Periodic Reporting for period 1 - AmnioticID (Understanding the identity of the amniotic fluid stem cells)
Período documentado: 2019-09-01 hasta 2021-08-31
This project’s primary aim was to pinpoint the tissue and processes originating the Amniotic Fluid Stem Cells (AFSCs), mesenchymal cells that can be consistently isolated from the human amniotic fluid. Since their identification in 2007, the AFSCs have been ascribed to the mesenchymal stem cells lineage and proposed as a tool for autologous regenerative medicine. However, despite several achievements and breakthroughs since their discovery in 2007 (De Coppi et al., Nature Botechnology 2007), none of the therapeutic approaches based on these cells has yet entered the clinic. This lack of functional translation can be ascribed to the poor knowledge on the origin and identity of the AFSCs. Hence, the objective of this project was to understand the cell’s identity and the biological processes leading to the AFSCs presence in the amniotic fluid. This would help in developing targeted therapeutic interventions that employ these cells for the functions that they are naturally programmed to exert, ultimately leading to the development of novel foetal and perinatal regenerative medicine strategies based on these cells. Overall, for this project I studied the transcriptomic profile and protein expression of the AFSC - in comparison to similar mesenchymal cells, isolated ad hoc from a library of foetal tissues. By highlighting the analogies and differences between these cell types, I aimed at inferring the foetal origin of the AFSCs, first by tissue, then by cell type and finally by anatomical location. To develop this project, I combined three available transcriptomic technologies (bulk RNA sequencing, single cell RNA sequencing and spatial transcriptomics). Together with protein validation assays like immunofluorescence, FACS analysis and western blot, this helped demonstrating the validity of my research hypothesis and further support my findings.
Based on the results presented in this report I can conclude that the AFSCs originate during human development by release from the foetal kidney. Firstly, I have compared the transcriptome of 9 lines of human AFSCs, against an ad hoc generated library of foetal MSCs from three fetuses at different developmental stages. These RNA sequencing findings were then refined using DEG analysis and validated with protein-based techniques (immunofluorescence and western blot) highlighted the renal identity of the AFSCs. I have then compared my AFSC dataset with a full-scale single cell atlas representing all the cells present during human foetal kidney development. These results indicated that the AFSCs have plausible origin in developing nephron / supporting mesenchyme. This was followed by a spatial transcriptomic validation experiment, where I have compared my AFSCs with sections of developing human kidney from 3 fetal specimens at 20PCW. This allowed me to pinpoint the exact anatomical region responsible for originating the AFSCs. Ultimately, I have analysed my dataset looking for the co-expression of Mesenchymal, Epithelial and EMT markers. This allowed me to formulate a model in which the AFSC origin, is ascribed to the release of epithelial cells from the developing renal tubule, and consequent transition to a mesenchymal phenotype.
Thanks to this project, I have demonstrated that the foetal origin of the AFSCs lie in the developing human kidney. In line with this, AFSCs show expression of markers that are typical of the renal lineage (e.g. PAX8, LHX1, LFNG). By comparing the transcriptomic profile of the AFSCs, with a collated single cell atlas and an ad hoc generated spatial transcriptomic dataset of the developing human kidney, I provided evidence that the renal tubule and supporting mesenchyme could be deemed responsible for the release of the AFSCs. In addition, I have observed for the first time that AFSCs co-express mesenchymal, epithelial and Epithelial-to-Mesenchymal Transition (EMT) markers. Based on these findings, I proposed a model whereby the AFSCs form initially through exfoliation from the developing renal tubule epithelia (Figure 5d). When exposed to the amniotic environment, and/or placed in culture, these cells would enter EMT and acquire their long-known mesenchymal signature. Moreover, during the progression of my MSCA fellowship i have received training, engaged in communication/dissemination and progressed my career development. Overall, the completion of this project led me to cover my first independent position as Lecturer in Stem Cell Science and Biomaterials at University College London, where I am now leading my independent research line, and a small research team.