Stem Cell Department, Royan Institute
Cellular and Molecular Biology Research Center, Shaheed Beheshti University of Medical Sciences
Introduction: The aim of this study was to differentiate human mesenchymal stem cells (hMSCs) into cartilage in a micromass culture system and study of their structure by light and electron microscopy.
Material and Methods: Human bone marrow cells obtained from volunteer patients were plated in 75-cm2 flasks and their MSCs were expanded through several sub-cultures. The passage 4 cells were used to establish micromass culture system for chondrogenic differentiation. For this purpose, 200,000 fibroblastic cells were placed in centrifuge tubes and pelleted at 250 g for 5 minutes. About 0.5 ml chondrogenic induction medium was then added to the pellet and the culture incubated in 5% CO2 at 37°C for 21 days. Then, some pellets were utilized to evaluate chondrogenic differentiation by either RT-PCR analysis of some cartilage marker molecules or specific staining for detecting cartilage matrix, and other pellets were used for light and electron microscopic study of differentiated tissue.
Results: Primary culture of the bone marrow cells were initially composed of the spindle- and round shaped cells, from which the spindle cells remained and expanded through several passages. At the end of differentiation period, RT-PCR analysis showed high production of collagen II and X and aggrecan mRNA inside the differentiated cells, and toluidine blue staining indicated intermediate accumulation of the metachromatic matrix among the induced cells. In general, light micrograph indicated a rather cellular state of the differentiated tissue in which the peripheral part had more metachromatic matrix than central zone. More detailed study of the sections revealed that induced aggregates of the cells were composed externally of very thin layer of elongated cells reminiscent of perichondrium and internally a mass of oval cells comprising the main part of the pellet. Ultra-thin sections showed that the cells in perichondrium-like layer were very similar to fibroblastic cells and those located centrally had a set of well-developed organelles, characteristic of highly active cells. Some fat cells were seen in central zone.
Conclusion: Cartilage tissue differentiated from MSCs in micromass culture system seemed to be structurally very similar to developing cartilage not to adult mature cartilage.