Periodic Reporting for period 2 - HUTER (HUMAN UTERUS CELL ATLAS)
Okres sprawozdawczy: 2021-01-01 do 2022-06-30
The Human Uterus Cell Atlas (HUTER) aimed to create a single-cell and spatial reference map of the human uterus and provide unprecedented insight to transcriptomic, genomic and spatial changes of this key female organ across both the menstrual cycle and the lifespan.
The overall objective is to better understand the human uterus to more effectively address conditions that impact women´s health, reproductive outcomes and maternal and foetal mortality and morbidity, and, by extension, negatively impact population well-being.
HUTER consortium have managed to successfully profile and study the cellular heterogeneity of the human uterus.
We have revealed the existence of previously uncharacterised cell states in the endometrium, including different epithelial populations driving regeneration of the endometrium during the menstrual cycle. Endometrial cells of the late secretory phase downregulate a gene signature in patients who suffered sPE in a previous pregnancy. In addition, we have reached for the first time, an unbiased identification of the cells residing in the myometrium, including at least 10 cell types, of which five poorly characterised in the literature so far.
The overall objective is to better understand the human uterus to more effectively address conditions that impact women´s health, reproductive outcomes and maternal and foetal mortality and morbidity, and, by extension, negatively impact population well-being.
HUTER consortium have managed to successfully profile and study the cellular heterogeneity of the human uterus.
We have revealed the existence of previously uncharacterised cell states in the endometrium, including different epithelial populations driving regeneration of the endometrium during the menstrual cycle. Endometrial cells of the late secretory phase downregulate a gene signature in patients who suffered sPE in a previous pregnancy. In addition, we have reached for the first time, an unbiased identification of the cells residing in the myometrium, including at least 10 cell types, of which five poorly characterised in the literature so far.
A total of 59 uterus samples (whole uterus and endometrial biopsies) from healthy donors and 25 endometrial biopsies from patients diagnosed with severe preeclampsia (sPE patients) and their healthy controls have been collected (Reproductive Medicine Collection, registered under reference C.0005866 in the national registry of biobanks in the ISCIII Health institute. Spain). During HUTER execution, samples processing has provide 117 cDNA libraries for sc-RNA sequencing, 37 paraffin blocks for Protein Atlas, 40 OCT blocks for Spatial Transcriptomics, 12 myometrial samples and 18 sPE & control endometrium sorted plates for sc-epigenomics and 53 snap frozen samples to ehole genomic sequencing (WSG).
HUTER partners have gained expertise in conducting the 10X Genomics Spatial Gene Expression protocols on uterine samples and profiling more than 1 million human uterine cells with single-cell or single-nuclei RNA-seq. Generated data has allowed a better characterisation of uterine tissues and helped to better define uterine cell subpopulations and cell-to-cell interactions. This information has revealed how cells organize into uterine tissues and allow the regulation of important processes such the menstrual cycle or embryo implantation, and how deregulation of those processes could contribute to infertility and disease.
Using the optimised scEM&T-seq protocol we generated single-cell transcriptomes and methylomes for the primary endometrial and myometrial samples. Processing of SMART-seq2 scRNA-seq libraries revealed that 56-89% of samples had worked efficiently. This project has generated single cell and in silico merged methylation atlases for endometrium and myometrium biopsies. Multi-omics protocols have been used to deconvolute samples into their major cell types using specific marker expression and determine their unique methylation profiles. Also cell-specific endometrial methylation signatures linked with aberrant profiles associated with sPE.
HUTER have also gained further expertise in all the technologies and pipelines that have been used for generation of high-resolution spatial maps. We generated Digital high-resolution images corresponding to all HUTER samples IHC stained, comprising in total 2,261 images. The spatial characterization of cell types in endometrium and myometrium was enhanced by the use of an immunofluorescence multiplex approach developed. Therefore, we have located in the tissue the previously uncharacterised cell states uncovered. One of the main focus has been on the novel epithelial progenitors previously defined in our single-cell transcriptomics dataset, as well as the microenvironment essential to maintain these progenitors.
The HUTER project also implemented an open-source platform making the first comprehensive single-cell and spatial atlas of the uterus across the lifespan accessible to the scientific community. The platform enabled data uploading and data query for raw data and analytical results from our single cell molecular characterisation, epigenomics, transcriptomics and spatial resolution images. The platform includes advanced visualization tools, providing intuitive visualization of data with high dimensionality, and facilitating comparative assessments. The HUTER platform performance has been validated by used to identify cell types, states and transcriptional fingerprints related to endometrial decidualization resistance in sPE patients. Using scRNA-seq results from scEM&T-seq dataset generated, we observed that the most robust differential expressed genes were located in within stromal-associated clusters.
Finally, HUTER jointly with other EU funded Consortia (pilot actions to build the foundations of the human cell atlas) have stablished alignment with different working groups of the Human Cell Atlas (HCA) initiative and HCA-DCP managers that will facilitate current and further progress in the field.
HUTER partners have gained expertise in conducting the 10X Genomics Spatial Gene Expression protocols on uterine samples and profiling more than 1 million human uterine cells with single-cell or single-nuclei RNA-seq. Generated data has allowed a better characterisation of uterine tissues and helped to better define uterine cell subpopulations and cell-to-cell interactions. This information has revealed how cells organize into uterine tissues and allow the regulation of important processes such the menstrual cycle or embryo implantation, and how deregulation of those processes could contribute to infertility and disease.
Using the optimised scEM&T-seq protocol we generated single-cell transcriptomes and methylomes for the primary endometrial and myometrial samples. Processing of SMART-seq2 scRNA-seq libraries revealed that 56-89% of samples had worked efficiently. This project has generated single cell and in silico merged methylation atlases for endometrium and myometrium biopsies. Multi-omics protocols have been used to deconvolute samples into their major cell types using specific marker expression and determine their unique methylation profiles. Also cell-specific endometrial methylation signatures linked with aberrant profiles associated with sPE.
HUTER have also gained further expertise in all the technologies and pipelines that have been used for generation of high-resolution spatial maps. We generated Digital high-resolution images corresponding to all HUTER samples IHC stained, comprising in total 2,261 images. The spatial characterization of cell types in endometrium and myometrium was enhanced by the use of an immunofluorescence multiplex approach developed. Therefore, we have located in the tissue the previously uncharacterised cell states uncovered. One of the main focus has been on the novel epithelial progenitors previously defined in our single-cell transcriptomics dataset, as well as the microenvironment essential to maintain these progenitors.
The HUTER project also implemented an open-source platform making the first comprehensive single-cell and spatial atlas of the uterus across the lifespan accessible to the scientific community. The platform enabled data uploading and data query for raw data and analytical results from our single cell molecular characterisation, epigenomics, transcriptomics and spatial resolution images. The platform includes advanced visualization tools, providing intuitive visualization of data with high dimensionality, and facilitating comparative assessments. The HUTER platform performance has been validated by used to identify cell types, states and transcriptional fingerprints related to endometrial decidualization resistance in sPE patients. Using scRNA-seq results from scEM&T-seq dataset generated, we observed that the most robust differential expressed genes were located in within stromal-associated clusters.
Finally, HUTER jointly with other EU funded Consortia (pilot actions to build the foundations of the human cell atlas) have stablished alignment with different working groups of the Human Cell Atlas (HCA) initiative and HCA-DCP managers that will facilitate current and further progress in the field.
HUTER has provided the foundations for innovating new diagnostic and prognostic tools for uterine dysfunctions, as it has provide the definition and organization of cell types in the human uterus main tissues (endometrium and myometrium). Large amount of single-cell sequencing data and high resolution spatial localisation of cell states based on transcriptomics and proteins has been generated, as well as valuable expertise in advanced single cell characterisation techniques (processing, characterisation, data integration and advanced analytics) applied specifically to the uterine tissues.
In addition to healthy uterine samples, HUTER has performed single-cell characterization of endometrial samples obtained from patients with preeclampsia and patients with endometriosis, providing first comparison between healthy and diseased endometrial tissue. Further comparisons are being the main objective of new research projects leaded by HUTER partners and potentially by other research teams, for the next years, such as the study of myometrial tumours, single-cell characterisation of main uterine pathologies, and the study and identification of endometrial progenitor cells. So that, new research in the field will be supported and empowered by HUTER delivered results.
Sustainability of HUTER platform (the computing infrastructure to run the repository and its functionalities + the data) is being considered by HUTER partners through the participation in new initiatives like Mission Cancer, Digital Europe, the European Health Data Space and Gaia-X that are pushing in the direction of populating and/or making available new and already existing health research data in EU. This may bring new opportunities for the HUTER Partners to contribute directly to other research initiatives in the future.
In addition to healthy uterine samples, HUTER has performed single-cell characterization of endometrial samples obtained from patients with preeclampsia and patients with endometriosis, providing first comparison between healthy and diseased endometrial tissue. Further comparisons are being the main objective of new research projects leaded by HUTER partners and potentially by other research teams, for the next years, such as the study of myometrial tumours, single-cell characterisation of main uterine pathologies, and the study and identification of endometrial progenitor cells. So that, new research in the field will be supported and empowered by HUTER delivered results.
Sustainability of HUTER platform (the computing infrastructure to run the repository and its functionalities + the data) is being considered by HUTER partners through the participation in new initiatives like Mission Cancer, Digital Europe, the European Health Data Space and Gaia-X that are pushing in the direction of populating and/or making available new and already existing health research data in EU. This may bring new opportunities for the HUTER Partners to contribute directly to other research initiatives in the future.