Single-cell characterization and metabolic profiling of in vitro cultured human skeletal progenitors with enhanced in vivo bone forming capacity

Abstract

Abstract Cell populations and their interplay provide the basis of a cell-based regenerative construct. Serum-free preconditioning can overcome the less predictable behavior of serum expanded progenitor cells, but the underlying mechanism and how this is reflected in vivo remains unknown. Herein, the cellular and molecular changes associated with a cellular phenotype shift induced by serum-free preconditioning of human periosteum-derived cells were investigated. Following BMP-2 stimulation, preconditioned cells displayed enhanced in vivo bone forming capacity, associated with an adapted cellular metabolism together with an elevated expression of BMPR2. Single-cell RNA sequencing confirmed the activation of pathways and transcriptional regulators involved in bone development and fracture healing, providing support for the augmentation of specified skeletal progenitor cell populations. The reported findings illustrate the importance of appropriate in vitro conditions for the in vivo outcome. In addition, BMPR2 represents a promising biomarker for the enrichment of skeletal progenitor cells for in vivo bone regeneration. Significance statement A critical number of in vitro expanded progenitor cells provide the key driving force in a cell-based regenerative construct. Standard expansion protocols highly affect the initial cellular phenotype due to the focus on fast expansion rather than on the maintenance of the progenitor potential. This article describes a serum-free preconditioning regime of in vitro expanded human periosteum-derived cells that lead to a progenitor cell with enhanced in vivo bone forming capacity at the single cell level. This phenotype shift was associated with an adapted cellular metabolism and activation of pathways and transcriptional regulators involved in bone development and fracture healing, illustrating the importance of appropriate in vitro conditions for the in vivo outcome.