This Li-Fraumeni syndrome iPSC-based osteosarcoma model can help answer key questions in the osteosarcoma research field about using patient iP cells in cancer modeling. The main advantage of this model is that it allows the generation unlimited cells at all stages of osteosarcoma development for mechanistic studies, biomarker identification, and drug screening. Ruoji will be demonstrating the cell culture procedure and I will be demonstrating the animal procedure.
After two weeks of mouse embryonic fibroblast, or MEF, co-culture wash the 80 to 90%confluent iPS cell culture with five milliliters of DPBS followed by the addition of one milliliter of cell detachment solution to the cells for a three minute incubation at room temperature. It is critical to maintain iPS cells in good conditions on MEFs for at least 14 days for the cells to be able to attach to a gelatin-coated plate for their differentiation into mesenchymal stem cells. When the cells have detached from the plate bottom add nine milliliters of MEF/MSC culture medium to the cells to neutralize the cell detachment solution and transfer the cell suspension into an uncoated 100 millimeter tissue culture plate.
Incubate the cells at room temperature for 30 minutes to allow the MEFs to attach to the plate and carefully collect the unattached iPS cell-containing supernatant. Centrifuge the iPS cell solution and resuspend the pellet in six milliliters of MSE differentiation medium. Then seed two milliliters of cells into each of three 0.1%gelatin-coated wells of a six-well culture plate and place the plate in the cell culture incubator for 28 days feeding the cells with fresh medium every two days to remove any unattached or dead cells.
On day 28 large masses of differentiated cells will be visible with fibroblast-like MSCs observed at the edges of the clusters. Wash the cell cultures with one milliliter of DPBS per well followed by their detachment with 0.5 milliliters of 0.25%Trypsin-EDTA per well at room temperature. After three minutes neutralize the Tyrypsin with 1.5 milliliters of MEF/MSE culture medium per well and pool the detached MSEs into a single 15 milliliter conical tube for their centrifugation.
Resuspend the pellet in three milliliters of MEF/MSE culture medium and seed the cells onto a 0.1%gelatin-coated 60 millimeter plate, changing the medium every two days until the cells reach 100%confluence. After splitting the 100%confluent culture at a one to three ratio the MSCs will proliferate quickly demonstrating a fibroblast-like morphology and formi&ng a swirl-like culture pattern when the culture reaches a high density. The iPS cell-derived MSCs can also be examined by a immunofluorescent staining for mesenchymal stem cell markers at any point during their differentiation.
To differentiate the MSE into osteoblasts plate the appropriate number of MSE from a 95%confluent cell culture onto a cell culture plate in the appropriate volume of MEF/MSC culture medium for 24 hours. Then begin replacing the supernatant with the appropriate volume of osteoblast differentiation medium the next day and every two days after until the end of the culture period. To detect the bone-associated alkaline phosphatase produced by preosteoblasts and the mineral deposition produced by mature osteoblasts perform alkaline phosphatase and Alizarin Red S staining, respectively, according to the manufacturer's protocol at the appropriate experimental time points.
To set up an in vivo tumorigenesis model with the differentiated osteoblasts seed the appropriate number of MSC into five 100 millimeter dishes to start osteoblastic differentiation. On day 14 of osteoblastic differentiation wash five culture plates of osteoblasts with five milliliters of DPBS and treat the cells with 1.5 milliliters of osteoblast detachment solution per dish. After a 30 minute incubation at 37 degrees celsius confirm detachment of the cells by light microscopy and neutralize the detachment solution with fresh osteoblast differentiation medium.
Pool the cells in a 50 milliliter conical tube for their centrifugation and resuspend the pellet in 25 milliliters of fresh osteoblast differentiation medium. Filter the cell solution through 70 micron strainer to remove any aggregated cells prior to counting and set aside 1 X 10 to the 7 cells per subcutaneous injection. Pellet the cells by centrifugation and reconstitute the cells at a 1 X 10 to the 7 osteoblasts per 50 microliters of ice-cold DPBS concentration.
Mix the osteoblasts with an equal volume of phenol-red free basement membrane matrix and place the cells on ice until their injection. After confirming a lack of response to toe pinch in an eight-week old female immuno-compromised nude mouse load the cells into a one milliliter syringe equipped with the 27 gauge needle and subcutaneously inject 100 microliters of the cells into the disinfected flank of the animal. Then monitor the mice with weekly palpitations at the injection site for the growth of subcutaneous nodular densities.
Tumor formation should be observed between six to 10 weeks after injection. Established LFS iPS cell clones should exhibit a typical human embryonic stem cell morphology and demonstrate a positive alkaline phosphatase activity. LFS iPS cells also highly express pluripotency factor mRNAs comparable to human embryonic stem cell lines and much higher than levels observed in parental fibroblasts.
Successfully differentiated LFS MSCs also express typical MSC markers including CD44, CD73, CD105 and CD166 as assessed by immunofluoresent staining. When the MSCs are subjected to osteogenic differentiation signals the differentiated cells begin to exhibit positive alkaline phosphatase activity by day nine and mineral deposition by day 21. The differentiating osteoblasts also demonstrate an upregulated expression of pre-osteoblast and mature osteoblast genes throughout their differentiation process.
Six to 10 weeks after osteoblast injection LFS osteoblast derived tumors demonstrate immature osteoblast characteristics including positive alkaline phosphatase activity, positive collagen matrix deposition, and negative mineralization. After watching this video you should have a good understanding of how to differentiate Li-Fraumeni Syndrome iPS Cells into osteoblasts and to generate an in vivo osteosarcoma model using patient-derived osteoblasts. In addition to osteosarcoma this Li-Fraumeni Syndrome iPS cell-based disease model can be also applied to other Li-Fraumeni Syndrome related malignancies such as soft tissue sarcoma, breast cancer, and brain tumors.
