Diabetes Volume 65, May 2016 1297

Jennifer E. Bruin,1 Nelly Saber,1 Shannon O’Dwyer,1 Jessica K. Fox,1 Majid Mojibian,1 Payal Arora,2 Alireza Rezania,2 and Timothy J. Kieffer1,3

Hypothyroidism Impairs Human Stem Cell–Derived Pancreatic Progenitor Cell Maturation in Mice

Diabetes 2016;65:1297–1309 | DOI: 10.2337/db15-1439

Pancreatic progenitors derived from human embryonic (T1D) (1,2) but is not widely available to most patients stem cells (hESCs) are a potential source of transplantable due to the inadequate supply of donor cells and burden of cells for treating diabetes and are currently being tested in immunosuppression. Pluripotent stem cells are a highly clinical trials. Yet, how the milieu of pancreatic progenitor scalable alternative cell source (3), and we have previously cells, including exposure to different factors after trans- demonstrated that human embryonic stem cell (hESC)– plant, may influence their maturation remains unclear. derived pancreatic progenitor cells can reverse hypergly- Here, we examined the effect of thyroid dysregulation on cemia in mouse models of streptozotocin-induced T1D the development of hESC-derived progenitor cells in vivo. (4–6) and high-fat diet–induced type 2 diabetes (7). How- STUDIES ISLET Hypothyroidism was generated in SCID-beige mice using an ever, glucose-responsive human insulin secretion was only iodine-deficient diet containing 0.15% propyl-2-thiouracil, achieved after a lengthy cell maturation period in vivo, and and hyperthyroidism was generated by addition of how environmental factors within the host may affect L-thyroxine (T4) to drinking water. All mice received this maturation process remains unclear. ViaCyte Inc. macroencapsulated hESC-derived progenitor cells, has initiated phase 1/2 clinical trials involving trans- and thyroid dysfunction was maintained for the duration – of the study (“chronic”) or for 4 weeks posttransplant plant of macroencapsulated hESC derived pancreatic pro- (“acute”). Acute hyperthyroidism did not affect graft genitor cells into patients with T1D. Thus, understanding function, but acute hypothyroidism transiently impaired how variability in the physiology of transplant recipients human C-peptide secretion at 16 weeks posttransplant. may affect the development of progenitor cells in vivo is Chronic hypothyroidism resulted in severely blunted basal important. human C-peptide secretion, impaired glucose-stimulated Patients with diabetes have a significantly higher risk of insulin secretion, and elevated plasma glucagon levels. developing thyroid disease than the general population (8). Grafts from chronic hypothyroid mice contained fewer Up to one-third of patients with T1D also have thyroid b-cells, heterogenous MAFA expression, and increased dysfunction, which can exacerbate the impaired metabolic glucagon+ and ghrelin+ cells compared to grafts from control and complications associated with diabetes, particu- euthyroid mice. Taken together, these data suggest that larly when the thyroid disorder is undetected (8). Moreover, long-term thyroid hormone deficiency may drive the differ- there is evidence to suggest that excessive or deficient levels entiation of human pancreatic progenitor cells toward of thyroid hormones may affect b-cell development and a-and«-cell lineages at the expense of b-cell formation. function. Maternal hypothyroidism caused impaired insulin secretion in neonatal rats as well as glucose intolerance and b-cell dysfunction in adult offspring (9). Systemic knockout Transplantation of cadaveric human b-cells can restore of Dio3 (the enzyme required for intracellular inactivation insulin-independence in patients with type 1 diabetes of thyroid hormones) caused significantly reduced islet area

1Laboratory of Molecular and Cellular Medicine, Department of Cellular & Phys- This article contains Supplementary Data online at http://diabetes iological Sciences, Life Sciences Institute, The University of British Columbia, .diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1439/-/DC1. Vancouver, British Columbia, Canada J.E.B. and N.S. contributed equally to this work. 2BetaLogics Venture, Janssen R&D, LLC, Raritan, NJ © 2016 by the American Diabetes Association. Readers may use this article as 3Department of Surgery, The University of British Columbia, Vancouver, British long as the work is properly cited, the use is educational and not for profit, and Columbia, Canada the work is not altered. Corresponding author: Timothy J. Kieffer, [email protected]. See accompanying article, p. 1155. Received 19 October 2015 and accepted 29 December 2015. 1298 Hypothyroidism Impairs hESC-Derived b-Cells Diabetes Volume 65, May 2016 and pancreatic insulin content compared with wild-type Animals mice at birth as well as glucose intolerance and impaired Male 7- to 8-week-old SCID-beige mice (C.B-Igh-1b/ insulin secretion during adulthood (10). Triiodothyronine GbmsTac-Prkdcscid-LystbgN7; Taconic, Hudson, NY) were (T3) has also been shown to have prosurvival effects on maintained on a 12-h light/dark cycle throughout the study. adult b-cells by protecting mice from streptozotocin-induced The first cohort of mice (7–8 weeks of age) was used to b-cell death and diabetes (11). Aguayo-Mazzucato et al. (12) characterize the different models of thyroid dysregulation: demonstrated that daily T3 injections from postnatal days chronic hypothyroid (n = 10), chronic hyperthyroid (n = 10), 1–7 in rats increased expression and nuclear localization of and euthyroid (n = 10); the treatment protocol is summa- Mafa, a transcription factor essential for b-cell maturation, rizedinFig.1A and described in detail below. A subset of whereas inhibition of postnatal thyroid hormone synthesis mice from this cohort was subsequently used for transplan- decreased Mafa levels in neonatal rat islets. Moreover, treat- tation,assummarizedinFig.2A. The chronic hypothyroid ment of isolated neonatal rat islets with T3 in vitro induced group was maintained on an iodine-deficient diet (n =8), glucose-stimulated insulin secretion, an effect that was and the euthyroid group was divided into two subgroups: blocked in the presence of dominant-negative Mafa, sug- euthyroid (group A; n = 5) and acute hypothyroid (n =4).A gesting that the effects of T3 on b-cell maturation are second group of mice (8 weeks of age) was used for a new via Mafa regulation (12). Consistent with these findings, hyperthyroid cohort (Fig. 2A): acute hyperthyroid (n =8) addition of T3 to differentiating hESCs increased gene versus euthyroid (group B; n =8).EuthyroidgroupsAandB expression of INS and MAFA and led to improved glucose- were analyzed separately for blood glucose and body weight stimulated insulin secretion in vitro (13). Taken together, tracking but subsequently combined for all further analysis. these studies suggest that thyroid hormone signaling may All experiments were approved by the UBC Animal Care play an important role in b-cell development, maturation, Committee and performed in accordance with the Canadian survival, and maintenance of adult b-cell function. CouncilonAnimalCareguidelines. Here, we investigated the effects of thyroid hormone Diets and L-Thyroxine Administration dysregulation on the maturation of encapsulated hESC- All mice received ad libitum access to a standard irradiated derived pancreatic progenitor cells in vivo. We hypothesized diet (Teklad Diet #2918; Harlan Laboratories, Madison, that hyperthyroidism may accelerate the development of WI) to allow for acclimatization after their arrival at UBC. hESC-derived pancreatic progenitor cells into mature insulin- At 12–13 weeks old, mice from the first cohort were ran- secreting cells, whereas hypothyroidism may hinder the domly selected to undergo one of the following treatments maturation process. Our findings indicate that chronic (summarized in Fig. 1A): 1) euthyroid, iodine control diet hypothyroidism impairs the development of hESC-derived (cat. #TD.08260; Harlan Laboratories) and normal drinking b-cells in vivo and instead promotes the formation of a-and water; 2) hypothyroid, iodine-deficient diet with 0.15% e-cells from pancreatic progenitor cells. Short-term exposure propylthiouracil (PTU) (cat. #TD.08259; Harlan Laborato- to hyperthyroidism for 4 weeks posttransplant did not affect ries) and normal drinking water; or 3) hyperthyroid, io- the development of glucose-dependent human insulin pro- dine control diet (cat. #TD.08260; Harlan Laboratories) duction from pancreatic progenitor cells in vivo. and drinking water containing various concentrations RESEARCH DESIGN AND METHODS of L-thyroxine sodium salt pentahydrate (T4) (cat. #T2501, Sigma-Aldrich): a) 12 mg/L: 50% PBS, 50% double-distilled In Vitro Differentiation of hESCs and Assessment of [dd]H O; b) 6 mg/L: 25% PBS, 75% ddH O; or c) 3 mg/L: Pancreatic Progenitor Cells 2 2 12.5% PBS, 87.5% ddH O. The H1 hESC line was obtained from WiCell Research 2 For the mice that received cell transplants, five treat- Institute, Inc. (Madison, WI). All experiments at The ment groups were monitored for 180 days posttransplant University of British Columbia (UBC) with H1 cells were (summarized in Fig. 2A). Euthyroid mice (groups A and B) approved by the Canadian Stem Cell Oversight Committee and the acute hyperthyroid mice received the iodine con- and the UBC Clinical Research Ethics Board. Pluripotent H1 trol diet for the duration of the study. The acute hyper- cells were differentiated into pancreatic progenitor cells for thyroid group received T4 drinking water (3 mg/L) for transplantation studies according to a 14-day, four-stage 1 week before and 4 weeks after transplantation. The acute protocol, as previously described (4). Expression of key pan- hypothyroid mice received the iodine-deficient diet with creatic progenitor cell markers was assessed before trans- 0.15% PTU for 4 weeks after transplantation, and the plant using flow cytometry, as previously described (5); chronic hypothyroid mice received the iodine-deficient antibody information is provided in Supplementary Table diet for the duration of the study. 1. To determine the effect of T3 on hESC development in vitro, H1 cells were differentiated according to our re- Transplantation of hESC-Derived Pancreatic cently published protocol (13), and T3 was added during Progenitor Cells stage (S)4 (500 nmol/L or 1,000 nmol/L final concentra- Euthyroid(groupA),chronic,andacutehypothyroidmice tion). Differentiated cells were assessed by quantitative received hESC-derived pancreatic progenitor cell transplants (q)PCR on S4 day 3 (S4D3), S5D3, and S6D3, as de- at 20–21 weeks of age (after 8–9 weeks of treatment regi- scribed below. mens;Fig.2A). Euthyroid (group B) and acute hyperthyroid diabetes.diabetesjournals.org Bruin and Associates 1299

Figure 1—Characterization of hyperthyroid and hypothyroid models in immunodeficient SCID-beige mice. A: Schematic of study timeline and treatment groups. OGTT, oral glucose tolerance test. B: Random fed plasma T3 levels in mice before administration of treatments (day [d]0), midhypothyroid treatment (d14), and midhyperthyroid treatment, using T4 in drinking water at a dosage of 12 mg/L (d7), 6 mg/L (d14), or 3 mg/L (d24). The dashed line indicates the lower limit of detection for the T3 assay. *P < 0.05 vs. euthyroid by t test. C and D:Bodyweight(C)and blood glucose levels (D) after a 4-h morning fast. The red (hypothyroid) and blue (hyperthyroid) bars at bottom of graphs indicate timing and dosage for treatment regimens. *P < 0.05 vs. euthyroid by two-way repeated-measures ANOVA with Dunnett post hoc test. E: Blood glucose levels during an OGTT (2 g/kg glucose) at day 22. *P < 0.05 vs. euthyroid by two-way repeated-measures ANOVA. Right: *P < 0.05 vs. euthyroid for area under the curve by one-way ANOVA. F: Mouse insulin levels after a 6-h morning fast. *P < 0.05 by one-way ANOVA. Data are presented as mean 6 SEM plus individual biological replicates.

mice received cell transplants at 10 weeks of age (Fig. 2A). Metabolic Assessments Allmicewereanesthetizedwithinhalableisoflurane, and All metabolic analyses were performed in conscious, ;5 3 106 hESC-derived pancreatic progenitor cells were restrained mice, and blood samples were collected via transplanted subcutaneously within a 20 mL TheraCyte mac- saphenous vein at the indicated time points. Specific assays roencapsulation device (TheraCyte Inc., Laguna Hills, CA) used to measure plasma analytes and detailed protocols for on the right flank, as previously described (4). metabolic testing are described in the Supplementary Data. 1300 Hypothyroidism Impairs hESC-Derived b-Cells Diabetes Volume 65, May 2016

Figure 2—Metabolic characterization of transplant recipients with thyroid dysfunction. A: Schematic of study timeline and treatment groups used to assess the effects of acute hyperthyroidism (Hyper) and acute or chronic hypothyroidism (Hypo) on the maturation of hESC-derived pancreatic progenitor cells in SCID-beige mice. ipArgTT, intraperitoneal arginine tolerance test; ipGTT, intraperitoneal glucose tolerance test; NC, normal chow. B: Random fed plasma T3 levels in mice at 28 days posttransplant (during acute thyroid treatments) and at 42 days posttransplant (after cessation of acute treatments). The dashed line indicates the lower limit of detection for the T3 assay. *P < 0.05 vs. euthyroid by unpaired two-tailed t tests. C: Tracking of body weight change (relative to day 256, before administration of chronic hypothyroid treatment) and blood glucose for euthyroid (group A) and chronic/acute hypothyroid mice before and after transplant. Red and green bars at the bottom indicate timing of iodine-deficient diet administration. D: Tracking of body weight change (relative to day 0, before administration of acute hyperthyroid treatment) and blood glucose for euthyroid (group B) and acute hyperthyroid mice before and after transplant. Blue bar at bottom indicates the timing of L-thyroxine administration. *P < 0.05 vs. euthyroid by two-way repeated measures ANOVA with Dunnett post hoc for panel C and by Bonferroni test for panel D. Data are presented as mean 6 SEM plus individual biological replicates.

Quantitative Real-Time PCR described (5) (n = 2 biological replicates per condition). A TheraCyte devices were harvested at 27 weeks posttrans- list of primers is provided in Supplementary Table 2. plant from all mice for qPCR analysis. Devices were cut longitudinally, and one-half was preserved in RNAlater Immunofluorescent Staining and Image Quantification Stabilization Solution (Life Technologies, Carlsbad, CA) Engrafted hESC-derived cells (half of TheraCyte device), and stored at 280°C until use. The other half of each pancreas tissue, and thyroid glands were harvested at device was stored in 4% paraformaldehyde, as described 27 weeks posttransplant, fixed in 4% paraformaldehyde, below. The procedure for isolating RNA from engrafted and stored in 70% ethanol before paraffin-embedding. tissue within devices and qPCR analysis has been de- All paraffinsections(5-mm thickness) were prepared scribed in detail elsewhere (7). Data were analyzed using by Wax-it Histology Services (Vancouver, British Columbia, Expression Suite 1.0.3 software (Thermo Fisher Scientific/ Canada). Immunofluorescent staining and imaging Life Technologies) and normalized to adult human islets were performed as previously described (14), and pri- (n = 2 donors) using the DDCt method. Gene expression mary antibody information is provided in Supplemen- in cultured hESC-derived cells was assessed as previously tary Table 3. Refer to Supplementary Data for details diabetes.diabetesjournals.org Bruin and Associates 1301 about quantification of the endogenous pancreas and ;99.5% PDX1+ and 70% NKX6.1+ before transplant engrafted tissue. (Supplementary Fig. 1), consistent with previous studies – Statistical Analysis (5 7,13). The progenitor population also contained ;14% endocrine cells, which coexpressed NKX2.2, but All statistics were performed using GraphPad Prism 2 were largely NKX6.1 (Supplementary Fig. 1). These pan- software (GraphPad Software Inc., La Jolla, CA). Details creatic progenitor cells were transplanted subcutaneously about individual statistical tests are provided in the within TheraCyte devices into mice with thyroid hormone Supplementary Data. For all analyses, P , 0.05 was dysregulation (as outlined in Fig. 2A). Our experimental considered statistically significant. Data are presented as mean 6 SEM with individual data points. groups enabled us to examine the effect of acute exposure to deficient or excessive thyroid hormone during the first 30 days posttransplant and chronic thyroid hormone de- RESULTS ficiency for the duration of the study. We chose not to Chronic Hypothyroidism Induced Weight Loss and include a chronic hyperthyroid treatment group because Hyperglycemia, Whereas Chronic Hyperthyroidism even the lowest dosage of T4 tested (3 mg/L) caused dan- Induced Hypoglycemia and Hyperinsulinemia in gerous hypoglycemia and death (Fig. 1D and E). To validate SCID-Beige Mice the efficacy of treatments, plasma T3 levels were measured Our first goal was to establish a protocol to induce fi at 28 days posttransplant (during the acute intervention) hyperthyroidism or hypothyroidism in immunode cient and were confirmed to be significantly lower in the acute SCID-beige mice (outlined in Fig. 1A). Initially, plasma T3 hypothyroid mice and significantly higher in the acute hy- levels were similar between groups, but T3 levels were perthyroid group compared with euthyroid controls (Fig. significantly reduced after 14 days of feeding with an 2B). On day 42 (12 days after the cessation of the acute iodine-deficient diet compared with an iodine control thyroid interventions), T3 levels were normal in the acute diet (euthyroid) (Fig. 1B). In contrast, the addition of hyperthyroid and hypothyroid mice but remained signifi- T4 to drinking water produced dose-dependent increases cantly lower in the chronic hypothyroid mice, reflecting in plasma T3 levels. A dosage of 12 mg/L for 7 days, their ongoing treatment with iodine-deficient diet (Fig. followed by 6 mg/L for 7 days, produced plasma T3 levels 2B). As further validation of the model, the thyroid gland in the hyperthyroid mice that were ;10- to 15-fold higher was examined at the end of the study. Thyroid weight than the euthyroid group (Fig. 1B) but much higher than (normalized to body weight) was significantly increased T3 levels reported in humans with clinical hyperthyroidism in both the chronic and acute hypothyroid groups com- (euthyroid: 3.3 6 0.7 ng/mL; hyperthyroid: 7.1 6 1.0 ng/mL pared with the euthyroid group, although this was most [15]). Therefore, the dosage of T4 was further reduced to pronounced in the chronic group (Supplementary Fig. 2A). 3 mg/L to achieve clinically relevant plasma T3 levels Moreover, we observed severe thyroid follicular atrophy (Fig. 1B) and in an effort to prevent the severe hypogly- and absence of colloid in the thyroid gland of mice with cemia observed with higher T4 dosages (Fig. 1D and E). chronic hypothyroidism (Supplementary Fig. 2B). Mice displayed an initial reduction in body weight Consistent with the first study (Fig. 1), acute hypothy- after administration of the iodine-deficient diet, which roidism resulted in a transient decrease in body weight, recovered and stabilized after 12 days of treatment but which quickly recovered once the mice were taken off the remained significantly lower than euthyroid controls (Fig. iodine-deficient diet at 30 days posttransplant (Fig. 2C). 1C). Hyperthyroidism had no effect on body weight (Fig. The initial weight loss in the chronic hypothyroid group 1C). Hypothyroidism caused significantly elevated blood plateaued after ;80 days, and body weight remained sta- glucose levels under fasting conditions (Fig. 1D) and after ble thereafter (Fig. 2C). The reduction in body weight was an oral glucose challenge (Fig. 1E), whereas hyperthyroidism associated with a significant decrease in fat pad weight resulted in significantly decreased blood glucose levels (relative to body weight) in the chronic hypothyroid mice at (Fig. 1D and E). Hyperthyroidism was also associated with 27 weeks posttransplant (Supplementary Fig. 3). Chronic hy- significantly higher fasting plasma insulin levels compared pothyroidism also led to elevatedfastingbloodglucoselevels, with euthyroid controls (Fig. 1F). Unfortunately, after 24 whereas acute hypothyroid mice remained normoglycemic days of T4 administration, 4 of 10 hyperthyroid mice throughout the study (Fig. 2C). Acute hyperthyroidism had died, and the remaining mice were consequently switched no effect on body weight but caused a transient decrease in to normal drinking water, resulting in return to normo- blood glucose levels during the T4 treatment period (Fig. 2D), glycemia within 2 weeks (Fig. 1D). consistent with the previous cohort of mice (Fig. 1D). From Thyroid Hormone Deficiency Hinders the Development this point forward, all data from the two euthyroid groups of hESC-Derived Progenitor Cells Into Mature were pooled because their body weight and blood glucose Insulin-Secreting Cells In Vivo levels were not significantly different. We next assessed the effects of excessive or deficient To assess the development of the hESC-derived grafts thyroid hormone levels on the development of hESC- under conditions of thyroid dysregulation, human C-peptide derived pancreatic progenitor cells in vivo. After the and blood glucose levels were measured in response to 14-day differentiation in vitro, hESC-derived cells were various secretagogues after transplant. Chronic hypothyroid 1302 Hypothyroidism Impairs hESC-Derived b-Cells Diabetes Volume 65, May 2016 mice exhibited elevated blood glucose levels during an posttransplant, respectively (Fig. 3C and D). Acute hypo- oral mixed-meal challenge at all ages examined (Fig. 3A thyroidism did not affect glycemia during any metabolic and C), as well as during intraperitoneal glucose (Fig. 3D) challenge (Fig. 3A, C, D, and G). and arginine (Fig. 3G)tolerancetestsat22and24weeks Beginning at 8 weeks posttransplant and persisting posttransplant, respectively. Mice with acute hyperthy- throughout the study, the chronic hypothyroid mice had roidism had decreased blood glucose levels after the meal significantly decreased human C-peptide levels compared challenge at 4 weeks posttransplant (during their T4 with euthyroid control mice under fasting conditions (data treatment period; Fig. 3A) but mild hyperglycemia dur- not shown) and at 40 min after the oral mixed meal (Fig. ing the meal and glucose challenges at 8 and 22 weeks 3B). In addition, mice with acute hypothyroidism had

Figure 3—Maturation and function of hESC-derived pancreatic progenitor cells in mice with thyroid dysfunction. Blood glucose (A) and plasma human C-peptide (B) levels 40 min after an oral mixed-meal challenge at 4, 8, 12, 16, and 25 weeks posttransplant. *P < 0.05 vs. euthyroid by one-way ANOVA. Blood glucose levels during an oral mixed-meal challenge at 8 weeks posttransplant (C) and an intraper- itoneal glucose tolerance test (ipGTT) at 22 weeks posttransplant (D). Plasma human C-peptide levels during the ipGTT at 22 weeks, expressed in ng/mL (E) or relative to basal levels at time 0 (F). C–F: Line graphs: *P < 0.05 vs. euthyroid by two-way repeated-measures ANOVA. Bar graphs: *P < 0.05 vs. euthyroid by one-way ANOVA. Blood glucose levels (G) and plasma levels of human insulin (H), glucagon (I), and GLP-1 (J) after a 4-h morning fast and 15 min after an intraperitoneal arginine challenge at 24 weeks posttransplant. Hyper, hyperthyroid; Hypo, hypothyroid. #P < 0.05 for 0 vs. 15 min by paired t test; *P < 0.05 vs. euthyroid by one-way ANOVA. Data are presented as mean 6 SEM plus individual biological replicates. diabetes.diabetesjournals.org Bruin and Associates 1303 a transient decrease in meal-stimulated human chronic hypothyroid compared with euthyroid mice, C-peptide levels compared with euthyroid mice at 16 weeks whereas the glucagon-to-insulin ratio in the endogenous posttransplant, which was recovered by 25 weeks (Fig. 3B). pancreas was not affected by chronic hypothyroidism (Fig. At 22 weeks posttransplant, chronic hypothyroid mice 6B). Moreover, thyroid hormone deficiency did not af- exhibited severely blunted human C-peptide secretion dur- fect the b-cell area per islet in the endogenous pancreas ing an intraperitoneal glucose challenge (Fig. 3E) and also (Fig. 6C). At 27 weeks posttransplant, proliferating cell displayed altered C-peptide secretion kinetics compared nuclear antigen+ (PCNA) endocrine cells were rare, and with euthyroid mice (Fig. 3F). Peak human C-peptide levels there were no obvious differences in the number of pro- were observed at 60 min after glucose administration in liferating insulin+ or glucagon+ cells between treatment the chronic hypothyroid mice (;2.5 times higher than groups (Supplementary Fig. 4). Despite the significant in- basal), whereas the other groups had reached similar peak crease in SST transcript levels in chronic hypothyroid C-peptide levels at 30 min and were approaching basal grafts (Fig. 4), there was no significant difference in the levels by 60 min (Fig. 3F). The acute hyperthyroid group fraction of somatostatin+ cells among groups (quantifica- also had significantly reduced human C-peptide levels at tion not shown; Supplementary Fig. 5). Pancreatic poly- 30 min after the glucose injection (Fig. 3E), but unlike peptide immunoreactivity was rare in grafts and did not the chronic hypothyroid group, they exhibited human appear to differ between groups, although this was not C-peptide secretion kinetics similar to those of euthyroid quantified (Supplementary Fig. 5). control mice (Fig. 3F). Consistent with the meal and glucose Interestingly, chronic hypothyroid grafts had approx- challenges, the chronic hypothyroid group also displayed imately an eightfold increase in GHRL transcript levels significantly reduced human insulin levels at fasting and (Fig. 4) and a significantly higher proportion of ghrelin+ 15 min after an arginine injection compared with euthy- cells relative to DAPI+ cells (Figs. 5C and 6A) compared roid mice at 24 weeks posttransplant (Fig. 3H). Interest- with euthyroid grafts. Moreover, while ghrelin+ cells were ingly, these mice also had higher plasma glucagon levels abundant in the hESC-derived engrafted tissue, they were postarginine (Fig. 3I)aswellaselevatedGLP-1levelsatfast- only rarely detected in the endogenous pancreas (Fig. 6D). ing and postarginine (Fig. 3J) compared with euthyroid mice. Chronic hypothyroid grafts contained approximately equal proportions of ghrelin+ and insulin+ cells, whereas + Chronic Hypothyroidism Affects the Endocrine euthyroid grafts had approximately four insulin cells for Composition of hESC-Derived Grafts but not the every one ghrelin+ cell (Fig. 6E). In the pancreas, the ratio Endogenous Pancreas of ghrelin-to-insulin immunoreactive cells was not af- TheraCyte devices were harvested at 27 weeks posttrans- fected by exposure to chronic hypothyroidism (Fig. 6E). plant for qPCR and histology analysis. Although the The difference in circulating acylated (active) or unacy- chronic hypothyroid group displayed decreased plasma lated (inactive) ghrelin levels among the groups was not human insulin and increased plasma glucagon levels (Fig. significant, but the ratio of unacylated-to-acylated ghrelin 3H and I), INS and GCG mRNA levels in these hESC- was approximately two times higher in the chronic hypo- derived grafts were not significantly different from the thyroid mice than in the euthyroid mice (Fig. 6F). euthyroid group (Fig. 4). However, chronic hypothyroid- ism resulted in significantly increased levels of SST, GHRL, Thyroid Hormone Deficiency Affects b-Cell Maturation and ISL1, as well as a pronounced reduction in IAPP and in hESC-Derived Grafts but not the Endogenous G6PC2 mRNA in hESC-derived grafts compared with Pancreas grafts from euthyroid controls (Fig. 4). Acute hyperthy- Chronic hypothyroidism resulted in a reduced number of roidism also caused a mild but significant reduction in hESC-derived b-cells (Figs. 5 and 6), which may explain IAPP and G6PC2 and elevated GCG mRNA levels relative the decreased absolute human C-peptide levels compared to euthyroid grafts (Fig. 4). with euthyroid mice (Fig. 3B, E, and H). However, the Given that the most pronounced effects on graft disrupted human insulin secretion kinetics in chronic hy- function and gene expression were a result of chronic pothyroid mice (Fig. 3F) suggested that the maturation hypothyroidism (Figs. 3 and 4), we focused our detailed status of individual hESC-derived b-cells may also be al- characterization of graft composition on the chronic hy- tered by thyroid hormone deficiency. On the basis of ev- pothyroid versus euthyroid mice. Thyroid hormone defi- idence that islet Mafa expression was regulated by thyroid ciency did not affect the overall formation of endocrine hormones in neonatal rats (12), we examined expression (synaptophysin+) or ductal (CK19+) cells from hESCs (Fig. of MAFA in grafts and the endogenous mouse pancreas. 5A) but appeared to shunt the cells toward a-cells at the Although MAFA transcript levels were not affected in expense of b-cells (Fig. 5B and C). Indeed, grafts from whole grafts (Fig. 4), we observed substantial heterogene- chronic hypothyroid mice contained a significantly lower ity in nuclear MAFA immunoreactivity within the hESC- fraction of insulin+ cells and more than twice as many derived insulin+ population from chronic hypothyroid glucagon+ cells compared with euthyroid grafts (Fig. 6A). mice but no difference in MAFA expression within the Thus, there was a significantly higher ratio of glucagon-to- endogenous pancreas of hypothyroid versus euthyroid insulin immunoreactive cells in hESC-derived grafts from mice (Fig. 7A). Similarly, hypothyroidism also caused 1304 Hypothyroidism Impairs hESC-Derived b-Cells Diabetes Volume 65, May 2016

Figure 4—qPCR analysis of hESC-derived grafts from mice with thyroid dysfunction. TheraCyte devices were harvested at 27 weeks posttransplant for qPCR analysis of key b-cell markers. Gene expression is expressed relative to levels in adult human islets (indicated by dashed red line and orange bars; n = 2 donors). *P < 0.05, **P < 0.01 vs. euthyroid for hESC-derived engrafted cells by one-way ANOVA. Data are presented as mean 6 SEM plus individual biological replicates.

decreased NKX2.2 immunoreactivity in the grafts, but no vivo study, we observed reduced GHRL, GCG, and ARX differences were observed among the groups in the en- mRNA during S5 after treatment with 500 nmol/L or dogenous pancreas (Fig. 7B). In addition, there was less 1,000 nmol/L T3 during S4 (Supplementary Fig. 6). T3 amylin immunoreactivity within the insulin+ cell popula- treatment also increased mRNA levels of INS and mature tion in the hypothyroid compared with euthyroid grafts b-cell markers, G6PC2, IAPP, and MAFA,duringS6 (Fig. 7C), confirming the significant reduction in overall (Supplementary Fig. 6). gene expression of IAPP in hypothyroid grafts (Fig. 4); amylin immunoreactivity was not affected in the b-cells DISCUSSION from the endogenous pancreas (Fig. 7C). The high incidence of thyroid disease in patients with The hESC-derived pancreatic progenitor cells were also T1D (8) means that hESC-derived pancreatic progenitor treated in vitro with T3 during S4 to determine if the cells transplanted into these patients may be exposed to effects on graft maturation in vivo might be a result of abnormal thyroid hormone levels in vivo. Notably, we direct action by thyroid hormones. Consistent with our in found that chronic thyroid hormone deficiency had a diabetes.diabetesjournals.org Bruin and Associates 1305

Figure 5—Immunofluorescent staining for pancreatic endocrine hormones in hESC-derived grafts and the endogenous pancreas from euthyroid or chronic hypothyroid mice at 27 weeks posttransplant. Low-magnification images of the hESC-derived grafts from mice with euthyroid or chronic hypothyroid treatments showing immunofluorescent staining for endocrine (synaptophysin; red) and ductal (CK19; green) populations (A) and insulin (red) and glucagon (green) cell populations (B). C: Representative high-magnification images of hESC- derived grafts from euthyroid or chronic hypothyroid mice stained for insulin (Ins; red), glucagon (Gcg; green), or ghrelin (green). D: Images of representative islets in the endogenous pancreas stained for insulin (red) and glucagon (green) or ghrelin (Ghrl, green). DAPI (49,6-diamidino- 2-phenylindole) nuclear staining is shown in gray for all images. All scale bars = 50 mm.

detrimental effect on hESC-derived b-cell development lineage commitment of differentiating endocrine cells, and was associated with higher numbers of hESC-derived thus resulting in a fate-switch toward a-cells and e-cells a- and e-cells. Beginning at 8 weeks posttransplant and at the expense of b-cells, or whether perhaps thyroid persisting throughout the study, grafts from chronic hormone is required for survival and/or expansion of hypothyroid mice secreted less than half as much human newly differentiated b-cells. T3-treated neonatal rats had C-peptide as grafts from euthyroid mice. Blunted human increased b-cell proliferation but no measureable change in C-peptide secretion was also observed in mice acutely ex- b-cell apoptosis relative to control rats (12). Proliferation posed to hypothyroidism, but this effect was transient and of hESC-derived endocrine cells was not affected by thyroid fully recovered at 25 weeks posttransplant. Chronic hypo- hormone deficiency in our study after 27 weeks, but it is thyroid mice also displayed impaired glucose-stimulated possible that impaired b-cell replication and/or increased insulin secretion kinetics, suggesting that the maturation b-cell apoptosis may have occurred in hypothyroid grafts at status of b-cells was affected by thyroid hormone defi- an earlier time point. ciency. The elevated plasma glucagon and GLP-1 levels in Thyroid hormone deficiency resulted in heterogeneous chronic hypothyroid mice pointed to preferential forma- protein expression of nuclear MAFA in hESC-derived tion of a-cells in hESC-derived grafts exposed to thyroid grafts as well as reduced NKX2.2 and amylin levels, hormone deficiency. Indeed, grafts harvested from chronic important markers of mature b-cells. IAPP (amylin) and hypothyroid mice contained approximately three times G6PC2 mRNA levels were also significantly reduced in fewer insulin+ cells and more than twice as many glucagon+ grafts from hypothyroid mice compared with euthyroid cells as grafts from euthyroid mice. It is unclear from these controls, whereas the observed differences in insulin+, studies whether thyroid hormone deficiency altered the glucagon+, and MAFA+ cells were not reflected at the level 1306 Hypothyroidism Impairs hESC-Derived b-Cells Diabetes Volume 65, May 2016

Figure 6—Quantification of the pancreatic endocrine populations in hESC-derived grafts and the endogenous pancreas from euthyroid vs. chronic hypothyroid mice at 27 weeks posttransplant. A: The number of cells that were immunoreactive (IR) for insulin (Ins), glucagon (Gcg), or ghrelin expressed relative to the total number of DAPI+ (49,6-diamidino-2-phenylindole) cells in the hESC-derived grafts from euthyroid vs. chronic hypothyroid mice. B: The ratio of glucagon+ cells to insulin+ cells in hESC-derived grafts and the endogenous pancreas. C:Total b-cell (i.e., insulin+) area per islet in the endogenous pancreas. D: The total number of cells immunoreactive for insulin or ghrelin that were counted in the hESC-derived grafts vs. the endogenous pancreas (every visible ghrelin+ or insulin+ cell was counted within each section). E: The ghrelin+-to-insulin+ cell ratio in the hESC-derived grafts and the endogenous pancreas. F: Plasma levels of active (acylated) and inactive (unacylated) ghrelin as well as the unacylated-to-acylated (Unacyl:Acyl) ghrelin ratio in mice from all treatment groups. A–E:*P < 0.05 by two- tailed unpaired t tests. F:*P < 0.05 vs. euthyroid by one-way ANOVA. Data are presented as mean 6 SEM plus individual biological replicates.

of gene expression. Because mRNA levels represent the induce Mafa expression and promote glucose-stimulated whole population of hESC-derived cells (including all en- insulin secretion in immature neonatal rat islets (12). docrine cell types, ductal cells, etc.), examining protein Ghrelin production by hESC-derived grafts also proved expression in individual insulin+ cells by immunofluores- to be interesting in this study. Grafts from euthyroid mice cent staining is a more accurate assessment of the hESC- contained substantially higher (200 times) ghrelin mRNA derived b-cell phenotype. The various lines of evidence levels compared with human islets and a 4:1 ratio of pointing to a b-cell deficiency in hypothyroid mice are insulin-to-ghrelin immunoreactive cells, whereas ghrelin+ consistent with our previous observations in nude rats cells were exceedingly rare (,0.5%) in the adult human implanted with hESC-derived progenitor cells (16). Inter- and mouse pancreas. This discrepancy was even further estingly, nude rats had significantly higher circulating T3 amplified in the chronic hypothyroid grafts, which con- levels than SCID-beige mice and also had improved glucose- tained ;1,250 times more ghrelin mRNA than human stimulated human insulin secretion, a higher proportion of islets and an ;1:1 ratio of insulin-to-ghrelin immunoreac- insulin-to-glucagon immunoreactivity in grafts, and more tive cells. In the human fetal pancreas, ghrelin+ cells con- consistent nuclear MAFA expression in hESC-derived stitute a relatively high proportion (;10%) of endocrine b-cells than SCID-beige mice (16). Neonatal rats with hy- cells (17). Therefore, the high ghrelin levels in chronic pothyroidism also exhibited decreased islet Mafa expres- hypothyroid grafts could reflect graft immaturity or may sion (12), which is consistent with our findings and simply indicate a shift in lineage commitment during suggests a potential role for thyroid hormone in regulat- development. Grafts from chronic hypothyroid mice also ing b-cell maturation. This is further supported by evi- contained less NKX2.2 immunoreactivity, which was pre- dence that thyroid hormones bind directly to the thyroid viously shown to be required for specification and main- hormoneresponseelementsintheMafa promoter to tenance of b-cell fate (18). Moreover, the absence of diabetes.diabetesjournals.org Bruin and Associates 1307

Figure 7—Characterization of b-cells in hESC-derived grafts compared with the endogenous pancreas of euthyroid vs. chronic hypothy- roid mice at 27 weeks posttransplant. Representative immunofluorescent staining images of TheraCyte devices containing hESC-derived grafts and islets from the endogenous pancreas for insulin (red) with MAFA (green; insets highlight the heterogeneity of MAFA immuno- reactivity in the chronic hypothyroid group) (A), NKX2.2 (green) (B), or amylin (green) (C). Images are shown with DAPI (49,6-diamidino-2- phenylindole) nuclear staining (gray) on the left or without DAPI on the right. All scale bars = 50 mm. 1308 Hypothyroidism Impairs hESC-Derived b-Cells Diabetes Volume 65, May 2016

NKX2.2 in mice resulted in a dramatic expansion of ghrelin- (21), whereas the opposite typically occurs with hypothy- producing cells at the expense of b-cells (18). Interest- roidism. Interestingly, we observed contradictory effects ingly, patients with hypothyroidism were reported to of thyroid hormones on blood glucose homeostasis in have elevated serum ghrelin levels (acylated and unacy- SCID-beige mice. Hyperthyroidism led to hypoglycemia, lated), which was normalized by T4 treatment (19). Al- whereas hypothyroidism caused hyperglycemia compared though the chronic hypothyroid mice in our study did not with euthyroid controls. Hypothyroidism also caused produce elevated circulating ghrelin levels, there was decreased body weight in mice, whereas in humans, hy- clearly an effect of thyroid hormone deficiency to induce pothyroidism generally leads to weight gain. Although our ghrelin locally within hESC-derived grafts. Pancreatic data differ from the clinical situation, these findings are ghrelin serves as a local regulator of insulin release consistent with previous studies examining hyperthyroid- even though it may not contribute to the circulating ism and hypothyroidism in rodents. For example, rodents ghrelin levels (20). Therefore, ghrelin may be acting in a that received subcutaneous T3 injections had lowered paracrine manner to impair insulin secretion in the hESC- fasting glucose levels compared with controls (12,22), derived grafts from chronic hypothyroid mice. and hypothyroidism in rats (induced via thyroidectomy Although thyroid hormone deficiency caused clear and PTU or methimazole treatment) caused decreased effects on the transplanted human pancreatic progenitor body weight (12,23) and increased serum glucose levels cells, the endogenous pancreas was largely unaffected (23). The differences between human and rodent thyroid in our study. It is possible that developing endocrine physiology that account for these opposing phenotypes cells are more susceptible to thyroid hormone dysregula- are currently unknown. tion than adult islet cells or that human pancreatic cells are Stem cell–derived pancreatic progenitor cells are cur- more susceptible than mouse cells, although we suspect the rently being transplanted into patients with T1D in a phase former is more likely. It is also possible that the effects on 1/2 clinical trial with the ViaCyte VC-01 cell product. Our the differentiation of hESC-derived cells are due to the findings raise the possibility that the maturation and ulti- profoundly disrupted metabolic control in the mice with mate function of the transplanted cells can be influenced hyper- or hypothyroidism. However, we previously by the hormonal and metabolic milieu of the cells. Specif- observed efficient graft maturation and robust human ically, we recommend that eligible patients are screened for C-peptide production in the setting of chronic hypergly- thyroid dysfunction and treated accordingly to reduce the cemia (4) and thus the impaired glycemic control in hypo- risk of off-target cell differentiation leading to compro- thyroid mice is unlikely to be the underlying cause of mised graft function. Alternatively, hESCs could be differ- impaired maturation in the current study. Thyroid hor- entiated to a more mature stage of development in vitro, as mones are also known to profoundly affect the stress re- we have recently described (13), to minimize the matura- sponse in vivo (12), which could also indirectly mediate tion period after transplantation. It is possible that more the observed effects of hypothyroidism on b-cell matura- mature hESC-derived cells will be less susceptible to altered tion. Interestingly, dexamethasone treatment of neonatal levels of thyroid hormones because they are closer to adult rat islets blocked the T3-mediated induction of Mafa cells, although this remains to be examined. mRNA and glucose-stimulated insulin secretion ex vivo (12). Our previous discovery that T3 treatment at late stages of hESC differentiation enhanced b-cell maturation Acknowledgments. The authors thank Diana Rosman-Balzer (Janssen in culture (13) supports the notion that the hESC-derived R&D, LLC) for her technical assistance with qPCR experiments and Ali Asadi (UBC Department of Cellular & Physiological Sciences) for his assistance with grafts may be directly affected by thyroid hormone levels. histology. Moreover, when S4 pancreatic progenitor cells were treat- Funding. This work was funded by the Canadian Institutes of Health Research ed with T3 in vitro, we observed a transient decrease in (CIHR) Regenerative Medicine and Nanomedicine Initiative, the Stem Cell GHRL, GCG, and ARX mRNA at S5, and elevated INS, Network, and JDRF. J.E.B. was funded by a JDRF postdoctoral fellowship, a G6PC2, IAPP, and MAFA mRNA levels at S6 of in vitro Canadian Diabetes Association (CDA) postdoctoral fellowship, the CIHR Trans- differentiation. These data are consistent with our in vivo plantation Training Program, and a L’Oréal Canada for Women in Science Re- study, in which reduced thyroid hormone levels caused search Excellence Fellowship. N.S. received funding from the Stem Cell Network. increased a-ande-cell formation along with impaired b-cell Duality of Interest. P.A. and A.R. are employees of Janssen R&D, LLC, maturation in hESC-derived grafts, including reduced G6PC2 and A.R. is also a shareholder. T.J.K. received financial support from Janssen and IAPP mRNA. Further studies are required to determine R&D, LLC, for the research described in this article. No other potential conflicts whether thyroid hormones are acting directly on developing of interest relevant to this article were reported. Author Contributions. J.E.B. and N.S. wrote the manuscript. J.E.B., human b-cells or whether the indirect effects of thyroid A.R., and T.J.K. contributed to conception and design of experiments. J.E.B., hormones on metabolism and/or the hypothalamic- N.S., S.O., J.K.F., M.M., P.A., A.R., and T.J.K. were responsible for acquisition, pituitary-adrenal axis may be mediating the observed analysis, and interpretation of data; contributed to the manuscript revisions; b changes in -cell development in vivo. and approved the final version of the manuscript. T.J.K. is the guarantor of Clinically, hyperthyroidism can lead to elevated blood this work and, as such, had full access to all the data in the study and takes glucose levels, possibly due to enhanced gluconeogenesis responsibility for the integrity of the data and the accuracy of the data and increased absorption of sugars from the intestine analysis. diabetes.diabetesjournals.org Bruin and Associates 1309

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