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

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.

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Matthias Hebrok
Apr 13, 2020 BD FACSDiva v. 6.0 software was used for flow cytometry data collection using BD LSRII. Figures with associated raw data: Figure 4A,B Figure S5A,B,C Figure S6A,B,C,D Figure S7A Figure S8 Statistical methods were not used to determine sample size. Sample sizes were chosen based on availability of experimental samples. These sample sizes are sufficient since the study is very thorough and we have used a variety of experimental techniques to confirm the findings Stem Cell differentiations that did not pass a minimum criteria of 85% SOX17+/FOXA2+ double positive cells at the early stage of defintive endoderm were excluded from further analysis. This criteria was preselected because the formation of definitive endoderm is the first step toward pancreatic differentiation. When cells don't pass the above criteria, the spheres do not form pancreatic endoderm efficiently and start falling apart on further culture.
At least 3 or more biological replicates were considered for the analysis using stage-matched controls as a reference. Using the above criteria, all replication attempts were successful. Additionally, we used a second hPSC cell line to confirm the major findings of the study. Samples of different stages of differentiation sent to other researchers confirmed our findings.
There was no randomization applicable for the in vitro experiments as cell lines had to be identified to establish independent clones . For transplantation experiments in vivo, mice were randomly assigned to receive either MAFB+/+ or MAFB-/-cells.
Investigators were blinded to samples collected from in vivo transplantation studies. Investigators were not blinded to group allocation as phenotypes were identifiable during the analysis. Note that full information on the approval of the study protocol must also be provided in the manuscript.

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