Xiaoyun Xie

Improving Cell Engraftment in Cardiac Stem Cell Therapy

Myocardial infarction (MI) affects millions of people worldwide. MI causes massive cardiac cell death and heart function decrease. However, heart tissue cannot effectively regenerate by itself. While stem cell therapy has been considered an effective approach for regeneration, the efficacy of cardiac stem cell therapy remains low due to inferior cell engraftment in the infarcted region. This is mainly a result of low cell retention in the tissue and poor cell survival under ischemic, immune rejection and inflammatory conditions. Various approaches have been explored to improve cell engraftment: increase of cell retention using biomaterials as cell carriers; augmentation of cell survival under ischemic conditions by preconditioning cells, genetic modification of cells, and controlled release of growth factors and oxygen; and enhancement of cell survival by protecting cells from excessive inflammation and immune surveillance. In this paper, we review current progress, advantages, disadvantages, and potential solutions of these approaches.

A prosurvival and proangiogenic stem cell delivery system to promote ischemic limb regeneration.

UNLABELLEDnStem cell therapy is one of the most promising strategies to restore blood perfusion and promote muscle regeneration in ischemic limbs. Yet its therapeutic efficacy remains low owing to the inferior cell survival under the low oxygen and nutrient environment of the injured limbs. To increase therapeutic efficacy, high rates of both short- and long-term cell survival are essential, which current approaches do not support. In this work, we hypothesized that a high rate of short-term cell survival can be achieved by introducing a prosurvival environment into the stem cell delivery system to enhance cell survival before vascularization is established; and that a high rate of long-term cell survival can be attained by building a proangiogenic environment in the system to quickly vascularize the limbs. The system was based on a biodegradable and thermosensitive poly(N-Isopropylacrylamide)-based hydrogel, a prosurvival and proangiogenic growth factor bFGF, and bone marrow-derived mesenchymal stem cells (MSCs). bFGF can be continuously released from the system for 4weeks. The released bFGF significantly improved MSC survival and paracrine effects under low nutrient and oxygen conditions (0% FBS and 1% O2) in vitro. The prosurvival effect of the bFGF on MSCs was resulted from activating cell Kruppel-like factor 4 (KLF4) pathway. When transplanted into the ischemic limbs, the system dramatically improved MSC survival. Some of the engrafted cells were differentiated into skeletal muscle and endothelial cells, respectively. The system also promoted the proliferation of host cells. After only 2weeks of implantation, tissue blood perfusion was completely recovered; and after 4weeks, the muscle fiber diameter was restored similarly to that of the normal limbs. These pronounced results demonstrate that the developed stem cell delivery system has a potential for ischemic limb regeneration.nnnSTATEMENT OF SIGNIFICANCEnStem cell therapy is a promising strategy to restore blood perfusion and promote muscle regeneration in ischemic limbs. Yet its therapeutic efficacy remains low owing to the inferior cell survival under the ischemic environment of the injured limbs. To increase therapeutic efficacy, high rate of cell survival is essential, which current approaches do not support. In this work, we tested the hypothesis that a stem cell delivery system that can continuously release a prosurvival and proangiogenic growth factor will promote high rates of cell survival in the ischemic limbs. The prosurvival effect could augment cell survival before vascularization is established, while the proangiogenic effect could stimulate quick angiogenesis to achieve long-term cell survival. Meanwhile, the differentiation of stem cells into endothelial and myogenic lineages, and cell paracrine effects will enhance vascularization and muscle regeneration.

Regulating myogenic differentiation of mesenchymal stem cells using thermosensitive hydrogels

UNLABELLEDnStem cell therapy has potential to regenerate skeletal muscle tissue in ischemic limb. However, the delivered stem cells experience low rate of myogenic differentiation. Employing injectable hydrogels as stem cell carriers may enhance the myogenic differentiation as their modulus may be tailored to induce the differentiation. Yet current approaches used to manipulate hydrogel modulus often simultaneously vary other properties that also affect stem cell differentiation, such as chemical structure, composition and water content. Thus it is challenging to demonstrate the decoupled effect of hydrogel modulus on stem cell differentiation. In this report, we decoupled the hydrogel modulus from chemical structure, composition, and water content using injectable and thermosensitive hydrogels. The hydrogels were synthesized from N-isopropylacrylamide (NIPAAm), acrylic acid (AAc), and degradable macromer 2-hydroxyethyl methacrylate-oligomer [oligolatide, oligohydroxybutyrate, or oligo(trimethylene carbonate)]. We found that using the same monomer composition and oligomer chemical structure but different oligomer length can independently vary hydrogel modulus. Rat bone marrow mesenchymal stem cells (MSCs) were encapsulated in the hydrogels with elastic expansion moduli of 11, 20, and 40 kPa, respectively. After 14 days of culture, significant myogenic differentiation was achieved for the hydrogel with elastic expansion modulus of 20 kPa, as judged from both the gene and protein expression. In addition, MSCs exhibited an elastic expansion modulus-dependent proliferation rate. The most significant proliferation was observed in the hydrogel with elastic expansion modulus of 40 kPa. These results demonstrate that the developed injectable and thermosensitive hydrogels with suitable modulus has the potential to deliver stem cells into ischemic limb for enhanced myogenic differentiation and muscle regeneration.nnnSTATEMENT OF SIGNIFICANCEnStem cell therapy for skeletal muscle regeneration in ischemic limb experiences low rate of myogenic differentiation. Employing injectable hydrogels as stem cell carriers may enhance the myogenic differentiation as hydrogel modulus may be modulated to induce the differentiation. Yet current approaches used to modulate hydrogel modulus may simultaneously vary other properties that also affect stem cell myogenic differentiation, such as chemistry, composition and water content. In this report, we decoupled the hydrogel modulus from chemistry, composition, and water content using injectable and thermosensitive hydrogels. We found that mesenchymal stem cells best differentiated into myogenic lineage in the hydrogel with elastic modulus of 20 kPa.

Sustained Release of a Peptide-based Matrix Metalloproteinase-2 Inhibitor to Attenuate Adverse Cardiac Remodeling and Improve Cardiac Function Following Myocardial Infarction

Following myocardial infarction (MI), degradation of extracellular matrix (ECM) by upregulated matrix metalloproteinases (MMPs) especially MMP-2 decreases tissue mechanical properties, leading to cardiac function deterioration. Attenuation of cardiac ECM degradation at the early stage of MI has the potential to preserve tissue mechanical properties, resulting in cardiac function increase. Yet the strategy for efficiently preventing cardiac ECM degradation remains to be established. Current preclinical approaches have shown limited efficacy because of low drug dosage allocated to the heart tissue, dose-limiting side effects, and cardiac fibrosis. To address these limitations, we have developed a MMP-2 inhibitor delivery system that can be specifically delivered into infarcted hearts at early stage of MI to efficiently prevent MMP-2-mediated ECM degradation. The system was based on an injectable, degradable, fast gelation, and thermosensitive hydrogel, and a MMP-2 specific inhibitor, peptide CTTHWGFTLC (CTT). The use of fast gelation hydrogel allowed to completely retain CTT in the heart tissue. The system was able to release low molecular weight CTT over 4 weeks possibly due to the strong hydrogen bonding between the hydrogel and CTT. The release kinetics was modulated by amount of CTT loaded into the hydrogel, and using chondroitin sulfate and heparin that can interact with CTT and the hydrogel. Both glycosaminoglycans augmented CTT release, while heparin more greatly accelerated the release. After it was injected into the infarcted hearts for 4 weeks, the released CTT efficiently prevented cardiac ECM degradation as it not only increased tissue thickness but also preserved collagen composition similar to that in the normal heart tissue. In addition, the delivery system significantly improved cardiac function. Importantly, the delivery system did not induce cardiac fibrosis. These results demonstrate that the developed MMP-2 inhibitor delivery system has potential to efficiently reduce adverse myocardial remodeling and improve cardiac function.

Transplantation of placenta-derived mesenchymal stem cells enhances angiogenesis after ischemic limb injury in mice

Mesenchymal stem cell‐based therapy has emerged as a promising approach for the treatment of peripheral arterial disease. The purpose of this study was to examine the potential effects of human placenta‐derived mesenchymal stem cells (PMSCs) on mouse hindlimb ischemia. PMSCs were isolated from human placenta tissue and characterized by flow cytometry. An in vivo surgical ligation‐induced murine limb ischemia model was generated with fluorescent dye (CM‐DiI) labelled PMSCs delivered via intramuscular injection. Our data show that PMSCs treatment significantly enhanced microvessel density, improved blood perfusion and diminished pathologies in ischemic mouse hindlimbs as compared to those in the control group. Further immunostaining studies suggested that injected PMSCs can incorporate into the vasculature and differentiate into endothelial and smooth muscle cells to enhance angiogenesis in ischemic hind limbs. This may in part explain the beneficial effects of PMSCs treatment. Taken together, we found that PMSCs treatment might be an effective treatment modality for treatment of ischemia‐induced injury to mouse hind limbs by enhancement of angiogenesis.

pH-Sensitive and Thermosensitive Hydrogels as Stem-Cell Carriers for Cardiac Therapy.

Stem-cell therapy has the potential to regenerate damaged heart tissue after a heart attack. Injectable hydrogels may be used as stem-cell carriers to improve cell retention in the heart tissue. However, current hydrogels are not ideal to serve as cell carriers because most of them block blood vessels after solidification. In addition, these hydrogels have a relatively slow gelation rate (typically >60 s), which does not allow them to quickly solidify upon injection, so as to efficiently hold cells in the heart tissue. As a result, the hydrogels and cells are squeezed out of the tissue, leading to low cell retention. To address these issues, we have developed hydrogels that can quickly solidify at the pH of an infarcted heart (6-7) at 37 °C but cannot solidify at the pH of blood (7.4) at 37 °C. These hydrogels are also clinically attractive because they can be injected through catheters commonly used for minimally invasive surgeries. The hydrogels were synthesized by free-radical polymerization of N-isopropylacrylamide, propylacrylic acid, hydroxyethyl methacrylate-co-oligo(trimethylene carbonate), and methacrylate poly(ethylene oxide) methoxy ester. Hydrogel solutions were injectable through 0.2-mm-diameter catheters at pH 8.0 at 37 °C, and they can quickly form solid gels under pH 6.5 at 37 °C. All of the hydrogels showed pH-dependent degradation and mechanical properties with less mass loss and greater complex shear modulus at pH 6.5 than at pH 7.4. When cardiosphere-derived cells (CDCs) were encapsulated in the hydrogels, the cells were able to survive during a 7-day culture period. The surviving cells were differentiated into cardiac cells, as evidenced by the expression of cardiac markers at both the gene and protein levels, such as cardiac troponin T, myosin heavy chain α, calcium channel CACNA1c, cardiac troponin I, and connexin 43. The gel integrity was found to largely affect CDC cardiac differentiation. These results suggest that the developed dual-sensitive hydrogels may be promising carriers for cardiac cell therapy.

Delivery of placenta-derived mesenchymal stem cells ameliorates ischemia induced limb injury by immunomodulation.

Background: Peripheral artery disease (PAD) is a major health burden in the world. Stem cell-based therapy has emerged as an attractive treatment option in regenerative medicine. In this study, we sought to test the hypothesis that stem cell-based therapy can ameliorate ischemia induced limb injury. Methods: We isolated mesenchymal stem cells derived from human placentas (PMSCs) and intramuscularly transplanted them into injured hind limbs. Treatment with PMSCs reduced acute muscle fibers apoptosis induced by ischemia. Results: PMSC treatment significantly enhanced regeneration of the injured hind limb by reducing fibrosis and enhancing running capacity when the animals were subjected to treadmill training. Mechanistically, injected PMSCs can modulate acute inflammatory responses by reducing neutrophil and macrophage infiltration following limb ischemia. ELISA assays further confirmed that PMSC treatment can also reduce pro-inflammatory cytokines, TNF-α and IL-6, and enhance anti-inflammatory cytokine, IL-10 at the injury sites. Conclusion: Taken together, our results demonstrated that PMSCs can be a potential effective therapy for treatment of PAD via immunomodulation.

Thermosensitive and Highly Flexible Hydrogels Capable of Stimulating Cardiac Differentiation of Cardiosphere-Derived Cells under Static and Dynamic Mechanical Training Conditions.

Cardiac stem cell therapy has been considered as a promising strategy for heart tissue regeneration. Yet achieving cardiac differentiation after stem cell transplantation remains challenging. This compromises the efficacy of current stem cell therapy. Delivery of cells using matrices that stimulate the cardiac differentiation may improve the degree of cardiac differentiation in the heart tissue. In this report, we investigated whether elastic modulus of highly flexible poly(N-isopropylamide) (PNIPAAm)-based hydrogels can be modulated to stimulate the encapsulated cardiosphere derived cells (CDCs) to differentiate into cardiac lineage under static condition and dynamic stretching that mimics the heart beating condition. We have developed hydrogels whose moduli do not change under both dynamic stretching and static conditions for 14 days. The hydrogels had the same chemical structure but different elastic moduli (11, 21, and 40 kPa). CDCs were encapsulated into these hydrogels and cultured under either native heart-mimicking dynamic stretching environment (12% strain and 1 Hz frequency) or static culture condition. CDCs were able to grow in all three hydrogels. The greatest growth was found in the hydrogel with elastic modulus of 40 kPa. The dynamic stretching condition stimulated CDC growth. The CDCs demonstrated elastic modulus-dependent cardiac differentiation under both static and dynamic stretching conditions as evidenced by gene and protein expressions of cardiac markers such as MYH6, CACNA1c, cTnI, and Connexin 43. The highest differentiation was found in the 40 kPa hydrogel. These results suggest that delivery of CDCs with the 40 kPa hydrogel may enhance cardiac differentiation in the infarct hearts.

Akt/eNOS signaling pathway mediates inhibition of endothelial progenitor cells by palmitate-induced ceramide.

Palmitate (PA) impairs endothelial progenitor cells (EPCs). However, the molecular mechanism underlying the suppressive function of PA remains largely unknown. Ceramide, a free fatty acid metabolite, mediates multiple cellular signals. We hypothesized that ceramide acts as an intermediate molecule to mediate inhibition of EPCs by PA. We first demonstrated that PA could inhibit the attachment, migration, and tube formation of EPCs through suppression of the Akt/endothelial nitric oxide (NO) synthase (eNOS) signaling pathway. In addition, we observed that PA could induce ceramide accumulation in EPCs. To test whether the accumulation of ceramide causes EPC dysfunction, the ceramide synthesis inhibitors myriocin and fumonisin B1 were used. We that found both inhibitors could effectively abolish PA-mediated EPC inhibition. Furthermore, the ceramide deacylation inhibitor N-oleoylethanolamine could augment the inhibitory effect of PA on EPCs, indicating that it is ceramide, not its metabolites, that mediates the suppression of EPCs by PA. We have previously shown that Akt/eNOS phosphorylation was reduced after PA treatment, which, in turn, hampered the normal bioavailability of NO, leading to impaired functions of EPCs. To test the role for ceramide in this process, a clinically used NO donor, sodium nitroprusside, was used. We found that sodium nitroprusside could rescue the suppressive effects of ceramide on EPCs, suggesting that ceramide-mediated EPC inhibition might be through reduction of NO production. Taken together, our findings indicated that ceramide-induced reduction of NO might be the molecular mechanism for PA-mediated EPC inhibition; thus, targeting either ceramide or NO production might be an effective means for improvement of EPC functions in diseases.

Stromal cell-derived factor-1α attenuates oleate-induced acute lung injury in rabbits.

The stromal cell-derived factor-1α/C-X-C chemokine receptor 4 (SDF-1/CXCR4) axis is involved in various aspects of tissue repair, regeneration and development. However, the role of SDF-1/CXCR4 in acute lung injury (ALI) remains largely unknown. The aim of the present investigation is to examine pathological changes in a rabbit model with ALI induced by oleic acid (OA) and to explore the protective effect of SDF-1α on ALI. Intravenous application (i.v.) of oleic acid (0.1 ml/kg/h for 2h) provoked pulmonary hemorrhage, edema, and protein leakage, resulting in severe ALI. When the rabbit received an infusion of SDF-1α (20 μg/kg/24h) for 30 min before OA treatment, SDF-1α seemed to significantly improve the pathologies associated with OA-induced ALI. While dissecting the molecular mechanisms underlying the beneficial effects of SDF-1α, we found that SDF-1/CXCR4 is expressed in uninjured lung tissues but is greatly reduced after OA treatment. Interestingly, intravenous delivery of SDF-1α could target an injured lung and rescue expression of CXCR4, which in turn activates anti-apoptotic proteins, Bcl-1 and Bcl-xl, but does not affect pro-apoptotic proteins, such as Bad and Bax. These data suggested that SDF-1α could protect rabbit lungs from AIL. The molecular mechanism might be associated with upregulating anti-apoptosis family expression through CXCR4. Thus, SDF-1/CXCR4 signaling pathway may be a promising target for treatment of patients with ALI.