Abstract
Given the limited regenerative capacities of most organs, strategies are needed to efficiently generate large numbers of parenchymal cells capable of integration into the diseased organ. Although it was initially thought that terminally differentiated cells lacked the ability to transdifferentiate, it has since been shown that cellular reprogramming of stromal cells to parenchymal cells through direct lineage conversion holds great potential for the replacement of post-mitotic parenchymal cells lost to disease. To this end, an assortment of genetic, chemical, and mechanical cues have been identified to reprogram cells to different lineages both in vitro and in vivo. However, some key challenges persist that limit broader applications of reprogramming technologies. These include: (1) low reprogramming efficiencies; (2) incomplete functional maturation of derived cells; and (3) difficulty in determining the typically multi-factor combinatorial recipes required for successful transdifferentiation. To improve efficiency by comprehensively identifying factors that regulate cell fate, large scale genetic and chemical screening methods have thus been utilized. Here, we provide an overview of the underlying concept of cell reprogramming as well as the rationale, considerations, and limitations of high throughput screening methods. We next follow with a summary of unique hits that have been identified by high throughput screens to induce reprogramming to various parenchymal lineages. Finally, we discuss future directions of applying this technology toward human disease biology via disease modeling, drug screening, and regenerative medicine.
Highlights
During development, cells become increasingly specialized to a terminally differentiated state
It was initially thought that terminally differentiated cells lacked the ability to transdifferentiate, it has since been shown that cellular reprogramming of stromal cells to parenchymal cells through direct lineage conversion holds great potential for the replacement of post-mitotic parenchymal cells lost to disease
open reading frame (ORF) overexpression approaches allow for strong overexpression of genes as well as the expression of specific isoforms or mutants, while CRISPR activation (CRISPRa) allows for endogenous gene expression to more physiological levels and ease of use given that generating single guide RNA (sgRNA)
Summary
Cells become increasingly specialized to a terminally differentiated state. Recent technological advances have allowed researchers to move past the trial and error approach and instead combined high throughput genetic or chemical perturbations with phenotypic or transcriptomic readouts to identify factors governing cell fate (Table I).[16,17,18,19,35,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61] These screens are either performed in arrayed format, where perturbations are maintained in separate culture conditions, or pooled format, where perturbations are assayed en masse. Consideration of the strengths and weakness of different methods for these components is critical to interpret results from high throughput screens and extract meaningful data
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