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Background: Embryonic stem (ES) cell–derived cardiomyocytes transplantation and tissue engineering together represent a promising approach for the treatment of myocardial infarction, despite the limited supply of cardiac myocytes. This study examines whether functional cardiomyocytes can be efficiently enriched from mouse embryonic stem (mES) cells. Methods: mES cells were induced by ascorbic acid to differentiate into cardiomyocytes. Beating cells were observed after 1 week and increased in number with time while under differentiation conditions. Furthermore, the differentiated cultures could be dissociated and enriched by Percoll gradient density centrifugation. Results: The beating cells expressed markers characteristic of cardiomyocytes, such as cardiac troponin T (cTnT). The enriched population contained 88.7% cardiomyocytes and showed expression of cardiomyocyte markers of troponin T and cardiac genes, including α-MHC, β-MHC, ANF and Nkx2.5. However, Oct-4, a marker of early-stage ES cells, was not expressed in the mES cell–derived cardiac cell clusters. Moreover, the mES cell–derived and Percoll-enriched cardiomyocytes responded appropriately to cardioactive drugs, as did normal neonatal rat cardiomyocytes. Conclusions: mES cell–derived functional cardiomyocytes can be enriched by the method of discontinuous Percoll gradient centrifugation. The ability to differentiate and enrich for functional mouse cardiomyocytes makes it possible for further development of these cells as a model of myocardial repair through cell transplantation or tissue engineering. [Copyright &y& Elsevier]

Objective: To clone the glial cell line-derived neurotrophic factor (GDNF) from the mouse testis, construct the eukaryotic expression vector and transfect this vector into Sertoli cells in order to use the gdnf-transfected Sertoli cells as the feeder layer to cultivate spermatogonial stem cells (SSCs)., Methods: Total RNA was extracted from the testes of normal mature mice and gdnf was cloned and amplified using RT-PCR, inserted into the eukaryotic expression vector and transfected into sertoli cells (TM4 cell line). Immunofluorescence with anti-GDNF antibodies was performed at 40 h following the transfection., Results: gdnf cDNA was cloned successfully, and GDNF expressed after transfected into Sertoli cells., Conclusion: This study provides a basis for culturing SSCs with gdnf-transfected Sertoli cells as the feeder layer.

Embryonic stem (ES) cells are pluripotent cells capable of extensive proliferation while maintaining their potential to differentiate into any cell type in the body. ES cells can therefore be considered a renewable source of therapeutically useful cells. While ES-derived cells have tremendous potential in many experimental and therapeutic applications, the scope of their utility is dependent on the availability of relevant cell quantities. Therefore, most of the researches are being focused on the differentiation of ES cells. ES cell aggregation is important for embryoid body (EB) formation and the subsequent generation of ES cell derivatives. EB has been shown to recapitulate aspect of early embryogenesis, including the formation of a complex three-dimensional architecture wherein cell-cell and cell-matrix interactions are thought to support the development of the three embryonic germ layers and their derivatives. Standard methods of EB formation include hanging drop and liquid suspension culture. Both culture systems maintain a balance between allowing ES cell aggregation necessary for EB formation and preventing EB agglomeration for efficient cell growth and differentiation. However, they are limited in their production capacity. In this paper, we established a new approach for the mass production of EBs in a scalable culture system. The rotary cell culture system (RCCS, STLV type) was adopted to produce EBs. The vessel was placed on its rotary base and the experiment started with a beginning rotation rate of approximately 8 r/min which has been previously determined empirically as the optimal initial speed to yield randomized gravitational vectors while minimizing fluid shear stress. To keep the aggregations pfloating in simulated microgravityq, the rotation rate was increased as the EBs visibly grew. The EB production efficiency was calculated when different cell densities were inoculated. The kinetic change of EBs was measured during the time course of EB formation. Compared with the traditional method of producing EBs with hanging drop, the multi-potential of the resulting EBs in RCCS was analyzed by the capability of cardiomyocyte genesis. The results showed that EBs could be produced by RCCS with high efficiency. The optimal cell density inoculated in RCCS was 10000 cells/ml, in which EB production was about twice higher than that in the suspending culture. Day 4-5 was the optimal time point for harvesting EBs. To clarify whether the differentiated potential of EBs might be affected by the microgravity produced by the rotary cell culture system, cardiogenic induction during ES cell differentiation was evaluated in our study. It was manifested by appearance of spontaneously and rhythmically contracting myocytes. In addition, immuno-histological and RT-PCR detection showed that the harvested EBs in RCCS exhibited the expected cardiac genesis and morphology. So, scalable production of EBs is obtained by RCCS. It will provide a useful approach to generate a large quantity of ES-derived cells for further research or application.

Objective: To construct tissue-engineered heart tissue (EHT) using liquid collagen as scaffold., Methods: Neonatal rat cardiac myocytes were isolated, cultured, and mixed with liquid collagen type I and matrix factors and then cast in circular molds to construct circular cardiac myocytes/collagen strand. After a 7-day culture in circular molds, the strands were removed, and subjected to 10% static stretch for another 7 days. Microscopy and transmission electron microscopy, routine HE staining and immunohistochemical staining were used to analyze the engineered heart tissue., Results: Beating areas could be seen on the surface of the EHTs at the second day after stretching; more beating areas could be seen thereafter. These areas beat stronger and stronger, and finally came to synchronzation. Histological and immunohistochemical analyses showed that the cardiac myocytes in the EHTs distributed evenly in the whole strand and the majority of the cells, with elongated nuclei, stretched along the stretching direction. The morphology of EHTs resembled that of the native adult cardiac tissue. Transmission electron microscopy revealed that the cardiac myocytes in EHTs contained arranged myofibrils oriented parallel to the longitudinal cell axis. Clearly defined sarcomeres and Z lines were observed., Conclusion: Liquid type I collagen is a good scaffold for generation of EHTs similar to the native heart tissue.

Objective: To study the isolation of human bone marrow mesenchymal stem cells (MSCs) and in vitro differentiation into chondrocytes as potential seed cell for condyle cartilage tissue engineering., Methods: Human MSCs were isolated by percoll solution from normal human bone marrow sample and cultured in flasks. Specific cell surface markers were identified by flow-cytometry. After the cells were treated with inductive medium containing insulin, transferrin, pyruvate, dexathemesone and TGF-beta for 7 - 14 days, microscopic, histological and immuno-histo-chemical studies were performed for chondrogenic phenotype identification., Results: Primary cultures of human MSCs express CD29 and CD44 positively and meanly, but CD34, CD45 and HLA-DR negatively. After 14 days of induction, the cells were positively stained by safranin O. Immunohistochemical analysis proved strong type II collagen expression., Conclusions: Percoll helps to generate a better isolation of MSCs from human bone marrow aspirates with a purity more above 95%. The isolated MSCs can be expanded and induced in vitro to differentiate into chondrocytes by inductive medium.