Abstract
Reprogramming using small molecules is an innovative technology to engineer cartilage due to its flexible nature. Human dermal fibroblasts (HDFs) and human mesenchymal stem cells (hMSCs) are valid starting sources for chondrogenic reprogramming. Using the Mogrify® algorithm, a previous study from the Kafienah group identified a small molecule cocktail, resulting in partial chondrogenic reprogramming of HDFs. To improve this process, this project investigated the effect of cell cycle synchronisation and cell passage number in the HDF reprogramming model. Also, a newly derived small molecule cocktail for the hMSC reprogramming was tested for cytotoxicity and reprogramming ability.PI staining confirmed 24 hours serum starvation can sufficiently induce cell cycle synchronisation in HDFs. However, the small molecule treated HDFs showed no significant difference in chondrogenesis as demonstrated by SOX9 and type II collagen expression between normal and cell cycle synchronised HDFs at each passage (low, middle, and high).
For the hMSC reprogramming, each small molecule did not show significant cytotoxicity. As a cocktail, a significant increase and maintenance of cell viability was observed, suggesting a beneficial composite effect of the small molecules.
Regarding reprogramming ability, the small molecule treated hMSCs in 2D culture did not show a significantly higher level of SOX9, aggrecan, type I and type X collagen than DMSO or rhTGF-β3 treatment, but a single super responder, which follows the pattern of rhTGF-β3, was identified. As 3D pellets, the chondrogenic matrix formed by the small molecule cocktail was less than the matrix formed by rhTGF-β3. The super responder was no exception.
In conclusion, cell cycle synchronisation and cell passage number did not have a significant impact on the HDF reprogramming model. The small molecule cocktail for the hMSC reprogramming did not show significant cytotoxicity, but reselection of type and number of small molecules is required to optimise its reprogramming ability.
| Date of Award | 27 Sept 2022 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Wael Kafienah (Supervisor) |