Previous approaches relied on highly invasive methods for obtaining mesenchymal stem cells in vitro, but those mesenchymal stem cells had a limitation of producing heterogeneous pool of 30%with efficiency ranging from 30 to 60%Here, we present a protocol for producing high number of rapidly proliferating mesenchymal stem cells that can produce a pure population of mature adipocytes by sorting using Nile red. When the iPSCs have reached 80%confluence, wash the cells with DPBS and add dissociation medium containing EDTA. Incubate the cells at 37 degrees Celsius for one minute.
After one minute, aspirate the dissociation reagent and place the cells at 37 degrees Celsius for an additional one minute. Next, remove the plate from the incubator. Add one milliliter of StemFlex media per well and carefully collect the cells in a 15-milliliter conical tube.
Centrifuge the cells. Remove the supernatant, and gently resuspend them in three milliliters of MSC differentiation media containing 10 micromoles of a rock inhibitor. Mix the cell suspension and distribute 500 microliters per well in a 24-well ultra-low attachment plate.
Then place the plate in an incubator at 37 degrees Celsius. After 24 hours, to induce the attained embryonic bodies, collect them in a 15-milliliter tube and allow them to settle down. After 15 minutes, remove the supernatant and add MSC differentiation medium supplemented with 10 micromolar retinoic acid.
Resuspend the embryonic bodies gently and distribute 500 microliters per well in the same 24-well ultra-low attachment plate. Then place the plate in the incubator. After 48 hours, collect the embryonic bodies in a 15-milliliter tube and allow them to settle down for 15 minutes.
Then remove the supernatant and add MSC differentiation medium supplemented with 0.1 micromolar retinoic acid. After resuspending the embryonic bodies, distribute 500 microliters per well in the same 24-well plate and place the plate in the incubator. Forty-eight hours after the last retinoic acid treatment, collect the embryonic bodies, remove the supernatant, and add DMEM low-glucose medium without cytokines.
Gently resuspend and distribute 500 microliters of the cell suspension per well in the same 24-well ultra-low attachment plate, then incubate the plate at 37 degrees Celsius. After 48 hours, to plate the iPSC-derived embryonic bodies, collect the embryonic bodies in a 15-milliliter tube, allow them to settle, then remove the supernatant, and resuspend in two milliliters of fresh MSC differentiation medium. Next, transfer the suspension to two wells of a basement membrane matrix-coated six-well plate and incubate the cells at 37 degrees Celsius.
After five days, replace the spent media with a fresh MSC differentiation medium containing 2.5 nanograms per milliliter of basic fibroblast growth factor and continue the MCS differentiation for up to 11 and 15 days. When the plated embryonic bodies have reached 80 to 90%confluence, passage them by washing with DPBS, adding Trypsin-EDTA, and incubating at 37 degrees Celsius for one minute. Remove the Trypsin-EDTA from the plate and incubate again at 37 degrees Celsius for one minute.
Next, add one milliliter media per well. After three minutes, collect the cells using MSC differentiation media in a 15-milliliter conical tube and centrifuge the cells. Then resuspend the cells in an MSC differentiation medium containing basic fibroblast growth factor and plate the cells on basement membrane matrix-coated plates at a ratio of one to three.
After the MSCs have reached 90%confluence, continue culturing them for another 48 hours to allow them to undergo a period of growth arrest. Then remove the medium and wash the cells with DBPS. After the wash, add complete adipocyte differentiation medium to the plate and incubate the cells at 37 degrees Celsius.
Change the medium every other day for 14 days. Differentiate iPSC-derived MSCs into adipocytes. To sort the adipocytes using Nile red, prepare Nile red working solution in DMSO as described in the text manuscript.
Before use, thaw the stock solution and reconstitute it in DPBS to attain a 300-nanomolar working solution concentration. On or after day 14 of adipocyte differentiation, discard the medium from the cells and wash using DPBS. Then add Nile red working solution.
Cover the plate and incubate the cells at 37 degrees Celsius. After 15 minutes, replace the Nile red solution with Trypsin-EDTA and incubate the cells at 37 degrees Celsius for four minutes. Then collect the cells using DMEM containing 5%FBS in a 15-milliliter conical tube and centrifuge the cells.
Remove the supernatant and resuspend the cells in DPBS at a density of 1 million cells per milliliter. Then using the FACS sorter, isolate the Nile red-positive cells using the FL-1 channel. Collect the sorted cells and reculture them in adipocyte differentiation media or proceed to RNA extraction followed by quantitative analysis of adipocyte differentiation markers.
Upon initiating differentiation, cells plated in suspension are round with defined cell borders and are small to medium size in diameter. The viability of embryonic bodies is observed by the rapid proliferation behavior giving rise to more MSCs. This rapid proliferation behavior, along with their peculiar and elongated morphology, is retained even after passaging the MSCs onto fresh matrix-coated plates.
Good differentiation produces reliable MSCs with greater than 90%expression efficiency of mesenchymal surface markers CD73, CD44, and CD90. In addition, assessment of cells for the absence of surface markers depicting the hematopoietic phenotype CD14, CD34, and CD19 showed less than 1%expression efficiency, High expression in cytoplasmic distribution of FABP4, a marker for terminally-differentiated adipocytes, indicates their developmental maturity. Additionally, high expression of adiponectin, another marker of adipocyte maturity, indicates that the adipocytes are functional enough to undergo lipid storage and adipogenesis in response to glucose signaling.
Upon staining, Nile red exclusively binds to the lipid-bearing mature adipocytes, making it an effective tool for sorting mature adipocytes using fluorescent-activated flow cytometry. The Nile red-positive cells show a significant upregulation of maturation markers by at least two-folds compared to unsorted cells. You have to be very gentle while collecting cells during embryonic body formation and mesenchymal cell dissociation as that would highly impact the survival efficiency post-dissociation of mesenchymal stem cells.
Pure population of adipocytes attained from mutation-carrying patients can be subjected to functional and the whole transcriptome sequencing to determine regulatory and signaling pathways affected by particular mutation without the data being affected by sample heterogeneity. This technique will allow scalable generation of mesenchymal stem cells in vitro, removing the need of harvesting them from human donors to readily differentiate them to adipocytes, chondrocytes, and osteocytes for understanding tissue-related disease pathogenesis.
