Loss of the transcription factor MAFB limits \u03b2-cell derivation from human PSCs

Ronan Russell,Simone Giacometti,Holger A Russ,Thomas G Hennings,Jennifer S Liu,Matthias Hebrok,Phichitpol P Carnese,Emily M Walker,Roland Stein

doi:10.1038/s41467-020-16550-9

Abstract

Next generation sequencing studies have highlighted discrepancies in β-cells which exist between mice and men. Numerous reports have identified MAF BZIP Transcription Factor B (MAFB) to be present in human β-cells postnatally, while its expression is restricted to embryonic and neo-natal β-cells in mice. Using CRISPR/Cas9-mediated gene editing, coupled with endocrine cell differentiation strategies, we dissect the contribution of MAFB to β-cell development and function specifically in humans. Here we report that MAFB knockout hPSCs have normal pancreatic differentiation capacity up to the progenitor stage, but favor somatostatin- and pancreatic polypeptide–positive cells at the expense of insulin- and glucagon-producing cells during endocrine cell development. Our results describe a requirement for MAFB late in the human pancreatic developmental program and identify it as a distinguishing transcription factor within islet cell subtype specification. We propose that hPSCs represent a powerful tool to model human pancreatic endocrine development and associated disease pathophysiology.

Highlights

Generation sequencing studies have highlighted discrepancies in β-cells which exist between mice and men
Utilizing the INS-GFP reporter human embryonic stem cell (hESC) line, in which GFP is knocked into one endogenous locus of the insulin gene[25], this protocol routinely generates >90% definitive endoderm (DE) cells co-expressing SOX17 and FOXA2, >70% pancreatic progenitor (PP) cells co-expressing PDX1 and NKX6.1, and 20–50% mono-hormonal β-like cells (β-like) co-expressing C-peptide and NKX6.1 as assessed by flow cytometry (FC) and immunofluorescence (IF) staining (Fig. 1b, c, Supplementary Fig. 1a)
We examined the expression of MAF BZIP Transcription Factor B (MAFB) over the time course of differentiation using qPCR and western blot analysis. mRNA levels of MAFB are first detected at the PP stage and increase in levels at the β-like stage (Fig. 1d)

Summary

Introduction

Generation sequencing studies have highlighted discrepancies in β-cells which exist between mice and men. While much effort has been spent focusing on optimizing directed differentiation of human cell types for disease modeling or the phenotypes associated with known genetic susceptibility genes[19,20], the underlying complex biological processes which established protocols recapitulate provides a unique platform for investigating the normal pancreatic developmental program as shown for PAX4 and ARX as well pan-pancreatic lineage determinants such as PDX118,21,22. These reports have demonstrated the feasibility of this approach and verified in a human context, the precise role of established transcription factors in β-cell biology. These findings suggest that species-specific differences in transcription factor activity may exist between mice and men and promote the use of human developmental models such as directed differentiation strategies to enhance our understanding of human biology

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