Periodic Reporting for period 3 - CellKarma (Dissecting the regulatory logic of cell fate reprogramming through integrative and single cell genomics)
Okres sprawozdawczy: 2021-03-01 do 2022-08-31
The concept that any cell type, upon delivery of the right “cocktail” of transcription factors, can acquire an identity that otherwise it would never achieve, revolutionized the way we approach the study of developmental biology. In light of this, the discovery of induced pluripotent stem cells (IPSCs) and cell fate conversion approaches stimulated new research directions into human regenerative biology. In particular, reprogramming to pluripotency offers an attractive avenue to explore: IPSCs can be produced from almost any type of somatic cell, have an indefinite self-renewal capability and a broad differentiation potential. Moreover, reprogrammed cells circumvent ethical and immunological issues related to the use of human embryonic stem cells (ESCs), providing a valuable resource for the development of patient-tailored therapies. However, the chance to successfully develop patient-tailored therapies is still very limited because reprogramming technologies are applied without a comprehensive understanding of the molecular processes involved. This project offered a multifaceted approach that combines a wide range of cutting-edge integrative genomic strategies to significantly advance our understanding of the regulatory logic driving cell fate decisions during human reprogramming to pluripotency. The overall goal of the proposed project is to define novel rules governing the transitions among different cellular states during the reprogramming to pluripotency at both transcriptional, epigenetic and genetic layers. This approach will identify the regulators and roadblocks of reprogramming to pluripotency and highlight the identity and plasticity of its intermediate stages. This will be done through a meticulous analysis at unprecedented resolution leading to the identification of single cell transcriptional trajectories (Aim 1), the dissection of the regulatory relationships among Transcription Factors (TFs) at promoter and enhancer regions (Aim 2) and the assessment of the impact of reprogramming-induced genetic mutations (Aim 3). Ultimately, this will lead to identifying modulators required to improve the quality of IPSC reprogramming (Aim 4). In summary, this project will expose novel determinants and yet unidentified molecular barriers of reprogramming to pluripotency and will be essential to unlock the full potential of reprogramming technologies for shaping cellular identity in vitro and to address pressing challenges of regenerative medicine.
The overall goal of the proposed project is to define novel rules governing the transitions among different cellular states during the reprogramming to pluripotency at both transcriptional, epigenetic and genetic layers. In the original proposal, I anticipated that in the first three years we would make significant progress towards the characterization of the major regulatory features leading to reprogramming dynamics, and indeed we successfully identified reprogramming intermediate stages (Aim 1), uncovering a transient population that we hypothesize to have a functional role in supporting the formation of IPSCs. Taking advantage of Gene Set Enrichment Analysis (GSEA), we observed that this population has a strong enrichment for matrisome-related signatures and proteins involved in many signaling pathways. Altogether, these findings suggest that during reprogramming not only fibroblasts are converted to a primed pluripotent phenotype, but also the supportive extracellular context is shaped accordingly. As for Aim 2, we set up the MPRA (Massive Parallel Reporter Assay) and it will soon be applied to dissect the promoter and enhancer activities of dynamic DNA regulatory regions. In addition, we successfully developed a scorecard to assess IPSC differentiation, and set up a single-cell ATACseq (scATACseq) procedure to define TF networks that shape the epigenetic identity of IPSCs. In the context of Aim 3, the Whole Exome Sequencing (WES) approach allowed us to identify a significantly higher rate of mutations in IPSCs, compared to un-reprogrammed somatic fibroblasts. Finally, the work done for Aim 4 generated some preliminary results that indicate significant conversion-specific changes for many TFs, thus implying an important role in cell fate decisions.
In summary, I developed all points described in each project task and also amplified the range of outcomes with novel and more efficient strategies. Some of such choices have also been undertaken as a natural adaptation to the ongoing scientific field changes and demands.
In summary, I developed all points described in each project task and also amplified the range of outcomes with novel and more efficient strategies. Some of such choices have also been undertaken as a natural adaptation to the ongoing scientific field changes and demands.
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.