Alp staining

To assess multilineage potential, cells were incubated under specific conditions to induce differentiation into osteocytes, chondrocytes, and adipocytes. After differentiation, the multilineage potential was evaluated as previously described [21 (link), 22 (link)]. Briefly, osteocyte formation was assessed by measuring the level of ALP staining (Sigma-Aldrich, St. Louis, MO, USA); chondrocyte formation was determined by safranin O staining (Sigma); adipocyte formation was assessed based on the staining of accumulated lipid vacuoles with oil red O (Sigma).

MC3T3 cells were seeded onto 24-wells plates with the density of 1 × 10⁴ cells/well. The period of culture medium renewal was 2 days. At the third and fifth day, cells were harvested, and then ALP staining (Sigma) was performed according to the manufacturer's protocol and observed via microscope (Nikon).

ALP staining (Sigma-Aldrich) was performed for 30 min at room temperature in the dark using the Vector Red Alkaline Phosphatase Substrate Kit I (Sigma-Aldrich), in accordance with the manufacturer’s protocol. For immunocytochemical staining, human iPSCs were fixed in 4% paraformaldehyde (USB) in phosphate-buffered saline (PBS) for 10 min at room temperature. After washing twice in PBS, the specimens were blocked with 1% BSA in PBS for 1 h at room temperature. The fixed specimens were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature. The fixed cells were stained with primary antibodies against SSEA-4 (1:100; Abcam, Cambridge, UK), OCT4 (1:100; Cell Signaling, Danvers, MA, USA), SOX2 (1:50; Cell Signaling), NANOG (1:100; Cell Signaling), ALP (1:100; Abcam), and GAPDH (1:10,000; Cell Signaling). The primary antibodies were detected with Alexa Fluor 488 (1:200; Invitrogen, Burlington, Canada) and Alexa Fluor 568 (1:200; Invitrogen) conjugated with secondary antibodies. Images were acquired using a fluorescence microscope (Axio Observer Z1; Carl Zeiss, Oberkochen, Germany).

Primary osteoblasts isolated from calvaria of neonatal Dicer^{gtRosaCreERT2} mice (postnatal day 3–5) by sequential digestions were cultivated as previously described38 (link). The cells were exposed to 1 μM 4-hydroxytamoxifen (4-OHT) (Sigma-Aldrich, St. Louis, USA) for 3 days and subsequently subjected to osteogenic induction medium (Alpha-MEM containing 10% fetal bovine serum and 1% penicillin/streptomycin, supplemented with 100 mg/ml sodium ascorbate and 5 mM beta-glycerol phosphate, Sigma-Aldrich, St. Louis, USA) with or without 1 μM Dexamethasone (Sigma-Aldrich, St. Louis, USA). Osteoblast differentiation was determined by ALP staining (Sigma-Aldrich, St. Louis, USA) and Alizarin Red staining (Sigma-Aldrich, St. Louis, USA) at indicated time points. Quantitative ALP staining was performed using ELF phosphatase detection kit (ATCC, UK), and quantified by using cellomics arrayscan V^TI automated microscope (Thermo-Fisher Scientific, Waltham, USA).
Embryonic bones were isolated at day E15.5 from Dicer^Runx2Cre mice and cultured for three days in osteogenic induction medium (as described for the calvaria derived osteoblasts) with or without 1 μM Dexamethasone (Sigma-Aldrich, St. Louis, USA).

Human amniotic fluid stem cells (hAFSCs)29 (link)30 (link)31 (link) were a gift from Shiaw-Min Hwang, PhD (Bioresource Collection and Research Center, Hsinchu, Taiwan). The primary hAFSCs were cultured in α-MEM (Sigma-Aldrich) supplemented with 20% FBS (Hyclone) and 4 ng/ml of bFGF (Peprotech), and then incubated at 37 °C in 5% CO₂. The hAFSCs at the 5th passage were grown to 70–90% confluence and shifted, for 14 and 28 days, to osteoblast differentiation medium (α-MEM supplemented with 10% FBS, 0.1 μM dexamethasone, 10 mM β-glycerol phosphate, 50 μM ascorbate) (Sigma-Aldrich) containing 200 ng/ml of IL-20 (R&D system), 2 μg/ml of 7E, or IL-20+7E. The culture medium was changed every 2 days for all differentiation experiments. Osteoblast differentiation was evaluated and confirmed using ALP staining (Sigma-Aldrich) 14 days and alizarin red S staining (Sigma-Aldrich) 28 days. ALP activity was measured using an ALP assay kit (ANASPCT) 14 days after the cells had been cultured.

After 24 h of seeding, 6-well plates were incubated with the TPF at concentrations of 0, 50, and 100 ng/ml for 48 h, followed by ALP staining (Sigma-Aldrich, St. Louis, MO) and ALP activity assay. For ALP staining, cells were rinsed twice with phosphate-buffered saline (PBS), fixed with 2% paraformaldehyde. ALP substrate mixture was then added and incubated for 15 min for color development. For the ALP activity assay, cell layers were scraped off culture plates in scraping buffer. Cell pellets were collected after a quick spin (14,000 rpm for 5 min). Cells were then lysed with lysis buffer and subjected to three freeze–thaw cycles. After centrifuge at 14,000 rpm for 5 min, supernatant was collected. Twenty millilitre supernatant from each sample was added to each well as duplicates in a 96-well plate and incubated with an assay mixture of p-nitrophenyl phosphate. Plates were then scanned for spectrophometric analysis using a plate reader (T60 U, PG Instruments Ltd., England) [22 (link), 23 (link)]. Absorbance was measured at 405 nm every 5 for 30 min. Activity was calculated of cells staining positive for ALP using an image analyzing system (KS 400; Carl Zeiss, Jean, Germany) were performed on culture day 6, as previously described [23 (link)].