UCSF UC San Francisco Previously Published Works Title Loss of the transcription factor MAFB limits β-cell derivation from human PSCs. Permalink https://escholarship.org/uc/item/0zb338n9 Journal Nature communications, 11(1) ISSN 2041-1723 Authors Russell, Ronan Carnese, Phichitpol P Hennings, Thomas G et al. Publication Date 2020-06-02 DOI 10.1038/s41467-020-16550-9 Peer reviewed eScholarship.org Powered by the California Digital Library University of California ARTICLE https://doi.org/10.1038/s41467-020-16550-9 OPEN Loss of the transcription factor MAFB limits β-cell derivation from human PSCs Ronan Russell1, Phichitpol P. Carnese1, Thomas G. Hennings1, Emily M. Walker 2, Holger A. Russ 1,3, ✉ Jennifer S. Liu1, Simone Giacometti1, Roland Stein2 & Matthias Hebrok 1 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 1234567890():,; (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 soma- tostatin- 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. 1 UCSF Diabetes Center, University of California San Francisco, San Francisco, CA 94143, USA. 2 Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA. 3Present address: Barbara Davis Center for Diabetes, School of Medicine, University of Colorado Denver, ✉ Aurora, CO 80045, USA. email: [email protected] NATURE COMMUNICATIONS | (2020) 11:2742 | https://doi.org/10.1038/s41467-020-16550-9 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16550-9 dentifying key factors that mediate islet cell function is critical In the present study, we generate isogenic MAFB loss of Ito understanding Type 1 and Type 2 Diabetes Mellitus (T1D, function hPSCs to dissect its contribution in human β-cell T2D) disease processes and to moving forward with cell-based development, using a stepwise differentiation protocol. MAFB interventions in humans1. Notably, the advent of in-depth loss of function cells display no major defects in the ability to sequencing studies has elucidated alternative transcription fac- specify early stages of pancreatic development. However, MAFB tor activity among closely related islet cell subtypes2,3 and addi- null cells have an increased propensity to generate somatostatin- tionally, uncovered numerous examples of heterogeneity within (δ) and pancreatic polypeptide- (γ) producing cells at the expense healthy β-cells4. It has long been recognized that differences in of glucagon (α) and insulin (β) producing cells. Our results cellular composition and architecture exist between human and identify an important role of MAFB within endocrine cell- mice adult islets, although the precise reasons and implications subtype specification decisions and demonstrate that it is a critical for such disparity have remained elusive. Numerous recent single- transcription factor for the generation of glucose-responsive β- cell RNA-sequencing studies have reported a discrepancy in the cells from hPSCs. This essential role of MAFB in human β-cell expression of a number of factors, highlighting potential species- identity is considerably different from what was concluded in specific regulatory differences among rodent and human β-cells/ genetically engineered mouse models (GEMMs). These findings islet counterparts3,5. suggest that species-specific differences in transcription factor One such factor is MAFB, which has been identified in activity may exist between mice and men and promote the use of numerous studies to be expressed within adult human β-cells, human developmental models such as directed differentiation while being absent from the murine counterparts. MAFB is a strategies to enhance our understanding of human biology. member of the large Maf family of transcription factors that share highly similar basic region/leucine zipper DNA binding motifs Results and N-terminal activation domains. MAFB is expressed in MAFB is expressed during β-like cell differentiation.We developing pancreatic α- and β-cells in the mouse, while its – employed our previously published β-cell directed differentiation expression becomes restricted to α-cells in the adult6 8 and protocol with modifications (Fig. 1a, Supplementary Tables 1 and MAFB knockout (KO) in the mouse pancreas merely delays β-cell 2)23,24. Utilizing the INS-GFP reporter hESC line, in which GFP maturation for a short period of time9,10. MAFB binds to the is knocked into one endogenous locus of the insulin gene25, this MAF responsive element (MARE) expressed within critical β-cell protocol routinely generates >90% definitive endoderm (DE) cells genes including PDX1 and INS. Kim and colleagues recently co-expressing SOX17 and FOXA2, >70% PP cells co-expressing showed that MAFB mRNA is expressed at robust levels both in α- PDX1 and NKX6.1, and 20–50% mono-hormonal β-like cells and β-cells in humans with no significant fluctuations in levels (β-like) co-expressing C-peptide and NKX6.1 as assessed by flow over age11,afinding that has since been demonstrated at protein cytometry (FC) and immunofluorescence (IF) staining (Fig. 1b, c, level12. Building on this, single-cell RNA-sequencing studies have Supplementary Fig. 1a). First, we examined the expression of identified that MAFB is robustly expressed in human adult β-cells – MAFB over the time course of differentiation using qPCR and as well as α- and δ-cells2,3,5,13 16. This is a notable difference to western blot analysis. mRNA levels of MAFB are first detected at the related factor MAFA, a β-cell specific transcription factor that the PP stage and increase in levels at the β-like stage (Fig. 1d). also binds to the insulin MARE site, activates insulin gene tran- MAFB protein was principally detected at the β-like stage, indi- scription7, and has been reported as a critical factor in adult β- cating that robust MAFB expression coincides with the onset of cells across species. MAFA expression increases in an age- hormone expression (Fig. 1e)7. dependent manner in both human and mouse β-cells and is the In order to examine the role of MAFB in the differentiation of predominant isoform expressed in functional mouse β-cells12, β-cells, we utilized an inducible (i)CRISPR gene editing system26 although it is not robustly expressed in human β-cells until after in the INS-GFP cell line along with three individual guide RNAs ~10 years of age3,11. Together, these data suggest a potential (gRNAs) (Fig. 1f, Supplementary Table 3). Three independent evolutionary discrepancy in the role of MAFB in the pancreatic MAFB heterozygous and homozygous clones with frameshift islet compartment and the use of MAFA as a marker of human β- mutations in the transactivation domain, leading to disruption of cell function has been questioned in recent studies17. the functional full-length protein, were established. Isogenic Directed differentiation of human pluripotent stem cells clonal cell lines which lacked genomic editing were employed as (hPSCs) into insulin producing β-like cells provides a powerful controls. All clonal hESC lines grew as expected compared with platform to investigate human biology. This approach has been the parental control line, suggesting the genomic editing and sub- shown to effectively recapitulate the sequential stages of islet cell cloning procedure did not compromise pluripotency. To verify development, in which hPSCs transition through a definitive the genotypes, cell lines were assayed via targeted sequencing of endoderm (DE) stage, multipotent pancreatic progenitor (PP) the genome-edited locus at the pluripotency stage, as well as by stage, and toward β-like cells. By combining this strategy with western blot and IF analysis for MAFB protein at the β-like cell genome editing approaches such as CRISPR/Cas9 (clustered stage (Fig. 1g–h, Supplementary Fig. 1b). Mono-allelic and bi- regularly interspaced short palindromic repeats), it is possible to allelic MAFB clonal lines harboring frameshift mutations will be dissect the specific contribution of pancreatic transcription fac- referred to as +/− and −/−, respectively, throughout the text and tors at precise stages in a human-specific context18. While much specific indel sequence information for each clone is outlined in effort has been spent focusing on optimizing directed differ- Supplementary Tables 4 and 5. entiation of human cell types for disease modeling or the phe- notypes associated with known genetic susceptibility genes19,20, the underlying complex biological processes which established Differentiation of MAFB KO hESCs to pancreatic β-like cells. protocols recapitulate also provides a unique platform for To control for potential off-target