ESPE Abstracts

Background: The growth hormone-insulin-like growth factor (GH-IGF) axis plays a crucial role in bone growth, as evidenced by the phenotypes of patients with pathogenic IGF1 receptor (IGF1R) gene variants. These mutations lead to pre- and post-natal growth impairment and short stature. However, the molecular mechanisms of IGFs on human growth plate chondrocytes remain unclear, mainly due to the lack of tissue samples from healthy children.

Objective: To explore expression levels of IGF1R and chondrocyte markers in chondrocytes derived from human induced pluripotent stem cells (hiPSCs) at various differentiation stages.

Methods: HiPSCs (201B7-Ff, RIKEN BioResource Research Center) were cultured under feeder-free conditions. We induced hiPSCs into sclerotome cells following protocols by Tani et al. (Cell Rep., 2023) and Pretemer et al. (Stem Cell Reports., 2021). Subsequently, spheroids were formed in U-bottom plates and differentiated into chondrocytes. RNAs were extracted at various stages (iPSC, sclerotome cell, chondrocyte Days 14, 28, and 57). Relative expression levels of pluripotency markers (NANOG, POU5F1), sclerotome cell markers (PAX1, PAX9, and FOXC2), chondrocyte markers (PTHLH, SOX9, ACAN, COL2A1, and RUNX2), and IGF1R were compared using quantitative PCR (qPCR) and the ΔΔCt method. Safranin O staining was also performed on chondrocyte Days 14, 28, and 57.

Results: qPCR showed high SOX9, ACAN, COL2A1, and RUNX2 expression at chondrocyte Day 28, with proliferative chondrocyte-like cells embedded in Safranin O-stained extracellular matrix. At chondrocyte Day 57, most cells exhibited enlarged cytoplasm within an extracellular matrix stained with Safranin O, indicating the presence of hypertrophic chondrocyte-like cells. Indeed, compared to Day 28, SOX9, ACAN, and COL2A1 expressions decreased, while RUNX2 expression tended to increase. IGF1R expression levels increased 2.65-fold at chondrocyte Day 28 and 1.8-fold at Day 57 compared to hiPSCs and sclerotome cells.

Conclusion: We have successfully induced reproducible differentiation of proliferative to hypertrophic chondrocyte cells from hiPSCs. Our qPCR results, which include changes in IGF1R expression, suggest differential roles of IGF1 between proliferative and hypertrophic chondrocytes. We are currently in the process of investigating downstream signaling in response to IGF1 stimulation in these cells. This will provide further insights into the molecular mechanisms of IGF action in the human growth plate.