Diabetes Volume 66, May 2017 1293 Mafa Enables Pdx1 to Effectively Convert Pancreatic Islet Progenitors and Committed Islet a-Cells Into b-Cells In Vivo Taka-aki Matsuoka,1 Satoshi Kawashima,1 Takeshi Miyatsuka,1,2 Shugo Sasaki,1 Naoki Shimo,1 Naoto Katakami,1 Dan Kawamori,1 Satomi Takebe,1 Pedro L. Herrera,3 Hideaki Kaneto,4 Roland Stein,5 and Iichiro Shimomura1 Diabetes 2017;66:1293–1300 | DOI: 10.2337/db16-0887 Among the therapeutic avenues being explored for re- including coronary and renal disease. A variety of innova- placement of the functional islet b-cell mass lost in tive approaches are being explored to produce b-cells from type 1 diabetes (T1D), reprogramming of adult cell types embryonic stem cells (1,2) and adult cell types (3–5). A into new b-cells has been actively pursued. Notably, supposition in these efforts involves producing conditions mouse islet a-cells will transdifferentiate into b-cells un- that correctly regulate the transcription factor networks der conditions of near b-cell loss, a condition similar to required in programming pancreatic progenitor cells into ISLET STUDIES a T1D. Moreover, human islet -cells also appear to poised b-cells and subsequently controlling mature islet cell func- for reprogramming into insulin-positive cells. Here we tion. These include transcription factors like Pdx1 (6–10), have generated transgenic mice conditionally expressing which is essential in the formation of early pancreatic ep- the islet b-cell–enriched Mafa and/or Pdx1 transcription ithelium, developing b-cells and adult islet b-cells, as well factors to examine their potential to transdifferentiate as neurogenin 3 (Ngn3) (11–13), which is required during embryonic pan–islet cell Ngn3-positive progenitors and embryogenesis for specification of all islet cell types (i.e., the later glucagon-positive a-cell population into b-cells. b-cells, glucagon hormone–producing a-cells, somatostatin Mafa was found to both potentiate the ability of Pdx1 d to induce b-cell formation from Ngn3-positive endocrine -cells, pancreatic polypeptide (PP) cells, and ghrelin e precursors and enable Pdx1 to produce b-cells from -cells). In addition, there are transcription factors like fl a-cells. These results provide valuable insight into the Mafa (14,15) that are in uential later during postnatal b fundamental mechanisms influencing islet cell plasticity -cell maturation and adult cell function. Indeed, ectopic in vivo. expression of Pdx1, Ngn3, and Mafa can reprogram pan- creatic exocrine cells (3) and intestinal cells (4) into func- tional b-like cells in vivo. While current therapies of insulin treatment afford T1D results from the specific loss of islet b-cells. In- glycemic control to patients with type 1 diabetes (T1D), terestingly, functional b-like cells are produced from en- these relatively static methods do not completely recapit- dogenous mouse islet a-cells (16) or d-cells (17) after near ulate the acute regulation of the endogenous islet b-cells total targeted destruction of this cell population, a model destroyed by autoimmune destruction. Consequently, pa- mimicking the disease state (16). Furthermore, epigenomic tients with T1D have a dramatically shortened life expec- findings suggest that human a-cells are poised for reprog- tancy due to serious long-term diabetes complications, ramming, with treatment to prevent histone 3 repressor 1Department of Metabolic Medicine, Osaka University Graduate School of Med- Corresponding author: Taka-aki Matsuoka, [email protected]. icine, Osaka, Japan Received 20 July 2016 and accepted 6 February 2017. 2Center for Molecular Diabetology, Juntendo University Graduate School of Med- This article contains Supplementary Data online at http://diabetes icine, Tokyo, Japan .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0887/-/DC1. 3Department of Genetic Medicine and Development, University of Geneva Faculty of Medicine, Geneva, Switzerland © 2017 by the American Diabetes Association. Readers may use this article as 4Department of Diabetes, Endocrinology and Metabolism, Kawasaki Medical long as the work is properly cited, the use is educational and not for profit, and the School, Okayama, Japan work is not altered. More information is available at http://www.diabetesjournals 5Department of Molecular Physiology and Biophysics, Vanderbilt University Med- .org/content/license. ical School, Nashville, TN 1294 Competency of Mafa for Reprogramming Islet Cells Diabetes Volume 66, May 2017 site marking at lysine 27 leading to the appearance of in- Olympus FV1000-D confocal microscope. The images sulin-positive–glucagon-positive bihormonal cells in human shown are representative of our analysis of at least three islets (18). independently derived mice unless otherwise specified. Here, we generated transgenic mice that allow condi- The total number of insulin-, glucagon-, PP-, and tionally and targeted expression of Mafa or Pdx1 to b-gal–positive cells in five sections per pancreas from at determine their contribution to b-cell generation from least three mice per genotype were manually counted in embryonic endocrine Ngn3-positive and committed gluca- the Ngn3-Cre (Figs. 1D and 2B) and Gcg-Cre (Fig. 3D) gon-positive progenitors. Earlier studies had established lines. Approximately 500 cells were counted in the that forced Pdx1 expression in this endocrine precursor 6-week-old pancreas and 200 cells in the P0.5 pancreas. population results in greater b-cell production at the ex- The data are presented as the ratio of each hormone- pense of a-cells, with no effect on d-cells or PP cells (19). positive cell or hormone-positive–to–b-gal–positive cell We found that Mafa not only was found to potentiate the for each genotype. ability of Pdx1 to reprogram Ngn3-positive endocrine progenitor cells to insulin-positive cells but also empow- Statistical Analysis ered Pdx1 to transdifferentiate committed glucagon- Statistical analysis was performed using one-way ANOVA positive a-cells to this cell fate. These results provide followed by the Fisher test. A value of P , 0.05 was further support for the essential role of Mafa and Pdx1 considered to be statistically significant. in the production of therapeutic b-cells for treatment of patients with T1D. RESULTS Mafa + Pdx1 More Efficiently Programs Endocrine RESEARCH DESIGN AND METHODS Ngn3-Positive Precursors to b-Cells Than Pdx1 Alone fl fl CAG-CAT-Pdx1 ag, CAG-CAT-Mafamyc, Cre, To explore how Mafa in uences the ability of Pdx1 to and ROSA26 Mice convert a-cells into b-cells, we developed mouse lines that fl CAG-CAT-Mafamyc (20), CAG-CAT-Pdx1 ag (21), Ngn3-Cre allow for conditional and heritably overproduction of ei- (12), Gcg-Cre (22), ROSA26-LacZ (23), and ROSA26-GFP (24) ther Mafa, Pdx1, or Mafa + Pdx1 upon expression of Cre mice have previously been described. All animal procedures recombinase (Fig. 1A). Antibodies to Mafa and Pdx1 and were approved by the Ethics Review Committee for Animal their incorporated antigenic tags were used to immunohis- Experimentation of the Osaka University Graduate School tochemically detect the levels of the transgenic proteins, myc flag of Medicine. termed Mafa and Pdx1 . Both of these islet b-cell– enriched proteins (27–29) were now found throughout Immunohistochemistry and Cell Quantification the islet cell population upon activation in embryonic en- Pancreata were dissected and fixed in 4% paraformalde- docrine precursors with Ngn3-driven Cre, which were hyde in PBS at 4°C, washed in PBS, immersed in sucrose termed the Ngn3-Cre;CAT-Mafa and Ngn3-Cre;CAT-Pdx1 solution, embedded and frozen in Tissue-Tek (O.C.T. Com- lines. For example, flag-tagged Pdx1 was produced in pound; Sakura), or processed routinely for paraffinembed- essentially all islet cells in P0.5 and 6-week-old Ngn3-Cre; ding. Frozen and paraffin blocks were sectioned at 6-mm CAT-Pdx1 mice; the penetrance rate was ;95% in the in- thickness and immunostained. The following primary an- sulin-positive and glucagon-positive cell populations (Sup- fl tibodies were used at the given dilutions: rabbit anti-MafA plementary Fig. 1). Notably, neither Mafamyc nor Pdx1 ag (1:500) (Bethyl Laboratories, Inc., Montgomery, TX); goat was expressed in the surrounding acinar or ductal cells of anti-MafA (25) (1:200); rabbit anti-Pdx1 (26) (1:1,000); the pancreas (Fig. 1B). While there was no overt effect on rabbit anti-Nkx6.1 (1:200) (Sigma-Aldrich, St. Louis, a-cell fate in Ngn3-Cre;CAT-Mafa islets (Fig. 1C and D), in- MO); rabbit anti-MafB (1:200) (Bethyl Laboratories, Inc.); sulin was expressed in ;10% of Ngn3-Cre;CAT-Pdx1 glucagon- goat anti-Arx (1:200) (Santa Cruz Biotechnology, Inc., positive cells at P0.5. These double hormone–positive cells Dallas, TX); rabbit anti-myc (1:200) (Cell Signaling Technol- resolved to become only insulin-positive by 6 weeks, with ogy,Inc.,Danvers,MA);rabbitanti-flag (1:100) (Affinity essentially no islet glucagon-positive cells remaining (Fig. BioReagents, Golden, CO); mouse anti-flag (1:500) (Trans- 1C), as previously reported (19). Moreover, there was no fl Genic Inc., Kobe, Japan); rabbit anti-Glut2 (1:200) (abcam, evidence for apoptosis within Mafamyc– and Pdx1 ag–positive Cambridge, U.K.); guinea pig anti-insulin (1:2,000) (DAKO, islet glucagon-positive or insulin-positive cells, with the Glostrup, Denmark); rabbit anti-glucagon (1:500) (DAKO); TUNEL-positive signal detected exclusively from red blood guinea pig anti-glucagon (1:200) (Millipore, St. Charles, MO); cells (Supplementary Fig. 2). These results suggested fl rabbit anti–b-galactosidase (b-gal) antibody (1:200) that resolution of the Mafamyc +Pdx1 ag–positive cells (Medical and Biological Laboratories, Nagoya, Japan); to monohormonal insulin production reflects transdiffer- chicken anti–b-gal antibody (1:200) (abcam); and chicken entiation of a-tob-like cells. anti–green fluorescent protein (GFP) antibody (1:500) Mafa expression in endocrine precursor cells augmented (abcam).