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

Cell populations and their interplay provide the basis of a cell-based regenerative construct. Serum-free preconditioning can overcome the less predictable behaviour 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 was investigated. Following BMP2 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. Human periosteum-derived cells (hPDCs), pooled from 4 individuals, were cultured in growth medium (GM), i.e. DMEM-GlutaMAX supplemented with 10% foetal bovine serum and 1% antibiotic-antimycotic solution until passage 5. The hPDCs were then preconditioned in chemically-defined medium (CDM) or further cultured in GM as a control. Preconditioned and control cells were collected after 6 days and loaded onto the 10X Chromium Platform.