Cardiomyocyte-specific role of miR-24 in promoting cell survival

Cardiomyocyte cell death is a major contributing factor to various cardiovascular diseases and is therefore an important target for the design of therapeutic strategies. More recently, stem cell therapies, such as transplantation of embryonic or induced pluripotent stem (iPS) cell-derived cardiomyocytes, have emerged as a promising alternative therapeutic avenue to treating cardiovascular diseases. Nevertheless, survival of these introduced cells is a serious issue that must be solved before clinical application. We and others have identified a small non-coding RNA, microRNA-24 (miR-24), as a pro-survival molecule that inhibits the apoptosis of cardiomyocytes. However, these earlier studies delivered mimics or inhibitors of miR-24 via viral transduction or chemical transfection, where the observed protective role of miR-24 in cardiomyocytes might have partially resulted from its effect on non-cardiomyocyte cells. To elucidate the cardiomyocyte-specific effects of miR-24 when overexpressed, we developed a genetic model by generating a transgenic mouse line, where miR-24 expression is driven by the cardiac-specific Myh6 promoter. The Myh6-miR-24 transgenic mice did not exhibit apparent difference from their wild-type littermates under normal physiological conditions. However, when the mice were subject to myocardial infarction (MI), the transgenic mice exhibited decreased cardiomyocyte apoptosis, improved cardiac function and reduced scar size post-MI compared to their wild-type littermates. Interestingly, the protective effects observed in our transgenic mice were smaller than those from earlier reported approaches as well as our parallelly performed non-genetic approach, raising the possibility that non-genetic approaches of introducing miR-24 might have been mediated via other cell types than cardiomyocytes, leading to a more dramatic phenotype. In conclusion, our study for the first time directly tests the cardiomyocyte-specific role of miR-24 in the adult heart, and may provide insight to strategy design when considering miRNA-based therapies for cardiovascular diseases

CellTag Indexing: Genetic barcode-based sample multiplexing for single-cell genomics

High-throughput single-cell assays increasingly require special consideration in experimental design, sample multiplexing, batch effect removal, and data interpretation. Here, we describe a lentiviral barcode-based multiplexing approach, CellTag Indexing, which uses predefined genetic barcodes that are heritable, enabling cell populations to be tagged, pooled, and tracked over time in the same experimental replicate. We demonstrate the utility of CellTag Indexing by sequencing transcriptomes using a variety of cell types, including long-term tracking of cell engraftment and differentiation in vivo. Together, this presents CellTag Indexing as a broadly applicable genetic multiplexing tool that is complementary with existing single-cell technologies

Single-Cell Resolution Mechanistic Analyses of Direct Lineage Reprogramming

End-stage organ failures remain a clinical challenge with an unmet need for medical therapies, with transplantation often being the only curative option. Despite advances in transplantation outcomes, organ shortage continues to limit the availability of cures to patients in need. The direct lineage reprogramming of one cell type to another is a promising avenue for therapy with the following advantages: (1) patient-specific cell sources, (2) direct conversion without reverting to pluripotency and the associated risk of teratoma formation, and (3) utilization of the cell type responsible for fibrotic scar formation for the engineering towards the desired cell fate. Nonetheless, many questions remain in the field, with open issues related to reprogramming trajectory, efficiency, and specificity. These issues contribute to the limited utility of directly reprogrammed cells, which oftentimes do not engraft target organs successfully or do so with only partial functionality. Of note, many reprogramming strategies are extremely inefficient and produce heterogeneous cells, with much of the molecular mechanism remaining unknown. A prototypical engineering approach is reprogramming fibroblasts to induced endoderm progenitors (iEPs). This lineage conversion is achieved by overexpressing transcription factor Hnf4α, and pioneer transcription factor Foxa1, 2, or 3. In addition to binding to their gene targets, pioneer transcription factors can bind compacted chromatin, increase target site accessibility, and recruit cooperative transcription factors. Pioneer transcription factor binding often precedes transcriptional activation during development and is thought to be important for establishing competence for developmental programs. Interestingly, when iEPs are transplanted into the mouse, they can engraft both liver and colon, suggesting that they consist of cells with hepatic and intestinal potentials. This is unsurprising, as both Foxa1/2/3 and Hnf4α are known to be important for endoderm development. However, not all cells become reprogrammed despite abundant overexpression of reprogramming factors. This raises important questions regarding the mechanism of reprogramming: What gene targets are bound by reprogramming factors? Are different outcomes due to differences in binding? Is pioneer transcription factor binding important for reprogramming success? My hypothesis for the observed inefficiency and heterogeneity of direct lineage reprogramming is that pioneer transcription factors bind developmental gene targets inefficiently and variably, influenced by variable chromatin contexts. To test my hypothesis, I utilized several recent technologies, including: single-cell RNA-sequencing, to measure transcriptional changes at a resolution needed to reveal population heterogeneity and reprogramming dynamics; our novel cell tracking approach ‘CellTagging’, to study the dynamics of clonal expansion during reprogramming and to track cell identity changes in a competitive transplant setting; and the single cell Calling Cards assay, to record transcription factor binding in different reprogramming trajectories and reveal important gene regulatory events that influence reprogramming outcomes during the early phases of reprogramming. We found that direct reprogramming is characterized by distinct paths: one leading to successful reprogramming, and a ‘dead-end’ trajectory. Trajectory bifurcation is deterministic early on, by day 13. This also led to the discovery of Mettl7a1, a putative methyltransferase identified to be associated with the successful trajectory, which increases reprogramming efficiency when added to the reprogramming cocktail. We later used CellTag Indexing to track cells from the reprogramming and dead-end trajectories transplanted into the colon, and discovered that successfully reprogrammed iEPs engraft via an intestinal stem cell state. Finally, active enhancer recording in early iEP reprogramming showed that the reprogramming trajectory is associated with rapid activation of target tissue-specific enhancers in regions that are repressed in the starting cell type, suggesting that overcoming the chromatin barrier might an important event for reprogramming outcome. In summary, I hope to have revealed some insight into the observed inefficiency and heterogeneity in direct lineage reprogramming. This is a nascent but promising field where much of the molecular mechanism is still poorly understood, with many more remaining questions to be answered before advances can be brought from bench to bedside. Further understanding the mechanism of direct reprogramming, by studying the early actions of pioneer transcription factors, may reveal additional roadblocks that are limiting reprogramming efficiency, and may one day lead to novel strategies for improved cell fate engineering and application in regenerative medicine

Cardiomyocyte‐specific role of miR‐24 in promoting cell survival

Cardiomyocyte cell death is a major contributing factor to various cardiovascular diseases and is therefore an important target for the design of therapeutic strategies. More recently, stem cell therapies, such as transplantation of embryonic or induced pluripotent stem (iPS) cell-derived cardiomyocytes, have emerged as a promising alternative therapeutic avenue to treating cardiovascular diseases. Nevertheless, survival of these introduced cells is a serious issue that must be solved before clinical application. We and others have identified a small non-coding RNA, microRNA-24 (miR-24), as a pro-survival molecule that inhibits the apoptosis of cardiomyocytes. However, these earlier studies delivered mimics or inhibitors of miR-24 via viral transduction or chemical transfection, where the observed protective role of miR-24 in cardiomyocytes might have partially resulted from its effect on non-cardiomyocyte cells. To elucidate the cardiomyocyte-specific effects of miR-24 when overexpressed, we developed a genetic model by generating a transgenic mouse line, where miR-24 expression is driven by the cardiac-specific Myh6 promoter. The Myh6-miR-24 transgenic mice did not exhibit apparent difference from their wild-type littermates under normal physiological conditions. However, when the mice were subject to myocardial infarction (MI), the transgenic mice exhibited decreased cardiomyocyte apoptosis, improved cardiac function and reduced scar size post-MI compared to their wild-type littermates. Interestingly, the protective effects observed in our transgenic mice were smaller than those from earlier reported approaches as well as our parallelly performed non-genetic approach, raising the possibility that non-genetic approaches of introducing miR-24 might have been mediated via other cell types than cardiomyocytes, leading to a more dramatic phenotype. In conclusion, our study for the first time directly tests the cardiomyocyte-specific role of miR-24 in the adult heart, and may provide insight to strategy design when considering miRNA-based therapies for cardiovascular diseases