Detailed reprogramming protocol

Induction of muscle-regenerative multipotent stem cells from human adipocytes by PDGF-AB and 5-azacytidine.

Work protocol: 

I. Primary adipocyte harvest and culture

1. Use 4-5 gm of freshly obtained human subcutaneous fat stored in normal saline supplemented with Penicillin/Streptomycin (P/S) antibiotics as starting material for adipocyte harvest. 
2. Rinse fat tissue specimen in PBS and then clean using scalpels and forceps to get rid of any blood, soft-connective or cauterized tissue and debris.
3. Mince the cleaned tissue with scalpels in 1-2 mL of 0.2% Collagenase I (to aid the digestion process).
4. Transfer the homogenized tissue to a 50 mL container and add 15 mL of 0.2% Collagenase I.
5. Incubate at 37˚C with shaking at 60 rpm for 40 minutes to facilitate enzyme-mediated digestion. During incubation, swirl the container intermittently to allow efficient exposure of the enzyme to majority of the tissue. 
6. Filter the digested tissue through a 40 µm strainer before inactivating with 1mL of 100% serum. 
7. The cell suspension is then spun at 300g for 5 min at 4˚C, which causes the suspension to separate into three distinct layers. Primary adipocytes are separated as the floating top layer, whereas the stromal vascular fraction (SVF) which contains the adipose derived MSCs (AdMSCs) pellets to the bottom, with buffer supernatant in between.
8. Seed the buoyant primary adipocytes in 35 mm dishes in adipocyte culture medium in a ceiling culture setup. This arrangement allows attachment of unilocular adipocytes to the cell culture dish surface over prolonged time in culture. Leave the dishes undisturbed for 8-10 days before subjecting the primary adipocytes to reprogramming.
9. Seed the SVF pellet obtained after spinning the homogenized cell-suspension in complete MSC medium in T25 flasks for culturing AdMSCs. AdMSCs are washed 2 days following seeding to deplete dead and non-adherent cells. Add fresh complete MSC medium every 3-4 days until the cells reach 70% confluence following which they can be passaged as per requirement at a density of 5000 cells per T25 flask.

II. Reprogramming of primary adipocytes into iMS cells

1. Use attached primary adipocytes for reprogramming.
2. Wash the cells thoroughly in PBS.
3. Culture adipocytes in reprogramming medium (complete MSC medium supplemented with 5µM AZA and 400ng/mL rhPDGF-AB) for 2 days.
4. Wash the cells thoroughly to get rid of AZA from the medium. Replace complete MSC medium supplemented with 400ng/mL rhPDGF-AB. Change medium every 3-4 days. During reprogramming, adipocytes tend to lose their fat droplets which are seen floating in the culture dish.
5. Culture cells in this medium for a total of 25 days to obtain reprogrammed iMS cells which exhibit a stromal cell-like morphology. 

III. In vitro expansion of reprogrammed iMS cells

1. After 25 days, detach the reprogrammed cells by trypsinization by adding 500uL of TrypLE per dish.
2. Wash cells with PBS thoroughly.
3. Seed cells at a density of 5000 cells per T25 flask and culture in complete MSC medium.
4. Change medium every 3-4 days.
5. Passage cells on reaching 70% confluence.
6. Re-seed as per requirement for downstream experiments.
    

Reagents required:

1. Normal saline supplemented with Penicillin/Streptomycin (P/S).
2. PBS.
3. 0.2% Collagenase I.
4. Serum (either Foetal calf serum/allogenic or autologous human serum as per experimental requirement)
5. Complete MSC culture medium (Alpha MEM + 20% serum + P/S).
6. Adipocyte culture medium (DMEM + 10% serum + P/S).
7. 5’- Azacitidine (AZA).
8. Recombinant human Platelet Derived Growth Factor – AB (rhPDGF-AB).
9. TrypLE enzyme.

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