Yanmeng Wang

The influence of chitosan hydrogel on stem cell engraftment, survival and homing in the ischemic myocardial microenvironment

One challenge of cellular cardiomyoplasty for myocardial infarction (MI) is how to improve MI microenvironment to facilitate stem cell engraftment, survival and homing for myocardial repair. The application of injectable hydrogels is an effective strategy. However, it has not been thoroughly investigated on the role of the injectable scaffolds, in improving MI microenvironment, providing space and guidance for cell survival, engraftment and homing. We explored an injectable chitosan hydrogel for stem cell delivery into ischemic heart and investigated the beneficial effects and mechanisms of the hydrogel. In vitro, H(2)O(2)-treatment was used to mimic reactive oxygen species (ROS) microenvironment. The influence of ROS and protection of chitosan components on adipose-derived mesenchymal stem cells (ADSCs) was analyzed too. In vivo, MI was induced by the left anterior descending artery ligation in SD rats. PBS, chitosan hydrogel, ADSC/PBS and ADSC/chitosan hydrogel were injected into the border of infracted hearts respectively. Multi-techniques were used to assess the beneficial effects of chitosan hydrogel after transplantation. We observed that ROS generated by ischemia would impair ADSC adhesion molecules, including integrin-related adhesion molecules integrin αV and β1, focal adhesion-related molecules p-FAK and p-Src, and corresponding ligands of host myocardium ICAM1 and VCAM1. Chitosan hydrogel could rescue these molecules through ROS scavenging and recruit key chemokine for stem cell homing, such as SDF-1. The results suggest that chitosan hydrogel could improve MI microenvironment, enhance stem cell engraftment, survival and homing in ischemic heart through ROS scavenging and chemokine recruitment, contributing to myocardial repair.

Improved myocardial performance in infarcted rat heart by co-injection of basic fibroblast growth factor with temperature-responsive Chitosan hydrogel

BACKGROUND Basic fibroblast growth factor (bFGF) stimulates neoangiogenesis. The sustained release of bFGF by using biomaterials helped to enhance its angiogenic activity in vivo. In this study we investigated the effects of co-injection of bFGF with temperature-responsive chitosan hydrogel on myocardial performance in a rat model of infarction. METHODS Myocardial infarction was induced in rats using coronary artery ligation. Temperature-responsive chitosan hydrogel was prepared and injected intramyocardially into the left ventricular wall of rat infarction models alone or together with bFGF. Detailed histologic analysis and echocardiography were used to determine the structural and functional consequences 4 weeks after injection. RESULTS Heart function improved significantly in the chitosan+bFGF group compared with the phosphate-buffered saline (PBS)+bFGF group with regard to left ventricular ejection fraction (LVEF) and LV fractional shortening (LVFS) 4 weeks after transplantation (p < 0.05, n = 8 per group). In addition, arteriole densities within the infarcted area improved significantly (p < 0.01) in the chitosan+bFGF group (259 +/- 22/mm(2)) compared with the PBS+bFGF group (95 +/- 18/mm(2); n = 8 per group) at 4 weeks after transplantation. Infarct size and fibrotic area decreased significantly (p < 0.05) in the chitosan+bFGF group (39.64 +/- 1.75% and 25.09 +/- 3.31%, respectively) compared with the PBS+bFGF group (48.91 +/- 1.39% and 48.0 +/- 3.83%, respectively; n = 8 per group). No significant difference (p > 0.05) was noted between the PBS and PBS+bFGF groups. CONCLUSIONS Co-injection of bFGF with temperature-responsive chitosan hydrogels enhanced the effects of bFGF on arteriogenesis, ventricular remodeling and cardiac function. Our findings suggest a new approach to improve infarcted repairs in the prevention of adverse remodeling after myocardial infarction.

Bioreactor Cultivation Enhances NTEB Formation and Differentiation of NTES Cells into Cardiomyocytes

Autogenic embryonic stem cells established from somatic cell nuclear transfer (SCNT) embryos have been proposed as unlimited cell sources for cell transplantation-based treatment of many genetic and degenerative diseases, which can eliminate the immune rejection that occurs after transplantation. In the present study, pluripotent nuclear transfer ES (NTES) cell lines were successfully established from different strains of mice. One NTES cell line, NT1, with capacity of germline transmission, was used to investigate in vitro differentiation into cardiomyocytes. To optimize differentiation conditions for mass production of embryoid bodies (NTEBs) from NTES cells, a slow-turning lateral vessel (STLV) rotating bioreactor was used for culturing the NTES cells to produce NTEBs compared with a conventional static cultivation method. Our results demonstrated that the NTEBs formed in STLV bioreactor were more uniform in size, and no large necrotic centers with most of the cells in NTEBs were viable. Differentiation of the NTEBs formed in both the STLV bioreactor and static culture into cardiomyocytes was induced by ascorbic acid, and the results demonstrated that STLV-produced NTEBs differentiated into cardiomyocytes more efficiently. Taken together, our results suggested that STLV bioreactor provided a more ideal culture condition, which can facilitate the formation of better quality NTEBs and differentiation into cardiomyocytes more efficiently in vitro.

Tumourigenesis in the infarcted rat heart is eliminated through differentiation and enrichment of the transplanted embryonic stem cells.

The therapeutic potential of embryonic stem cells (ESCs) in ischaemic heart disease has been widely explored. However, tumourigenesis upon implantation interferes with the clinical application of ESC transplantation. This study aims to evaluate the influence of differentiation and enrichment of transplanted ESCs on tumourigenesis in infarcted rat hearts.

The reconstruction of lung alveolus‐like structure in collagen‐matrigel/microcapsules scaffolds in vitro

This study attempted to use collagen–Matrigel as extracellular matrix (ECM) to supply cells with three‐dimensional (3D) culture condition and employ alginate‐poly‐l‐lysine‐alginate (APA) microcapsules to control the formation of alveolus‐like structure in vitro. We tested mice foetal pulmonary cells (FPCs) by immunohistochemistry after 2D culture. The alveolus‐like structure was reconstructed by seeding FPCs in collagen–Matrigel mixed with APA microcapsules 1.5 ml. A self‐made mould was used to keep the structure from contraction. Meanwhile, it provided static stretch to the structure. After 7, 14 and 21 days of culture, the alveolus‐like structure was analysed histologically and immunohistochemically, or by scanning transmission electron microscopy (TEM). We also observed these structures under inverted phase contrast microscope. The expression of pro‐surfactant protein C (SpC) was detected by reverse transcription‐polymerase chain reaction (RT‐PCR). We obtained fibroblasts, epithelial cells and alveolar type II (AE2) cells in FPCs. In the reconstructed structure, seeding cells surrounding the APA microcapsules constructed alveolus‐like structures, the size of them ranges from 200 to 300 μm. In each reconstructed lung tissue sheet, microcapsules had integrity. Pan‐cytokeratin, vimentin and SpC positive cells were observed in 7‐ and 14‐day cultured structures. TEM showed lamellar bodies of AE2 cells in the reconstructed tissues whereas RT‐PCR expressed SpC gene. Primary mice FPCs could form alveolus‐like structures in collagen–Matrigel/APA microcapsules engineered scaffolds, which could maintain a differentiated state of AE2 cells.

Efficient Isolation of Cardiac Stem Cells from Brown Adipose

Cardiac stem cells represent a logical cell type to exploit in cardiac regeneration. The efficient harvest of cardiac stem cells from a suitable source would turn promising in cardiac stem cell therapy. Brown adipose was recently found to be a new source of cardiac stem cells, instrumental to myocardial regeneration. Unfortunately, an efficient method for the cell isolation is unavailable so far. In our study we have developed a new method for the efficient isolation of cardiac stem cells from brown adipose by combining different enzymes. Results showed that the total cell yield dramatically increased (more than 10 times, P < .01) compared with that by previous method. The content of CD133-positive cells (reported to differentiate into cardiomyocytes with a high frequency) was much higher than that in the previous report (22.43% versus 3.5%). Moreover, the isolated cells could be the efficiently differentiated into functional cardiomyocytes in optimized conditions. Thus, the new method we established would be of great use in further exploring cardiac stem cell therapy.

Transplantation of Sertoli-Islet Cell Aggregates Formed by Microgravity: Prolonged Survival in Diabetic Rats

Transplantation of pancreatic islets is a potentially attractive treatment for type I diabetes. We generated the transplantable, tissue-like aggregates composed of Sertoli cells and islets in rotating wall vessel bioreactors, SICA (Sertoli-islet cell aggregates), to improve their biological function in vitro and in vivo. The isolated islet equivalent and Sertoli cells were purified from Wistar rats and cocultured for 5 days in bioreactor to generate SICA. The SICA, islets aggregates, and fresh isolated islets were transplanted under the kidney capsule of diabetic Sprague-Dawley (SD) rats, respectively. The functions of different grafts were ascertained by blood glucose level measurements and an in vivo glucose tolerance test. In response to elevated glucose, insulin secretion from SICA was 1.4-fold higher (P < 0.05, n = 5) than islet aggregates cultured alone. Of the rats that received SICA, 90% (9/10) remained normoglycemic at 60 days post-transplantation, and the survival significantly increased compared with recipients bearing homotypic islets aggregates or freshly isolated islets. The former responded similarly with healthy rats to the glucose tolerance test. Our results support the usefulness of SICA for the treatment of type 1 diabetes without any immunosuppressive agents.