Induction of human mesenchymal stem cells in vitro and related identification / 中国耳鼻咽喉头颈外科

ABSTRACT

OBJECTIVE To establish the isolation and identification system of human mesenchymal stem cells(hMSCs)in vitro, and then to establish a stable cultured system in which the hMSCs can be induced to osteoblasts, chondroblasts and lipoblasts in vitro. To study the possibility if hMSCs can be used as the seed cells in bone tissue engineering.This study would lay a foundation for the clinical application of tissue engineering. METHODS 5ml bone marrow of the patient was aspirated from iliac crest. The human MSCs were isolated using Percoll gradient centrifugation. The hMSCs were cultured in DMEM medium in vitro, FACS(fluorescence axtivated cell sorter)was used to identify the phenotype of hMSCs. The hMSCs were cultured in vitro, and induced to osteoblasts, chondrocytes and lipocytes. The changes of phenotype were tested by mmunohistochemisty and molecular biology technique. RESULTS The hMSCs were isolated by Percoll gradient centrifugation from patient’s bone marrow. The expression rate of MSC markers including CD105, CD166, CD29 were(78?6)%, (43?7)%, (69? 12)% respectively. The phenotype of osteoblasts induced from hMSCs was verified by the mineralized nodes formation when cultured in vitro and Col, and OCN expression showed by immunohistochemistryand RT-PCR. The phenotype of chondrocytes induced from hMSCs was verified by type, collagen, SOX9 expression with immunohistochemical staining technique and type, collagen, aggrecan mRNA expression with RT-PCR. The lipocytes’ phenotype was verified by positive result of oil red O staining and PPAR 2mRNA expression by RT-PCR. CONCLUSION The highly pure hMSCs can be harvested by means of density gradient centrifugation, and the hMSCs have multi-potentiality of cell differentiation via induced culture in vitro. The third passage of hMSCs can be induced to express osteoplast phenotype and meet the qualitative and quantitative demands of seed cells when bone tissue engineering was used in clinical practice.