INS GFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells
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We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS+) cells derived in vitro.
Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS+ cells.
Differentiation of INS GFP/w hESCs using published protocols demonstrated that all GFP+ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP+ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS GFP/w hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP+ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP+ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS+ cells.
INS GFP/w hESCs are a valuable tool for investigating the nature of early INS+ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS+ cells from differentiating hESCs.
KeywordsDiabetes Gene targeting GFP Human embryonic stem cells Insulin
Bone morphogenetic protein 4
Green fluorescent protein
Fibroblast growth factor 2
Human embryonic stem cell
Hepatocyte growth factor
ISL LIM homeobox
Fibroblast growth factor 7
NK2 homeobox 2
NK6 homeobox 1
Paired box 6
Pancreatic and duodenal homeobox 1
Rho associated kinase
Type 1 diabetes is an autoimmune disease characterised by destruction of beta cells in the pancreas, deficient insulin production, and persistent high levels of blood glucose. Treatment with exogenous insulin, although life-saving, does not restore physiological control of blood glucose, leaving people with type 1 diabetes at risk of long-term complications. Control can be improved by islet transplantation (reviewed by Speight et al. ), but this treatment option will always be limited by the scarcity of cadaveric donor tissue.
Beta cells derived from the differentiation of human embryonic stem cells (hESCs) in vitro potentially represent an inexhaustible source of insulin-producing cells for the treatment of type 1 diabetes. Several laboratories have demonstrated that hESC-derived endocrine cells can regulate blood glucose in a diabetic mouse model, providing proof of principle for future clinical application (for example, see studies by Kroon et al.  and Jiang et al.  and a review by van Hoof et al. ). However, while attempts to generate INS+ cells from pluripotent stem cells have been encouraging, the biology of this process remains poorly understood. In this light, better tools and reagents to facilitate the understanding of beta cell development are required.
We describe the generation and characterisation of two independently derived hESC lines in which sequences encoding green fluorescent protein (GFP) have been targeted to the insulin locus (INS GFP/w hESCs). We demonstrate the utility of these lines by characterising the transcriptional signature of hESC-derived insulin-producing (INS+) cells generated using established differentiation protocols. Analysis of these data in conjunction with immunofluorescence studies confirms that such cells display an immature phenotype, with the majority of INS+ cells also producing glucagon. We used INS GFP/w hESCs to develop a novel 96-well format spin embryoid body (EB) differentiation protocol for the differentiation of hESCs to INS+ pancreatic endoderm. This method is based on a protocol originally developed for the differentiation of hESCs to mesodermal populations  and uses a defined wholly recombinant protein-based medium (APEL) . Characterisation of INS+ cells generated with this platform reveals that, unlike INS+ cells derived with previous methods, a substantial proportion also produce the beta cell-associated marker, NK6 homeobox 1 (NKX6.1), suggesting that the EB environment is conducive to ongoing differentiation. INS GFP/w hESCs are therefore a valuable tool for investigating and refining the generation of INS+ cells from pluripotent stem cells in vitro.
Generation and identification of targeted INSGFP/W hESCs
hESC culture and differentiation
hESCs were cultured and passaged as reported elsewhere . The differentiation of hESCs into INS+ cells was performed using several different protocols. Adherent, flat culture differentiations based on the work of D’Amour et al.  and Kroon et al.  (referred to as ‘flat cultures’). Spin EB differentiations (referred to as ‘spin EBs’) , were set up in APEL medium . Differentiation of spin EBs under pancreatic-specific conditions was as follows. EBs were formed by the forced aggregation of 2,000 (HES3) or 3,500 (MEL1) hESCs in APEL (the protein-free hybridoma medium component was omitted from this formulation) containing 10 ng/ml bone morphogenetic protein 4 (BMP4) and 150–200 ng/ml activin A (batch dependent) in low-attachment 96-well plates. After 3 days, medium was replaced with APEL containing 200–400 ng/ml noggin (batch dependent). At day 6, medium was replaced with APEL containing 1 × 10−5 mol/l retinoic acid (RA). At day 9, the medium was changed to APEL without polyvinyl alcohol (AEL) containing 1 × 10−5 mol/l RA, 100 μmol/l glucagon-like peptide 1 (GLP1), 1 × B27 and 10 mmol/l nicotinamide. At day 15 of differentiation, EBs were transferred to gelatinised, adherent 96-well plates, and insulin production was induced in AEL containing 10 mmol/l nicotinamide and 50 ng/ml IGF-I. With this system, most EBs contained INS-GFP+ cells by day 30 of differentiation. In addition, INS-GFP+ cells were also differentiated according to a protocol developed by Nostro and colleagues , referred to as the ‘Nostro protocol’. Recombinant human activin A, fibroblast growth factor 10 (FGF10), fibroblast growth factor 7 (KGF), IGF-I and hepatocyte growth factor (HGF) were purchased from R&D Systems (Minneapolis, MN, USA). Basic FGF (FGF2) was purchased from Peprotech (Rocky Hill, NJ, USA). Wingless-type MMTV integration site family, member 3A (WNT3A) and noggin were purchased from R&D Systems or provided by the Australia Stem Cell Centre (Melbourne, VIC, Australia). KAAD-cyclopamine was purchased from Toronto Research Chemicals (North York, ON, Canada); all-trans RA, nicotinamide, SB431542 and GLP1 were purchased from Sigma-Aldrich (St Louis, MO, USA).
Live cell imaging and immunofluorescence
Live cell imaging of spin EBs in a 96-well plate format was performed with a Leica TCS NT inverted microscope, and images were processed with ImageJ software. For immunofluorescence analysis of flat cultures, differentiated cells were fixed for 15 min in 4% (wt/vol.) paraformaldehyde in PBS, permeablised in 0.2% (vol./vol.) Triton X-100 at room temperature for 10 min, and blocked for 60 min in 10% (vol./vol.) goat serum. Primary antibodies were incubated overnight at 4°C, and secondary antibodies were incubated for 1 h at 24°C. The following antibodies were used: rabbit anti-pancreatic and duodenal homeobox 1 (PDX1) (kindly provided by C. Wright, Vanderbilt University, Nashville, TN, USA); mouse anti-NK2 homeobox 2 (NKX2-2) (Developmental Studies Hybridoma Bank (DSHB; Iowa City, IA, USA; clone 74.5A5); mouse anti-NKX6.1 (DSHB); mouse anti-ISL LIM homeobox (ISL)1/2 (DSHB clone 39.4D5); mouse anti-paired box 6 (PAX6) (DSHB); guinea pig anti-insulin (Dako, Glostrup, Denmark; clone A0564); rabbit anti-C-peptide (Millipore; clone 4020; note that this antibody detects both C-peptide and proinsulin); rabbit anti-glucagon (Dako; clone A0565); anti-glucagon (Sigma; clone K79bB10); rat anti-somatostatin (Millipore; clone MAB354). Secondary antibodies used were Alexa-488- and Alexa-568-conjugated goat antibodies against mouse, rat, rabbit and goat (Invitrogen) and a tetramethyl rhodamine iso-thiocyanate (TRITC)-conjugated antibody against guinea pig (Sigma).
For wholemount immunofluorescence of spin EBs , differentiated EBs were removed from 96-well plates and fixed for 90 min on ice in 4% (wt/vol.) paraformaldehyde in PBS and permeablised in 1% (vol./vol.) Triton X-100 at room temperature for 90 min. EBs were blocked for 90 min in 10% (vol./vol.) goat serum. Incubation of primary and secondary antibodies was as described above. All washes were for 15 min in PBS/10% FCS.
Flow cytometric analysis
For flow cytometric analysis and sorting of live cells, hESCs differentiated in flat cultures or as spin EBs were dissociated with TrypLE-select (Invitrogen) to give a single-cell suspension and purified as described previously . High-throughput flow cytometric analysis of cells in 96-well plates was performed with an LSR II multi-laser benchtop flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and FACS plots processed using Gatelogic software (www.inivai.com/GatelogicHome.html).
By using flow cytometry we purified day 20 INS-GFP+ cells generated with the flat culture protocol and then added between 2 × 103 and 5 × 103 INS-GFP+ cells to each well of a low-attachment 96-well tray in APEL medium containing 10 μmol/l rho-associated, coiled-coil containing protein kinase 1 (ROCK) inhibitor Y27632 [7, 14]. FGF10, HGF, FGF2, BMP4, KGF and noggin (10–100 ng/ml) were added singly or in combination at the time of aggregation or after 24 h. Medium containing the ROCK inhibitor was replaced with APEL containing combinations of the above growth factors after aggregates had formed (usually 24–72 h). Half of the medium was changed every 3–4 days over a 3-week period. When used, 20 μl Matrigel (diluted 2:1 in APEL medium) was added directly to reaggregated INS-GFP+ cells. After Matrigel polymerisation, 100 μl APEL medium containing combinations of the above growth factors was added to each well. Medium was refreshed periodically as described above. Intracellular flow cytometric analysis was performed as described by Nostro et al. . Bromodeoxyuridine (BrdU) incorporation measured by flow cytometry was performed according to the manufacturer’s (BD Biosciences) instructions.
Gene expression analysis
RNA preparation, Illumina microarray analysis and real-time quantitative PCR was performed essentially as described previously . Briefly, total RNA for each sample was amplified, labelled and hybridised to human WG-6v2, human HT12v3 or HT12v4 BeadChips according to Illumina standard protocols (Illumina, San Diego, CA, USA) at the Australian Genome Research Facility. Initial data analysis was performed using GenomeStudio version 2010.3 (Illumina), using average normalisation across all the samples. Alternatively, data were analysed using R/BioConductor using algorithms within the lumi package  (function: bgAdjust.affy and quantile normalisation ). Subsequent data analysis was performed using MultiExperiment Viewer [17, 18]. Hierarchical clustering was performed using Pearson correlation with average linkage clustering. Differentially expressed genes were subjected to functional clustering analysis using the DAVID public database (Database for Annotation, Visualization and Integrated Discovery) [19, 20].
We targeted a GFP reporter gene to the INS locus of HES3 and MEL1 hESCs, creating a reagent to enable detailed study of the potential and characteristics of INS+ cells (Fig. 1a). Undifferentiated INS GFP/w hESCs had a normal karyotype (46XX for the HES3-derived line and 46XY for the MEL1-derived line), produced stem cell markers and generated teratomas containing derivatives of the three primary germ layers upon transplantation into immunodeficient mice (electronic supplementary material [ESM] Fig. 1). Southern blotting analysis indicated that both lines contained a single GFP insertion, while PCR and DNA sequencing confirmed that the targeting vector had been integrated by homologous recombination (Fig. 1a–c). Differentiation of INS GFP/w hESCs in flat culture (ESM Fig. 2) revealed that INS-GFP+ cells appeared as small clusters, which co-stained with insulin and C-peptide (Fig. 1d–f), confirming the fidelity of the reporter gene. Immunofluorescence experiments also demonstrated that INS-GFP+ cells co-produced somatostatin and glucagon (Fig. 1g, h), confirming previous reports  that early hESC-derived INS+ cells are polyhormonal. This conclusion was supported by flow cytometry analysis, which showed that ~80% of INS-GFP+ cells co-produced glucagon, and ~20% produced somatostatin (Fig. 1i–l). INS-GFP+ cells producing neither hormone constituted a minor proportion of the population; however, it is possible that these cells produced other hormones that were not assayed (e.g. pancreatic polypeptide [PPY], ghrelin [GHRL]).
The growth and differentiation potential of IPAs was subsequently examined in vitro. We first tested the ability of previously reported pancreatic growth factors to either sustain INS-GFP production over a 3-week period or promote expansion of the GFP+ population. Extended cultures of IPAs in APEL medium alone revealed that INS-GFP production waned rapidly after IPA formation (data not shown). We also observed that factors previously reported to have a role in expansion of the pancreatic primordium, such as HGF and FGF10 [22, 23, 24], promoted the slow growth of the population overall, particularly in the presence of Matrigel (Fig. 2d and data not shown). However, none of the factor combinations tested (see Methods) sustained GFP production for more than 3 weeks nor promoted expansion of the INS-GFP+ pool. Instead, GFP− cells (either derived from INS+ cells or representing contaminants in the original sorted population) eventually became the predominant cell type within the IPAs (Fig. 2d), raising the possibility that INS-GFP+ cells generated in flat cultures were postmitotic. To address this, we performed BrdU incorporation analysis of cells differentiated using the flat culture protocol. This analysis indeed showed that, at later differentiation stages, few INS-GFP+ cells and/or their immediate precursors incorporated BrdU (Fig. 2e), consistent with the notion that these cells were essentially non-proliferative.
Comparison of GFP+ and GFP− fractions indicated that GFP+ cells had upregulated a suite of genes that confirmed the commitment of this population to endocrine differentiation (Fig. 3b). Genes that were substantially upregulated in the GFP+ fraction included those for hormones traditionally associated with pancreatic endocrine cells (GCG, INS, SST, PPY), a suite of known pancreatic transcription factor genes (NKX2.2 [also known as NKX2-2], ARX, NEUROD1, MAFB) as well as a number of genes associated with type 1 diabetes (HLA, GAD, PTPRN). This analysis also revealed that, within this restricted set, INS-GFP+ samples were most similar to islets, consistent with their endocrine nature.
INS GFP/w hESCs reported on here represent a novel reagent for the study of beta cell differentiation in vitro. By directly tagging the INS locus, cells can be viably isolated for further studies or followed in real time, allowing their growth and response to culture manipulations to be directly monitored. We have used these cells to generate a set of gene profiling data that will serve as a baseline for future studies on hESC-derived endocrine cells. These data, in conjunction with immunofluorescence studies, reveal that INS-GFP+ cells generated with two distinct but related protocols [11, 12] have hallmarks of immature endocrine cells. This conclusion was drawn from the observation that most INS-GFP+ cells in late-stage (day 20–25) cultures produced other endocrine hormones, most commonly glucagon. Although cells producing multiple hormones are present in the developing human pancreas, their relative abundance as a fraction of the hormone-positive population is minor . Several theories have been proposed to account for polyhormonal cells in cultures of differentiating hESCs. The preponderance of INS+ cells that express other hormones may indicate a bona fide differentiation-intermediate population, further development of which is arrested because culture conditions are not appropriate. Alternatively, the appearance of this cell type may signify that current culture conditions drive the generation of an in vitro artefact that lacks the capacity for further differentiation along the beta cell lineage. BrdU labelling experiments suggest that the polyhormonal cells generated under these conditions are postmitotic, a conclusion consistent with other studies suggesting that the major source of new islets during development is not pre-existing hormone-producing cells [27, 28, 29, 30].
Taken together, the above observations emphasise that further work will be required before mature beta cells can be readily generated from hESCs. Therefore, methodologies that lend themselves to testing large numbers of variables will assist efforts to refine or reconstruct hESC to beta cell differentiation protocols. In this context, we used INS GFP/w hESCs to develop a 96-well format spin EB differentiation protocol that used the recombinant protein-based medium, APEL . This platform has a number of advantages that will facilitate further exploration of pathways governing pancreatic differentiation of hESCs. First, the 96-well format is compatible with high-throughput methodologies that enable the simultaneous assessment of large numbers of variables. Second, because APEL contains only recombinant proteins (albumin, transferrin and insulin), inconsistencies arising from batch to batch variation intrinsic to media components such as BSA are minimised. Using the spin EB platform, we observed that INS-GFP+ cells appeared in the context of a variety of morphologically distinct structures, some of which appeared to derive and/or separate from the main mass of the EB and resembled islet-like clusters described by others [3, 31]. Interestingly, islet-like clusters in both reports contained a substantial fraction of INS+ cells that did not co-produce either glucagon or somatostatin, mirroring our findings with spin EB-derived INS-GFP+ cells. This phenotype would be consistent with the idea that spin EB-derived INS-GFP+ cells represented a more mature stage of development, a conclusion supported by the substantial number of INS-GFP+ cells in spin EBs producing the later-stage beta cell differentiation marker, NKX6.1 .
Nevertheless, with the notable exception of INS, GCG and SST, our microarray data indicated that INS-GFP+ cells generated using all of the protocols (flat, Nostro or spin EB) expressed relatively low levels of genes recently reported by Dorrell and colleagues  to be associated with mature alpha or beta cells (ESM Fig. 7). This observation reinforces the notion that establishing culture conditions that promote appropriate maturation represents a significant hurdle for the generation of functional beta cells in vitro.
Our microarray data also suggested that spin EB-derived INS-GFP+ cells expressed lower levels of transcripts associated with non-pancreatic endodermal cell types and higher levels of HOX genes—genes with a known role in setting axial position within the developing embryo. In this light, it is tempting to speculate that the spin EB environment may provide differentiative cues that more precisely specify positional identity and/or more closely resemble those in the embryo. While further studies are necessary to determine if INS-GFP+ cells can be further differentiated, the INS GFP/w hESCs and 96-well spin EB format protocol described here represent new tools for optimising generation of beta cells in vitro for the treatment of type 1 diabetes.
We thank R. Mayberry, K. Koutsis and A. Bruce for the provision of hESCs, S. Hawes for the provision of human fetal pancreas RNA, FlowCore for flow cytometric sorting, and Monash Micro Imaging for confocal microscopy and time-lapse video services. All work involving experimentation with human ES cell lines was performed under approval of the Monash University Human Ethics Committee (2002-225MC). This work was supported by the Australian Stem Cell Centre (ASCC), The National Health and Medical Research Council (NHMRC) of Australia and the Juvenile Diabetes Research Foundation. M. C. Nostro was supported by a postdoctoral fellowship from the McEwen Centre for Regenerative Medicine. L. C. Harrison is a Senior Principal Research Fellow, and A. G. Elefanty and E. G. Stanley are Senior Research Fellows of the NHMRC.
SJM contributed to the conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing and final approval of the manuscript. XL, JVS, CEH, QCY, SML, MCN, DAE and SF contributed to the collection and assembly of data, data analysis and interpretation and critical revision, and gave final approval of the manuscript. LCH and GK contributed to provision of study materials, data analysis and interpretation and critical revision, and gave final approval of manuscript. AGE and EGS contributed to conception and design, data analysis and interpretation, manuscript writing and financial support, and gave final approval of manuscript.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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