Human anterior cruciate ligament-derived mesenchymal stem cells for regenerative medicine applications

Abstract

Background: The anterior cruciate ligament (ACL) has poor healing capabilities and is the most commonly injured knee ligament. Although ACL repair is being highly studied, the current treatment involves reconstructive surgery utilizing autografts or allografts, which have limitations. The use of Mesenchymal Stem Cells (MSCs) as a possible therapeutic option has grown. ACL-derived MSCs are likely to be the best source because studies have shown that target tissue derived stem cells will better differentiate into the target tissue than the stem cells derived from non-target ones. However, the existing literature discusses only the isolation of a mixed population of MSCs. Here we present the isolation, differentiation and characterization of human ACL-derived MSCs according to the International Society for Cellular Therapy (ISCT) criteria.The ACL tissue was enzymatically digested. Separation of MSCs from the crude mixture of cells was then performed by fluorescence activated cell sorting (FACS) analysis. The isolated population were passaged in specific induction medium to differentiate them into adipocyte, osteocytes and chondrocytes. The cells were then further characterized with respect to their growth curve, population doubling time, colony forming ability, anchorage independent growth, and cell surface markers. The cells were finally examined for their tumorigenic potential by cell cycle analysis.Immunoprofiling via FACSs showed an average isolation rate for cells carrying MSCs markers of 5.5%. Cells exhibited spindle-shaped morphology, and immunocytochemistry confirmed the expression of appropriate cell surface markers. The growth curve showed distinct lag and log phase. Over agar assay demonstrated no anchorage independent growth, but clonogenic potential was observed post-culture on plastic Petri dishes. The cells showed a population doubling time of about 1.5 days. Oil Red O, Alizarin Red S, and Alcian Blue staining confirmed adipogenic, osteogenic and chondrogenic differentiation, respectively. Cell cycle analysis displayed more ACL-derived MSCs in G/G phase compared to BMSCs, showing that the isolates were non-tumorigenic. The presence of MSCs within the human ACL was confirmed via ISCT criteria, paving the way for their potential use for future ACL reconstructions. Although BMSCs have been the choice for regenerative purposes, making use of MSCs derived from ACL ligament will cut down the burden of trauma one has to undergo to obtain the Bone Marrow. Moreover, it is more convenient to harvest MSCs from otherwise discarded ACL. Finally, MSCs derived from the target tissue are believed to better differentiate to the ligament tissue than the bone derived MSCs.