CD146 defines commitment of cultured annulus fibrosus cells to express a contractile phenotype

Abstract

Characterization of cells is important for facilitating cell-based therapies for degenerative diseases of intervertebral discs. For this purpose, we analyzed mouse annulus fibrosus cells by flowcytometory to detect phenotypic change in their primary cultures. After examination of sixteen cell surface proteins, we focused on CD146 that solely increased during culture expansion. CD146 is known to be a marker for mesenchymal stem cells and for their vascular smooth muscle commitment with expression of contractile phenotype enhanced by SM22a. We sorted CD146þ cells to elucidate their characteristics and the key factors that play a role in this change. Whole cell cultures showed the ability for tripotent differentiation toward mesenchymal lineages, whereas sorted CD146þ cells did not. Expression of CD146 was elevated by addition of transforming growth factor b1, and sorted CD146þ cells expressed higher levels of mRNA for SM22a and Elastin than did CD146À cells. Morphologically, CD146þ cells more broadly deposited extracellular type I collagen than CD146À cells and showed filamentous actin bundles traversing their cytoplasm and cell-cell junctions. Moreover, CD146þ cells demonstrated significantly higher gel contraction properties than CD146À cells when they were embedded in collagen gels. Human annulus fibrosus CD146þ cells also showed higher contractility. Immunohistochemistry determined CD146þ cells localized to the outermost annulus layers of mouse intervertebral disc tissue with co-expression of SM22a. These results suggest that increment of CD146 expression indicates gradual change of cultured annulus fibrosus cells to express a contractile phenotype and that transforming growth factor b1 enhances this cellular commitment. ß

Annulus fibrosus cell sheets limit disc degeneration in a rat annulus fibrosus injury model

“…In recent years, functional cellular markers for AF cells, such as CD146 and Mohawk have been identified, offering opportunities for AF cell repair. Despite the many studies that have been performed on transplanting cells to AF, no definitive transplantation methods have been established .…”

Annulus fibrosus cell phenotypes in homeostasis and injury: implications for regenerative strategies

“…Schematics and representative microscopic images of the different cell types and their locations in the healthy adult AF. (A) Peripheral cells, (B) outer annulocytes, (C) inner annulocytes, and (D) interlamellar cells …”

“…A single layer of cells expressing the markers CD146 and transgelin (SM22ɑ) was identified in the AF periphery in both mouse and human IVD (Table ) . While there is speculation that these CD146 + cells may be a population of resident stem cells, their function is still not clear, and they may also serve important functions in wound healing responses and contractility …”

Critical aspects and challenges for intervertebral disc repair and regeneration—Harnessing advances in tissue engineering

“…In terms of AF repair, the lack of specific markers for functional AF cells has hampered similar developments. Recently, new markers such as CD146 and Mohawk (MKX) have been identified that characterize the functional AF cell phenotype, opening the door for future cell engineering approaches. Notably these technologies may represent new strategies for unlimited generation of functional IVD‐like cells; though in vivo studies will be needed to assess the therapeutic values of cell engineering developments for IVD repair and regeneration.…”