Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Cord blood mononuclear cells, or CBMC, have emerged as a potential cell source for regenerative medicine, because human leukocyte antigen typing is essential to the cell banking system. CBMC-induced pluripotent stem cells, or CMBC-iPSCs, can be differentiated into keratinocytes and fibroblasts. To generate 3D skin organoid, we're using dermatologic research.

Begin by culturing CBMC-iPSC onto vitronectin-coated 100-milliliter plates at 37 degrees Celsius and 10%carbon dioxide. When the cells have reached 80%confluency, wash the cultures with PBS, and treat them with one milliliter of one-millimolar EDTA per plate for two minutes at 37 degrees Celsius and 5%carbon dioxide. When the cells have detached, stop the reaction with three milliliters of E8 medium per plate, and collect the cells by centrifugation.

Re-suspend the pellets in five milliliters of E8 medium for counting, and transfer one-times-10-to-the-sixth cells to a 15-milliliter conical tube for centrifugation. Re-suspend the transferred cells with 2.5-milliliters of embryonic body formation medium, supplemented with 10-micromolar rho-associated kinase, or ROCK inhibitor. Use a pipette to transfer 125-microliter droplets of cells onto an uncoated culture plate lid.

When all of the droplets have been placed, turn over the dish to allow the droplets to hang from the lid for 24 hours at 37 degrees Celsius and 5%carbon dioxide. The next day, wash the lid with fresh E8 medium, and transfer the embryonic body solution to a 50-milliliter conical tube. Allow the embryonic bodies to settle for one minute at room temperature, before aspirating the supernatant and re-suspending the embryonic bodies with fresh E8 medium for their culture in a 90-millimeter Petri dish at 37 degrees Celsius and 5%carbon dioxide until their differentiation.

For CBMC-iPSC keratinocyte differentiation, replace the E8 medium from the embryonic body culture with fresh E8 medium, supplemented with one nanogram per milliliter of bone morphogenetic protein 4 for a 24-hour incubation at 37 degrees Celsius and 5%carbon dioxide. The next day, harvest the embryonic bodies into a 50-milliliter conical tube as demonstrated, and allow the bodies to settle for one minute at room temperature. Next, replace the supernatant with six milliliters of keratinocyte differentiation medium 1, or KDM1, supplemented with 10 micromolar ROCK inhibitor, and transfer the embryonic bodies to a Type IV Collagen-coated 100-millimeter dish.

On days zero through eight of culture, replace the medium every other day with fresh KDM1, supplemented with three-micromolar retinoic acid and 25 nanograms per milliliter each of bone morphogenetic protein 4 and epidermal growth factor. Between days nine through 12, replace the medium every other day with KDM2, supplemented with three-micromolar retinoic acid, 25 nanograms per milliliter of bone morphogenetic protein 4, and 20 nanograms per milliliter of epidermal growth factor. Between days 13 through 30, change the medium every other day to KDM3, supplemented with 10 nanograms per milliliter of bone morphogenetic protein 4, and 20 nanograms per milliliter of epidermal growth factor.

For CBMC-iPSC fibroblast differentiation, re-suspend the embryonic body culture in six milliliters of fibroblast differentiation medium 1, supplemented with 10-micromolar ROCK inhibitor, and transfer the embryonic body suspension to a basement membrane matrix-coated 100-millimeter dish. After three days of incubation, treat the culture with 0.5-nanomolar bone morphogenetic protein 4 for four days, before changing the medium to fibroblast differentiation medium 2. On day seven of culture, change the medium to FDM2 every other day for one week.

On day 14 of culture, detach the cells with one-millimolar EDTA as demonstrated, and collect the cells in three milliliters of fibroblast differentiation medium 2 for centrifugation. Re-suspend the cells in five milliliters of fibroblast differentiation medium 1 for counting, and transfer two-times-10-to-the-sixth cells to a non-coated dish. On day 21 of culture, transfer two-times-10-to-the-sixth cells from the culture to a Type I Collagen-coated 100-millimeter dish in fresh fibroblast differentiation medium 1.

On day 28 of culture, transfer two-times-10-to-the-six of the iPSC-derived fibroblasts to a new non-coated dish. For three-dimensional skin organoid generation, harvest the iPSC-derived fibroblasts from culture, and transfer two-times-10-to-the-fifth cells to a 15-milliliter conical tube for centrifugation. Re-suspend the fibroblast pellet in 1.5 milliliters of fibroblast differentiation medium 1, and 1.5 milliliters of neutralized Type I Collagen solution.

Incubate the cells on an insert in a six-well microplate for 30 minutes at room temperature. When the solution has solidified, add two milliliters of medium to the top of the insert, and three milliliters to the bottom of the well. Then place the fibroblast matrix into the incubator for five to seven days, until the gelation is complete, and the matrix no longer contracts.

At the end of the incubation, harvest the iPSC-derived keratinocytes, and transfer one-times-10-to-the-sixth cells to a 15-milliliter conical tube for centrifugation. Re-suspend the pellet in 50 to 100 microliters of low calcium epithelial medium 1, and replace the medium over the fibroblast matrix with the keratinocyte cell solution. After 15 minutes at 37 degrees Celsius, replace the medium at the top of the insert with two milliliters of epithelial medium 1, and the medium in the bottom well with three milliliters of the same, and return the plate to the incubator.

After two days, replace the medium in the insert and the well with normal calcium epithelial medium 2 for two additional days of culture. On day four of culture, remove all of the medium, and add three milliliters of cornification medium to the bottom well only, to generate an air-liquid interface. Then return the plate to the incubator for up to 14 days before harvesting the 3D skin organoid for downstream analysis.

CBMC-iPSC-derived keratinocytes have morphology similar to primary keratinocytes, and the expression of keratinocyte markers is increased in these cells. CBMC-iPSC-derived fibroblasts have morphology similar to primary fibroblasts, and the expression of pluripotent stem cell marker Oct-4 is down-regulated, while fibroblast markers are up-regulated. The thickness of the 3D skin organoid increases during 3D culture, confirming that the 3D skin organoid is generated from iPSC-derived keratinocytes and fibroblasts.

After two weeks, a transplanted 3D skin organoid graft efficiently incorporates into a recipient mouse skin, as confirmed by HNE analysis. Further, keratinocyte maturation and epidermal differentiation markers are expressed in the CBMC-iPSC-derived 3D skin organoids, demonstrating a functional differentiation, efficient grafting, and effective healing of the mouse skin defects. We use in the past with the high calcium medium that induces stratified layers of 3D skin organoid to mimic near skin.

analysis shows that the skin is stratified. CBMC-iPSCs are a potential cell source for skin grafts, and CBMC-iPSC-derived 3D skin organoids can be used in studies related to dermatology, cosmetic screening, and regenerative medicine.