Genetic Manipulation of Calcium Release-Activated Calcium Channel 1 Modulates the Multipotency of Human Cartilage-Derived Mesenchymal Stem Cells

Figure 1

Modulation of Ca²⁺ in CRAC-manipulated MSCs. The following experiments were conducted at 7 days after gene transfection of wild-type MSCs, pcDNA3.1-Orai1-transfected MSCs (M1-MSCs), and CRACM1-specific gRNA vector and linear EF1a-GFP-P2A-Puro donor-cotransfected MSCs (KOM1-MSCs). (a) PCR amplification of reverse transcription products produced the expected band following genetic modification. Molecular marker (lane 1); CARCM1 expression (523 bp) in MSCs, M1-MSCs, and KOM1-MSCs (lanes 3, 4, and 5, respectively); and GAPDH expression (214 bp) in MSCs, M1-MSCs, and KOM1-MSCs (lanes 7, 8, and 9, respectively) are shown. (b) CRACM1 mRNA expression in MSCs, M1-MSCs, and KOM1-MSCs (a.u. (arbitrary units); and ). Results are expressed as (). (c) The relative expression of CRACM1 to housekeeping GAPDH in MSCs, M1-MSCs, and KOM1-MSCs using quantitative real-time PCR. Relative fold of CRACM1 expression was achieved using the comparative Ct method (2^-ΔΔCt) ( and ). (d) Time sequential patterns of Ca²⁺ imaging in single MSCs, M1-MSCs, and KOM1-MSCs. The imaging period was 200 s without stimulation, followed by 500 s after stimulation. After a 200 s baseline measurement, cells were slowly perfused with TG (0.5 μM) and then perfused with a CaCl₂ solution (2 mM) at the 400 s time point (scale bar: 10 μm). (e) Typical Ca²⁺ influx patterns of MSCs, M1-MSCs, and KOM1-MSCs shown in (c). (f) Maximum increases in fluorescent intensity values of MSCs, M1-MSCs, and KOM1-MSCs. Quantification was performed using images acquired from 100–120 cells of each group (). Results are expressed as . (g) Initial rate of Ca²⁺ influx (in the first 15 s after Ca²⁺ addition) into MSCs, M1-MSCs, and KOM1-MSCs. Quantification was performed using images acquired from 100–120 cells of each group ( and ). Results are expressed as

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