Characteristics of bone marrow-derived mesenchymal stem cells of different species: An in vitro comparison study / 中国组织工程研究

ABSTRACT

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BMSCs) can not only differentiate into multiple nonhematopoietic cell lineages, but seek out damaged tissues and repair them as well. Hence, they were largely studied for their potential clinical use. However, their biological characteristics have not been fully discovered. OBJECTIVE: To compare the biological characteristics of BMSCs of different species cultured in vitro, in order to provide basis for the clinical research of stem cell therapy.DESIGN: Randomized controlled observation was designed.SETTING: Medical Research Department of General Hospital of Guangzhou Military Area.MATERIALS The experiment was performed in Medical Research Department of General Hospital of Guangzhou Military Area from June 2004 to July 2005. Thirty SD rats weighing (160±20) g, aged 35 to 40 days, 30 Kunming mice weighing (16.0±2.0) g, aged about 40 days, 8 New Zealand white rabbits weighing (2.0±0.2) kg, aged 80 to 90 days and 10 healthy volunteers (25-32 years old) were selected. All the animals were of clean grade, which were purchased from the Animal Center of Southern Medical University.METHODS: The BMSCs of mice and rats were prepared according to the protocol developed in the Caplan laboratory, while those of rabbits and human were isolated from bone marrow suspension obtained by iliac puncture.The morphology of BMSCs was observed by light microscope and transmission electron microscope. Cell growth curve was tested by MTT. Expression of Stro-1 was analyzed by immunofluorescence cytochemistry and flow cytometry. To evaluate the specific response of BMSCs to osteogenic supplements(10 nmol/L dexamethasone, 10 mmol/L β-glycerophosphate,and 50 mg/L ascorbic acid), the activity of alkaline phosphatase (AKP) activity was tested by a commercial kit. Expression of osteocalcin was examined by immunocytochemistry and hydroxyapatite crystals were shown by von Kossa staining. Adipogenic differentiation was evaluated by estimating percent of cells containing Oil Red-O- stained oil droplets.MAIN OUTCOME MEASURES: ① Morphological observation and growth situation of BMSCs. ② Expression of Stro-1 BMSC marker. ③ Differentiation in osteogenic medium.RESULTS: ①The morphology of adherent BMSCs ofthose four species observed by optic microscopy was obviously different. When they became mature or aged, the mouse cells turned into flat shape, irregularly polygonal, fell to pieces and deposited on the flask-bottom, while the rat, rabbit and human cells would enlarge and become polygonal, vacuoles would appear in their cytoplasm, finally, the cells were detached from the flask-bottom, floating off like cotton wool. The cultures of different species also had some commonness, such as poly-layer growing manner, without contact inhibition and consisting of two groups. Cells of one group grew into colonies from single cells and expanded quickly, while cells of the other group were sporadic and did not proliferate. Electron microscopy revealed that all of the primary cells had microvilli and that they could be divided into two subpopulations according to their ultrastructures. Some cells were rich in organelles and most chromatin was euchromatin, while the other subpopulation cells had much fewer organelles and more heterochromatin. Growth curves of BMSCs of different species were almost the same. ② The positive rate of human adherent bone marrow-derived cells for Stro-1 BMSC marker was (91.4±8.3) %, and that of mouse adherent cells was (83.5±6.2) % .③Treated with osteogenic supplements, mouse BMSCs differentiated into adipose tissue, rabbit ones died, while rat and human ones differentiated into osteocytes. BMSCs also demonstrated spontaneous differentiation in vitro.CONCLUSION: Mouse, rat, rabbit and human BMSCs can be easily expanded in vitro, although the harvest of the current method is a mixture of mesenchymal cells with various maturities, most of which are poor-differen-tiated cells. BMSCs of those species are different in morphology and response to the same inductive supplements. Therefore, in order to establish a kind of stem cell therapy, it is necessary not only for evidence from animal experiments but for that from human experiments as well.