Human basic fibroblast growth factor

Liposuction was obtained from a healthy adult donor, under Institutional Review Board (IRB) approval (protocol number IRB00119905) and a waiver informed consent. Liposuction was stored at 4°C and processed within 48 hours. ASCs were obtained according to the previously published method.⁹, ¹⁴, ²⁷, ²⁸ Equal volume phosphate‐buffered saline (PBS) was used to wash the lipoaspirate. Washed liposuction was digested at 37°C for 60 minutes with 1 mg/mL collagenase II in Dulbecco modified Eagle medium (DMEM) containing 3.5% bovine serum albumin (Sigma‐Aldrich, St. Louis, Missouri) under agitation. After centrifugation, supernatants containing adipocytes were removed. Meanwhile, the cell pellet was resuspended and incubated in red blood cell lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM ethylenediaminetetraacetic acid [EDTA]) at room temperature (RT) for 10 minutes. Next, after centrifugation, cells were resuspended with PBS and filtered at 40 μm. Cells were cultured at 37°C in a humidified atmosphere containing 95% air and 5% CO₂ and with the standard growth medium consisted of DMEM (Gibco, Grand Island, New York), 10% fetal bovine serum (FBS) (Gibco), 1% penicillin/streptomycin (Gibco), and 2 mg/mL human basic fibroblast growth factor (R&D System, Minneapolis, Minnesota).

HCC cells were cultured in 6-well ultra-low attachment surface dishes (Corning) at the cell density of 2,000 cells/well with stem-cell medium, serum-free DMEM/F12 (Life Technologies) supplemented with N-2 supplement (Life Technologies), 10 ng/mL recombinant human epithelial growth factor (R&D Systems), 10 ng/mL human basic fibroblast growth factor (R&D Systems). Two weeks after cell seeding, sphere formation was observed under a light microscope. A cell cluster with the diameter longer than 100 µm was regarded as a sphere.

Sphere culture was performed as described previously. [23 ] Briefly, 10³ cells were plated, and the number of spheres in an area of 100 μm in diameter was counted on day 14. The sphere culture medium was serum-free DMEM/F12 (Life Technologies) supplemented with N-2 supplement (Wako, Osaka, Japan), 20 mg/ml recombinant human epithelial growth factor, 10 mg/ml human basic fibroblast growth factor (R & D Systems), 4 μg/ml heparin (AY pharma, Tokyo, JAPAN) and 1% penicillin and streptomycin.

Bone-marrow MSCs (BM-MSC) were isolated from the right leg of SAMP8 and SAMR1 mice. Femur and tibia were dissected, and diaphyses were flushed with PBS. Cell suspensions were filtered and plated at a concentration of 1x10⁶ cells/cm² in DMEM medium supplemented with 10% FBS (Hyclone, Thermo Fisher Scientific), 2mM glutamine, 100U/mL penicillin, 100mg/mL streptomycin (Lonza, Levallois-Perret, France) and 2ng/mL human basic fibroblast growth factor (R&D Systems, Lille, France). Medium was changed daily to remove red blood cells and non-adherent cells. BM-MSCs were stored at -80°C after passage 1.

NSI mice were maintained under specific pathogen-free conditions. Bilateral tibialis anterior (TA) muscles of 6- to 8-month-old mice were locally pretreated with cardiotoxin (10 μL, 10 μM/50 μL, Sigma-Aldrich, St. Louis, MO, USA) 24 h prior to transplantation to stimulate the migration of engrafted cells and the formation of new myofibers. After the mice were anesthetized by abdominal injection of 5% chloral hydrate, the TA muscle was exposed, and 1 × 10⁶ DMD–MDSCs resuspended in 10 μL Matrigel (Corning Inc., Corning, NY, USA) supplemented with 0.1 μg human basic fibroblast growth factor (R&D Systems, Minneapolis, MN, USA) were slowly injected at an oblique angle into five to seven sites in the muscle by using a 10 μL 33G microsyringe (Hamilton Co., Reno, NV, USA). The skin was then sutured closed. NSI mice injected with Matrigel served as the negative control. TA muscles were harvested 30 days post transplantation for immunohistochemical analysis.

The procedure has been previously described [53 (link)]. Briefly, bone marrow from tibias and femurs of C57BL/6 mice were collected. Cell suspensions were seeded at a concentration of 10⁶ cells/cm² in modified minimum essential Eagle's medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, Thermo Fisher Scientific), 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (Lonza, Levallois-Perret, France) and 2 ng/ml human basic fibroblast growth factor (bFGF) (R&D Systems, Lille, France). MSCs were CD29⁺, Sca1⁺, CD73⁺. Functionally, MSCs had the capacity to differentiate into adipocytes, chondrocytes and osteoblasts when cultured under specific and appropriated conditions [53 (link)].