Purpose

Cartilage regeneration is the future of regenerative medicine, as this can provide a massive relief to degenerative diseases such as osteoarthritis and rheumatoid arthritis. My research project involves working on reprogrammed human induced pluripotent stem cells (hiPSCs) acquired from patients suffering from a rare genetic disease known as pseudo chondrodysplasia. This will also give an insight into production of defective cartilage through various phases of growth. Using Crispr Cas9 approach, gene editing of the mutant type hiPSCs will be carried out in future.

This poster show different techniques been utilized in our laboratory to convert induced pluripotent stem cells to mesenchymal stem cells which closely resembles chondrocytes and ultimately forming a growth plate like cartilage structure. Pluripotency was checked through immunofluorescence, flow cytometry and Q-PCR as this will determine the fate of the hiPSCs.

Methods and Materials

Materials:

Reprogrammed iPSCs, Tesr 8 media, MeSen Pro RS media, chondro basal media, growth factors

Method: Stem cells were grown in TESR-8 media and were differnetiated by adding a mesodemal media Mesen Pro RS and cutured untill they form MSC like cells these were then condensed into 3 dimensional growth plate like cartilage pellets by using basal media and growth factors like TGF-b and BMP and maintained for 21 days.

Results

The healthy normal pellets stained positive with alcian blue and Safrinin O and the mutant pellets when growth factors were added on addition there has been a slight rescue of the abnormal phenotype. Cartilage ,markers like collagen 2, aggrecan and COMP were detetced in the healthy pellets and was reduced in untreated mutant pellets but on addition of growth factors to the mutant pellets there was an increase in the cartilage markers.

Conclusion

growth factors increases the growth plate like cartilage formation.

hiPSCs from healthy person formed normal cartilage and hiPSCs from disease patient formed abnormal cartilage.