| Summary: | 博士 === 國立陽明大學 === 臨床醫學研究所 === 99 === Somatic or adult stem cells can be obtained from a variety of adult human tissues. The aim of this study is to investigate two types of stem cells, first, the isolation of stem cells from the human parathyroid gland and second, the mechanism of how extracellular matrix stiffness regulates the osteogenic differentiation bone marrow mesenchymal stem cells (MSCs).
Although many types of stem cells has been isolated from different tissues, it was not clear whether human parathyroid glands, which secrete parathyroid hormones and are essential in maintaining homeostasis levels of calcium ions in the circulation, contained stem cells. We aimed to investigate the possibility of isolating such parathyroid-derived stem cells (PDSC). The potentials of stem cells from the parathyroid include treatment of hypoparathyroidism and osteoporosis. Surgically removed parathyroid glands were obtained with informed consent. After enzymatic digestion and primary culture, plastic adherent, fibroblast-like cells appeared in culture and a morphologically homogeneous population was derived from subsequent limiting dilution and clonal expansion. Karyotyping was normal and doubling time of clonal cell growth was estimated to be 70.7 ± 14.5 h (mean ± standard deviation). The surface phenotype of the cells was positive for CD73, CD166, CD29, CD49a, CD49b, CD49d, CD44, CD105, and MHC class I, and negative for CD34, CD133, CD117, CD114, CD31, CD62P, EGF-R, ICAM-3, CD26, CXCR4, CD106, CD90 and MHC class II, similar to mesenchymal stem cells (MSC). Detectable levels of telomerase activity along with pluripotency Sall4 and Oct4 gene expression were observed from the isolated PDSCs. Expression of calcium-sensing receptor gene along with alpha-smooth muscle actin was induced and cellular uptake of extracellular calcium ions was observed. Furthermore, PDSCs possessed osteogenic, chondrogenic and adipogenic differentiation potentials. Our results reveal that PDSCs were similar phenotypically to MSCs and further studies are needed to formulate induction conditions to differentiate PDSCs into parathyroid hormone-secreting chief cells.
Since the characterized PDSCs possess similar function to MSCs in morphology, surface phenotype and osteogenic potential. We further took advantage of studying a well-characterized source of MSCs from the human bone marrow to investigate the effects of matrix elasticity on osteogenic fate determination. Bone marrow-MSCs (BMMSCs) cultured on extracellular matrices with different stiffness have been shown to possess diverse lineage commitment due to the extracellular mechanical stimuli sensed by the cells. We further delineated how matrix stiffness affects intracellular signaling through the mechano-transducers Rho-kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulate the osteogenic phenotype of MSCs.
BMMSCs were cultured in osteogenic medium on tunable polyacrylamide hydrogels coated with type I collagen with elasticities corresponding to Young’s modulus of 7.0±1.2 and 42.1±3.2 kPa. Osteogenic differentiation was increased on stiffer matrices as evident by type I collagen, osteocalcin, Runx2 gene expressions and alizarin red S stain for mineralization. Western blot analysis demonstrated an increase in kinase activities of ROCK, FAK and ERK1/2 on stiffer matrices. Inhibition of FAK, an important mediator of osteogenic differentiation and inhibition of ROCK, a known mechano-transducer of matrix stiffness during osteogenesis, resulted in decreased expression of osteogenic markers during osteogenic induction. In addition, FAK affects osteogenic differentiation through ERK1/2, while ROCK regulates both FAK and ERK1/2. Furthermore, alpha2 integrin was up-regulated on stiffer matrices during osteogenic induction and its knockdown by siRNA down-regulated osteogenic phenotype through ROCK, FAK and ERK1/2.
Taken together, our result provides evidence that MSC-like cells exist in the human parathyroid and serves as an alternative source for stem cells. Hopefully its discovery can develop new and effective treatments in hypoparathyroidism and osteoporosis. In addition, we have delineated the mechanism whereby matrix rigidity affects the osteogenic outcome of BMMSCs through mechano-transduction events that are mediated by alpha2 integrin to downstream ROCK-FAK-ERK1/2. The regulation of targets in the signaling axis could provide researchers means to ameliorate osteogenic differentiation protocols.
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