Characterization of Human Embryonic Stem Cell (hESC)-derived Teratomas via Single Cell RNA-Sequencing

Although the advent of human pluripotent stem cell (hPSC)-based 2D and 3D models has enabled great strides in our understanding of human development and disease, the limitations of these models, particularly the absence of cellular interactions across different lineages as well as vascular networks, make it difficult to accurately recapitulate molecular and physiological processes in vitro. Towards addressing this, we have characterized human embryonic stem cell (hESC)-derived teratomas in order to investigate their potential as alternative 3D modeling systems. As teratomas are naturally comprised of cells representing each of the three germ layers (endoderm, mesoderm, ectoderm) and vascularized, we believe such a model may enable recapitulation of cell-cell interactions and developmental processes otherwise difficult to study in a context relevant to human biology. As an initial step, we employed single cell RNA-sequencing (SC RNA-seq) methods for characterization of the cellular composition, diversity, and consistency of cell populations across hESC-derived teratomas. We found that as expected, cell lineages derived from each of the three germ layers (endoderm, mesoderm, ectoderm), as well as both early progenitor and more mature differentiated cell types, were present, along with different cell states. Additionally, we found that methodologies used to process teratomas play a critical role in scRNA-seq data interpretation. However, despite intrinsic teratoma heterogeneity, teratoma samples in general showed close alignment and representation across clusters, indicating a level of predictability to this system. Taken together, the results indicate that teratomas hold potential as a novel alternative modeling system for the study and recapitulation of normal and diseased multi-lineage human development

Characterization of Human Embryonic Stem Cell (hESC)-derived Teratomas via Single Cell RNA-Sequencing

Although the advent of human pluripotent stem cell (hPSC)-based 2D and 3D models has enabled great strides in our understanding of human development and disease, the limitations of these models, particularly the absence of cellular interactions across different lineages as well as vascular networks, make it difficult to accurately recapitulate molecular and physiological processes in vitro. Towards addressing this, we have characterized human embryonic stem cell (hESC)-derived teratomas in order to investigate their potential as alternative 3D modeling systems. As teratomas are naturally comprised of cells representing each of the three germ layers (endoderm, mesoderm, ectoderm) and vascularized, we believe such a model may enable recapitulation of cell-cell interactions and developmental processes otherwise difficult to study in a context relevant to human biology. As an initial step, we employed single cell RNA-sequencing (SC RNA-seq) methods for characterization of the cellular composition, diversity, and consistency of cell populations across hESC-derived teratomas. We found that as expected, cell lineages derived from each of the three germ layers (endoderm, mesoderm, ectoderm), as well as both early progenitor and more mature differentiated cell types, were present, along with different cell states. Additionally, we found that methodologies used to process teratomas play a critical role in scRNA-seq data interpretation. However, despite intrinsic teratoma heterogeneity, teratoma samples in general showed close alignment and representation across clusters, indicating a level of predictability to this system. Taken together, the results indicate that teratomas hold potential as a novel alternative modeling system for the study and recapitulation of normal and diseased multi-lineage human development

Apoptotic dysregulation mediates stem cell competition and tissue regeneration

Abstract Since adult stem cells are responsible for replenishing tissues throughout life, it is vital to understand how failure to undergo apoptosis can dictate stem cell behavior both intrinsically and non-autonomously. Here, we report that depletion of pro-apoptotic Bax protein bestows hair follicle stem cells with the capacity to eliminate viable neighboring cells by sequestration of TNFα in their membrane. This in turn induces apoptosis in “loser” cells in a contact-dependent manner. Examining the underlying mechanism, we find that Bax loss-of-function competitive phenotype is mediated by the intrinsic activation of NFκB. Notably, winner stem cells differentially respond to TNFα, owing to their elevated expression of TNFR2. Finally, we report that in vivo depletion of Bax results in an increased stem cell pool, accelerating wound-repair and de novo hair follicle regeneration. Collectively, we establish a mechanism of mammalian cell competition, which can have broad therapeutic implications for tissue regeneration and tumorigenesis

Thy1 marks a distinct population of slow-cycling stem cells in the mouse epidermis

Abstract The presence of distinct stem cells that maintain the interfollicular epidermis is highly debated. Here, we report a population of keratinocytes, marked by Thy1, in the basal layer of the interfollicular epidermis. We find that epidermal cells expressing differential levels of Thy1 display distinct transcriptional signatures. Thy1+ keratinocytes do not express T cell markers, express a unique transcriptional profile, cycle significantly slower than basal epidermal progenitors and display significant expansion potential in vitro. Multicolor lineage tracing analyses and mathematical modeling reveal that Thy1+ basal keratinocytes do not compete neutrally alike interfollicular progenitors and contribute long-term to both epidermal replenishment and wound repair. Importantly, ablation of Thy1+ cells strongly impairs these processes, thus indicating the non-redundant function of Thy1+ stem cells in the epidermis. Collectively, these results reveal a distinct stem cell population that plays a critical role in epidermal homeostasis and repair