Periodic Report Summary 1 - CELL FATE (Single-Cell Epigenetics in Cell Fate Determination)
Summary description of the project objectives
The demonstration of induced pluripotency and direct lineage conversion of cells has led to remarkable insights regarding the cellular mechanisms that mediate cell state transitions. However, approaches to further elucidate the molecular mechanisms behind induced reprogramming are hindered by the inefficiency of induction and the interplay of reprogramming mechanisms with other cellular processes such as the cell cycle, which leads to variability in induced cell populations and limits the conclusiveness of bulk analyses. For this project we propose to develop mass cytometry-based methods to monitor mRNA levels and ideally gene locus-specific epigenetic modifications in single-cells. Thereby, the multi-dimensionality of mass cytometry enables simultaneous measurements of up to 40 additional single-cell parameters, such as surface markers and intracellular phosphorylation sites. This platform will be used to profile cell populations at specific time-points during reprogramming of human somatic cells into induced pluripotent stem cells. The obtained data will be organized into clusters of similar cell phenotypes resulting in continuous single-cell maps that chart marker expressions during induced reprogramming. In combination, these analyses will lead to a more detailed understanding of the hierarchical and dynamic organization of reprogramming and will ideally define events that are rate-limiting for the process. This fine-grained view of reprogramming will then be used to define minimal combinations of early cellular markers that are likely to predict cell fate. In the final stage of the project, long-term single-cell imaging will be used to monitor cells with these previously defined properties to confirm their specific clonal future with absolute certainty.
Description of the work performed since the beginning of the project
As described in the proposal, the major objectives for the first 12 months of the project were the development of mass cytometry-based methods to measure RNA molecules and (gene-locus specific) epigenetic modifications as well as the validation of antibodies to monitor epigenetic reprogramming by mass cytometry. To reach these goals different strategies have been tried to detect nucleic acids (RNA) and the proximity of modified histones with defined DNA sequences (epigenetic modifications) in single cells using proximity ligation-based approaches in combination with mass cytometry. Thereby, it was crucial to develop methods that were compatible with traditional antibody staining of proteins so that protein epitopes could be measured simultaneously with RNA and/or gene-specific epigenetic modifications. Accordingly, many different conditions have been tried to simultaneously perform nucleic acid hybridizations and antibody staining in single cells and to stabilize antibody staining prior to nucleic acid hybridizations. In parallel, antibodies have been validated to monitor the cell cycle status, transcription factor expression, surface marker expression, and phosphorylation-based signaling in millions of individual cells during reprogramming. The development of a novel method to measure RNA by mass cytometry also enabled the measurement of many gene products on the transcript level instead of the protein level so that any surface marker or transcription factor can be measured without the need for suitable antibodies. The potential of this novel technology has immediately been demonstrated through applications with complex populations of cells at different time points during cellular reprogramming.
Description of the main results achieved so far
The main result of the project achieved so far has been the development of a method to efficiently measure RNA in single cells by flow and mass cytometry. Currently, this method enables the simultaneous measurement of up to 40 different transcripts and proteins by mass cytometry. The method is based on the principles of proximity ligation and rolling circle amplification that lead to remarkable sensitivity and specificity. This method can now be used to measure transcripts and the corresponding proteins simultaneously, which enables the study of the interplay between transcription and translation. Alternatively, transcripts can be measured instead of proteins so that panels for RNA-only experiments can be designed at a fraction of the time and costs involved in the design of antibody-based experiments and without the limitation of antibody availability for genes of interest. Such RNA-only experiments are especially useful for the study of reprogramming cellular populations as signal to noise ratios for some of the pluripotency-associated transcription factors were shown to be better on the transcript level compared to antibody-based protein measurements and antibodies for several genes of interest were not commercially available. The adaptation of this technology to mass cytometry has been enabled through the development of suitable detection reagents based on the efficient production and purification of metal-oligonucleotide conjugates. Multiplexing has been achieved through interference-free signal amplification based on specific oligonucleotide hybridizations and enzymatic steps. The final technology has been patented in the US and a corresponding scientific manuscript is currently accepted for publication. An adaptation of this technology has been shown to enable the detection of the interaction of proteins with DNA (in a non-sequence-specific manner) and a corresponding scientific manuscript has been prepared.
Expected final results and their potential impact
This project has been terminated prematurely after 12 months since the researcher carrying out the corresponding experiments has been offered a position at a pharmaceutical company in Europe based on the results obtained so far. Accordingly, no major additional results are expected in the future but the results obtained during the initial phase of the project already have a far-reaching impact and are presented in the form of scientific publications and a patent. Most importantly, the mass cytometry-based method for simultaneous detection of transcripts and proteins has been accepted for publication in a scientific journal with a broad readership and has already been shared with flow and mass cytometry laboratories around the world. This technology is a game changer especially for laboratories working with mass cytometry since it drastically reduces the costs and time associated with the design and validation of highly multiplexed panels compared to traditional antibody-based experiments. Technological cornerstones of this method are the multiplexed amplification of proximity-based ligation events and the detection of amplified products via metal-chelated oligonucleotides. These innovations can easily be adapted for the multiplexed detection of hybridized oligonucleotides in related applications as has been demonstrated with the development of an additional technology for the detection of interactions of proteins with DNA. Furthermore, specific conditions have been established for the simultaneous detection of nucleic acids (through hybridization of labeled oligonucleotides) and protein epitopes (through antibody staining). The optimized conditions, such as gentle hybridization conditions and antibody crosslinking steps, are of general utility in a multitude of molecular biology protocols.
The demonstration of induced pluripotency and direct lineage conversion of cells has led to remarkable insights regarding the cellular mechanisms that mediate cell state transitions. However, approaches to further elucidate the molecular mechanisms behind induced reprogramming are hindered by the inefficiency of induction and the interplay of reprogramming mechanisms with other cellular processes such as the cell cycle, which leads to variability in induced cell populations and limits the conclusiveness of bulk analyses. For this project we propose to develop mass cytometry-based methods to monitor mRNA levels and ideally gene locus-specific epigenetic modifications in single-cells. Thereby, the multi-dimensionality of mass cytometry enables simultaneous measurements of up to 40 additional single-cell parameters, such as surface markers and intracellular phosphorylation sites. This platform will be used to profile cell populations at specific time-points during reprogramming of human somatic cells into induced pluripotent stem cells. The obtained data will be organized into clusters of similar cell phenotypes resulting in continuous single-cell maps that chart marker expressions during induced reprogramming. In combination, these analyses will lead to a more detailed understanding of the hierarchical and dynamic organization of reprogramming and will ideally define events that are rate-limiting for the process. This fine-grained view of reprogramming will then be used to define minimal combinations of early cellular markers that are likely to predict cell fate. In the final stage of the project, long-term single-cell imaging will be used to monitor cells with these previously defined properties to confirm their specific clonal future with absolute certainty.
Description of the work performed since the beginning of the project
As described in the proposal, the major objectives for the first 12 months of the project were the development of mass cytometry-based methods to measure RNA molecules and (gene-locus specific) epigenetic modifications as well as the validation of antibodies to monitor epigenetic reprogramming by mass cytometry. To reach these goals different strategies have been tried to detect nucleic acids (RNA) and the proximity of modified histones with defined DNA sequences (epigenetic modifications) in single cells using proximity ligation-based approaches in combination with mass cytometry. Thereby, it was crucial to develop methods that were compatible with traditional antibody staining of proteins so that protein epitopes could be measured simultaneously with RNA and/or gene-specific epigenetic modifications. Accordingly, many different conditions have been tried to simultaneously perform nucleic acid hybridizations and antibody staining in single cells and to stabilize antibody staining prior to nucleic acid hybridizations. In parallel, antibodies have been validated to monitor the cell cycle status, transcription factor expression, surface marker expression, and phosphorylation-based signaling in millions of individual cells during reprogramming. The development of a novel method to measure RNA by mass cytometry also enabled the measurement of many gene products on the transcript level instead of the protein level so that any surface marker or transcription factor can be measured without the need for suitable antibodies. The potential of this novel technology has immediately been demonstrated through applications with complex populations of cells at different time points during cellular reprogramming.
Description of the main results achieved so far
The main result of the project achieved so far has been the development of a method to efficiently measure RNA in single cells by flow and mass cytometry. Currently, this method enables the simultaneous measurement of up to 40 different transcripts and proteins by mass cytometry. The method is based on the principles of proximity ligation and rolling circle amplification that lead to remarkable sensitivity and specificity. This method can now be used to measure transcripts and the corresponding proteins simultaneously, which enables the study of the interplay between transcription and translation. Alternatively, transcripts can be measured instead of proteins so that panels for RNA-only experiments can be designed at a fraction of the time and costs involved in the design of antibody-based experiments and without the limitation of antibody availability for genes of interest. Such RNA-only experiments are especially useful for the study of reprogramming cellular populations as signal to noise ratios for some of the pluripotency-associated transcription factors were shown to be better on the transcript level compared to antibody-based protein measurements and antibodies for several genes of interest were not commercially available. The adaptation of this technology to mass cytometry has been enabled through the development of suitable detection reagents based on the efficient production and purification of metal-oligonucleotide conjugates. Multiplexing has been achieved through interference-free signal amplification based on specific oligonucleotide hybridizations and enzymatic steps. The final technology has been patented in the US and a corresponding scientific manuscript is currently accepted for publication. An adaptation of this technology has been shown to enable the detection of the interaction of proteins with DNA (in a non-sequence-specific manner) and a corresponding scientific manuscript has been prepared.
Expected final results and their potential impact
This project has been terminated prematurely after 12 months since the researcher carrying out the corresponding experiments has been offered a position at a pharmaceutical company in Europe based on the results obtained so far. Accordingly, no major additional results are expected in the future but the results obtained during the initial phase of the project already have a far-reaching impact and are presented in the form of scientific publications and a patent. Most importantly, the mass cytometry-based method for simultaneous detection of transcripts and proteins has been accepted for publication in a scientific journal with a broad readership and has already been shared with flow and mass cytometry laboratories around the world. This technology is a game changer especially for laboratories working with mass cytometry since it drastically reduces the costs and time associated with the design and validation of highly multiplexed panels compared to traditional antibody-based experiments. Technological cornerstones of this method are the multiplexed amplification of proximity-based ligation events and the detection of amplified products via metal-chelated oligonucleotides. These innovations can easily be adapted for the multiplexed detection of hybridized oligonucleotides in related applications as has been demonstrated with the development of an additional technology for the detection of interactions of proteins with DNA. Furthermore, specific conditions have been established for the simultaneous detection of nucleic acids (through hybridization of labeled oligonucleotides) and protein epitopes (through antibody staining). The optimized conditions, such as gentle hybridization conditions and antibody crosslinking steps, are of general utility in a multitude of molecular biology protocols.