臍帯血由来誘導多能性幹細胞からの軟骨形成ペレット形成

The overall goal of this procedure is to generate chondrogenic pellets from cord blood-derived induced pluripotent stem cells. With this protocol, we can generate a relatively large quantity of chondrogenic pellets. The generated chondrogenic pellets can be used for studies or disease modeling, drug screening, and researcher to medicine to farther our understanding of the nature of cartilage.

The human cartilage last year belated to repair. Therefore, cartilage degeneration must be treated by conservative treatment. With unlimited self renew-ability and multi potency, human iPSCs have been highlighted as new replacement cell source for cartilage repair.

Demonstrating the procedure will be Eugene Nam and Arie Alice Ream who are graduate students from my laboratory. To begin the protocol, collect blood cells in a 15 milliliter conical tube, and count them using a hemocytometer. Prepare three times 10 to the fifth cells and centrifuge them for five minutes at 515 times G at room temperature.

Discard the supernatant by suction, and re suspend the cells in 0.5 milliliters of blood cell medium. Next, transfer the cells to a well on a non-coated, 24 well plate, and add the Sendai virus mixture following the manufacturer's recommendations. Centrifuge the plate for 30 minutes at 1, 150 times G, and 30 degrees celsius.

After centrifugation, incubate the cells overnight. The next day, transfer the transude cells to a matrix coded well. Centrifuge the plate.

After centrifugation, remove the supernatant, add one milliliter of iPSC medium, and maintain the cells. Change the medium daily, and replace it with fresh iPSC medium. Maintain the human induce pluripotent stem cells, or HIPSCs.

Change the medium daily, replacing it with fresh essential eight, or E8 medium. Prepare two times 10 to the 6th HIPSCs in a vitronectin encoded 100 milliliter and E8 medium. Next, remove the E8 medium from the culture dish and wash the cells with sterile phosphate buffered saline, or PBS.

Add one milliliter of one millimolar of EDTA, and incubate the dish for two minutes. Then harvest the cells with three milliliters of E8 medium, and transfer them to a new 15 milliliter conical tube. Centrifuge the cells at 250 times G at room temperature for two minutes.

Aspirate the supernatant without disturbing the cell palate, and re suspend the cells in five milliliters of E8 medium. After counting the cells with a hemocytometer, prepare two times 10 the 6th cells for each 100 millimeter Petri dish. Re suspend the prepared cells in a 10 milliliter one to one mixture of E8 and EB medium, with 10 micromolar row associated carnase, or rock inhibitor.

Incubate the cells overnight for aggregation. On the following day, harvest the aggregated EBs by pipeting. Remove the supernatant, and re suspend the EBs in 10 milliliters of fresh E8 medium.

Enlarge the generated EBs for five days, performing daily changes with fresh E8 medium. For further maturation, change the culture medium to E7 medium. Maintain the EBs at 37 degrees celsius and 5%carbon dioxide for another five days, performing daily medium changes with fresh E7 medium.

Add six milliliters of 1%gelatin to a 100 millimeter dish, and incubate the dish for 30 minutes. Remove the gelatin, and dry this dish completely for two to three hours before use. Transfer the EBs to a 50 milliliter conical tube.

Allow the EBs to settle to the bottom of the conical tube. Remove the supernatant without disturbing the EBs. Next, re suspend the EBs in 10 milliliters of pre-warmed shilbecco's modified equals medium, or DMEM, with 20%fetal bulbing serum, or FBS.

Transfer the EBs to the gelatin coating 100 millimeter dish. Add 10 micromolar of rock inhibitor. Incubate and maintain the cells for seven days.

Change the medium every other day without adding rock inhibitor. Aspirate the culture medium from the dish. Immediately apply one milliliter of one millimolar EDTA to the dish, and incubate the cells for two minutes.

Next, harvest the cells using five milliliter of DMEM with 20%FBS, and transfer them to a new 15 milliliter conical tube. Centrifuge the cells for two minutes at 250 time G, at room temperature. Discard the supernatant, and re suspend the pellet in 10 milliliter of DMEM with 20%FBS.

Filter and discard the cell clumps using a 40 micrometer cell strainer, and harvest the single cells. Count the single cells using a hemocytometer. Centrifuge the cells.

Remove the supernatant, and seed three times 10 to the fifth cells per pellet in a 15 milliliter conical tube with 300 microliters of chondrogenic differentiation medium, or CDM. For pellet formation, centrifuge the cells, then incubate the cells overnight. Within three days, pellets will exhibit flattened spheroidal morphologies.

Maintain the pellets for 21 days. Before embedding, prepare and melt paraffin at 58 degrees celsius. Fix the pellets in one milliliter of 4%paraformaldehyde for two hours at room temperature in a 1.5 milliliter tube.

Place one layer of gauze onto the cassette and transfer the fixed pellets using a pipette. Cover the pellet by folding the gauze and closing the cassette lid. Initiate dehydration in 100 milliliters of 70%ethanol.

Dehydrate the pellets through sequential terminate washes in 80%and 95%ethanol. Transfer to pellets to 100%ethanol for 10 minutes. For clearing, exchange the solution with a 100 milliliter one to one mixture of ethanol and xylene for 10 minutes.

Next, exchange the one to one solution with a one to two mixture of ethanol and xylene for 10 minutes. Clear the remaining ethanol by incubating the pellets in 100%xylene. For paraffin infiltration, incubate the pellets in sequential xylene and paraffin mixtures.

Perform the whole paraffin infiltration process at 58 degrees celsius. Incubate the pellets in 100 milliliters of a two to one mixture of xylene and paraffin for 30 minutes. Next, exchange the solution for 100 milliliters of a one to one mixture of xylene and paraffin and incubate for 30 minutes.

Then exchange the solution for 100 milliliters of a one to two mixture of xylene and paraffin and incubate for 30 minutes. For the final infiltration, transfer the pellets to the first bath of 100%paraffin and incubate the pellet for two hours. Transfer the pellets to the second bath of 100%paraffin and incubate overnight at 58 degrees celsius.

The next day, use tweezers to gently transfer the pellets to a mold. Add paraffin to the mold from the paraffin dispenser. Solidify the paraffin for 30 minutes at four degrees celsius.

Slice the sections at seven micrometers and transfer the sections onto the slide. Allow the slides to dry overnight, and store the slides at room temperature until they are ready for use. Before differentiation, iPSC colonies were expanded.

The expanded iPSCs were assembled as EBs to initiate differentiation. The generated EBs were attached to gelatin coated dishes to induce outgrowth cells. After 21 days of differentiation, small bead like chondrogenic pellets were obtained and used for further characterization.

The quality of the chondrogenic pellets were histologically evaluated on day 21, with BMSC chondrogenic pellets used as the positive control. The accumulation of ECM proteins secreted by the differentiated chrondrocites was confirmed by Alcian blue, in Toluiodine blue staining. Chondrogenic pellets generated from CDMC HIPSCs expressed higher COL2A1 than CBMSC HIPCs derived pellets.

The expression of collagen type one, a marker for fibrotic cartilage, was lower in CBMC HIPSC derived pellets compared to the expression of COL2A1. The gene expression of cartilage extracellular matrix proteins on day 21 chondrogenic pellets was confirmed by real time PCR. The aggracan expression of CBMC HIPSC derived pellets was similar to BMSC derived pellets.

We provided a detailed method for the differentiation of cord blood mononuclear cell derived iPSCs into chrondrogenic pellets. Once mastered, this technique can be done in two months if it is performed properly. After watching this video, you should have a good understanding of how to generate chondrogenic pellets from iPSCs.