Date of Award

8-8-2024

Document Type

Thesis

School

School of Chemical & Biotechnology

Programme

Ph.D.-Doctoral of Philosophy

First Advisor

Dr.Sowmya Parameswaran

Second Advisor

Dr.K.Uma Maheswari

Keywords

Retinoblastoma Gene, Mesenchymal Stem Cells, Retinal Differentiation, Adipogenic Differentiation, Osteogenic Differentiation

Abstract

The Retinoblastoma (RB1) gene, predominantly recognized as a tumour suppressor, plays a crucial role in early growth and development. Mice lacking the retinoblastoma gene do not survive and exhibit defects in the differentiation of various tissues, underscoring its involvement in cellular differentiation. This suggests that the functions of the RB1 gene extend beyond its role as a tumour suppressor, encompassing cell cycle regulation, maintenance of genomic stability, cellular senescence, and cellular differentiation.

While the tumour suppressor function of the RB1 has been extensively studied, its role in cellular differentiation remains incompletely understood. Existing literature, primarily based on animal models, has provided inconclusive results on RB1’s role in differentiation, highlighting the need for human studies. This study aimed to investigate the role of RB1 in cellular differentiation using patient-derived stem cells from retinoblastoma patients.

Mesenchymal stem cells (MSCs) isolated from the orbital adipose tissue of the retinoblastoma patients were cultured and characterised as Orbital Adipose Mesenchymal Stem cells (OAMSCs). The RB1+/- OAMSCs were differentiated into osteogenic and adipogenic lineages to investigate the role of RB1. Furthermore, to study the role of RB1 retinal differentiation, OAMSCs were reprogrammed to induced pluripotent stem cells (iPSCs) and then subjected to stepwise retinogenesis. The study revealed that the xvi

monoallelic loss of RB1 does not alter the mesenchymal phenotype of the MSCs, but impacts its proliferation and differentiation. In adipogenesis, the monoallelic loss of RB1 accelerated and increased the differentiation, with a bias towards brown adipocytes; in osteogenesis, it increased differentiation and proliferation but maintained genomic integrity, possibly preventing or delaying tumourigenesis.

However, in retinogenesis, RB1 monoallelic loss did not impact proliferation or differentiation but influenced metabolism, as evidenced by enhanced glycolytic activity and ATP production. This metabolic adaptation likely meets altered energy demands and cellular requirements in RB1+/- retinal organoids.

These findings highlight that RB1 exerts different roles in distinct cell types. While promoting proliferation and differentiation in adipogenic and osteogenic lineages, RB1's monoallelic loss in retinal cells primarily influences metabolic processes rather than differentiation. This underscores the multifaceted role of RB1 in cellular differentiation and emphasizes the importance of cell-type-specific studies to fully elucidate RB1’s diverse functions.

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