Periodic Reporting for period 1 - SCAPA (Functional analysis of Alternative Polyadenylation during neuronal differentiation at single cell resolution)
Période du rapport: 2019-04-01 au 2021-03-31
Understanding how stem cells give rise to all the cell types that compose an organism is a fundamental question in biomedical sciences. With the development of single-cell transcriptomics, now we can identify the genes expressed in individual cells and understand how much gene expression across cells. Furthermore, we can also use it to characterize the dynamic changes in gene expression with time and understand how cellular differentiation is regulated at the transcriptomic level. This technology has already been used to characterize the cell composition of organs and tissues, developmental stages, and even whole organisms. Yet, it has not been used to profile the function of gene regulatory mechanisms in cell differentiation.
The aim of this project was to take advantage of the recently developed single-cell transcriptomics to understand the functional impact of regulatory proteins in the differentiation of neurons derived from mouse embryonic stem cells (mESCs).
The results of this project will advance our knowledge on how gene regulation affects the differentiation of neurons and will provide specific information of some regulators. This information will be a steppingstone to better understand how different gene regulatory mechanisms influence neuronal differentiation. Ultimately, this information will be useful for the future development of personalized medicine therapies based on inducible pluripotent stem cells.
The aim of this project was to take advantage of the recently developed single-cell transcriptomics to understand the functional impact of regulatory proteins in the differentiation of neurons derived from mouse embryonic stem cells (mESCs).
The results of this project will advance our knowledge on how gene regulation affects the differentiation of neurons and will provide specific information of some regulators. This information will be a steppingstone to better understand how different gene regulatory mechanisms influence neuronal differentiation. Ultimately, this information will be useful for the future development of personalized medicine therapies based on inducible pluripotent stem cells.
Since the beginning of the project, we have been working on optimizing the experimental protocols and developing new computational pipelines and tools for the analyses of the expected data. These include:
• Optimize the culture and differentiation conditions from mouse embryonic stem cells to neurons. We have morphologically characterized the differentiation of stem cells and characterized the different populations obtained using single-cell transcriptomics.
• Develop new mouse embryonic stem cell transgenic lines to perform functional studies. We have generated 3 stable cell lines that can by use to knock down the expression of genes using the CRISPR-Cas9 system. We are currently characterizing the function of the constructs and testing if they can be used to perform functional studies.
• Develop new pipelines and computational tools to analyze the single-cell transcriptomics data generated. We have already stablished computational pipelines for the standard single-cell transcriptomics analyses and we are still developing computational tools to perform isoform quantification.
Regarding exploitation and dissemination, given that the project is not finished yet, little has been done. I have participated in the European Researcher’s Night (27/09/19) and in the PRBB Open day (05/10/19) to present the work that I am doing with single-cell transcriptomics to the general public. Additionally, I have presented the work about the development of computational pipelines in the RNA society meeting (Krakow, June 2019).
• Optimize the culture and differentiation conditions from mouse embryonic stem cells to neurons. We have morphologically characterized the differentiation of stem cells and characterized the different populations obtained using single-cell transcriptomics.
• Develop new mouse embryonic stem cell transgenic lines to perform functional studies. We have generated 3 stable cell lines that can by use to knock down the expression of genes using the CRISPR-Cas9 system. We are currently characterizing the function of the constructs and testing if they can be used to perform functional studies.
• Develop new pipelines and computational tools to analyze the single-cell transcriptomics data generated. We have already stablished computational pipelines for the standard single-cell transcriptomics analyses and we are still developing computational tools to perform isoform quantification.
Regarding exploitation and dissemination, given that the project is not finished yet, little has been done. I have participated in the European Researcher’s Night (27/09/19) and in the PRBB Open day (05/10/19) to present the work that I am doing with single-cell transcriptomics to the general public. Additionally, I have presented the work about the development of computational pipelines in the RNA society meeting (Krakow, June 2019).
The grant was terminated early after ~6 months as I was offered a position as independent group leader. Thus, there are not more expected additional results at the end of the project.