The reprogramming of skin or other easy to obtain cell types into therapeutically useful cells holds great promise for regenerative medicine, but the practical usefulness of this technology has been limited because typically only a small percentage of cells are successfully converted to the target cell type and these are often immature or incompletely specified. Single-cell expression analysis has revealed that most reprogrammed cells start with very similar expression profiles but then acquire a wide variety of different fates, many of which are developmental dead ends. Why does a cell population that is initially homogenous produce a myriad of different cell fates, with only a few cells achieving the targeted fate? This question is extremely difficult to answer with existing methods. The main focus of our proposal is to develop self-reporting Calling Cards, a new technology that can answer this question by simultaneously measuring transcription factor binding and genome-wide mRNA levels from thousands of single cells. We will demonstrate the utility of self-reporting Calling Cards by mapping the binding and function of the pioneer factor Foxa1 and its cofactor Hnf4a during the reprogramming of fibroblasts into induced endoderm progenitors (iEPs). We hypothesize that the direct targets of these TFs are stochastically expressed in cells undergoing lineage reprogramming, and that by forcing the expression of target genes that are usually transcribed only in successfully converted cells, we can improve overall target cell yield and maturity.
The conversion of skin cells into therapeutically useful cell types could lead to new treatments for a number of diseases. However, most protocols for reprogramming skin cells are inefficient, and the cells that are produced often do not fully recapitulate the properties of the target cell type. We propose to develop a technology that will analyze the reprogramming process in a novel way to understand why this is so. We will apply this technology to investigate what molecular pathways go awry in the reprogramming of skin cells to liver and intestine progenitor cells. The methods that we develop here will be broadly applicable to a number of open questions in biology and medicine.
| Biddy, Brent A; Kong, Wenjun; Kamimoto, Kenji et al. (2018) Single-cell mapping of lineage and identity in direct reprogramming. Nature 564:219-224 |
