Abstract
Background The current treatment for postoperative hypoparathyroidism has shortcomings, such as repeated blood monitoring for dosage adjustment, uncertain long-term efficacy, and the high price of recombinant parathyroid hormone therapy. Adipose-derived stem cells can undergo adipogenic and osteogenic differentiation in vitro and are considered a novel source of parathyroid-like cells, but the idea lacks theoretical basis and feasibility. We aimed at establishing a protocol for differentiating adipose-derived stem cells into parathyroid-like cells for treating hypoparathyroidism. Materials/Methods Adipose-derived stem cells were isolated and purified from the inguinal adipose tissue of Sprague Dawley rats. Adipogenic differentiation and osteogenic differentiation of the cells were identified by oil red O and alizarin red S staining, respectively. The adipose-derived stem cells were stimulated by sonic hedgehog (SHH) and activin A. The differentiation of the adipose-derived stem cells to parathyroid-like cells was confirmed by the detection of parathyroid hormone and the related parathyroid markers. Results Adipose-derived stem cells were successfully isolated and purified from the rat adipocytes. The adipogenic and osteogenic differentiation capabilities of the adipose-derived stem cells were determined. SHH and activin A stimulated parathyroid hormone secretion by the adipose-derived stem cells and significantly increased the expression of calcium-sensing receptor (CaSR), parathyroid hormone, and glial cells missing homolog 2 (GCM2) in the cells in a time- and concentration-dependent manner. Conclusion We successfully differentiated rat adipose-derived stem cells into parathyroid-like cells, which will pave a new route to curing hypoparathyroidism.
Highlights
Background. e current treatment for postoperative hypoparathyroidism has shortcomings, such as repeated blood monitoring for dosage adjustment, uncertain long-term efficacy, and the high price of recombinant parathyroid hormone therapy
Adipogenic differentiation and osteogenic differentiation of the cells were identified by oil red O and alizarin red S staining, respectively. e adipose-derived stem cells were stimulated by sonic hedgehog (SHH) and activin A. e differentiation of the adipose-derived stem cells to parathyroid-like cells was confirmed by the detection of parathyroid hormone and the related parathyroid markers
After incubation with lipid inducer for 7 days (Figure 1(a)), 14 days (Figure 1(b)), and 21 days (Figure 1(c)), the rat adipose-derived stem cells (ADSCs) were stained with oil red O, and the lipid droplets were observed under an inverted microscope. e number of lipid droplets in the cells increased gradually with time, proving that the lipid induction of ADSCs was successful
Summary
E cells were cultured in low-glucose DMEM containing 10% FBS, 0.1 μmol/L dexamethasone (Sigma, St. Louis, MO), 50 μmol/L ascorbic acid (Sigma, St. Louis, MO), 10 mmol/L β-glycerophosphate (Sigma, St. Louis, MO), and 0.01 μmol/ L vitamin D3 (Sigma, St. Louis, MO). Total RNA was extracted from cells that had been collected and lysed in TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. E cells were cultured in low-glucose DMEM containing 10% FBS, 1 μmol/L dexamethasone (Sigma, St. Louis, MO), 2.7. Western blotting was performed to quantify CaSR, GCM2, and PTH protein levels. E cultured cells were washed with ice-cold PBS and lysed in lysis buffer containing protease inhibitor cocktail (KGP 250 kit, Keygen Biotech, Nanjing, China) for 30 min on ice. Protein levels were quantified using the bicinchoninic acid (BCA) method. A difference was considered significant when p < 0.05
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