In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

The induction of somite derivatives from induced pluripotent stem cells, or iPSC, is a critical step for using pluripotent stem cells for regenerative therapy or disease research applications. This technique can be used to differentiate human iPS cells into somite derivatives such as myotome, dermatome, sclerotome, and syndetome cells under chemically defined conditions. Using iPSC established from a patient suffering from an intractable musculoskeletal disorder, this method can be used for modeling the phenotype of the disease and for testing the new drug therapies.

This method can also be applied to furthering our understanding of the biology and the mechanisms underlying the development of paraxial mesoderm during human embryogenesis. Four days prior to starting presomitic mesoderm induction, coat a 10-centimeter dish with four milliliters of extracellular matrix solution for an overnight incubation at four degrees Celsius. The next morning, replace the extracellular matrix solution with eight milliliters of feeder-free cell culture medium.

To begin feeder-free culturing, wash the human iPSC culture with PBS, and add one milliliter of CTK solution to the culture dish. When the cells begin to detach from the dish bottom, wash the culture two times with fresh PBS to fully remove all of the SNL feeder cells before adding one milliliter of feeder-free cell culture medium to the dish. Use a cell scraper to manually detach the stem cells, and transfer the cells to a 15-milliliter conical tube.

Gently pipette the cell solution three times with a 1, 000-microliter pipette tip, and transfer the cells to the extracellular matrix-coated culture dish. Then, return the cells to the cell culture incubator for three days. At the end of the incubation, replace the feeder-free cell culture medium with eight milliliters of presomitic mesoderm induction medium, and initiate presomitic mesoderm differentiation in the cell culture incubator for the next four days, changing the medium on day three.

At the end of the incubation, isolate the delta-like protein 1 positive cells by flow cytometry, and collect the sorted cells by centrifugation. Resuspend the pellet in one milliliter of somitic mesoderm induction medium for counting, and seed one times 10 to the fifth cells into each well of an extracellular matrix solution-coated 12-well plate containing one milliliter of somitic mesoderm induction medium supplemented with 10 micromolar of the rho kinase, or ROCK, inhibitor Y-27632. Then, return the cells to the cell culture incubator for four more days, changing the medium not containing the inhibitor on days one and three of culture.

For syndetome differentiation from sclerotome cells, wash the sclerotome cells with PBS before detaching the cells with 0.2 milliliters of cell dissociation reagent per well. After three minutes at room temperature, add 0.8 milliliters of chemically defined basal medium to each well, and detach the cells with a cell scraper. Transfer the cells to a 15-milliliter conical tube for centrifugation, and resuspend the pellet in one milliliter of syndetome induction medium one for counting.

Seed five times 10 to the fourth cells into each well of an extracellular matrix solution-coated 24-well plate containing one milliliter of syndetome induction medium one, and return the plate to the cell culture incubator for three days. On day three of syndetome induction, replace the medium with syndetome induction medium two, and return the plate to the incubator for 18 days, changing the medium every three days. After presomitic mesoderm differentiation, over 85%of the cells are positive for delta-like protein 1, a marker of presomitic mesoderm, but negative for PAX3, a marker of somitic mesoderm.

This population becomes PAX3 positive somitic mesoderm cells after four days of somitic mesoderm induction. Further, staining of cadherin-11, a marker of epithelialized somitic mesoderm, only accumulates at the cell-to-cell junctions, following the addition of CHIR99021. Differentiation toward dermomyotome, myotome, dermatome, sclerotome, and syndetome can be assessed by immunocytochemistry and PAX3 fluorescence.

Similar to human dermal fibroblasts, iPSC-derived dermatome cells produce collagen and hyaluronic acid as assessed by ELISA. To demonstrate the comparable reactivity of human iPSC-derived syndetome and human adult tenocytes, a mechanical stretch stimulation assay can be performed. Before iPSC passaging, confirm that the culture confluency is between 70 to 80%and the split cells at one-to-two to one-to-four ratio accordingly.

This passaging is critical for successful iPSCs differentiation. For future cell-based therapies using this method, it is necessary to establish a novel germ-free condition that does not include any components derived from non-human animals.