory mediators that were induced by bleomycin, and subsequent survival benefit. No evidence of 7ND-MSC-induced toxicity was observed during or after treatment. Thus, inhibiting the effects of macrophages may greatly enhance the ability of MSCs to effect lung repair in ARDS.

Cell-based therapies in pulmonary hypertension: who, what, and when?

pulmonary hypertension (PH), diagnosed when mean pulmonary arterial pressure exceeds the upper limits of normal (i.e., >25 mmHg) at rest (2), occurs in a variety of clinical situations and is associated with a broad spectrum of histological patterns and abnormalities. PH is currently classified into five distinct World Health Organization (WHO) groups, based on common clinical parameters, potential etiological mechanisms, and responses to treatment (22). Although any form of PH can contribute to increased patient morbidity and mortality, pulmonary arterial hypertension (PAH) (WHO group 1) is a particularly severe and progressive form associated with right heart failure and premature death (1). At present, therapeutic approaches to stabilize or reverse this debilitating condition involve treatment with one or a combination of up to three specific classes of agents, including prostacyclin analogs, endothelin-1 receptor antagonists, and/or phosphodiesterase-5 inhibitors. Retrospective (meta)analyses of these therapeutic strategies have demonstrated a reduction in mortality with their use (7, 12); however, many experts believe that current PAH treatment is inadequate given the persistently high mortality rate and functional hemodynamic impairment in many patients. These observations have led to continued intensive investigation into pathogenetic mechanisms and many proposals for additional alternative new therapies (20, 24). Among the potential new therapies, increasing interest in the role of endothelial progenitor cells (EPCs) as a cell-based therapy has emerged. However, issues remain regarding what group of PH patients are most likely to benefit from treatment, at what point in the disease is treatment most likely to be successful, and what types of cells should be utilized for therapy.

Micro‐CT‐based bone ceramic scaffolding and its performance after seeding with mesenchymal stem cells for repair of load‐bearing bone defect in canine femoral head

Osteonecrosis of the femoral head is a debilitating and painful orthopedic condition characterized by joint collapse. Salvage of the femoral head is highly desirable to preserve the contour and mechanical properties and prevent joint collapse. This study aimed to develop a new tissue-engineering approach for treatment of large bone defect in femoral head, that is, after osteonecrosis. The biphasic calcium phosphate (BCP) ceramic scaffolds were fabricated by a 3D gel-lamination technique based on micro-computed tomography (micro-CT) images of the cancellous bone microarchitecture of femoral heads. After seeding with autologous bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, the cell-scaffold composite was implanted into a bone defect surgically induced in canine femoral head via trapdoor procedure, which was a common procedure for treatment of osteonecrosis. A total of 24 adult dogs were randomly divided into three groups (n = 8 each) for implantation of the BCP scaffold with or without with BMSCs, and also the autologous bone chips for comparisons. All animals were sacrificed at 30 weeks postoperatively and processed for radiological and histological evaluations. The contour of the femoral head was well preserved with implantation of BCP scaffolds with or without BMSCs, whereas joint collapse was found after treatment with autologous bone chips. The osteointegration and new bone formation was significantly greater with BCP scaffold implantation with than without BMSC seeding and showed greater strength and compressive modulus in the repair site. Micro-CT-based bone ceramic scaffolds seeding with BMSC might be a promising way to repair bone defects in the femoral head.

Mesenchymal stem cells for repair of the airway epithelium in asthma

The airway epithelium is constantly faced with inflammatory and potentially injurious stimuli. Following damage, rapid repair mechanisms involving proliferation and differentiation of resident progenitor and stem cell pools are necessary in order to maintain a protective barrier. In asthma, evidence pointing to a compromised ability of the epithelium to properly repair and regenerate is rapidly accumulating. The consequences of this are presently unknown but are likely to have a significant impact on lung function. Mesenchymal stem cells have the potential to serve as a universal source for replacement of specific cells in several diseases and thus offer hope as a potential therapeutic intervention for the treatment of the chronic remodeling changes that occur in the asthmatic epithelium. However, controversy exists regarding whether these cells can actually home to and engraft within the airways and contribute to tissue function or whether this mechanism is necessary, since they can have potent paracrine immunomodulatory effects. This article focuses on the current knowledge about specific stem cell populations that may contribute to airway epithelial regeneration and discusses the use of mesenchymal stem cells as a potential therapeutic intervention.

A novel biomimetic composite scaffold hybridized with mesenchymal stem cells in repair of rat bone defects models

In this study, the in vivo bone-regenerative potential of a novel bioactive glass-collagen-hyaluronic acid-Phosphatidylserine (BG-COL-HYA-PS) composite scaffold hybridized with mesenchymal stem cells (MSCs) was investigated in a rat bone defect model. HrGFP-labeled MSCs were cultured for 2 weeks on the BG-COL-HYA-PS scaffold before implantation into the defect. A cell-free scaffold and an untreated defect were used as controls. The regeneration process was evaluated by histology, X-ray, and mechanical rigidity experiments at different time points post-implantation. The results revealed that BG-COL-HYA-PS scaffold exhibited a low inflammatory response and foreign body response within 3 weeks. At week 6, those responses disappeared following the resorption of scaffolds and the formation of new bone. Compared with the pure scaffold or empty group, the introduction of MSCs into the porous scaffold dramatically enhanced the efficiency of the new bone formation and biomechanical property of the femur. In addition, the transplanted MSCs could survive for up to 3 weeks or longer. The results demonstrated that the BG-COL-HYA-PS scaffold was biocompatible and osteoconductive and the transplanted MSCs with the scaffold enhanced the healing of the bone defect.

Low Oxygen Tension and Synthetic Nanogratings Improve the Uniformity and Stemness of Human Mesenchymal Stem Cell Layer

A free-standing, robust cell sheet comprising aligned human mesenchymal stem cells (hMSCs) offers many interesting opportunities for tissue reconstruction. As a first step toward this goal, a confluent, uniform hMSC layer with a high degree of alignment and stemness maintenance needs to be created. Hypothesizing that topographical cue and a physiologically relevant low-oxygen condition could promote the formation of such an hMSC layer, we studied the culture of hMSCs on synthetic nanogratings (350 nm width and 700 nm pitch) and either under 2 or 20% O(2). Culturing hMSCs on the nanogratings highly aligned the cells, but it tended to create patchy layers and accentuate the hMSC differentiation. The 2% O(2) improved the alignment and uniformity of hMSCs, and reduced their differentiation. Over a 14-day culture period, hMSCs in 2% O(2) showed uniform connexon distribution, secreted abundant extracellular matrix (ECM) proteins, and displayed a high progenicity. After 21-day culture on nanogratings, hMSCs exposed to 2% O(2) maintained a higher viability and differentiation capacity. This study established that a 2% O(2) culture condition could restrict the differentiation of hMSCs cultured on nanopatterns, thereby setting the foundation to fabricate a uniformly aligned hMSC sheet for different regenerative medicine applications.

Human Bone Marrow Mesenchymal Stem Cells: A Systematic Reappraisal Via the Genostem Experience

Genostem (acronym for "Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side") has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.

Potential of Human Umbilical Cord Matrix and Rabbit Bone Marrow–Derived Mesenchymal Stem Cells in Repair of Surgically Incised Rabbit External Anal Sphincter

Anal sphincter defects and fecal incontinence are complicated surgical problems. We investigated the ability of human umbilical cord matrix (hUCM) and rabbit bone marrow (rBM) stem cells to improve anal sphincter incontinence due to induced sphincter defects without surgical repair. We harvested hUCM cells from human Wharton's jelly and rBM stem cells from rabbit femurs and tibias. To induce sphincter defects, we made an incision in the external anal sphincter. Rabbits were randomly allocated to 5 groups to receive either no intervention (n = 3) or injections of 10 hUCM cells in medium (10 microL RPMI-1640), rBM cells in medium, medium only, or normal saline (n = 7 per group), 2 weeks after sphincterotomy. Transplanted cells were tracked in the injured sphincters by prelabeling with bromodeoxyuridine. Electromyography was performed before and 2 weeks after the external anal sphincterotomy, and 2 weeks after cell transplantation. We also evaluated the proliferation and differentiation of injected cells with histopathologic techniques. Electromyography showed significant improvement in sphincter function 2 weeks after local injection of rBM stem cells compared with pretreatment values and controls. Moderate, nonsignificant improvement was observed with hUCM cell injection. Cells with incorporated bromodeoxyuridine were detected at the site of injury after transplantation of hUCM and rBM. Histopathologic evaluation showed normal or muscle-dominant sphincter structure in all animals receiving rBM and fibrous-dominant sphincter structure in most animals receiving hUCM. Stem cell injection at the site of injury can enhance contractile function of the anal sphincter without surgical repair. Transplantation of stem cells, particularly bone marrow mesenchymal cells, may provide an effective tool for treating anal sphincter injuries in humans.

Mesenchymal stem cell repair capabilities are defective in rheumatoid arthritis in relation with in vivo exposure to inflammation

Mesenchymal stem cell repair capabilities are defective in rheumatoid arthritis in relation with in vivo exposure to inflammation

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.

Mo-P3:232 Monolayered mesenchymal stem cells repair scarred myocardium while growing in situ

Mo-P3:232 Monolayered mesenchymal stem cells repair scarred myocardium while growing in situ

Human Adult Bone Marrow Mesenchymal Stem Cells Repair Experimental Conduction Block in Rat Cardiomyocyte Cultures

We evaluated whether human adult bone marrow-derived mesenchymal stem cells (hMSCs) could repair an experimentally induced conduction block in cardiomyocyte cultures. Autologous stem cell therapy is a novel treatment option for patients with heart disease. However, detailed electrophysiological characterization of hMSCs is still lacking. Neonatal rat cardiomyocytes were seeded on multi-electrode arrays. After 48 h, abrasion of a 200- to 450-microm-wide channel caused conduction block. Next, we applied adult hMSCs (hMSC group, n = 8), human skeletal myoblasts (myoblast group, n = 7), rat cardiac fibroblasts (fibroblast group, n = 7), or no cells (control group, n = 7) in a channel-crossing pattern. Cross-channel electrical conduction was analyzed after 24 and 48 h. Intracellular action potentials of hMSCs and cardiomyocytes were recorded. Immunostaining for connexins and intercellular dye transfer (calcein) assessed the presence of functional gap junctions. After creation of conduction block, two asynchronously beating fields of cardiomyocytes were present. Application of hMSCs restored synchronization between the two fields in five of eight cultures after 24 h. Conduction velocity across hMSCs (0.9 +/- 0.4 cm/s) was approximately 11-fold slower than across cardiomyocytes (10.4 +/- 5.8 cm/s). No resynchronization occurred in the myoblast, fibroblast, or control group. Intracellular action potential recordings indicated that conduction across the channel presumably occurred by electrotonic impulse propagation. Connexin-43 was present along regions of hMSC-to-cardiomyocyte contact, but not along regions of cardiomyocyte-to-myoblast or cardiomyocyte-to-fibroblast contact. Calcein transfer from cardiomyocytes to hMSCs was observed within 24 h after co-culture initiation. Human mesenchymal stem cells are able to repair conduction block in cardiomyocyte cultures, probably through connexin-mediated coupling.

Bone Marrow–Derived Mesenchymal Stem Cells in Repair of the Injured Lung

We sought to determine whether an intact bone marrow is essential to lung repair following bleomycin-induced lung injury in mice, and the mechanisms of any protective effects conferred by bone marrow-derived mesenchymal stem cell (BMDMSC) transfer. We found that myelosupression increased susceptibility to bleomycin injury and that BMDMSC transfer was protective. Protection was associated with the differentiation of engrafted BMDMSC into specific and distinct lung cell phenotypes, with an increase in circulating levels of G-CSF and GM-CSF (known for their ability to promote the mobilization of endogenous stem cells) and with a decrease in inflammatory cytokines. In vitro, cells from injured, but not from normal, mouse lung produced soluble factors that caused BMDMSC to proliferate and migrate toward the injured lung. We conclude that bone marrow stem cells are important in the repair of bleomycin-injured lung and that transfer of mesenchymal stem cells protects against the injury. BMDMSC localize to the injured lung and assume lung cell phenotypes, but protection from injury and fibrosis also involves suppression of inflammation and triggering production of reparative growth factors.

927. Human Mesenchymal and Hematopoietic Stem Cell Contribution to Cardiac Tissue Regeneration after Injury

Cardiomyocyte regeneration by stem cells from non-cardiac tissues could be a future treatment for cardiovascular diseases. However, there is little direct evidence that adult stem cells can regenerate a damaged heart. In vitro assays of stem cells from the bone marrow compartment using 5-azacytidine suggests that these stem cells can adopt the cardiomyocyte morphology and express cardiac-specific markers. One of the most commonly used in vivo models for myocardial infarction is coronary ligation. In this model, the left anterior descending (LAD) coronary artery is tied off to create an area of ischemia in the left ventricle. Using this model, it remains a controversy as to whether stem cells from the bone marrow, either hematopoietic or mesenchymal, can contribute and/or enhance cardiomyocyte regeneration following damage. Our lab has previously reported that co-transplantation of hematopoietic stem cells (HSC) with mesenchymal stem cells (MSC) greatly enhances the complete hematopoietic lineage reconstitution in myeloablated immunodeficient mice. To address the role of HSC and/or MSC in repair for cardiovascular diseases, we have developed a heart injury model using doxorubicin, a chemotherapeutic drug that has been shown to cause cardiac toxicity. Hematopoietic and mesenchymal cells are injected next to the ischemic area immediately after the LAD surgery, or intraperitoneally/intravenously two days after the doxorubicin injection. LAD-injured mice are then assessed functionally by echocardiography, to obtain ejection fraction, end systolic volume, end diastolic volume and wall motion score indexes. Our preliminary studies showed show low but persistent engraftment of human cells in the cardiac tissue in 100% of surviving transplants. An additional aim of our studies is to ask whether co-transplantation of HSC and MSC could give a functional phenotype suggestive of cardiovascular repair. To distinguish HSC from MSC following their co-transplantation, we transduced HSC with a retroviral construct expressing YFP and MSC either with a monocistronic retroviral construct expressing GFP alone or with a bicistronic retroviral construct expressing a chemotatic/proliferation factor (hepatocyte growth factor) and GFP. The use of the bicistronic construct is to determine whether secretion of HGF in vivo might enhance the survival of either HSC, MSC, or both, in the damaged heart. Moderate but detectable improvements in cardiac function can be seen in these mice. Whether the exogenous human HSC and/or MSC had transdifferentiated into cardiomyocyte-like cells or whether the injected cells indirectly recruit endogenous cardiomyocyte stem cells to proliferate and repair the cardiac damage is currently being investigated. Collectively, the use of retroviral gene transfer of different fluorescent markers to track stem cells in vivo in this cardiac-damaged xenotransplantation model will provide a more definitive answer as to which stem cells of the bone marrow compartment enhance cardiac repair. The use of retroviral gene transfer of specific soluble factors, such as HGF, could provide additional survival and/or differentiation support of stem cells in vivo.

Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats

Bone marrow derived mesenchymal stem cells (MSC) have been shown to be progenitor cells for mesenchymal tissues. These cells may also provide a potential therapy for bone repair. Our previous studies showed that MSC engineered with the gene for bone morphogenetic protein 2 (BMP-2), a growth factor for bone cells, were capable of differentiating into osteoblast lineage and inducing autologous bone formation in several animal models. Culturing individual MSC for autologous implantation, however, remains problematic. The number of human MSC with osteogenic potential decreases with age, and, in certain diseases, the patient’s marrow may be damaged or the healthy cells reduced in number. In this study, we used rats with a femoral segmental defect to investigate whether allogeneic BMP-2 engineered MSC would facilitate bone healing. We show that BMP-2 engineered allogeneic MSC can repair critical bone defects to the same degree as rats treated with BMP-2 engineered autologous MSC, if the allogeneic group receives short-term treatment with immunosuppressant FK506. We also show that allogeneic gene transferred MSC are directly involved in bone repair, in addition to acting as gene deliverers.