Novel single cell multiomics approaches to overcome barriers to interspecies chimerism for generating exogenic organs and cells

Abstract

There is a massive shortage of organ donors available for transplantation therapy, and millions of people die every year waiting for donor organs. The fields of interspecies chimerism and blastocyst complementation have shown promise towards addressing this organ shortage. The goal of these fields is to develop transplantable human organs in host animals like pigs, that can then be extracted and used in the clinic for transplantation therapy. While these fields hold great promise, there are still barriers that need to be overcome.One of the major barriers to successful interspecies chimerism is the mismatch in the developmental stages of the donor and the host cells, in the chimeric embryo. In Aim 1, we interrogated scRNA sequenced datasets of early embryos and stem cells from four commonly used donor and host species- human, marmoset, mouse, and pig. We found that the human blastocyst (E6/E7) best matched with the gastrulating mouse embryo (E6-E6.75), the marmoset late inner cell mass, and the pig late blastocyst. Another major barrier to successful chimerism is the mismatch in developmental speeds between the donor and host cells in the chimeric embryos. To address this barrier, there is first a need to define developmental speed at the single cell level. In Aim 2, we developed a novel single cell (sc) multiomics approach to simultaneously analyze replication timing (RT) and gene expression, to define developmental speed. RT is an epigenetic cell-type specific feature, that allows us to analyze how genes replicate, early or late, during the S-phase of the cell cycle. We optimized this approach in a well-established cancer cell line HepG2 and for the first time, demonstrated RT and gene expression interactions within single cells. We were able to capture conserved patterns between cells and cell-to-cell variations. Mouse is the most commonly used host embryo in chimerism studies. Hence, in aim 3, we analyzed wildtype (WT) early mouse preimplantation embryos using the sc-multiomics approach. Analysis of the WT mouse embryos provided us with novel chromatin accessibility, genome, and transcriptome trends that could be observed for the first time, using the sc- multiomics approach. The techniques used in this aim can be used to analyze donor and host cells in chimeric embryos. This provides us with molecular targets for enhancing interspecies chimerism and driving the field towards its end goal of generating exogenic human organs for transplantation therapy in the clinic.