Tesi etd-04032019-112951
Link copiato negli appunti
Tipo di tesi
Perfezionamento
Autore
DE CESARI, CHIARA
URN
etd-04032019-112951
Titolo
Molecular and functional characterization of rod precursors for regenerative therapy of the retina.
Settore scientifico disciplinare
BIO/11
Corso di studi
SCIENZE MEDICHE - Translational Medicine
Commissione
relatore Dott.ssa ANGELONI, DEBORA
Parole chiave
- Nessuna parola chiave trovata
Data inizio appello
13/06/2019;
Disponibilità
completa
Riassunto analitico
Retinitis pigmentosa (RP) is a complex set of hereditary diseases leading to the death of photoreceptors, and ultimately to blindness. Presently, there is no cure for RP. Once photoreceptors have degenerated, gene or pharmacological therapies are impractical. However, recent results have shown that cell transplantation approaches may rescue visual functions by replacing lost photoreceptors.
Following the path of regenerative medicine, human iPSCs may be instructed to generate 3D retinal organoids able to differentiate into photoreceptors for disease modeling in vitro, and in perspective for transplantation. However, several problems should be solved to reach this goal.
One of the fundamental challenges is the difference between retinal rod precursors and precursors generated from optic cups. In addition, it is fundamental to improve the number of integrated cells after the transplant, and the evaluation of their functional impact.
In this thesis, we characterized, from a molecular and electrophysiological point of view, mouse rod precursors to better understand their intrinsic characteristics and highlight the differences between several developmental time points.
In order to identify the sub-population of retinal rod precursors suitable for transplantation we used a multifaceted experimental approach.
First, we selected genes the expression of which changes between post-natal day (P) 4, P6 and P8 of retinal development (as retrieved in literature and in transcriptome studies of our collaborators). Those genes were three early expressing genes: c-Kit, Mcam and Tnafsf9; and two late expressing genes: Rho and Hcn1.
Second, we evaluated with single cell qRT-PCR the expression of the aforementioned genes in putative rod precursors that were explanted from retinas at different post-natal ages (P0, P4) and kept in culture for 2, 4 and 8 days respectively, at different plating density. Each cell that was studied with qRT-PCR, had been characterized electrophysiologically before the lysis.
Third, to evaluate the correspondence between gene expression data and protein synthesis, we evaluated with immunofluorescence the presence of proteins encoded by selected genes of interest.
Unexpectedly, we found that the activation of genes characteristic of mature rods, like Rho, is not dependent on the silencing of genes characteristic of a state of immaturity, like c-Kit. Additionally, the comparison between electrophysiological analysis of Ih current (a cationic current that flows through HCN1 channels) and single cell qRT-PCR highlighted a latency time between Hcn1 expression and protein trafficking in the plasma membrane. Moreover, our results highlighted the fundamental role that plating density plays on the fate of dissociated retinal cells. In fact, cells kept at a lower plating density maintain a higher expression of early genes for a longer time in respect of those kept at a higher plating density. This information could be extremely important for the selection of appropriate cells, in anticipation of future transplants.
Finally, we have begun generating 3D retinal organoids with the intent to characterize them molecularly and functionally. The final goal is to understand the differences between retinal rod precursors explanted from post-natal eye and precursors generated from retinal organoids, to evaluate the possibility of using organoids as a source of cells for transplantation.
Following the path of regenerative medicine, human iPSCs may be instructed to generate 3D retinal organoids able to differentiate into photoreceptors for disease modeling in vitro, and in perspective for transplantation. However, several problems should be solved to reach this goal.
One of the fundamental challenges is the difference between retinal rod precursors and precursors generated from optic cups. In addition, it is fundamental to improve the number of integrated cells after the transplant, and the evaluation of their functional impact.
In this thesis, we characterized, from a molecular and electrophysiological point of view, mouse rod precursors to better understand their intrinsic characteristics and highlight the differences between several developmental time points.
In order to identify the sub-population of retinal rod precursors suitable for transplantation we used a multifaceted experimental approach.
First, we selected genes the expression of which changes between post-natal day (P) 4, P6 and P8 of retinal development (as retrieved in literature and in transcriptome studies of our collaborators). Those genes were three early expressing genes: c-Kit, Mcam and Tnafsf9; and two late expressing genes: Rho and Hcn1.
Second, we evaluated with single cell qRT-PCR the expression of the aforementioned genes in putative rod precursors that were explanted from retinas at different post-natal ages (P0, P4) and kept in culture for 2, 4 and 8 days respectively, at different plating density. Each cell that was studied with qRT-PCR, had been characterized electrophysiologically before the lysis.
Third, to evaluate the correspondence between gene expression data and protein synthesis, we evaluated with immunofluorescence the presence of proteins encoded by selected genes of interest.
Unexpectedly, we found that the activation of genes characteristic of mature rods, like Rho, is not dependent on the silencing of genes characteristic of a state of immaturity, like c-Kit. Additionally, the comparison between electrophysiological analysis of Ih current (a cationic current that flows through HCN1 channels) and single cell qRT-PCR highlighted a latency time between Hcn1 expression and protein trafficking in the plasma membrane. Moreover, our results highlighted the fundamental role that plating density plays on the fate of dissociated retinal cells. In fact, cells kept at a lower plating density maintain a higher expression of early genes for a longer time in respect of those kept at a higher plating density. This information could be extremely important for the selection of appropriate cells, in anticipation of future transplants.
Finally, we have begun generating 3D retinal organoids with the intent to characterize them molecularly and functionally. The final goal is to understand the differences between retinal rod precursors explanted from post-natal eye and precursors generated from retinal organoids, to evaluate the possibility of using organoids as a source of cells for transplantation.
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