ANALYSIS OF THE IN VIVO FREQUENCY AND THE IN VITRO OSTEOGENIC DIFFERENTIATION OF IMMUNOPHENOTIPICALLY DEFINED HUMAN SKELETAL STEM AND PROGENITOR CELL SUBPOPULATIONS

Abstract

Skeletal stem and progenitor cells are responsible for bone maintenance and regeneration. Recently, a more specific set of phenotypic markers was described to identify the multipotential stem cell population (SSC, PDPN+ CD146− CD164+ CD73+), which gives rise to a PDPN+ CD146+ Bone, Cartilage and Stromal Progenitor (BCSP), which in turn originates a PDPN− CD146+ osteoprogenitor (OP) or a PDPN+ CD146− chondroprogenitor (CP). However, little is known about the frequency of these cell populations in bones of distinct anatomical locations, whether these phenotypic markers are stable enough to be used as distinctive markers during in vitro expansion, and whether these populations differ in osteogenic potential, three relevant questions for the development of characterized and effective Advanced Medicinal Therapeutic Cellular Products (AMTCPs) for application in innovative therapies for bone diseases. Therefore, the objective of this study was to investigate the frequency of SSCs and its progeny in the bone marrow collected from different bone sources and the stability of their immunophenotypic profile following in vitro expansion by FACS, their osteogenic differentiation potential in vitro, and how these profiles correlate with that of unfractionated Bone Marrow Stromal Cells (BMSCs). Following approval (nº 21768719.0.0000.5257), human hip (n = 19) and humerus (n = 5) samples were collected. In hip samples, SSCs accounted for 0.13% ± 0,20% of total nucleated non-hematopoietic cells, BCSPs were 4.78% ± 10.47%, CPs were 7.38% ± 14.12%, and OPs were 9.94% ± 12.20%. In humerus, SSCs and CPs were only detected in one sample (0,2% e 12%, respectively). BCSPs were 0.3% ± 0.15% and a higher frequency of OPs (25.05% ± 5.30%, p = 0.04) was detected in comparison to hip samples. Following PDPN− CD146+ (OP) and PDPN+ CD146− (SSCs and CPs) sorting, it was observed that their immunophenotypic profiles converged during expansion to PDPN+ CD146+, becoming similar to BMSCs. However, the expanded subpopulations differed in osteogenic capacity, with the PDPN+ CD146− (SSCs and CPs) producing a lower mineralized matrix area revealed by Alizarin Red staining than the BMSCs and OPs. Further in vivo functional assays will be performed to confirm these findings, that may lay the foundation to produce better AMTCPs.