A VERSATILE SCREENING TECHNOLOGY TO DETERMINE TRANSCRIPTION FACTOR COMBINATIONS FOR REPROGRAMMING CELLULAR IDENTITY

Background & Aim Cellular plasticity enables reprogramming of cell identity through ectopic expression of transcription factors (TFs) that induce and stabilize gene regulatory networks specifying discrete cellular states. For example, transient expression of the 'Yamanaka factors' reprograms diverse cell types to iPSCs. In turn, these cells can be programmed into further, more developed, lineages using different TFs cocktails. However, facile identification of TF combinations for cell reprogramming remains elusive, mainly because current screening methods are highly multiplexed and therefore challenging to deconvolute. Even if TF switches and effectors of reprogramming can be identified, the circuitry of these components is not immediately apparent since TF expression is not temporally resolved during the screen. We aim to develop a high throughput mRNA-based screening method to test all possible combinations of (2, 3, 4… n) TFs from a predetermined subset to discover functional combinations, and to resolve the order/timing in which they orchestrate reprogramming. Methods, Results & Conclusion We will describe a screen covering 10,000 combinations of 10 TFs involved in pancreatic development, to discover combinations of three or four TFs that reprogram iPSC-derived pancreatic progenitor cells into insulin producing beta cells. We will present results showing re-discovery of the known beta-cell reprogramming cocktail comprising Pdx-1, Ngn-3 and Maf A, in addition to several novel transcription factor compositions. The ability to discover and map out genetic circuits for rational cell reprogramming will enable diverse applications in fields ranging from synthetic biology to regenerative medicine. Cellular plasticity enables reprogramming of cell identity through ectopic expression of transcription factors (TFs) that induce and stabilize gene regulatory networks specifying discrete cellular states. For example, transient expression of the 'Yamanaka factors' reprograms diverse cell types to iPSCs. In turn, these cells can be programmed into further, more developed, lineages using different TFs cocktails. However, facile identification of TF combinations for cell reprogramming remains elusive, mainly because current screening methods are highly multiplexed and therefore challenging to deconvolute. Even if TF switches and effectors of reprogramming can be identified, the circuitry of these components is not immediately apparent since TF expression is not temporally resolved during the screen. We aim to develop a high throughput mRNA-based screening method to test all possible combinations of (2, 3, 4… n) TFs from a predetermined subset to discover functional combinations, and to resolve the order/timing in which they orchestrate reprogramming. We will describe a screen covering 10,000 combinations of 10 TFs involved in pancreatic development, to discover combinations of three or four TFs that reprogram iPSC-derived pancreatic progenitor cells into insulin producing beta cells. We will present results showing re-discovery of the known beta-cell reprogramming cocktail comprising Pdx-1, Ngn-3 and Maf A, in addition to several novel transcription factor compositions.