Diabetologia (2013) 56:1987–1998 DOI 10.1007/s00125-013-2955-4
ARTICLE
Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice Jennifer E. Bruin & Alireza Rezania & Jean Xu & Kavitha Narayan & Jessica K. Fox & John J. O’Neil & Timothy J. Kieffer
Received: 11 December 2012 / Accepted: 7 May 2013 / Published online: 16 June 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Aims/hypothesis Islet transplantation is a promising cell therapy for patients with diabetes, but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However, the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore, we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo. Methods The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the
kidney capsule or within Theracyte (TheraCyte, Laguna Hills, CA, USA) devices into diabetic mice. Results Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing >80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices, despite lacking direct contact with the host environment, and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells. Conclusions/interpretation Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device, yielding glucose-responsive insulin-producing cells capable of reversing diabetes.
Jennifer E. Bruin and Alireza Rezania contributed equally to this work.
Keywords Cell therapy . Diabetes . Encapsulation . Human embryonic stem cells . Insulin . Islets
Electronic supplementary material The online version of this article (doi:10.1007/s00125-013-2955-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users. J. E. Bruin : J. K. Fox : T. J. Kieffer (*) Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Room 5308-2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3 e-mail:
[email protected] A. Rezania : J. Xu : K. Narayan : J. J. O’Neil BetaLogics Venture, Janssen R&D LLC, Raritan, NJ, USA T. J. Kieffer Department of Surgery, University of British Columbia, Vancouver, BC, Canada
Abbreviations CXCR4 Chemokine [C-X-C motif] receptor 4 GLP-1R Glucagon-like peptide 1 receptor H&E Haematoxylin and eosin hESCs Human embryonic stem cells MAFA v-Maf musculoaponeurotic fibrosarcoma oncogene homolog A (avian) NKX2.2 NK2 homeobox 2 NKX6.1 NK6 homeobox 1 S4 Stage 4 STZ Streptozotocin UBC University of British Columbia
1988
Introduction Islet transplantation is an effective cell therapy for patients with type 1 diabetes as it re-establishes endogenously regulated production of insulin [1, 2]. However, widespread adoption of this clinical treatment is limited by the shortage of donor islets and the requirement for life-long immunosuppression [3, 4]. The obstacle of insufficient cell supply may be overcome with the use of pluripotent, self-renewable human embryonic stem cells (hESCs). Indeed, numerous groups have generated immature pancreatic insulin-producing cells in vitro from hESCs using step-wise differentiation protocols that mimic pancreatic development [5–16]. However, mature glucoseresponsive insulin-producing cells have only been achieved following transplantation of hESC-derived pancreatic progenitor cells. This was first demonstrated in healthy mice [11] and subsequently, by our group, in mice with streptozotocin (STZ)induced diabetes [17]. In our studies, human insulin levels were sufficient after several months in vivo to wean diabetic mice from exogenous insulin and by 30 weeks the engrafted insulin-secreting cells were v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (avian) (MAFA)-positive and glucose-responsive [17]. Unfortunately, the development of pancreatic endocrine cells was also accompanied by sporadic growth of mesodermal cells [5, 11, 17], illustrating the risk of transplanting immature hESC-derived cells. Presuming that hESCs can bridge the gap between cell supply and clinical demand, the issue of immunosuppression must be addressed before a cell-replacement therapy can be widely implemented for treating diabetes. Although safer immunosuppressants are being developed [3], those currently used for human islet transplantation are associated with complications, including impaired beta cell regeneration and function [18–21]. Moreover, it is difficult to justify the use of chronic immunosuppression in most patients, particularly newly diagnosed children. An ideal diabetes stem cell therapy would incorporate an immuno-isolation barrier to protect engrafted cells from the host immune system, thus eliminating the need for immunosuppression. Planar macroencapsulation devices (e.g. Theracytes [TheraCyte, Laguna Hills, CA, USA]) may be suitable, as they support neovascularisation via their outer membranes, while containing the engrafted cells within cell-impermeable inner membranes (0.4 μm pore size) [22]; this arrangement prevents contact with the surrounding host environment, including immune cells and vasculature. However, the relatively hypoxic environment is a potential concern, as pancreatic islets are accustomed to a rich vascular supply [23, 24]. Indeed, previous studies have demonstrated poor survival and function of adult human islets within Theracyte devices, whereas macroencapsulated human fetal islet-like cell clusters developed efficiently into glucoseresponsive beta cells after 5 months in immunodeficient mice [25]. These data suggest that immature pancreatic cells may be
Diabetologia (2013) 56:1987–1998
compatible with an encapsulation environment, though Matveyenko and colleagues detected only fibrotic tissue following transplantation of hESC-derived pancreatic progenitor cells within Theracyte devices in nude rats [26]. Given that the majority of hESC development occurs in vivo with a progenitor cell therapy approach, it is essential to understand how the surrounding host environment may affect maturation, particularly in an effort to translate this work towards a clinical setting. In the current studies, our goals were to determine how variables in the pre- and posttransplant environments may influence the maturation of hESC-derived progenitor cells into functional endocrine cells in vivo. We first examined the impact of various cell preparation techniques on progenitor cell maturation under the kidney capsule and then compared the influence of a normoglycaemic vs a hyperglycaemic environment with and without exogenous insulin therapy. We also assessed the capacity of hESC-derived progenitor cells to survive and mature within macroencapsulation devices, and the potential influence of exendin-4 therapy post-transplant. Finally, we describe a revised differentiation protocol aimed to improve the efficiency of generating pancreatic progenitor cells and reduce the development of off-target cells.
Methods In vitro differentiation of hESCs The H1 hESC line was obtained from WiCell Research Institute (Madison, WI, USA). All experiments at the University of British Columbia (UBC) with H1 cells were approved by the Canadian Stem Cell Oversight Committee and the UBC Clinical Research Ethics Board. For the studies described in Figs 1, 2, 3 and 8, H1 cells were differentiated according to our previously published 14 day, four-stage protocol [17]. For all other studies, H1 cells were cultured on 1:30 diluted Matrigel (BD BioSciences, Franklin Lakes, NJ, USA; catalogue [cat.] no. 356231) in mTeSR-1 (Stem Cell Technologies, Vancouver, BC, Canada; cat. no. 05850) as described in the electronic supplementary material (ESM). The revised differentiation protocol is summarised in ESM Fig. 1 and details are described in the ESM text. Quantitative RT-PCR Gene expression was assessed in stage 4 (S4) cells using custom Taqman Arrays (Applied Biosystems; Foster City, CA, USA), as previously described [17]. Data were always normalised to an internal housekeeping gene (GAPDH) and then calculated as the fold change relative to undifferentiated H1 cells using the ΔΔCt method. In Fig. 2a, the ΔΔCt data are presented as suspension relative to monolayer clusters to accommodate a wide range of data on a single graph. Details of the primers are provided in ESM Table 1.
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Fig. 1 Cell preparation (single cells vs overnight suspension clusters) prior to transplant affects the development of insulin-producing cells in vivo. S4 pancreatic progenitor cells were transplanted under the kidney capsule of diabetic SCID-beige mice in three separate cohorts (sample size indicated below bar graphs). (a–c) Immunofluorescent staining of the different cell preparations prior to transplant for insulin, glucagon and synaptophysin; scale bars, 200 μm. Cells in studies A and B were prepared as overnight suspension clusters (a, b) and in study C as
single cells (c). (d–f) Human C-peptide levels after an overnight fast (white bars) and 45 min meal (black bars) for each cohort between 4 and 16 weeks post-transplant (studies A, B and C are shown in d, e and f, respectively). By 16 weeks post-transplant, human C-peptide levels were significantly increased in both cohorts where cells had been transplanted as cell clusters (d, e). In contrast, pancreatic progenitor cells that had been transplanted as a single cell suspension failed to develop into insulin-secreting cells by 16 weeks post-transplant (f)
Viability/cytotoxicity assay In all studies, cell viability was tested immediately before transplant using a viability/ cytotoxicity assay (Life Technologies, Burlington, ON, Canada; cat. no. L3224), according to manufacturer’s instructions.
(C.B-Igh-1b/GbmsTac-Prkdcscid-LystbgN7; Taconic, Hudson, NY, USA) were maintained on a 12 h light/dark cycle with ad libitum access to a standard irradiated diet (Teklad Diet no. 2918; Harlan Laboratories, Madison, WI, USA). A summary of all animal studies is provided in ESM Table 2.
Animal studies All experiments were approved by the UBC Animal Care Committee. In vivo studies described in Figs 2b and 5e–f were performed at Janssen R&D and approved by the Janssen Institutional Animal Care and Use Committee. Male 8–10-week-old SCID-beige mice
Fig. 2 Cell preparation (monolayer clusters vs overnight suspension clusters) prior to transplant affects the development of insulin-producing cells in vivo. S4 pancreatic progenitor cells were dispersed into clusters (
