Enrichment of Human Embryonic Stem Cell-Derived NKX6.1 ...

REGENERATIVE MEDICINE

Enrichment of Human Embryonic Stem Cell-Derived NKX6.1-Expressing Pancreatic Progenitor Cells Accelerates the Maturationof Insulin-Secreting Cells In Vivo

ALIREZA REZANIA,a JENNIFER E. BRUIN,b JEAN XU,a KAVITHA NARAYAN,a JESSICA K. FOX,b JOHN J. O’NEIL,a

TIMOTHY J. KIEFFERb,c

aBetaLogics Venture, Janssen R & D LLC, Raritan, New Jersey, USA; bLaboratory of Molecular and CellularMedicine, Department of Cellular and Physiological Sciences, Life Sciences Institute; cDepartment of Surgery,University of British Columbia, Vancouver, British Columbia, Canada

Key words: Diabetes • Beta cells • Human embryonic stem cells • Cell therapy • Transplantation

ABSTRACT

Human embryonic stem cells (hESCs) are considered apotential alternative to cadaveric islets as a source oftransplantable cells for treating patients with diabetes.We previously described a differentiation protocol to gen-erate pancreatic progenitor cells from hESCs, composedof mainly pancreatic endoderm (PDX1/NKX6.1-positive),endocrine precursors (NKX2.2/synaptophysin-positive,hormone/NKX6.1-negative), and polyhormonal cells(insulin/glucagon-positive, NKX6.1-negative). However,the relative contributions of NKX6.1-negative versusNKX6.1-positive cell fractions to the maturation of func-tional b-cells remained unclear. To address this question,we generated two distinct pancreatic progenitor cell pop-ulations using modified differentiation protocols. Prior totransplant, both populations contained a high proportionof PDX1-expressing cells (!85%–90%) but were distin-guished by their relatively high (!80%) or low (!25%)expression of NKX6.1. NKX6.1-high and NKX6.1-lowprogenitor populations were transplanted subcutaneouslywithin macroencapsulation devices into diabetic mice.

Mice transplanted with NKX6.1-low cells remainedhyperglycemic throughout the 5-month post-transplantperiod whereas diabetes was reversed in NKX6.1-highrecipients within 3 months. Fasting human C-peptidelevels were similar between groups throughout the study,but only NKX6.1-high grafts displayed robust meal-,glucose- and arginine-responsive insulin secretion asearly as 3 months post-transplant. NKX6.1-low recipientsdisplayed elevated fasting glucagon levels. Theracytedevices from both groups contained almost exclusivelypancreatic endocrine tissue, but NKX6.1-high grafts con-tained a greater proportion of insulin-positive andsomatostatin-positive cells, whereas NKX6.1-low graftscontained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high, but not NKX6.1-low graftsexpressed nuclear MAFA. Collectively, this study demon-strates that a pancreatic endoderm-enriched populationcan mature into highly functional b-cells with only aminor contribution from the endocrine subpopulation.STEM CELLS 2013;31:2432–2442

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

Diabetes is characterized by chronic high blood glucose levelsresulting from deficient insulin-producing pancreatic b-cells.There are numerous experimental strategies aimed to restoreendogenous regulated insulin production in patients with type1 diabetes [1]. To date, islet cell transplantation is the mosteffective clinical therapy [2,3], but widespread application isseverely limited by the shortage of cadaveric donor islets [4].Human embryonic stem cells (hESCs) may be a potentialrenewable source of insulin-producing cells to bridge the gapbetween cell supply and clinical demand [5]. Step-wise differ-

entiation protocols designed to mimic pancreatic developmenthave successfully generated insulin-producing cells fromhESCs in vitro, but these cells resemble fetal endocrine cellsrather than adult pancreatic b-cells [6–17]. Mature, glucose-responsive insulin-secreting cells have only been producedfrom hESCs following transplantation of immature pancreaticprogenitor cells; this was first demonstrated in healthy mice[12] and subsequently by our group in mice with diabetes[17,18]. We demonstrated that graft-derived human insulinlevels gradually increased over time and after a 3-month mat-uration period, previously insulin-dependent diabetic miceachieved normal fasting blood glucose levels without exoge-nous insulin treatment [17]. After a lengthy maturation period

Author contributions: A.R.: conception and design, collection and/or assembly of data, data analysis and interpretation; final approval ofmanuscript; J.E.B.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, andfinal approval of manuscript; J.X.: conception and design, collection and/or assembly of data, and data analysis and interpretation; K.N.,J.F., and J.J.O.: collection and/or assembly of data; T.J.K.: conception and design, data analysis and interpretation, and final approval ofmanuscript. A.R., J.E.B., and J.X. contributed equally to this article.

Correspondence: Timothy Kieffer, Ph.D., Room 5308-2350 Health Sciences Mall, University of British Columbia, Vancouver, BritishColumbia V6T 1Z3, Canada. Telephone: 604-822-2156; Fax: 604-822-2316; e-mail: [email protected] Received December 19, 2012;Revised June 9, 2013; accepted for publication July 1, 2013; first published online in STEM CELLS EXPRESS July 29, 2013. VC AlphaMedPress 1066-5099/2013/$30.00/0 doi: 10.1002/stem.1489

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of approximately 30 weeks in vivo, human insulin secretionwas regulated by various secretagogues, including meal, glu-cose, and arginine challenges, and a corresponding improve-ment in glucose tolerance was observed along with theappearance of insulin/V-maf musculoaponeurotic fibrosarcomaoncogene homolog A (MAFA) co-positive b-cells within kid-ney capsule grafts [17].

The pancreatic progenitor cells generated with our previousdifferentiation protocol were composed of mainly immaturepolyhormonal cells (insulin/glucagon-positive, NKX6.1-nega-tive), endocrine precursor cells (NKX2.2/synaptophysin-posi-tive, hormone/NKX6.1-negative), and pancreatic endoderm-likecells (NKX6.1/PDX1-positive, insulin-negative) [17]. Theexpression of NKX6.1 distinguishes these populations and isthought to mark the progenitor cells that arise during the sec-ond transition of pancreas development, as opposed to the pol-yhormonal cells that arise during the first transition [19].Results from several recent studies suggest that polyhormonalcells may develop into pancreatic a-cells following transplanta-tion [6,16,20]. In contrast, when hESC-derived pancreaticendoderm cells were purified by fluorescence-activated cellsorting (FACS) and transplanted into mice, functional islet-likeclusters developed in vivo, although less efficiently than anunsorted progenitor cell population [6]. Thus, it remainsunclear whether the NKX6.1-negative fraction contributes tomaturation of the NKX6.1-positive cell fraction. Here, weexamined the relative contributions of each pancreatic progeni-tor subpopulation to the development of mature insulin-secreting cells in vivo by transplanting cell populations thatwere enriched, but not FACS-purified, for either pancreaticendoderm or pan-endocrine cells.

MATERIALS AND METHODS

In Vitro Differentiation of hESCsThe H1 hESC line was obtained from WiCell Research Institute,Inc. (Madison, WI, http://www.wicell.org). All experiments at theUniversity of British Columbia (UBC) with H1 cells were approvedby the Canadian Stem Cell Oversight Committee and UBC ClinicalResearch Ethics Board. H1 cells were cultured on 1:30 dilutedMatrigel (BD BioSciences, San Diego, http://www.bdbiosciences.-com; Cat#356231) in mTeSR-1 (Stem Cell Technologies, Vancou-ver, BC, Canada, http://www.stemcell.com; Cat#05850). Atapproximately 70%–80% confluence, cultures were rinsed with 13Dulbecco’s phosphate-buffered saline (DPBS) without Mg21 andCa21 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com;Cat#14190) followed by incubation with Versene (EDTA), 0.02%(LONZA, http://www.lonza.com, Cat#17–711E) for 12 minutes atroom temperature. Released single cells were rinsed with mTeSR-1and spun at 1,000 rpm for 5 minutes. The resulting cell pellet wasresuspended in mTeSR-1 medium supplemented with Y-27632 (10mM; Sigma-Aldrich; St. Louis, MO, http://www.sigmaaldrich.com;Cat#Y0503) and the single cell suspension was seeded at approxi-mately 1.3 3 105 cells per centimeter square. Cultures were fedevery day and differentiation was initiated 48 hours following seed-ing, resulting in approximately 90% starting confluence. The follow-ing differentiation protocol variations were used to generate either“NKX6.1-high” or “NKX6.1-low” cell populations (summarized inFig. 1A):

Stage 1: Definitive Endoderm (3 Days). Undifferentiated H1cells plated on 1:30 Matrigel-coated surfaces were exposed toRPMI 1640 medium (Invitrogen; Cat#22400) supplemented with1.2 g/L sodium bicarbonate (Sigma, MO; Cat# S6297), 0.2% fetalbovine serum (FBS; Hyclone, Logan, UT, http://www.hyclone.-com; Cat# SH30071.02), 100 ng/mL activin-A (AA; Pepro-tech,

Rocky Hill, NJ, http://www.peprotech.com), and 20 ng/mL ofWnt3A (R&D Systems, Minneapolis, http://www.rndsystems.com)for day 1 only. For the next 2 days, cells were cultured in RPMIwith 0.5% FBS, 1.2 g/L sodium bicarbonate, and 100 ng/mL AA.

Stage 2: Primitive Gut Tube (3 Days). Stage 1 cells wereexposed to Dulbecco’s modified Eagle’s medium (DMEM)-F12medium (Invitrogen) supplemented with 2 g/L sodium bicarbon-ate, 2% FBS, and 50 ng/mL of fibroblast growth factor 7 (Pepro-tech) for 3 days.

Stage 3: Posterior Foregut (4 Days). Cultures were contin-ued for 4 days in DMEM-HG (high-glucose) medium (Invitrogen)supplemented with 0.25 mM SANT-1 (Sigma-Aldrich), 2 mM reti-noic acid (RA; Sigma-Aldrich), 100 ng/mL of Noggin (R&D Sys-tems), and 1% (v/v) B27 (Invitrogen). At this stage, NKX6.1-lowcells received 1 mM ALK5 inhibitor II (ALK5i; Axxora, SanDiego, CA, http://www.axxora.com). ALK5i was not added to theNKX6.1-high cultures during stage 3.

Stage 4: Pancreatic Endoderm/Endocrine Precursors (5Days). Stage 3 cells were then cultured for 4 days in DMEM-HG medium supplemented with 100 ng/mL Noggin and 1% B27.For NKX6.1-high cells, 500 nM TPB (PKC activator; (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)ben-zolactam, EMD, http://www.emdmillipore.com/, Chemicals Inc,Gibbstown, NJ) was added to the stage 4 media. For NKX6.1-low cells, 1 mM ALK5i was added.

For the last day of culture, all cells were treated with 5 mg/mLdispase for 5 minutes at 37"C, pipetted gently to break into cellclumps (<100 mm) and transferred into Spinner Flasks (Corning,Acton, MA, http://www.corning.com). Cell clusters were spun at80–100 rpm overnight in suspension with DMEM-HG supplementedwith 0.2 mM ALK5i, 100 nM LDN-193189 (LDN; bone morphoge-netic protein (BMP) receptor inhibitor, Stemgent, CA, https://www.stemgent.com/, Cat# 04–0074) and 1% B27.

Flow CytometryStage 4 hESC-derived cells were assessed before transplant toensure that the starting population met the appropriate quality con-trol standards. Cell clusters were released into a single-cell suspen-sion and either stained directly for surface markers or fixed andstained for various intracellular markers, as described previously[17]. Refer to Supporting Information Table 1 for antibody details.

Quantitative Reverse Transcriptase Polymerase ChainReactionGene expression was assessed in NKX6.1-high and NKX6.1-lowprogenitor cells using custom Taqman Arrays (Applied Biosys-tems, Foster City, CA, http://www.appliedbiosystems.com), aspreviously described [17]. Data were analyzed using SequenceDetection Software (Applied Biosystems) and normalized toundifferentiated H1 cells using the DDCt method.

Animal StudiesAll experiments were approved by the UBC Animal Care Commit-tee. Male 8–10-week-old SCID-beige mice (C.B-Igh21b/GbmsTac-Prkdcscid-LystbgN7) were obtained from Taconic (Hudson, NY) andmaintained on a 12-hour light/dark cycle with ad libitum access to astandard irradiated diet (Harlan Laboratories, Teklad Diet #2918;Madison, WI). Mice were rendered diabetic by a multiple low-dosestreptozotocin (STZ) regimen of daily intraperitoneal STZ injectionsfor 5 consecutive days (50 mg/kg per day).

Mice were anesthetized with inhalable isoflurane and receivedapproximately 5 million hESC-derived stage 4 cells (NKX6.1-high or NKX6.1-low) transplanted subcutaneously on the rightflank within a single 20 mL Theracyte planar macroencapsulationdevice (TheraCyte Inc., Laguna Hills, CA; n 5 9 mice

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Figure 1. Characterization of NKX6.1-high and NKX6.1-low populations prior to transplantation in macroencapsulation devices. (A): Schematicsummary of the 15-day in vitro differentiation protocols used to generate NKX6.1-low (green) and NKX6.1-high (brown) cells. (B): FACS quan-tification of differentiated hESC-derived populations at stage 4, day 4 prior to transplant. NKX6.1-low (n 5 2) and NKX6.1-high (n 5 3) popula-tions were defined primarily based on their respective NKX6.1-expressing cell fractions. NKX6.1-low cells contained a relatively high proportionof endocrine cells (synaptophysin-positive and NKX2.2-positive) compared to NKX6.1-high cells. Representative FACS plots from both groupsare provided in panel (C). (D): qPCR for expression levels of various pancreatic endocrine genes (hormones and transcription factors) inNKX6.1-high cells (n 5 6) relative to NKX6.1-low cells (n 5 4). Dashed red line indicates equal expression in both groups. (E–G): NKX6.1-lowand NKX6.1-high cells were loaded into Theracyte devices pre-transplant and immunostained for insulin, glucagon and NKX6.1 (panel (E); scalebars 5 100 mm), NKX2.2 (panel (F); scale bars 5 50 mm) and DAPI (panel (G); scale bars 5 50 mm). Abbreviations: DMEM-HG, Dulbecco’smodified Eagle’s medium-high glucose; DAPI, 40,6-diamidino-2-phenylindole; FACS, fluorescence activated cell sorting; FBS, fetal bovineserum; FGF, fibroblast growth factor; hESC, human embryonic stem cells; LDN, LDN-193189; qPCR, quantitative polymerase chain reaction;TPB, (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam.

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transplanted per group). These devices support neovascularizationvia woven outer membranes, while containing the engrafted cellswithin cell impermeable inner membranes [18,21]. Although inour previous studies progenitor cells were transplanted under thekidney capsule [16,17], we found that macroencapsulated progen-itor cells develop into human C-peptide secreting cells with simi-lar efficiency to cells implanted under the kidney capsule(Supporting Information Fig. 1; [18]). All mice received a singles.c. injection of enrofloxacin, Baytril, at the time of transplanta-tion (10 mg/kg; Bayer Animal Health; Shawnee Mission, KS).

All metabolic analyses were performed in conscious,restrained mice on the indicated days. Blood glucose and bodyweight were monitored one to two times weekly following a 4hour morning fast. For monthly meal challenges, blood glucosewas measured and blood was collected after an overnight fast (16hours) and then again after a 45 minute feeding period with nor-mal chow. For the glucose tolerance test (GTT), mice receivedan oral bolus of glucose (2 g/kg, 30% solution; V!etoquinol, Lav-altrie, QC) and the arginine tolerance test (ArgTT), mice receivedi.p. arginine (2 g/kg, 40% solution; Sigma-Aldrich) following a4 hour morning fast. For all tests, blood glucose was tested viasaphenous vein using a handheld glucometer (Lifescan, Burnaby,BC), and saphenous blood samples were collected at the indicatedtime points using heparinized microhematocrit tubes. Plasma wasstored at 230"C and later assayed using a human C-PeptideELISA (Alpco Diagnostics, Salem, NH) or MSD Multi-SpotAssay for human glucagon-like peptide-1 (GLP-1), insulin, andglucagon (K15160C-2; Meso Scale Discovery, Gaithersburg,MD), according to manufacturer’s instructions.

ImmunohistochemistryhESC-derived cells (pre-transplant and post-transplant) withinTheracyte devices were fixed overnight in 4% PFA and stored in70% ethanol prior to paraffin-embedding and sectioning (5 mmthickness; Wax-it Histology Services, Vancouver, Canada).Hematoxylin and eosin (H&E) and Masson’s Trichrome stainingwere performed using standard protocols (Wax-it Histology Serv-ices), and all grafts were examined by an independent patholo-gist. H&E/Trichrome slides were scanned using the ScanScopeCS system (Aperio, Vista, CA). Immunofluorescent staining wasperformed as previously described [16]; primary antibodies aredetailed in Supporting Information Table 2. Images were capturedusing the ImageXpressMICRO Imaging System and analyzedusing MetaXpress Software (Molecular Devices Corporation,Sunnyvale, CA, http://www.moleculardevices.com).

Endocrine cells were quantified within Theracyte devices har-vested at 17–21 weeks post-transplant (n 5 3 per group). Engraftedcells were coimmunostained for insulin, glucagon, and somatostatin,and whole slide fluorescence scanning was performed as describedabove. Individual images were stitched together to recreate the fulllength of each Theracyte device and the entire region between theinner membranes was quantified using MetaXpress software. Theendocrine fraction was determined as the total number of insulin-,glucagon-, and/or somatostatin-positive cells relative to the totalnumber of 40,6-diamidino-2-phenylindole (DAPI)-positive cellswithin the inner membranes. The endocrine population was furthersubdivided into the number of insulin-positive, glucagon-positive,or somatostatin-positive cells relative to the total number of endo-crine cells (insulin, glucagon, and/or somatostatin-positive), regard-less of the number of hormones present per cell. Finally, the numberof single hormonal (insulin-only, glucagon-only, or somatostatin-only) and polyhormonal (insulin/glucagon, insulin/somatostatin,glucagon/somatostatin, or insulin/glucagon/somatostatin) cells werequantified as a proportion of the total number of endocrine cells (i.e.,insulin, glucagon, and/or somatostatin-positive).

Statistical AnalysisAll statistics were performed using GraphPad Prism software(GraphPad Software Inc., LA Jolla, CA). Specific statistical tests

for each experiment are described in the figure legends. Student-Newman-Keuls post hoc test was used for comparisons betweengroups with all one-way ANOVAs. For all analyses, p < .05 wasconsidered statistically significant

RESULTS

Characterization of NKX6.1-High and NKX6.1-LowCells Pre-transplantOur revised in vitro differentiation protocols were designed togenerate two different mixed populations of pancreatic progen-itor cells (see Materials and Methods section for details andFig. 1A for summary schematic). These two cell populationswere thoroughly characterized by FACS (Fig. 1B, 1C), quanti-tative polymerase chain reaction (Fig. 1D), and immunofluo-rescent staining (Fig. 1E–1G) prior to transplant. Thepancreatic endoderm-enriched population was designated asNKX6.1-high and the endocrine-enriched population asNKX6.1-low, since they contained approximately 80% versus25% NKX6.1-positive cells, respectively (Fig. 1B, 1C).NKX6.1 expression was inversely proportional to the endo-crine compartment; NKX6.1-high (pancreatic endoderm-enriched) and NKX6.1-low (endocrine-enriched) populationscontained approximately 11% and 60% synaptophysin-positivecells, respectively (Fig. 1B, 1C). With the exception ofNKX6.1 and PDX1, pancreatic endocrine transcription factorswere generally upregulated in the NKX6.1-low cell populationcompared to the NKX6.1-high population (Fig. 1B, 1D). Bothpopulations contained approximately 90% PDX1-positive cells(Fig. 1B) and PDX1 gene expression was similar (Fig. 1D).Immunofluorescent staining confirmed the profound differencein NKX6.1 expression between the two progenitor populations(Fig. 1E) and the enrichment of polyhormonal endocrine cellsin the NKX6.1-low population (Fig. 1E–1G). Insulin/glucagoncopositive cells were NKX6.1-negative (Fig. 1E) and NKX2.2-positive (Fig. 1F) prior to transplant.

Metabolic Profile of NKX6.1-High and NKX6.1-LowCells Following TransplantMacroencapsulated NKX6.1-high and NKX6.1-low cells weretransplanted into mice with STZ-induced diabetes (averagefasting blood glucose of 17.6 6 0.94 mM at the time of trans-plant). Mice that received NKX6.1-high cells exhibited signifi-cantly lower fasting blood glucose levels compared to micethat received NKX6.1-low cells throughout the study beginningat 3 weeks post-transplant (Fig. 2A) and also during ArgTTand GTT at 17 and 20 weeks, respectively (Figs. 2C, 2F). Byapproximately 90 days post-transplant, NKX6.1-high cells hadcompletely reversed STZ-induced hyperglycemia, whereasmice with NKX6.1-low cells remained moderately hyperglyce-mic until the endpoint of the study at 135 days (Fig. 2B).Blood glucose tracking data for individual animals are providedin Supporting Information Figure 2A and individual fastingglucose values pre-transplant (day 2) and post-transplant (day113) are provided in Supporting Information Table 3.

Overnight fasted human C-peptide levels were similarbetween groups at all ages, whereas only recipients ofNKX6.1-high cells displayed meal-stimulated humanC-peptide secretion (1.7- and 2.4-fold increase after feeding at3 and 4 months, respectively; Fig. 2B). Refer to SupportingInformation Figure 2B and Supporting Information Table 3for individual human C-peptide tracking data during monthlymeal challenges. NKX6.1-high cells secreted significantlyhigher levels of human insulin/C-peptide at 15–30 minutes

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following arginine and glucose challenges, unlike NKX6.1-low cells, which were not responsive to either secretagogue invivo (Fig. 2D, 2G). Fasting glucagon levels were significantlyhigher in NKX6.1-low versus NKX6.1-high mice; there wasno difference in arginine-stimulated glucagon secretionbetween groups (Fig. 2E).

Characterization of Grafts Derived from NKX6.1-High and NKX6.1-Low CellsAt 5 months post-transplant, there were no obvious morpholog-ical differences in graft histology between NKX6.1-high andNKX6.1-low cells by H&E or Trichrome staining (Fig. 3;n 5 3 per group). An independent pathology analysis of H&E-stained Theracyte devices indicated that grafts from NKX6.1-

low and NKX6.1-high mice were largely composed of pancre-atic endocrine cells (confirmed by quantification of % insulin,glucagon, and/or somatostatin-positive cells in Fig. 4B;NKX6.1-low: 67.3% 6 1.3%, NKX6.1-high: 73.5% 6 2.3%),as well as some loose mesenchymal-like cells (Fig. 3A, 3B,3E, 3F). Based on Masson’s trichrome staining, these regionsof “loose meschenchymal tissue” (generally located adjacent tothe inner membranes) were identified as collagen fibers (Fig.3C, 3D, 3G, 3H). Notably, none of the grafts analyzed in thisstudy contained mature bone or cartilage tissue, unlike graftsgenerated with our previous differentiation protocol [17].

Immunofluorescent staining of macroencapsulated cellsrevealed dramatic differences in the pancreatic endocrine sub-populations between groups (Fig. 4). NKX6.1-low cells

Figure 2. In vivo metabolic tracking of NKX6.1-low and NKX6.1-high cell function following transplantation in macroencapsulation devices.(A): Fasting blood glucose levels (mM) in diabetic mice (low-dose STZ model) transplanted with macroencapsulated NKX6.1-low or NKX6.1-high cells. Blood glucose levels were significantly lower in mice with NKX6.1-high cells compared to NKX6.1-low cells after 21 days post-transplant (*, p < .05, t test NKX6.1-high vs. NKX6.1-low). (B): NKX6.1-low and NKX6.1-high cells secreted similar levels of human C-peptide after an overnight fast at all ages, but at 3 and 4 months post-transplant, NKX6.1-high cells secreted significantly higher levels of humanC-peptide following a 45-minute meal challenge (*, p < .05, paired t test, fast vs. fed). (C–E): Response to an ipArgTT (2 g/kg) at 17 weekspost-transplant. (C): Blood glucose (*, p < .05, t test; NKX6.1-high vs. NKX6.1-low), (D) human insulin secretion (*, p < .05, paired t test; 0vs. 15 minutes), and (E) glucagon secretion (*, p < .05, one-way ANOVA; fasting glucagon NKX6.1-high vs. NKX6.1-low, sham). Sham micewere nontransplanted, nondiabetic SCID-beige controls. (F): Blood glucose levels and (G) human C-peptide secretion in response to an oral GTT(2 g/kg) at 20 weeks post-transplant. AUC values are shown to the right (*, p < .05, t test; NKX6.1-high vs. NKX6.1-low). Abbreviations: AUC,area under the curve; ArgTT, arginine tolerance test; GTT, glucose tolerance test; ipArgTT, intraperitoneal arginine tolerance test; STZ,streptozotocin.

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developed into mainly glucagon-positive cells (96.5% 6 0.4%),whereas NKX6.1-high cells became mainly insulin-positive cells(88.7% 6 5.7%); both groups contained a similar number ofsomatostatin-positive cells (28.6% 6 4.6% and 34.4% 6 5.4% inNKX6.1-low and NKX6.1-high grafts, respectively) (Fig. 4).Quantification of the insulin-only, glucagon-only, andsomatostatin-only populations, as well as the various polyhormo-nal populations are provided in Figure 4B and 4C. Paradoxically,devices from NKX6.1-high mice, which secreted human insulinin response to various secretagogues (Fig. 2B, 2D, 2G), also con-tained approximately twice as many insulin-positive cells thatcoexpressed at least one other hormone (Fig. 4). Both groups con-tained a small ghrelin-positive population and rare cells express-ing pancreatic polypeptide (Fig. 5).

We next examined the transcription factor profiles ofendocrine cells derived from NKX6.1-high and NKX6.1-lowcells (Fig. 6). Insulin-positive cells in both groups expressednuclear NKX6.1 (Fig. 6A, 6B) and PDX1 in the nucleus andcytoplasm (Fig. 6C). Insulin-positive cells that were negativefor PDX and NKX6.1 generally coexpressed either glucagonor somatostatin (Fig. 6A–6C, double arrows). Notably, MAFAexpression was distinctly nuclear in insulin-expressing cellsfrom NKX6.1-high grafts versus strictly cytoplasmic ininsulin-expressing cells from NKX6.1-low grafts (Fig. 6D).Otherwise, endocrine cells were similar between groups, with

somatostatin-positive cells generally expressing nuclearHhex1 (Fig. 6E) and glucagon-positive cells expressingnuclear ARX (Fig. 6F), regardless of insulin coexpression.

Finally, we examined other markers found in maturepancreatic b-cells to determine if there were additional dif-ferences between insulin-positive cells derived fromNKX6.1-high versus NKX6.1-low populations. In bothgroups, insulin-positive cells uniformly expressed glucosetransporter 1 (GLUT1) and sporadically expressed amylin(Fig. 7A, 7B, respectively). Prohormone convertase (PC) 1/3was robustly expressed in nearly all insulin-positive cellsfrom NKX6.1-high grafts but only in a subset of insulin-positive cells from NKX6.1-low mice (Fig. 7C).Surprisingly, neither group appeared to express PC2 in theinsulin-positive cell population, although this enzyme washighly expressed in glucagon-positive cells (Fig. 7D). TheKATP channel sulfonylurea receptor, SUR1, was uniformlyexpressed in insulin- and glucagon-positive cells from bothgroups, and if anything, at a higher level in insulin-positivecells from NKX6.1-low mice (Fig. 7E). SerpinB10 wasrecently reported to be exclusively expressed in mature b-cells within adult human pancreas [22]. We observed robustexpression of serpinB10 in the majority of insulin-positivecells from both NKX6.1-high and NKX6.1-low grafts andnever in glucagon-positive cells (Fig. 7F).

Figure 3. Morphology of engrafted NKX6.1-high and NKX6.1-low cells within Theracyte devices at 5 months post-transplant. H&E staining (panels(A), (B), (E), (F)) and Masson’s Trichrome staining (panels (C), (D), (G), (H)) of harvested Theracyte devices at 5 months post-transplant derivedfrom: (A–D) NKX6.1-low cells (enriched for polyhormonal endocrine cells) or (E–H) NKX6.1-high cells (enriched for pancreatic endoderm cells). ForH&E panels, low magnification images through the full length of devices are shown on top and magnified images below; corresponding scale bar dimen-sions are indicated on each image. Pancreatic endocrine and ductal cells are indicated on images. For trichrome staining, two magnified regions areshown to the right, illustrating varying degrees of collagen staining (blue) within the inner membranes.

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DISCUSSION

This study demonstrates that hESC-derived progenitor popula-tions containing a relatively high proportion of NKX6.1-expressing precursor cells may be more suitable for transplan-tation into patients with diabetes than a population enrichedfor immature endocrine cells. Progenitor cells enriched forNKX6.1-positive cells matured more quickly in vivo intoglucose-responsive insulin-secreting cells compared to anendocrine-enriched progenitor population. Moreover, the insu-lin secretion kinetics displayed by NKX6.1-high cells withinonly 3 months of transplantation are attractive from a clinicalcell therapy perspective. Not only did appropriate secreta-gogues, including glucose, stimulate rapid secretion of humaninsulin but also circulating C-peptide levels returned to base-line within an hour of a glucose challenge, thus reducing therisk of hypoglycemia.

Based on previous work by Kelly et al. [6], we predictedthat the hESC-derived PDX1/NKX6.1 copositive subpopulationof pancreatic progenitor cells may have the developmentalpotential to become glucose-responsive b-cells following trans-plantation. We also suspected that hESC-derived polyhormonalcells likely contribute important developmental cues for endo-crine cell maturation and thus should not be eliminated byFACS purification. Therefore, we generated two mixed pancre-atic progenitor populations using modified in vitro differentia-tion protocols (summarized in Fig. 1A): (a) NKX6.1-high cellswere enriched for pancreatic endoderm cells and contained a rel-atively minor endocrine component, whereas (b) NKX6.1-lowcells were enriched for polyhormonal endocrine cells andexpressed high levels of pancreatic endocrine transcription fac-tors (except for NKX6.1). It is also possible that the presence ofnonpancreatic cells may differ between progenitor populationsand potentially influence the development of pancreatic endo-crine cells post-transplant. Following transplant, NKX6.1-highand NKX6.1-low populations both developed into mainly pan-creatic endocrine cells (!70% insulin, glucagon, and/or somato-statin-positive), although the endocrine subpopulations differeddramatically between groups. Moreover, NKX6.1-high graftsnot only contained a higher proportion of insulin-positive cellspost-transplant but these cells also exhibited an advanced matu-ration state compared to insulin-positive cells from NKX6.1-lowgrafts. For instance, both groups secreted similar basal levels ofhuman C-peptide, but NKX6.1-high cells secreted insulin inresponse to meal, glucose, and arginine challenges, and showeda remarkable capacity to treat diabetes. Notably, we observedmeal responsiveness much earlier with NKX6.1-high cells thanin our previous studies (at 12 vs. 30 weeks [17]) and the level ofstimulation was considerably more robust (2.4-fold vs. 1.3-foldmeal stimulus [17]). NKX6.1-high cells also displayed excellentinsulin secretion kinetics, including rapid secretion within 15minutes of a glucose challenge and return to baseline levels by60 minutes. This is preferable over the lagging insulin secretionkinetics reported previously from hESC-derived cells, in whichglucose-induced insulin secretion was observed by 60 minutes,but return to baseline insulin levels was not reported [6,12,17].These findings begin to address important concerns about poten-tial insulin overproduction by a surrogate b-cell lacking adynamic “off switch” in response to falling glycemia [23].Indeed, mice in the Kroon et al. [12] studies displayed signifi-cant hypoglycemia relative to controls (nonfasting levels of 55mg/dL compared to 139 mg/dL). Hypoglycemia was neverobserved in mice engrafted with NKX6.1-high cells, either dur-ing a glucose challenge or weekly blood glucose tracking, likelydue to the ability of engrafted cells to appropriately reduce insu-lin secretion.

Figure 4. Endocrine profiles of macroencapsulated NKX6.1-lowand NKX6.1-high cells at 5 months post-transplant. (A): HarvestedTheracyte devices were stained for insulin (red), glucagon (blue),somatostatin (green), and DAPI (white). Individual channels areshown for each magnified region in the bottom right of all panels.Two different animals are shown from each treatment group.Scale bars 5 100 mm. (B): Quantification of the % endocrine cells(insulin-, glucagon-, and/or somatostatin-positive) relative to thetotal number of DAPI-positive nuclei within devices (n 5 3/group).Grafts from both groups contained a similar proportion of endocrinecells. (C): Quantification of individual endocrine subpopulations,expressed as a proportion of the total insulin-/glucagon-/somatosta-tin-positive population (n 5 3 per group). Grafts derived fromNKX6.1-low cells contained significantly more glucagon-positivecells overall, as well as more glucagon-only and glucagon/somato-statin copositive cells compared to NKX6.1-high cells. In contrast,NKX6.1-high grafts contained significantly more insulin-positiveand insulin-only cells compared to NKX6.1-low grafts. There wasalso a trend toward more polyhormonal insulin-expressing cells inNKX6.1-high cells compared to NKX6.1-low cells. *, p < .05, ttest. Abbreviation: DAPI, 40-6-diamidino-2-phenylindole.

2438 Treatment of Diabetes with Human Stem Cells

We next sought to understand what distinguishes insulin-expressing cells derived from NKX6.1-high versus NKX6.1-lowprogenitor populations, since the increased number of insulin-secreting cells observed post-transplant in NKX6.1-high graftscould not explain the differences in insulin secretion kinetics.GLUT1 (the predominant glucose transporter in human b-cells[24,25]) and SUR1 (regulatory subunit of the KATP channel)were each similarly expressed in insulin-positive cells from bothgroups and did not correspond with differences in insulin secre-tion kinetics. This supports recent findings that gene expressionfor glucose transporter and ATP-sensitive K1 channel subunitswas similar between immature neonatal and glucose-responsiveadult rat b-cells [26]. We also examined amylin and PC1/3expression, as our previous study demonstrated that these b-cellspecific proteins were absent from immature hESC-derived insu-lin-positive cells in the early post-transplant period [17]. Here,we observed a similar pattern of expression for amylin betweengroups, excluding this as an important maturation factor in oursystem. Interestingly, PC1/3 expression was more uniform ininsulin-positive cells from NKX6.1-high grafts compared toNKX6.1-low grafts, whereas PC2 expression was relatively lowin b-cells of both groups. Loss of PC1/3 expression has beenreported to cause more severe defects in proinsulin processingthan PC2 deficiency [27]. Considering that PC1/3 expression isrequired and sufficient for processing of proinsulin [27], the lackof PC1/3 expression in many insulin-positive cells fromNKX6.1-low grafts may lead to proinsulin processing defectsbut still would not explain the immature insulin secretionkinetics in these mice. Finally, we examined a relatively newputative marker of mature b-cells, SerpinB10 [22], and con-firmed that it was specifically localized to insulin-expressingcells but was not differentially expressed between NKX6.1-lowand NKX6.1-high grafts.

We believe that impaired MAFA nuclear translocationmay be a key defect in insulin-secreting cells derived fromNKX6.1-low progenitor cells. MAFA is a known regulator ofglucose-stimulated insulin secretion and key gene required formature b-cell function [28–30]. Indeed, immature neonatal ratb-cells lacking glucose-stimulated insulin secretion not onlyexpress low levels of MAFA relative to adult b-cells but alsoexpression is almost exclusively restricted to the cytoplasm ofb-cells until postnatal day 15 [30], thus resembling theinsulin-positive cells in NKX6.1-low grafts. Furthermore,overexpression of nuclear MAFA in neonatal (postnatal day2) rodent b-cells promoted glucose-induced insulin secretion,suggesting that MAFA nuclear localization is a key regulatorof b-cell maturity during normal pancreas development [30].Altered MAFA localization may also account for a loss of b-cell function during the pathogenesis of diabetes. Cytoplasmiclocalization of MAFA was observed in b-cells from hypergly-cemic db/db mice [31] and humans with type 2 diabetes [32].Moreover, overexpression of an endogenous antioxidantenzyme was able to preserve intranuclear MAFA expressionas well as ameliorate hyperglycemia in the db/db mousemodel [31]. Although the physiological mechanisms that con-trol MAFA localization are still unknown, the abundantnuclear MAFA expression could be a key factor driving theimproved insulin secretion kinetics in b-cells derived fromNKX6.1-high pancreatic progenitor cells.

Previous evidence in mice suggested that the timing ofNGN3 induction may control the competency for pancreaticprogenitors to generate specific endocrine cell types [33].Therefore, we predicted that the timing of NGN3 inductionduring hESC differentiation would also influence the produc-tion of insulin-producing cells in vivo. Indeed, our findingssuggest that the manner and timing of NGN3 induction in

Figure 5. Ghrelin and pancreatic polypeptide expression in Theracyte devices at 5 months post-transplant. Macroencapsulated human embryonic stemcell-derived pancreatic endocrine cells contained a small ghrelin-positive population and rare cells that expressed pancreatic polypeptide (white arrows).Cells derived from NKX6.1-low cells are shown on the left and NKX6.1-high cells on the right. Scale bars 5 100 mm. Abbreviation: DAPI, 40-6-diami-dino-2-phenylindole.

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developing hESCs does control the relative proportions of pan-creatic endocrine lineages. NGN3 timing was manipulated inour studies by adjusting exposure to ALK5i and PKC activator.We had previously reported that combined inhibition of trans-forming growth factor beta (TGF)b/BMP signaling withALK5i/Noggin during stage 4 caused dramatic induction ofpancreatic endocrine markers, including NGN3 [16,17],whereas addition of a PKC activator reduced NGN3 whileincreasing NKX6.1 expression [17]. Here, the addition ofALK5i during stages 3–4 and absence of the PKC activatorduring stage 4 in the NKX6.1-low protocol not only reducedNKX6.1 expression at stage 4 but also caused a dramaticinduction of NGN3 during stages 3–4 (!20-fold inductioncompared to NKX6.1-high cells). In contrast, induction ofNGN3 was delayed in NKX6.1-high cells due to the absenceof ALK5i during stage 3; PKC activation also reduced NGN3and increased NKX6.1 expression. Similar to mice in which

early induction of NGN3 favored a-cell development [33],early induction of NGN3 during stage 3 in the NKX6.1-lowpopulation resulted in the development of mainly mature a-cells in vivo, as indicated by both circulating glucagon levelsand the overwhelming number of glucagon/ARX co-positivecells within the harvested devices. Our observations also sup-port previous studies [6,16,20], which collectively indicate thatpolyhormonal cells may be lineage committed toward an a-cellfate and challenge a long-accepted lineage tracing study, whichconcluded that fetal insulin/glucagon copositive cells did notcontribute to adult a- or b-cell populations [34]. We alsoobserved an unexpected population of cells expressing eitherinsulin/glucagon or insulin/somatostatin derived from NKX6.1-high cells at 5 months post-transplant, particularly around theedges of encapsulation devices. The insulin/somatostatin copos-itive population is unusual, as this cell type is not typicallyobserved during human fetal pancreas development [35] and

Figure 6. Transcription factor expression in endocrine cells from macroencapsulated NKX6.1-low and NKX6.1-high cells at 5 months post-transplant. (A, B): NKX6.1 was expressed in insulin-positive cells from both groups (single arrowheads) but was generally not expressed incells coexpressing either insulin/glucagon (A) or insulin/somatostatin (B), as indicated by double arrowheads. (C): Insulin-positive cells inboth groups expressed PDX1 in the cytoplasmic and nuclear compartments (single arrowheads); insulin/glucagon co-positive cells did notexpress PDX1 (double arrowheads). (D): Insulin-positive cells in grafts derived from NKX6.1-low cells expressed MAFA in the cytoplasm,but not the nucleus, whereas NKX6.1-high grafts contained insulin-positive cells with nuclear MAFA expression (single arrowheads). MAFAwas not expressed in polyhormonal insulin-positive cells (double arrowheads). (E, F): In both groups somatostatin-positive cells were co-positive for Hhex1 (E) and glucagon-positive cells for ARX (F), even when insulin was also expressed in the same cell (double arrowheads).Unihormonal insulin-positive cells were negative for both Hhex1 (E) and ARX (F), as indicated by single arrowheads. For all panels, magni-fied regions illustrate three channels on top, blue/green/DAPI channels in the middle and red/green/DAPI channels on the bottom. Scalebars 5 50 mm. Abbreviation: DAPI, 40-6-diamidino-2-phenylindole.

2440 Treatment of Diabetes with Human Stem Cells

appeared to resemble mature d-cells rather than b-cells (hex-positive, NKX6.1-negative). The potential contribution of thesepolyhormonal cells to the function of surrounding unihormonalinsulin-secreting cells remains to be determined.

CONCLUSION

Taken together, these studies indicate that when enriched (butnot purified), hESC-derived pancreatic endoderm cells may bea suitable cell therapy product for treating patients with diabe-tes. Within a relatively short time frame (3 months), theNKX6.1-enriched population developed into insulin-secreting

cells that responded to various physiological secretagoguesand were capable of reducing insulin secretion when neces-sary. Moreover, these cells expressed key markers of maturepancreatic b-cells, including robust nuclear MAFA expres-sion. In contrast, the hESC-derived population enriched forpolyhormonal endocrine cells developed into mostly maturea-cells following transplant and produced immature pancreaticb-cells, that did not secrete insulin in a glucose-responsivemanner or express nuclear MAFA. These data support theconcept that hESCs may be a feasible alternative to cadavericislets for transplantation within macroencapsulation devicesinto patients with type 1 diabetes.

Figure 7. Characteristics of insulin-expressing cells in NKX6.1-low and NKX6.1-high cells post-transplant. NKX6.1-low and NKX6.1-high grafts at 5months post-transplant were immunostained for insulin, glucagon, DAPI, and various markers of mature pancreatic b-cells, including: (A) GLUT1, (B)amylin, (C) PC 1/3, (D) PC2, (E) SUR1, and (F) serpin peptidase inhibitor (serpin) B10. Insulin-positive cells from both groups expressed similar levelsof GLUT1 (A), amylin (B), SUR1 (E) and serpinB10 (F) but were generally negative for PC2 (D). PC1/3 was more uniformly expressed in insulin-positive cells from NKX-high grafts than in NKX6.1-low grafts, where many PC1/3-negative insulin-expressing cells were observed (C). Scalebars 5 100 mm. Abbreviations: DAPI, 40-6-diamidino-2-phenylindole; GLUT1, glucose transporter 1; PC, prohormone convertase; SUR1, sulfonylureareceptor.

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ACKNOWLEDGMENTS

T.J.K. was supported by a senior scholarship from theMichael Smith Foundation for Health Research. J.E.B. wasfunded by a JDRF postdoctoral fellowship, CIHR postdoctoralfellowship, and the CIHR Transplantation Training Program.J.E.B. also received a L’Or!eal Canada for Women in ScienceResearch Excellence Fellowship. We thank Mr. Ali Asadi forhis technical expertise with immunofluorescent staining andStem Cell Technologies for their financial support; Dr. Clif-ford Bogue from the Yale University School of Medicine forkindly providing the hex antibody. This work was funded bythe Canadian Institutes of Health Research (CIHR) Regenera-

tive Medicine and Nanomedicine Initiative, the Stem CellNetwork (SCN) and the Juvenile Diabetes Research Founda-tion (JDRF).

DISCLOSURE OF POTENTIAL

CONFLICTS OF INTEREST

A.R., J.X., K.N., and J.J.O. are employees of Janssen R&D,LLC; T.J.K. received financial support from Janssen R&D,LLC, for the research described in this article. No other poten-tial conflicts of interest relevant to this article were reported.

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