Periodic Reporting for period 1 - RePLASTIC (Uncovering the Regulators of Cellular Plasticity by Direct Reprogramming)
Reporting period: 2022-08-01 to 2024-07-31
Summary of the context and overall objectives of the project
Developmental specification is characterized by the transition of sequential cell identities that display different degrees of plasticity, ultimately leading to the generation of the numerous different cell types found in an organism. This process starts with a highly plastic cell that drops its plasticity capacity as it commits and acquires a certain cell identity, transitioning through progenitors until differentiated somatic cells. The key determinants that rule cell specification are the transcription factors, which are assisted by chromatin regulators and RNA modifiers that help to keep cell identity. In alternative to the developmental pathways, the attainment of another somatic cell type identity can also be achieved by cell reprogramming, evidencing how highly plastic cells can be. Additionally, cancer cells also exhibit remarkable plasticity, allowing them to switch lineages, develop stem-like properties, drive invasiveness, and resist treatments. Yet the principles governing plasticity in processes like development, reprogramming, and cancer cells remain poorly understood.
Altogether, the key interrogations are how cellular identities are committed to a particular cell type, what defines a cell type distinguishable from other cells, and how degrees of plasticity can account for such differences. The emergence of technologies like direct reprogramming approaches, deep sequencing, and gene editing convey an excellent time in science to tackle these questions. Focused on developing an innovative strategy, the general aim of this project is to use an integrative approach to uncover molecular drivers of degrees of cellular plasticity that allow or restrict interconversion between cellular identities. To achieve this, I will take advantage of the unique reprogramming systems developed and used in the host laboratory to induce unipotent dendritic cells type 1 (iDC1), multipotent hematopoietic stem and progenitor cells (iHSPC), and pluripotent stem cells (iPSC) from human dermal fibroblasts (HDF) coupled with CRISPR/Cas9 knock-out (KO) screens targeting chromatin and RNA modifiers. This methodology will be employed to induce genetic perturbation and identify critical regulators that impact the reprogramming outcomes, therefore critical for cellular plasticity. With an interdisciplinary approach taking a leaf from the fields of gene editing, stem cells and immunology, this work will allow us to address for the first time how different degrees of plasticity are established de novo. By investigating the facilitators or barriers of plasticity at the overlap of the three cellular conversions I will identify 1) players implicated in cell state interconversion and “universal” plasticity, 2) molecular drivers conferring the potential to generate additional cell types (increased plasticity), 3) genes controlling entry to the hematopoietic lineage and 4) genes in the unipotency-pluripotency transition that could contribute to interesting revelations about this two states of plasticity. These findings will collectively reveal a world of possibilities for interrogating the complex interplay of TFs, chromatin remodelers and RNA modifiers on the basic principles of cellular identity and degrees of plasticity, relevant knowledge for several biological processes like cell reprogramming, cancer, regeneration and development.
Altogether, the key interrogations are how cellular identities are committed to a particular cell type, what defines a cell type distinguishable from other cells, and how degrees of plasticity can account for such differences. The emergence of technologies like direct reprogramming approaches, deep sequencing, and gene editing convey an excellent time in science to tackle these questions. Focused on developing an innovative strategy, the general aim of this project is to use an integrative approach to uncover molecular drivers of degrees of cellular plasticity that allow or restrict interconversion between cellular identities. To achieve this, I will take advantage of the unique reprogramming systems developed and used in the host laboratory to induce unipotent dendritic cells type 1 (iDC1), multipotent hematopoietic stem and progenitor cells (iHSPC), and pluripotent stem cells (iPSC) from human dermal fibroblasts (HDF) coupled with CRISPR/Cas9 knock-out (KO) screens targeting chromatin and RNA modifiers. This methodology will be employed to induce genetic perturbation and identify critical regulators that impact the reprogramming outcomes, therefore critical for cellular plasticity. With an interdisciplinary approach taking a leaf from the fields of gene editing, stem cells and immunology, this work will allow us to address for the first time how different degrees of plasticity are established de novo. By investigating the facilitators or barriers of plasticity at the overlap of the three cellular conversions I will identify 1) players implicated in cell state interconversion and “universal” plasticity, 2) molecular drivers conferring the potential to generate additional cell types (increased plasticity), 3) genes controlling entry to the hematopoietic lineage and 4) genes in the unipotency-pluripotency transition that could contribute to interesting revelations about this two states of plasticity. These findings will collectively reveal a world of possibilities for interrogating the complex interplay of TFs, chromatin remodelers and RNA modifiers on the basic principles of cellular identity and degrees of plasticity, relevant knowledge for several biological processes like cell reprogramming, cancer, regeneration and development.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
The first aim of the project was to develop a powerful platform for gene editing in primary human dermal fibroblasts (HDFs). I used at least 3 different donors of HDF, contemplating both female and male, and an age range between 22-55 years old. This was an important experimental decision to account for donor diversity and to increase the robustness of the results. I established HDFs constitutively expressing the Cas9 protein, as well as a resistance cassette for the antibiotic blasticidin for the selection of Cas9-only cells. I assessed the appropriate Cas9 dose by nock-out (KO) activity in the different donors, ensuring the as well the cell viability. As for the CRISPR/Cas9 sgRNA library, I designed a custom-made, pooled sgRNA library targeting several candidate chromatin remodelers, as well as RNA modifiers and splicing factors (SF), called KORE library (Knock-Out RNA and Epigenetic regulators). The list of genes comprises 1,367 genes, including controls, selected from published databases and from collaborators. For each gene, 5 targeting sgRNAs were retrieved and non-targeting guides were also included as controls to evaluate noise and screening success. In total, the KORE library comprises a total of 7,400 guides. These were cloned as a pool into a lentiviral vector that contains a chimeric guide RNA backbone and a GFP cassette as a reporter. To ensure that each cell receives only one sgRNA construct, I tittered the lentivirus particles to achieve a functional MOI of approximately 0.3-0.4. Cas9-HDFs from all donors were transduced with the KORE library and later selected for GFP-positive cells by fluorescence-activated cytometry sorting (FACS) to enrich the cell population to use in future experiments.
After the incorporation of the KORE library and gene KO, I performed cell reprogramming experiments to generate representative cells with different degrees of plasticity from the engineered HDF cells. I followed specific protocols to obtain iDC1, iHSPC and iPSC using polycistronic vectors, all factors inducing a certain cell fate in a single vector. By ectopic expression of the human TFs PU.1 IRF8, BATF3 (PIB), I achieved direct conversion to iDC1, an example of unipotent cells. In the case of iHSPC, the expression of GATA2, GFI1B, and FOS is sufficient to obtain multipotent cells committed to the hemogenic fate. Moreover, the enforced expression of OCT4, SOX2, KLF4, and c-MYC TFs instructs reprogramming to iPSC. Once reprogramming was completed for each protocol (iDC1: day 9, iHSPC: day 15, iPSC: day 20), the endpoint bulk cell population was classified into different subpopulations of non-reprogrammed or reprogrammed cells by immunostaining of validated cell surface markers (iDC1: CD45, HLA-DR; iHSPC: CD9, CD49f; iPSC: CD13, EpCAM, TRA-1-60, SSEA4) and cell sorting. A day 0 sample (D0) was also collected as baseline, for unreprogrammed cells that were edited since they contain the Cas9 and the KORE library. Once all samples were collected, I continued with mapping the gene perturbations by next-generation sequencing (NGS). In this sense, genomic DNA was extracted and gRNA cassette amplified, and finally sequenced the library to evaluate sgRNA enrichment.
Later on, I focused on the data exploration and identification of candidate genes by the analysis of hits retrieved from performing deep sequencing of the samples collected after the reprogramming experiments. I classified different cell populations regarding their reprogramming efficiency and identified groups of genes. On one hand, genes depleted in the reprogrammed cells could act as negative regulators or barriers to the reprogramming process. In this scenario, silencing of these genes would hypothetically benefit the reprogramming outcome. On the opposite, another list containing positive regulators or conversion facilitators, namely genes depleted in non-reprogrammed cells. Here, the lack of expression would be detrimental for reprogramming, and cells will not interconvert even in a favourable scenario. These barriers and facilitators were proposed as molecular targets that could be functionally important in plasticity regulation.
For the bioinformatic analysis, I applied the MAGeCKFlute pipeline for the identification of the plasticity-associated genes. First, I normalized the endpoint hits with the baseline sample D0 for every sorted population, to then calculate the sgRNA abundance-based rank difference between reprogrammed and non-reprogrammed cells to retrieve lists of genes. To hone the list of top candidates, I checked the correlation between the replicated screens, looking across donors. Taking into consideration a threshold of 1.5 standard deviation from the median, I collected the lists of top barriers and facilitators from the 3 reprogramming systems. Since I used the same sgRNA library in all the reprogramming systems, I carried out a comparative analysis to explore the relationship between sets of regulators previously sorted and examine genes shared in the intersections of unipotency, multipotency, and pluripotency. Through this assessment, I anticipated uncovering a list of chromatin and RNA modifiers that play a significant role in cellular plasticity. The genes identified in all the reprogramming systems were described as ‘universal’ regulators of reprogramming and represent master regulators of cell identity modification. Additionally, genes shared among pluripotent and multipotent cells were classified as regulators for increased plasticity given their possible role as molecular drivers that allow or preclude the generation of multiple cell types. Lineage-commitment regulators were identified at the intersection between multipotency and unipotency. In this case, involved in hematopoiesis specification, or inversely, in preserving the multipotent state, a context frequently associated with blood-related diseases.
The results obtained validated the experimental pipeline decided on the first aim and provided evidence of the feasibility of the approach. For iDC1 reprogramming, I listed 113 barriers and 72 candidate genes as facilitators. In the case of iHSPC, there were 95 barriers and 111 facilitators identified. Finally, for iPSC, the candidate genes came to 82 barriers and 125 facilitators. Regarding the barriers in the overlapping analysis, when doing 2 at a time comparisons, I identified 24 genes shared among iPSC-iHSPC, 33 genes among iHSPC-iDC, and 27 genes between iDC-iPSC. Finally, I was able to uncover 4 shared hits that could be described as possible universal barriers of cell plasticity.
Lastly, I proposed the validation of the top hits from the ranked lists of candidate genes by secondary screens. I focused on some of the interesting candidate genes including the ones present in the intersections from the integrative analysis, but also significant hits from the iDC1 and the iHSPCs screenings since they have a clear translational impact on cancer-related cell therapies. After shortlisting the genes, I selected the 2 sgRNAs that performed best for each candidate gene and validated them for the previously observed reprogramming outcome from the screening with the pooled KORE library. These guides were simultaneously cloned in a multiplex vector containing Cas9 expression as well, for lentiviral delivery. To start with, I selected barrier and facilitator candidates from the iDC1 screening and repeated the reprogramming experiments and flow cytometry analysis for a comprehensive phenotyping exploration at the respective endpoint. In addition, I analyzed genomic DNA disruption by Sanger sequencing with custom primers and ICE analysis for efficient gene KO.
Robust hits from the intersections of plasticity will be further studied to delve into their molecular mechanism and functionality. I plan to perform RNA-sequencing experiments to study the transcriptional changes that these KO have at a global scale on the cells. I will also perform ATAC-seq and ChIP-seq experiments to investigate the chromatin state and possible targets in the genome for the studied proteins.
After the incorporation of the KORE library and gene KO, I performed cell reprogramming experiments to generate representative cells with different degrees of plasticity from the engineered HDF cells. I followed specific protocols to obtain iDC1, iHSPC and iPSC using polycistronic vectors, all factors inducing a certain cell fate in a single vector. By ectopic expression of the human TFs PU.1 IRF8, BATF3 (PIB), I achieved direct conversion to iDC1, an example of unipotent cells. In the case of iHSPC, the expression of GATA2, GFI1B, and FOS is sufficient to obtain multipotent cells committed to the hemogenic fate. Moreover, the enforced expression of OCT4, SOX2, KLF4, and c-MYC TFs instructs reprogramming to iPSC. Once reprogramming was completed for each protocol (iDC1: day 9, iHSPC: day 15, iPSC: day 20), the endpoint bulk cell population was classified into different subpopulations of non-reprogrammed or reprogrammed cells by immunostaining of validated cell surface markers (iDC1: CD45, HLA-DR; iHSPC: CD9, CD49f; iPSC: CD13, EpCAM, TRA-1-60, SSEA4) and cell sorting. A day 0 sample (D0) was also collected as baseline, for unreprogrammed cells that were edited since they contain the Cas9 and the KORE library. Once all samples were collected, I continued with mapping the gene perturbations by next-generation sequencing (NGS). In this sense, genomic DNA was extracted and gRNA cassette amplified, and finally sequenced the library to evaluate sgRNA enrichment.
Later on, I focused on the data exploration and identification of candidate genes by the analysis of hits retrieved from performing deep sequencing of the samples collected after the reprogramming experiments. I classified different cell populations regarding their reprogramming efficiency and identified groups of genes. On one hand, genes depleted in the reprogrammed cells could act as negative regulators or barriers to the reprogramming process. In this scenario, silencing of these genes would hypothetically benefit the reprogramming outcome. On the opposite, another list containing positive regulators or conversion facilitators, namely genes depleted in non-reprogrammed cells. Here, the lack of expression would be detrimental for reprogramming, and cells will not interconvert even in a favourable scenario. These barriers and facilitators were proposed as molecular targets that could be functionally important in plasticity regulation.
For the bioinformatic analysis, I applied the MAGeCKFlute pipeline for the identification of the plasticity-associated genes. First, I normalized the endpoint hits with the baseline sample D0 for every sorted population, to then calculate the sgRNA abundance-based rank difference between reprogrammed and non-reprogrammed cells to retrieve lists of genes. To hone the list of top candidates, I checked the correlation between the replicated screens, looking across donors. Taking into consideration a threshold of 1.5 standard deviation from the median, I collected the lists of top barriers and facilitators from the 3 reprogramming systems. Since I used the same sgRNA library in all the reprogramming systems, I carried out a comparative analysis to explore the relationship between sets of regulators previously sorted and examine genes shared in the intersections of unipotency, multipotency, and pluripotency. Through this assessment, I anticipated uncovering a list of chromatin and RNA modifiers that play a significant role in cellular plasticity. The genes identified in all the reprogramming systems were described as ‘universal’ regulators of reprogramming and represent master regulators of cell identity modification. Additionally, genes shared among pluripotent and multipotent cells were classified as regulators for increased plasticity given their possible role as molecular drivers that allow or preclude the generation of multiple cell types. Lineage-commitment regulators were identified at the intersection between multipotency and unipotency. In this case, involved in hematopoiesis specification, or inversely, in preserving the multipotent state, a context frequently associated with blood-related diseases.
The results obtained validated the experimental pipeline decided on the first aim and provided evidence of the feasibility of the approach. For iDC1 reprogramming, I listed 113 barriers and 72 candidate genes as facilitators. In the case of iHSPC, there were 95 barriers and 111 facilitators identified. Finally, for iPSC, the candidate genes came to 82 barriers and 125 facilitators. Regarding the barriers in the overlapping analysis, when doing 2 at a time comparisons, I identified 24 genes shared among iPSC-iHSPC, 33 genes among iHSPC-iDC, and 27 genes between iDC-iPSC. Finally, I was able to uncover 4 shared hits that could be described as possible universal barriers of cell plasticity.
Lastly, I proposed the validation of the top hits from the ranked lists of candidate genes by secondary screens. I focused on some of the interesting candidate genes including the ones present in the intersections from the integrative analysis, but also significant hits from the iDC1 and the iHSPCs screenings since they have a clear translational impact on cancer-related cell therapies. After shortlisting the genes, I selected the 2 sgRNAs that performed best for each candidate gene and validated them for the previously observed reprogramming outcome from the screening with the pooled KORE library. These guides were simultaneously cloned in a multiplex vector containing Cas9 expression as well, for lentiviral delivery. To start with, I selected barrier and facilitator candidates from the iDC1 screening and repeated the reprogramming experiments and flow cytometry analysis for a comprehensive phenotyping exploration at the respective endpoint. In addition, I analyzed genomic DNA disruption by Sanger sequencing with custom primers and ICE analysis for efficient gene KO.
Robust hits from the intersections of plasticity will be further studied to delve into their molecular mechanism and functionality. I plan to perform RNA-sequencing experiments to study the transcriptional changes that these KO have at a global scale on the cells. I will also perform ATAC-seq and ChIP-seq experiments to investigate the chromatin state and possible targets in the genome for the studied proteins.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
I generated a universal and scalable genetic perturbation platform based on the CRISPR/Cas9 toolbox in human fibroblasts for general use across lineage interconversion and with the possibility of being applied to other reprogramming systems.
I established several lists of candidate genes that can play a role in modulating cell plasticity in different reprogramming systems. These lists were overlapped to reveal shared genes among them, identifying hits that can have a broader impact across cell plasticity establishment and maintenance. This integrative analysis was the most critical experiment in the project since the vast amount of results obtained will open new questions and hypotheses to explore ahead, contributing to shaping the development and finalizing the project.
As a step forward, through an exhaustive validation experiment, I was able to validate the candidate genes SND1 and HIRA as barriers of iDC1 reprogramming. The follow-up experiments will tackle their mechanism of action during this conversion. This approach will be taken to the other reprogramming platforms, not only regarding their specific barriers in each system but also on the common hits among them. Additionally, the uncovered genes will be studies on different contexts like in vivo reprogramming and cancer.
I established several lists of candidate genes that can play a role in modulating cell plasticity in different reprogramming systems. These lists were overlapped to reveal shared genes among them, identifying hits that can have a broader impact across cell plasticity establishment and maintenance. This integrative analysis was the most critical experiment in the project since the vast amount of results obtained will open new questions and hypotheses to explore ahead, contributing to shaping the development and finalizing the project.
As a step forward, through an exhaustive validation experiment, I was able to validate the candidate genes SND1 and HIRA as barriers of iDC1 reprogramming. The follow-up experiments will tackle their mechanism of action during this conversion. This approach will be taken to the other reprogramming platforms, not only regarding their specific barriers in each system but also on the common hits among them. Additionally, the uncovered genes will be studies on different contexts like in vivo reprogramming and cancer.