We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.
The overall goal of this procedure is to generate chondrogenic lineage cells from human peripheral blood-derived induced pluripotent stem cells, or IPS cells. This method can help answer the key questions in the cartilage regeneration field about using patient derived peripheral blood cells as C cells for the generation of the IPS cells for cartilage repair. The main advantages of this technique are that it is easily reproducible, and the chondrocyte differentiation occurs on the serum and xeno-free conditions.
Begin by plating human IPS cells in four milliliters human IPSC medium onto a 60 millimeter tissue culture dish containing a monolayer of irradiated mouse embryonic fibroblast feeder cells. Culture the cells at 37 degrees Celsius in five percent carbon dioxide. When the stem cells are 80 to 90 percent confluent, dissociate the cells with dispace solution for subculturing.
After two to three passages, use a fire drawn glass needle to dissect the 80 to 90 percent confluent undifferentiated IPSC colony into smaller fragments, and transfer no more than 100 cell culture pieces into a new nonadherent 100 millimeter petri dish containing 10 milliliters of embryoid body formation medium. Mechanical dissociation is better than automatic digestion because it reduces cell damage to now small fragments of cells to be transferred to the new culture container, and announce manual disposal of the feeder cells, which repress human IPS cell differentiation. Return the human IPS cells to the cell culture incubator.
After two days, tilt the dish to let the embryoid bodies settle, and carefully replace three milliliters of the embryoid body formation medium with four milliliters of fresh basal culture medium. After 10 days, embryoid bodies will have formed. Coat a new 100 millimeter dish with four milliliters of 0.1 percent gelatin for 30 minutes at 37 degrees Celsius.
Transfer the embryoid bodies with the supernatant into a 15 milliliter conical tube, and let the bodies settle for four to five minutes. Aspirate all but the last 0.5 milliliters of medium, and resuspend the embryoid bodies in 10 milliliters of fresh basal culture medium. When the gelatin has solidified, discard the gelatin.
Then seed the cells onto the gelatin-coated dish, and place the dish in the cell culture incubator. After 10 days in culture, the embryoid bodies will have differentiated into fibroblastic cell outgrowths. To induce chondrocyte differentiation, wash the culture with DPBS and detach the cells with three milliliters of 0.25 percent trypsin EDTA at 37 degrees Celsius for five minutes.
When the cells have lifted from the bottom of the plate, neutralize the reaction with four milliliters of fresh basal culture medium, and gently titrate the culture five to 10 times. Filter the cell suspension through a 70-micron nylon mesh strainer, and collect the cells by centrifugation. Then seed the cells onto a new gelatin-coated, 100 millimeter plate in 10 milliliters of fresh basal culture medium.
When the culture reaches 90 to 100 percent confluency, harvest the cells with three milliliters of 0.25 percent trypsin EDTA as just demonstrated for counting. Transfer a three times 10 to the fifth aliquot of cells into a 15 milliliter polypropylene tube for centrifugation. And resuspend the pellet in one milliliter of chondrogenic differentiation medium.
Then, centrifuge the cells again, and place the tube in the cell culture incubator for 21 days without disturbing the pellet. Chondrogenic cells may assemble in vitro, producing characteristic extracellular metrics when under high density culture conditions. After 21 days in culture, a human IPS cell chondrogenic pellet will have formed.
After 21 days of culture, the human IPSC chondrogenic cells are positive for alcian blue and toluidine in blue staining, indicating a successful chondrogenic differentiation of the HIPSC pellet. Immunohistochemical analysis for collagen two and collagen 10 further confirms their chondrocyte-like phenotype. Sulfated glycosaminoglycan analysis reveals that the sulfated glycosaminoglycan contents of the cells are significantly upregulated in human IPSC chondrogenic pellets, compared to human IPSC fibroblastic-like cells, embryoid bodies, and undifferentiated human IPS cells.
Further, human IPSC chondrogenic cells demonstrate a high gene expression level of chondroprogenitor lineage and fully differentiated chondrocyte markers, suggesting the achievement of a successful chondrogenic differentiation. A multistep culture method used to differentiate human IPSCs into chondrocytes, including spontaneous differentiation via EB formation, cell outgrowth from EBs, monolayer cell culture after cell culture, and 3D pellet culture. The chondrocyte phenotype is assessed by histological analysis, biochemical analysis, and chondrogenic gene expression.
After watching this video, you should have a good understanding of how to use a multistep culture method for inducing human IPS cell differentiation into chondrocytes. This technique could be more patient specific and cost effective.
