Human Embryonic Stem Cells Facilitate Isolation of in Vitro Derived Insulin-Producing Cells

Diabetologia (2012) 55:694–706 DOI 10.1007/s00125-011-2379-y ARTICLE INSGFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells S. J. Micallef & X. Li & J. V. Schiesser & C. E. Hirst & Q. C. Yu & S. M. Lim & M. C. Nostro & D. A. Elliott & F. Sarangi & L. C. Harrison & G. Keller & A. G. Elefanty & E. G. Stanley Received: 27 July 2011 /Accepted: 20 October 2011 /Published online: 26 November 2011 # The Author(s) 2011. This article is published with open access at Springerlink.com Abstract spin embryoid body (EB) differentiation protocol that used Aims/hypothesis We aimed to generate human embryonic the recombinant protein-based, fully defined medium, stem cell (hESC) reporter lines that would facilitate the APEL. Like INS-GFP+ cells generated with other methods, characterisation of insulin-producing (INS+) cells derived those derived using the spin EB protocol expressed a suite in vitro. of pancreatic-related transcription factor genes including Methods Homologous recombination was used to insert ISL1, PAX6 and NKX2.2. However, in contrast with sequences encoding green fluorescent protein (GFP) into previous methods, the spin EB protocol yielded INS-GFP+ the INS locus, to create reporter cell lines enabling the cells that also co-expressed the beta cell transcription factor prospective isolation of viable INS+ cells. gene, NKX6.1, and comprised a substantial proportion of GFP/w Results Differentiation of INS hESCs using published monohormonal INS+ cells. protocols demonstrated that all GFP+ cells co-produced Conclusions/interpretation INSGFP/w hESCs are a valuable insulin, confirming the fidelity of the reporter gene. INS- tool for investigating the nature of early INS+ progenitors in GFP+ cells often co-produced glucagon and somatostatin, beta cell ontogeny and will facilitate the development of confirming conclusions from previous studies that early novel protocols for generating INS+ cells from differentiat- hESC-derived insulin-producing cells were polyhormonal. ing hESCs. INSGFP/w hESCs were used to develop a 96-well format Keywords Diabetes . Gene targeting . GFP. Human embryonic stem cells . Insulin S. J. Micallef and X. Li contributed equally to this study. Electronic supplementary material The online version of this article Abbreviations (doi:10.1007/s00125-011-2379-y) contains peer-reviewed but unedited BMP4 Bone morphogenetic protein 4 supplementary material, which is available to authorised users. : : : : : BrdU Bromodeoxyuridine S. J. Micallef X. Li J. V. Schiesser C. E. Hirst Q. C. Yu EB Embryoid body : : : * S. M. Lim D. A. Elliott A. G. Elefanty E. G. Stanley ( ) GFP Green fluorescent protein Monash Immunology and Stem Cell Laboratories (MISCL), Level 3, Building 75, STRIP1, West Ring Road, Monash University, FGF2 Fibroblast growth factor 2 Clayton, Victoria 3800, Australia hESC Human embryonic stem cell e-mail: [email protected] HGF Hepatocyte growth factor INS Insulin M. C. Nostro : F. Sarangi : G. Keller McEwen Centre for Regenerative Medicine, IPA Insulin-positive aggregates University Health Network, ISL ISL LIM homeobox Toronto, ON, Canada KGF Fibroblast growth factor 7 NKX2-2 NK2 homeobox 2 L. C. Harrison The Walter and Eliza Hall Institute of Medical Research, NKX6.1 NK6 homeobox 1 Parkville, VIC, Australia PAX6 Paired box 6 Diabetologia (2012) 55:694–706 695 PDX1 Pancreatic and duodenal homeobox 1 ongoing differentiation. INSGFP/w hESCs are therefore a RA Retinoic acid valuable tool for investigating and refining the generation ROCK Rho associated kinase of INS+ cells from pluripotent stem cells in vitro. Introduction Methods Type 1 diabetes is an autoimmune disease characterised by Generation and identification of targeted INSGFP/W destruction of beta cells in the pancreas, deficient insulin hESCs The INS-targeting vector comprised a 10.7 kb 5′ production, and persistent high levels of blood glucose. homology arm, GFP coding sequences, a loxP flanked Treatment with exogenous insulin, although life-saving, phosphoglycerol kinase (PGK)-promoter-neomycin resis- does not restore physiological control of blood glucose, tance cassette and 2.9 kb 3′ homology arm. The 5′ leaving people with type 1 diabetes at risk of long-term homology arm was derived from a bacterial artificial complications. Control can be improved by islet transplan- chromosome (RP11 889I17) encompassing the human tation (reviewed by Speight et al. [1]), but this treatment insulin locus using ET cloning as described previously option will always be limited by the scarcity of cadaveric [7]. The 3′ homology arm was derived by PCR using the donor tissue. same bacterial artificial chromosome clone as a template. Beta cells derived from the differentiation of human The vector was digested with the restriction enzyme embryonic stem cells (hESCs) in vitro potentially represent PacI before electroporation into the hESC lines HES3 an inexhaustible source of insulin-producing cells for the (http://www.escellinternational.co/) and MEL1 (Millipore, treatment of type 1 diabetes. Several laboratories have Billerica, MA, USA) as described previously [8]. Targeted demonstrated that hESC-derived endocrine cells can regu- hESC clones were identified by a PCR-based screening late blood glucose in a diabetic mouse model, providing strategy using primer P1, a forward primer in the neomycin proof of principle for future clinical application (for resistance gene, in conjunction with P2, a reverse primer example, see studies by Kroon et al. [2] and Jiang et al. located immediately 3′ of genomic sequences encompassed [3] and a review by van Hoof et al. [4]). However, while by the targeting vector. The fidelity of homologous attempts to generate INS+ cells from pluripotent stem recombination within the 5′ arm was confirmed by PCR cells have been encouraging, the biology of this process using P3, a forward primer located immediately 5′ of remains poorly understood. In this light, better tools and genomic sequences included in the targeting vector, in reagents to facilitate the understanding of beta cell conjunction with P4, a reverse primer in the GFP gene. By development are required. these criteria, a number of clones were identified in which We describe the generation and characterisation of two the vector was correctly integrated into the INS locus in independently derived hESC lines in which sequences both HES3 and MEL1 lines. One HES3-derived and one encoding green fluorescent protein (GFP) have been MEL1-derived INSGFPNeo/w clone was expanded, and the targeted to the insulin locus (INSGFP/w hESCs). We neomycin resistance cassette removed as described previ- demonstrate the utility of these lines by characterising the ously [9]. Single-cell cloning was performed by single-cell transcriptional signature of hESC-derived insulin-producing deposition using a FACSaria FACS station as described (INS+) cells generated using established differentiation previously [9]. Several colonies representing each primary protocols. Analysis of these data in conjunction with clone were picked and screened for the loss of the immunofluorescence studies confirms that such cells neomycin resistance cassette by PCR. Southern blot display an immature phenotype, with the majority of INS+ analysis using a probe encompassing the coding sequences cells also producing glucagon. We used INSGFP/w hESCs to of enhanced green fluorescent protein (EGFP) (Invitrogen, develop a novel 96-well format spin embryoid body (EB) Carlsbad, CA, USA) was performed on EcoRV-digested differentiation protocol for the differentiation of hESCs to genomic DNA from each cell line (Fig. 1b). As this enzyme INS+ pancreatic endoderm. This method is based on a cuts only once within the vector, the presence of a single protocol originally developed for the differentiation of band indicated that each cell line contained a single hESCs to mesodermal populations [5] and uses a defined integration of the targeting vector. The DNA fragments wholly recombinant protein-based medium (APEL) [6]. generated by PCR using the primers P1 and P2, P3 and P4 Characterisation of INS+ cells generated with this platform were cloned and sequenced to establish that the targeting reveals that, unlike INS+ cells derived with previous vector had been correctly integrated into the INS locus. methods, a substantial proportion also produce the beta cell-associated marker, NK6 homeobox 1 (NKX6.1), hESC culture and differentiation hESCs were cultured and suggesting that the EB environment is conducive to passaged as reported elsewhere [10]. The differentiation of 696 Diabetologia (2012) 55:694–706 a e1 e2 e3 bc INS locus GFP/w GFP/w GFP/w PacI NS INS Targeting vector GFP NeoR HES3 MEL1 HES3 MEL1INS I 5` 5`3`3` loxP loxP GFPNeoR 10 kb INS GFP NeoR 10 kb 11.1kb 4 kb 4 kb P3 P4 P5 P1 P2 Cre recombinase SphI SphI GFP INS GFP 17.7kb 5` arm 3` arm f ggccaaggg cttccgcagg GFP gagctc gcgcggggct 5` Junction 3` Junction INS-GFP C-pep/INS-GFP/DAPI d g INS-GFP INS-GFP Bright field /GFP INS-GFP GCG/INS-GFP/DAPI e h INS-GFP INS INS/INS-GFP/DAPI INS-GFP SOM/INS-GFP/DAPI ij kl + 15 70 GFP 57 7 193 192 2412 FL4 Glucagon Glucagon Glucagon GFP GFP Somatostatin Somatostatin Fig. 1 Generation and characterisation of INSGFP/w hESCs. a Vector INS locus. d Images showing clusters of GFP+ cells in differentiated used to target the INS locus in hESCs. The G418 antibiotic resistance cultures of INSGFP/w hESCs. Scale bar, 100 μm. e–h Immunofluorescence (NeoR) cassette, flanked by loxP sites (black triangles), was removed analysis of flat cultures showing that INS-GFP+ cells produce insulin, with Cre recombinase. PCR primers (P1, P2) and (P3, P4) were used C-peptide (C-pep), glucagon (GCG) and somatostatin (SOM). Scale bar, to identify targeted clones. The sequences of PCR fragments shown 10 μm. i Flow cytometry analysis showing that INS-GFP+ cells can be below represent the junctions between the extremities of the targeting clearly distinguished from the GFP− population. j–l Intracellular flow vector (red text) and the INS locus (black text).

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