Innovation in skeletal muscle tissue regeneration: Obtaining fibroadipogenic progenitor cells from infrapaletar fat.

Abstract

Introduction. Infrapatellar fat pads (IPFPs), also known as Hoffa's pad, are extra-synovial intracapsular structures with a structure similar to subcutaneous fat. The IPFPs function to promote synovial fluid distribution, absorb joint loads, and secrete pro-inflammatory mediators and growth factors with endocrine-paracrine-autocrine activity in cartilage and the synovial membrane. Recently, they have been proposed as a source of mesenchymal stem cells (MSCs), which have a greater capacity for differentiation into cartilage. Fibroadipogenic progenitor cells (FAPs) have been shown to be stromal mesenchymal cells identified in skeletal muscle with a high regenerative capacity for muscle and tendons, but not in IPFPs for therapeutic purposes. Objective. To identify MSCs and FAPs in primary cultures of human IPFPs using flow cytometry. Method. The study was approved by the INRLGII Research Committee (No. 83/23). Five IPFP samples were taken from patients who underwent arthroscopic knee surgery (both sexes, age range 18–51 years). Each sample was fragmented and digested with 0.3% collagenase in 1% antibiotic/antifungal DMEM/F12 culture medium, under constant agitation at 200 rpm at 37°C for 2 hours. Subsequently, the cells were filtered (70 mm sieve) and seeded at a density of 1–1.5 x 10⁴ per cm² with DMEM/F12, 10% SBF, and 1% AA. Once confluent, the cells were passaged with TrypLE Express trypsin, expanding to passage 2. During each passage (Primary Culture, P1 and P2), the expression of the markers CD90 FITC, CD105 PE, and CD73 APC for MSCs and CD140α RB780 for FAPs, CD45 FITC, CD34 PE, and HLA-DR APC were analyzed to determine the absence of hematopoietic stem cells (HSCs). 1 x 10⁵ cells were labeled, the data were acquired using a FACS DIVA II flow cytometer, and analyzed with Flowjo software. Results. The expression of the MSC markers CD90, CD105, and CD73 was, on average, greater than 90% for all three passages. The HSC markers CD45, CD34, and HLA-DR were 5% or less in all three culture stages. In the case of FAPs, CD140α expression was 62% in CP, 70% in P1, and 61% in P2, indicating that more than 50% of the cells were FAPs. Conclusion: In IPFP, MSCs and FAPs were identified by flow cytometry, suggesting that IPFP could be a viable option for obtaining cells that can impact the cellular treatment of musculoskeletal tissue injuries.