Yusaku Kodaka1,2, 3, Yoko Asakura1,2, 3, and Atsushi Asakura1,2, 3

1Stem Cell Institute2Paul and Sheila Wellstone Muscular Dystrophy Center3Department of Neurology, University of Minnesota Medical School, Minneapolis, MN

BioTechniques, Vol. 63, No. 2, August 2017, pp. 7276

Supplementary Material

Abstract

Viral vectormediated foreign gene expression in cultured cells has been extensively used in stem cell studies to explore gene function. However, it is difficult to obtain high-quality stem cells and primary cells after viral vector infection. Here, we describe a new protocol for high-efficiency retroviral infection of primary muscle stem cell (satellite cell) cultures. We compared multiple commercially available transfection reagents to determine which was optimal for retroviral infections of primary myoblasts. Centrifugation force was also tested, and a spin infection protocol with centrifugation at 2800 g for 90 min had the highest infection efficiency for primary myoblasts. We confirmed that infected muscle stem cells maintain cell proliferation and the capacity for in vitro and in vivo myogenic differentiation. Our new, efficient retroviral infection protocol for muscle stem cells can be applied to molecular biology experiments as well as translational studies.

Skeletal muscle regeneration is mediated by muscle stem cells called satellite cells (1), which are normally mitotically quiescent in adult muscle. After muscle injury or exercise, quiescent satellite cells undergo activation, followed by proliferation. Proliferating satellite cells, which are myogenic precursor cells, eventually exit the cell cycle and fuse with each other to form multinucleated myotubes. Isolated satellite cells from skeletal muscle can be cultured in vitro as satellite cellderived primary myoblasts (2,3). These primary myoblasts are used for in vitro models of skeletal muscle cell differentiation, self-renewal of satellite cells (4), in vivo satellite cell transplantation (5), and multi-lineage differentiation (6). As opposed to immortalized myoblast cell lines such as C2C12 cells, animal or human primary myoblasts can be utilized for cell transplantation as well as studies of stem cell biology (4,7).

METHOD SUMMARY

Here, we compared multiple commercially available transfection reagents with different infection protocols and determined that a spin infection protocol with centrifugation had the highest infection efficiency for primary myoblasts. The infected cells continued to proliferate and retained the capacity for in vitro and in vivo myogenic differentiation.

One drawback of primary myoblasts is that they need more complex culture conditions to maintain their proliferation and differentiation abilities. The use of high serum conditions for cell growth is an example of this. Furthermore, the efficiency of DNA transfection and viral infection for primary myoblasts is lower than for C2C12 cells (8,9). Retroviral or lentiviral infection has been used for obtaining stable foreign gene expression that enables long-term experiments, including in vivo cell transplantation of myogenic cells (2,10-12). However, the viral supernatant normally contains low levels of nutrients and growth factors, which inevitably induces cell cycle exit followed by myogenic differentiation. Therefore, a method for high-efficiency viral infection without the need for culturing with the viral supernatant is critical for maintaining the ability of primary myoblasts to proliferate and differentiate (13).

For efficient retroviral infection, a spin infection protocol has been established for several cell types, including hematopoietic progenitor cells (14-17). To adapt the spin infection method to primary myoblasts, we identified optimal conditions for both transfection reagents and centrifugation time and force.

All animal experimental protocols were approved by Institutional Animal Care and the Use Committee of the University of Minnesota. Satellite cellderived primary myoblasts such as CD31(-), CD45(-), Sca-1(-), and integrin 7(+) cells were isolated from skeletal muscles of 2 month-old mice (C57BL6, Charles River Laboratories, Wilmington, MA) by MACS separation (Miltenyi Biotec, San Diego, CA) as described previously (3). Myoblasts were maintained on collagen-coated dishes in growth medium (GM) [Hams/F10 (Sigma- Aldrich, St., Louis, MO), 20% FBS, 20 ng/ mL basic FGF (R&D Systems, Minneapolis, MN), and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA)] (7). Proliferating myoblasts in GM were defined as Day 0. Myogenic differentiation was induced by replacing GM with differentiation medium (DM) [DMEM (Sigma-Aldrich), 5% horse serum, and 1% penicillin/streptomycin] for 3 days.

Retroviral supernatants were produced by transfection of pMX-GFP (Cell Biolabs, San Diego, CA) or a pMX-mCherry retroviral vector into a 293T Platinum-E Retroviral Packaging Cell Line (Plat-E) (Cell Biolabs). One day before transfection, Plat-E cells were cultured in DMEM with 10% FBS and without antibiotics until they reached 70%90% confluency. Various transfection reagents were used: Lipofectamine (Thermo Fisher Scientific, Waltham, MA), Lipofectamine 2000 (Thermo Fisher Scientific), Lipofectamine LTX (Thermo Fisher Scientific), TransIT-293 (Mirus Bio LLC, Madison, WI), TransIT-2020 (Mirus Bio LLC), TransIT-LT1 (Mirus Bio LLC), PolyJet (SignaGen Laboratories, Rockville, MD), and LipoJet (SignaGen Laboratories). Five microliters of each transfection reagent was suspended in 200 l DMEM without FBS and with 5 g of pMX-GFP or pMX-mCherry plasmid DNA for 20 min at room temperature (RT). PlatE cells (6 105) were plated on collagen-coated 3 cm dishes 1 day before transfection. The next day, the medium was replaced with 800 ml DMEM with 10% FBS and 200 ml DMEM using the transfection complex described above. After incubation for 24 h, the medium was changed to 1 mL new DMEM with 10% FBS. Retroviral supernatants were then harvested 24 h after the medium change. Syringe filters (0.45 mm) (Millipore Sigma, Billerica, MA) were used to remove any cells from the retroviral supernatants. Primary myoblasts (1 105) were plated on collagen-coated 3 cm dishes for 24 h before the viral plating infection. Retroviral supernatants were used for viral infection of primary myoblasts with 10 g/mL polybrene (Millipore Sigma) for 4 h, and cells were then cultured in GM for 48 h. For the spin infection, myoblasts were treated with 0.25% trypsin-EDTA (Thermo Fisher Scientific), and 1 105 myoblasts were then transferred into 1.5 mL microcentrifuge tubes. The cells were centrifuged and then resuspended with the retroviral supernatant with 10 g/ mL polybrene. After the myoblast spin infection was performed at RT under appropriate centrifugation conditions, the cell pellets were resuspended with GM and plated on collagen-coated 3 cm dishes. GFP expression was examined 2 days after spin infection. Dead cells were counted by trypan blue (Thermo Fisher Scientific) staining. 5-ethynyl-2-deoxyuridine (EdU) was added to culture plates 3 h before fixation of the cells. EdU staining was performed using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific). Immunostaining was performed with anti-GFP (AB3080; Millipore Sigma; RRID:AB_91337), anti-myosin heavy chain (MHC) (MF 20; Developmental Study Hybridoma Bank, Iowa City, IA; RRID:AB_2147781), anti-myogenin antibody (F5D; Developmental Study Hybridoma Bank; RRID:AB_2146602), or anti-phospho-histone H3 antibody (pHisH3) (D2C8; Cell Signaling, Danvers, MA; RRID:AB_10694226), followed by Alexa 488-conjugated anti-rabbit IgG (A-21206; Thermo Fisher Scientific; RRID:AB_2535792) and Alexa 568-conjugated anti-mouse IgG (A10037; Thermo Fisher Scientific; RRID:AB_2534013) or Alexa 488-conjugated anti-mouse IgG (A-21202; Thermo Fisher Scientific; RRID:AB_141607). DAPI (Sigma-Aldrich) was used for counterstaining of nuclei.

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