저산소 환경이 인간 지방조직유래 줄기세포의 주변 분비와 분화 능력에 미치는 영향

Abstract

Stem cells are undifferentiated cells which have ability to renew themselves through mitotic cell division and differentiate into a diverse range of specialized cell types by appropriate signals or stimuli. The two broad types of mammalian stem cells are embryonic stem cells and adult stem cells. Adult stem cells are found throughout the body after embryonic development. Among available adult stem cells, adipose-derived stem cells (ASCs) can be obtained safely from fat with little pain. As fat is a widely dispersed tissue, its availability provides the advantage of use without the expansion of the cells. These merits make such ASCs a promising source for regenerative medicine, a medical field of tissue or organ repair or replacement after loss of function due to age, disease, or damage. In most cases, the damaged tissues have insufficient blood supply, causing unusual cellular microenvironments. Low oxygen concentration is one of the influential factors in damaged tissue, so it is important to understand stem cell properties under hypoxic conditions for successful cell therapy. In this context, this study was conducted to characterize hASCs and to investigate the effect of hypoxia on paracrine secretion and differentiation. In chapter Ⅰ, it is shown that hASCs established in the present study have stem cell properties, self-renewal and multipotency. The hASCs were established from 45 donors to characterize for stem cell properties. The mean isolation yield of hASCs was 0.93 × 105 cells/ml and decreased with aging. However, morphology and proliferation rate were not significantly different by age. Established hASCs had normal karyotypes containing 22 pairs of autosomal chromosomes and one pair of sex chromosome. RT-PCR analysis showed hASCs expressed OCT-4, SCF, ADAM-12, AFP, and BMP-4 but not expressed NCAM and HLA Class Ⅱ (HLA-DR). Immunophenotyping of hASCs showed positive expression for CD9, CD34 and CD90 and negative expression for CD31, CD45 and CD117. Moreover, hASCs had adipogenic, osteogenic and chondrogenic potentials. In chapter Ⅱ, hASCs were transplanted into animal models to examine their regenerative potential. In rabbit intervertebral disc degeneration model, hASCs differentiated into chondrocytes and replaced nucleus pulposus region with cartilage matrix. Height of degenerated disc was recovered, too. In mouse adipose transplantation model, injected tissues were exposed to hypoxia and degraded. After 24 weeks, tissue weight was decreased very significantly. However, delayed degradation and resorption of tissues were observed in hASCs injected group. Moreover, hASCs differentiated into endothelial cells and mediated adipocytes size maintenance. In chapter Ⅲ, the effect of hypoxia on hASCs and relation of PI3K/Akt signaling were investigated. Low oxygen gas mixture (1% O2) inducing hypoxia activated PI3K-Akt-HIF1α signaling without morphological changes. And angiogenic factors, HGF and VEGF secretion to cell culture medium were increased. While expression of stem cell marker genes was decreased during hypoxic culture but chondrogenesis and angiogenesis were enhanced. After chondrogenic differentiation, safranin O staining and immunocytochemistry showed hASCs produced more proteoglycans and collagens by hypoxia. After angiogenic differentiation, Matrigel assay showed area of tube-like structures were 6.5-fold increase in hypoxia. However, enhanced paracrine secretion and differentiation were not observed in LY294002 treated cells. This chapter demonstrated low O2 concentration promotes paracrine secretion and differentiation of hASCs via PI3K/Akt signaling. Taken together, these results demonstrate that hypoxic culture conditions enhanced paracrine secretion and differentiation of hASCs through PI3K/Akt activation. Hypoxia promoted angiogenesis of hASCs by increased paracrine secretion and tube formation capacity. Besides, more cartilage matrix molecules were synthesized during chondrogenic differentiation under hypoxia. Properties of hASCs under hypoxic condition may mediate regeneration of damaged tissues of animal model study. Therefore, hASCs appear to be a promising cell source for treatment of ischemic tissues in regenerative medicine.