Background/Purpose: Multipotent mesenchymal stem cells (MSCs) are widely used in regenerative medicine to repair damaged tissues. Regulating Ca²⁺ release-activated Ca²⁺ (CRAC) channel-mediated intercellular Ca²⁺ signaling is critical to the functional modulation of MSCs. CRACM1 in the plasma membrane is an important molecular regulator of the CRAC channel. Our study aimed to elucidate the possible effects of CRAC channel regulation on the multiple differentiation potential of MSCs.

Methods: To increase Ca²⁺ influx, we overexpressed CRACM1 by transfecting PCDNA3-CRACM1 into MSCs (m1-MSCs). Genomic CRACM1-knockout MSCs (m1-ko-MSCs) were perpared using CRISPR/Cas9 technique. YM-58483 was used as a CRAC blocker. The inhibitory effect of YM-58483 was verified by imaging. Wild-type MSCs, m1-MSCs, m1-ko-MSCs, and YM-58483-treated MSCs were differentiated to adipocytes, osteocytes, or chondrocytes. Fatty acid-binding protein 4 was used as an adipocyte biomarker and osteocalcin was used for the detection of osteocytes. Aggrecan was used as a chondrogenic differential marker.

Results: The results sugges YM-58483 inhibited Ca²⁺ influx in MSCs in a dose-dependent manner, as demonstrated by Ca²⁺ imaging of fura-4-loaded cells. Downregulation of CRACM1 function in MSCs suppressed the differentiation of osteocytes, but not of adipocytes. A increase tendency on the potential of chondrogenic differentiation was observed in intracellular Ca²⁺-downregulated MSCs compared to wild-type MSCs. Overexpressing CRACM1 had no effect on the differentiation of osteocytes, but suppressed adipogenic and chondrogenic differentiation, while comparing with that observed in wild-type cells.

Conclusion: The results suggest that MSC differentiation potential is related to intercellular Ca²⁺ signaling pathways. In future study, we aim to research the systemic chondrocyte differentiation in vivo and, eventually, to carry out experiments in animal models to further contribute to the growing field of regenerative medicine.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/regulation-of-multiple-differentiation-potential-in-mesenchymal-stem-cells-via-an-intercellular-ca2-signaling-pathway/