Glutamine

hBMSCs were purchased from Procell Life Science & Technology (CP-H166, Wuhan, China) and Cyagen Biosciences (HUXMA-01001, Guangzhou, China) and cultured in MSC medium (Cyagen, China) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and streptomycin, 1% glutamine (Cyagen, China) in humidified air of 5% CO₂ at 37°C. The purchased cells were accompanied by quality reports, including flow cytometry identification, which revealed that the hBMSCs were positive for CD29, CD44, CD73, and CD105, and negative for CD34, CD11b, and CD45. The purchased hBMSCs could differentiate into osteoblasts, adipocytes, and chondrocytes under specific inductive conditions. When cells reached 80-90% confluence, subculture was performed at a ratio of 1:2 or 1:3, and the medium was replaced every 2 days. After being cultured to P2-P4, the cells were plated in a 6-well plate at a density of about 1×10⁵ cells/well. When cell confluence reached roughly 70%, hBMSCs osteogenic induction medium (Cyagen, China) containing dexamethasone, vitamin C, and β-sodium glycerophosphate was added, and was changed every 3 days.

BMSCs in passage 2 were replated in the standard medium at 1×10⁵ cells/cm² in 6-well plates. Cells were incubated at 37°C in a 5% CO₂ humidified incubator. After the cells were 100% confluent, the standard medium was carefully aspirated off, and 2 ml Adipogenic Differentiation Medium A (induction medium) containing 10% FBS, 20 µg/ml insulin, 10 µM IBMX, 10 µM rosiglitazone, 10 µM dexamethasone, 1% penicillin/streptomycin, and 1% glutamine were added (Cyagen). Three days later, the Adipogenic Differentiation Medium A was replaced with Adipogenic Differentiation Medium B (maintenance medium), containing 10% FBS, 20 µg/ml insulin, 1% penicillin/streptomycin and 1% glutamine (Cyagen). Then, 24 h later, the medium was changed back to induction medium. To optimally differentiate BMSCs into adipogenic cells, the cycle of induction and maintenance was repeated three times. After three cycles, the cells were cultured in maintenance medium for an additional 7 days by replacing the medium every 3 days. After differentiation, Adipogenic Differentiation Medium was removed and the cells were rinsed twice with 0.1 M DPBS. Cells were fixed with 4% formaldehyde solution for 30 min. Then the cells were rinsed twice with 0.1 M DPBS and stained with oil red O (Cyagen) for 30 min at room temperature (RT). Cells were visualized under a light microscope and images were captured.

OriCell Wistar rat MSCs (Cat.^# RAWMX-01001, Cyagen, Santa Clara, CA, USA), derived from bone marrow of Wistar rats, were cultured in 25 cm² tissue culture flasks (Corning, Corning, NY, USA) under 37 ºC in a 5% CO₂ humidified incubator. The BM-MSCs were maintained in the OriCell MSC growth media (Cat.^# GUXMX-90011, Cyagen, Santa Clara, CA, USA), consisting 88% OriCell MSC basal media, 10% MSC-qualified fetal bovine serum (Cyagen, Santa Clara, CA, USA), 1% penicillin-streptomycin (Cyagen, Santa Clara, CA, USA), and 1% Glutamine (Cyagen, Santa Clara, CA, USA). The cells were passaged at 1:3 upon 90% confluency. The BM-MSCs at passage 3 with optimal stability and multi-differentiation capability were used for lentivirus transduction.

First, MSCs were grown in complete medium for adipogenic differentiation. When the cells reached 80% confluence, the medium was changed to adipogenic medium that consisted of rosiglitazone, 3-isobutyl-1-methylxanthin (IBMX), glutamine, dexamethasone and insulin (Cyagen Biosciences, Suzhou, China). Osteogenic differentiation was induced in MSCs by DMEM containing dexamethasone, ascorbic acids and β-glycerophosphate (Cyagen Biosciences, Suzhou, China). For Oil Red O staining, MSCs were fixed with 4% formaldehyde. After being washed with PBS, the cells were incubated with Oil Red O solution (Cyagen Biosciences Suzhou, China) for 30 min. The cells were then washed and examined under a microscope. For Alizarin Red S staining, the cells were incubated with Alizarin Red Solution (Cyagen Biosciences, Suzhou, China) for 30 min. Images were acquired by a microscope (Supplementary Figure S1B).

BMSCs were harvested from 6- to 8-week-old mice as described previously [27] (link). Mice were euthanized and both femurs and tibiae were aseptically removed. Then the ends of the femurs and tibiae were cut and the bone marrow was flushed out with 5 ml C57B/6 Mouse Mesenchymal Stem Cell Growth Medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% glutamine (Cyagen, San Francisco, CA, U.S.), here called standard medium. The cells were cultured in standard medium at 37°C in a 5% CO₂ humidified incubator. BMSCs were allowed to adhere to the plastic support for 24 h before the first medium change. Nonadherent cells were removed by flushing with 0.1 M DPBS and the standard medium was replaced every 3 days. Cells in passage 2 were used for the experiments. For Wnt stimulation, cells were cultured in standard medium with 100 ng/ml recombinant mouse protein Wnt-3a (R&D system Inc., Minneapolis, MN, U.S.).

Human BMSCs were inoculated at 2.5–4 × 10⁴/cm² in 24-well plates. When the culture reached 100% confluence, the medium was changed to the Human Mesenchymal Stem Cell Osteogenic Differentiation Basal Medium complemented with 10% FBS, 1% Penicillin-streptomycin, 1% Glutamine, 0.2 mM ascorbate, 10 mM β-glycerophosphate, and 0.1 μM dexamethasone (all from Cyagen Biosciences Inc.). Media were changed every two days and the cells were cultured for 14–21 days. The calcium accumulation was assessed using alizarin red sulfate (AR-S; Cyagen Biosciences Inc.) staining and quantified as described previously [12 (link)].

Briefly, human BMSCs (passage 1) were purchased from 307-Ivy Translation Medicine Center (Beijing, China) and grown in hMSC basal media (HUXMA-90011, Cyagen, USA) supplemented with 10% fetal bovine serum (Cyagen, USA) and 1% penicillin-streptomycin (Cyagen, USA) and glutamine (Cyagen, USA) at 37°C with 5% CO₂ and saturated humidity. We used 0.25% trypsin without EDTA (Solarbio Science & Technology Co. Ltd., Beijing, China) to digest cells when the cells had grown to a confluence of 70–80% in the flask (431464U, Corning, USA). The media were changed every two days, and cells at passage 3–5 were used for the following experiments. The BMSC phenotypes are identified (S Figure 2).