Periodic Reporting for period 4 - CellKarma (Dissecting the regulatory logic of cell fate reprogramming through integrative and single cell genomics)
Periodo di rendicontazione: 2022-09-01 al 2024-08-31
This project tackles a challenging question in biology: how to reprogram human cells to become pluripotent stem cells (IPSCs) efficiently and safely. Reprogramming involves transforming specialized adult cells into IPSCs, potentially becoming any cell type, making them extremely promising in research and medicine. IPSCs could be used to repair damaged tissues or to model diseases in the lab for study. However, reprogramming is still a slow and complex process that doesn’t always yield high-quality results. Understanding the steps and specific gene networks involved is critical to improving the speed and reliability of this process.
Current reprogramming methods face several challenges. First, few cells fully complete reprogramming, and the remaining cells may not function correctly. Second, the genetic and epigenetic processes behind reprogramming aren’t completely understood. This lack of understanding means it’s difficult to know what exactly happens as cells move through different stages of reprogramming. The process is also risky, as unwanted mutations can arise, making cells less safe for therapeutic use.
This project addresses these challenges using advanced multi-omics techniques—analyzing the genes, proteins, and regulatory networks involved in reprogramming—to identify the intermediate states and factors influencing cellular transitions to pluripotency. This approach aims to gain better control over the reprogramming process, ensuring cells achieve full pluripotency without unintended side effects.
This research holds significant societal potential, particularly in regenerative medicine and personalized health care. The ability to produce high-quality IPSCs can transform treatment options for many conditions, such as neurodegenerative diseases, spinal cord injuries, and heart disease, by providing a source of cells that can repair damaged tissues. IPSCs could be used to replace damaged cells or tissues, potentially restoring function to organs that have been damaged by disease or injury.
Moreover, IPSCs provide a new way to study diseases in the lab. Traditional models often rely on animal studies, which don’t always accurately predict human responses to treatments. By creating patient-specific models using IPSCs, scientists can explore how different genetic backgrounds influence disease progression and drug responses, leading to more effective and targeted therapies. This also allows for faster, more ethical drug testing and development, as drugs can be tested directly on human cells.
The societal and economic impacts are also worth noting. Efficient and accessible reprogramming could make stem cell-based therapies more affordable, reaching a wider population. Additionally, research of this kind supports the biotech and healthcare sectors, creating new job opportunities and potential collaborations with the industry. This work also provides essential foundations for spin-offs, like NEGEDIA s.r.l. which focuses on using genomics in disease diagnosis. All of these developments contribute to advancing medical science and improving public health.
This project has four main goals, each addressing a core issue in cell reprogramming and aiming to make IPSCs a practical tool for therapeutic use.
The first objective is to identify and describe the intermediate cellular states during reprogramming. This means discovering what cell types appear as cells change and how these states affect the overall process. Understanding these states will help researchers control the transitions more precisely, moving cells toward pluripotency efficiently.
The second goal is to explore the regulatory networks of transcription factors (TFs) that guide the reprogramming process. Transcription factors are proteins that help control gene activity. Using techniques like MPRA (which tests how DNA segments promote gene expression) and scATACseq (which analyzes DNA accessibility in single cells), the project aims to identify the genes and regulatory pathways needed for successful reprogramming. This can help scientists refine reprogramming by focusing on key TFs that improve efficiency.
The third goal is to find and address "roadblocks" in reprogramming. These roadblocks can be mutations or gene regulation issues preventing cells from reprogramming. By comparing DNA in different reprogrammed cells, this project aims to pinpoint common mutations and genetic changes that act as barriers. Understanding these obstacles will allow scientists to modify protocols to minimize these issues, making the resulting cells safer for clinical use.
The fourth objective is to improve the quality of reprogrammed cells by developing new tools and methods. For example, using a microfluidic platform speeds up the testing and validation of candidate genes that may improve reprogramming outcomes. Using a scoring system (TERA score), the project will rank transcription factors based on their ability to guide cell identity changes, making the reprogramming process more predictable and controlled.
Overall, this project aims to create a valuable resource that maps key transcription factors, their targets, and the molecular pathways involved in reprogramming. By integrating all this data, the project not only advances our understanding of cell biology but also provides practical tools for developing effective cell-based therapies in the future.
Current reprogramming methods face several challenges. First, few cells fully complete reprogramming, and the remaining cells may not function correctly. Second, the genetic and epigenetic processes behind reprogramming aren’t completely understood. This lack of understanding means it’s difficult to know what exactly happens as cells move through different stages of reprogramming. The process is also risky, as unwanted mutations can arise, making cells less safe for therapeutic use.
This project addresses these challenges using advanced multi-omics techniques—analyzing the genes, proteins, and regulatory networks involved in reprogramming—to identify the intermediate states and factors influencing cellular transitions to pluripotency. This approach aims to gain better control over the reprogramming process, ensuring cells achieve full pluripotency without unintended side effects.
This research holds significant societal potential, particularly in regenerative medicine and personalized health care. The ability to produce high-quality IPSCs can transform treatment options for many conditions, such as neurodegenerative diseases, spinal cord injuries, and heart disease, by providing a source of cells that can repair damaged tissues. IPSCs could be used to replace damaged cells or tissues, potentially restoring function to organs that have been damaged by disease or injury.
Moreover, IPSCs provide a new way to study diseases in the lab. Traditional models often rely on animal studies, which don’t always accurately predict human responses to treatments. By creating patient-specific models using IPSCs, scientists can explore how different genetic backgrounds influence disease progression and drug responses, leading to more effective and targeted therapies. This also allows for faster, more ethical drug testing and development, as drugs can be tested directly on human cells.
The societal and economic impacts are also worth noting. Efficient and accessible reprogramming could make stem cell-based therapies more affordable, reaching a wider population. Additionally, research of this kind supports the biotech and healthcare sectors, creating new job opportunities and potential collaborations with the industry. This work also provides essential foundations for spin-offs, like NEGEDIA s.r.l. which focuses on using genomics in disease diagnosis. All of these developments contribute to advancing medical science and improving public health.
This project has four main goals, each addressing a core issue in cell reprogramming and aiming to make IPSCs a practical tool for therapeutic use.
The first objective is to identify and describe the intermediate cellular states during reprogramming. This means discovering what cell types appear as cells change and how these states affect the overall process. Understanding these states will help researchers control the transitions more precisely, moving cells toward pluripotency efficiently.
The second goal is to explore the regulatory networks of transcription factors (TFs) that guide the reprogramming process. Transcription factors are proteins that help control gene activity. Using techniques like MPRA (which tests how DNA segments promote gene expression) and scATACseq (which analyzes DNA accessibility in single cells), the project aims to identify the genes and regulatory pathways needed for successful reprogramming. This can help scientists refine reprogramming by focusing on key TFs that improve efficiency.
The third goal is to find and address "roadblocks" in reprogramming. These roadblocks can be mutations or gene regulation issues preventing cells from reprogramming. By comparing DNA in different reprogrammed cells, this project aims to pinpoint common mutations and genetic changes that act as barriers. Understanding these obstacles will allow scientists to modify protocols to minimize these issues, making the resulting cells safer for clinical use.
The fourth objective is to improve the quality of reprogrammed cells by developing new tools and methods. For example, using a microfluidic platform speeds up the testing and validation of candidate genes that may improve reprogramming outcomes. Using a scoring system (TERA score), the project will rank transcription factors based on their ability to guide cell identity changes, making the reprogramming process more predictable and controlled.
Overall, this project aims to create a valuable resource that maps key transcription factors, their targets, and the molecular pathways involved in reprogramming. By integrating all this data, the project not only advances our understanding of cell biology but also provides practical tools for developing effective cell-based therapies in the future.
The project focused on uncovering the molecular mechanisms behind human cell reprogramming to pluripotency, utilizing advanced techniques in single-cell multiomics. One of the key findings was the identification of intermediate cellular states that play a crucial role in this process. The innovative use of a microfluidic platform significantly enhanced the efficiency of the reprogramming strategy, allowing for the investigation of how non-reprogrammed cells can contribute to the pluripotency of others by secreting essential factors. This research highlighted that these transient populations, previously thought to be by-products, are essential for successful reprogramming, this project revealed the importance of the transcription factor ESRRB in regulating naive pluripotency. By employing RNA sequencing and chromatin immunoprecipitation sequencing (ChIP-seq), the study demonstrated that ESRRB facilitates the transition from naive to formative states of pluripotency, thereby providing a new perspective on how these cellular states evolve.
The project established high-throughput enhancer assays and developed a new tool named SCRAMseq, which allows for the dissection of gene variant effects at a single-cell level. This methodological advancement enables a deeper understanding of gene regulatory networks and their roles in cell fate decisions, paving the way for future research in stem cell biology.
Overall, the results have significant implications for regenerative medicine and therapeutic applications, as they elucidate the regulatory circuits involved in cellular reprogramming and differentiation. This project advanced the understanding of stem cell biology and fostered interdisciplinary collaboration, leading to the establishment of a spin-off company focused on genomic diagnostics .
These findings were published in leading scientiing Nature Communications and Nature Cell Biology, showcasing the project's contribution to the field and its potential impact on future research and clinical applications
The project established high-throughput enhancer assays and developed a new tool named SCRAMseq, which allows for the dissection of gene variant effects at a single-cell level. This methodological advancement enables a deeper understanding of gene regulatory networks and their roles in cell fate decisions, paving the way for future research in stem cell biology.
Overall, the results have significant implications for regenerative medicine and therapeutic applications, as they elucidate the regulatory circuits involved in cellular reprogramming and differentiation. This project advanced the understanding of stem cell biology and fostered interdisciplinary collaboration, leading to the establishment of a spin-off company focused on genomic diagnostics .
These findings were published in leading scientiing Nature Communications and Nature Cell Biology, showcasing the project's contribution to the field and its potential impact on future research and clinical applications
In the original proposed project, my hypothesis was that the presence of transient developmental signatures identified during human reprogramming to pluripotency might represent either the accumulation of cells that are unable to overcome selective barriers, or alternative cellular stages that will never promote a successful reprogramming outcome. As described later for Aim 1 outcomes, thanks to the preliminary albeit robust results involving the extracellular matrix (ECM) contribution I can assess that the matrisome alternative stage we identified is of crucial importance for a successful reprogramming outcome. I anticipate that such a mechanism can be at the basis of several, if not all, cellular conversions.
Furthermore, from a more technically-related point of view, I originally planned to use single-cell approaches to obtain transcriptional information on a maximum of 3.000 genes in individual cells. My team set up a successful protocol that allowed the profiling at high resolution of up to 6.000 genes per cell, thus further increasing the information on ongoing molecular dynamics.
In addition, we validated an approach to obtain transcriptomic and epigenomic data from the same experiment, combining the single-cell RNAseq (scRNAseq) with scATACseq approach.
In Aim 4, I originally proposed to validate candidates by modulating their expression in a primary reprogramming model but the implementation of the microfluidic approach allowed the discovery of novel candidates and the simultaneous direct validation, thus accelerating the overall project performance. Therefore, I decided to use the TERA (Transcription Factor Epigenetic Remodeling Activity) score to carry on a de novo discovery on a list of 300 TFs, ranked on their ability to act on cellular plasticity. On such TFs, we already performed a comprehensive dosage profiling using RNAseq, ATACseq, morphology assay (immunofluorescence-based) and CUT&RUN (cleavage under targets and release using nuclease) sequencing approach to study each TF binding to DNA.
Furthermore, from a more technically-related point of view, I originally planned to use single-cell approaches to obtain transcriptional information on a maximum of 3.000 genes in individual cells. My team set up a successful protocol that allowed the profiling at high resolution of up to 6.000 genes per cell, thus further increasing the information on ongoing molecular dynamics.
In addition, we validated an approach to obtain transcriptomic and epigenomic data from the same experiment, combining the single-cell RNAseq (scRNAseq) with scATACseq approach.
In Aim 4, I originally proposed to validate candidates by modulating their expression in a primary reprogramming model but the implementation of the microfluidic approach allowed the discovery of novel candidates and the simultaneous direct validation, thus accelerating the overall project performance. Therefore, I decided to use the TERA (Transcription Factor Epigenetic Remodeling Activity) score to carry on a de novo discovery on a list of 300 TFs, ranked on their ability to act on cellular plasticity. On such TFs, we already performed a comprehensive dosage profiling using RNAseq, ATACseq, morphology assay (immunofluorescence-based) and CUT&RUN (cleavage under targets and release using nuclease) sequencing approach to study each TF binding to DNA.