The structure of Human Mesenchymal Stem CellsDifferentiated into Cartilage in MicromassCulture System

Introduction: the aim of this study was to differentiate humanmesenchymal stem cells (hmscs) into cartilage in a micromass culturesystem and study of their structure by light and electron microscopy.material and methods: human bone marrow cells obtained from volunteerpatients were plated in 75-cm^2 flasks and their mscs were expandedthrough several sub-cultures. the passage 4 cells were used to establishmicromass culture system for chondrogenic differentiation. for this purpose,200,000 fibroblastic cells were placed in centrifuge tubes and pelleted at 250g for 5 minutes. about 0.5 ml chondrogenic induction medium was thenadded 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 byeither rt-pcr analysis of some cartilage marker molecules or specificstaining for detecting cartilage matrix, and other pellets were used for lightand electron microscopic study of differentiated tissue.results: primary culture of the bone marrow cells were initially composed ofthe spindle- and round shaped cells, from which the spindle cells remainedand expanded through several passages. at the end of differentiation period,rt-pcr analysis showed high production of collagen ii and x and aggrecanmrna inside the differentiated cells, and toluidine blue staining indicatedintermediate accumulation of the metachromatic matrix among the inducedcells. in general, light micrograph indicated a rather cellular state of thedifferentiated tissue in which the peripheral part had more metachromaticmatrix than central zone. more detailed study of the sections revealed thatinduced aggregates of the cells were composed externally of very thin layerof elongated cells reminiscent of perichondrium and internally a mass of ovalcells comprising the main part of the pellet. ultra-thin sections showed thatthe cells in perichondrium-like layer were very similar to fibroblastic cells andthose located centrally had a set of well-developed organelles, characteristicof highly active cells. some fat cells were seen in central zone.conclusion: cartilage tissue differentiated from mscs in micromass culturesystem seemed to be structurally very similar to developing cartilage not toadult mature cartilage.