purchase A recently developed gene editing technology using

A recently developed gene-editing technology using RNA-guided endonuclease (Gaj et al., 2013), which allows the desired gene to be inserted into a genomic safe harbor (GSH) area (Papapetrou et al., 2011), should be considered to avoid undesirable random genomic insertion of EOS-C(3+)-KR in the generation of hPSCs that do not give rise to teratomas by simple light exposure. Especially, when applied to generate KR-human iPSCs, guided insertion of EOS-C(3+)-KR into the GSH area of parental purchase allowed for red fluorescence to be emitted when reprogramming was complete to properly identify iPSC colonies (Hotta et al., 2009), similar to the mouse model reported in the current study (Figure 1D).
Experimental Procedures Details of the methods are available in the Supplemental Experimental Procedures.
Acknowledgments
Introduction Patients with type 1 diabetes suffer from a severe deficiency in insulin production by pancreatic islets as a result of immune-mediated destruction of pancreatic β cells. Insulin independence can be achieved by transplantation of cadaveric human islets (Shapiro, 2011), but because of the scarcity of donor tissue, the field is exploring the potential use of scalable human embryonic stem cell (hESC)-derived pancreatic cells as an alternative cell source. We have demonstrated previously that hESC-derived pancreatic progenitor cells develop over several months in vivo into insulin-secreting cells capable of reversing hyperglycemia in a mouse model of type 1 diabetes (Rezania et al., 2012, 2013; Bruin et al., 2013). Interestingly, the maturation process was accelerated when mice were exposed to chronic hyperglycemia but unaffected by exposure to long-term insulin therapy, short-term exendin-4 treatment, oral anti-diabetic medications, or high-fat diets (Bruin et al., 2013, 2015). In addition, we recently reported a revised differentiation protocol that generated glucose-responsive insulin-secreting cells in vitro and required a much shorter maturation period (∼6 weeks) following transplantation to reverse hyperglycemia in mice (Rezania et al., 2014). Given the uncertainty surrounding the complex host environment and variables that may affect the maturation process in vivo, advancing the differentiation protocols in vitro prior to transplantation may be advantageous. Nevertheless, hESC-derived pancreatic progenitor cells are currently being tested for safety, tolerability, and efficacy in a phase 1/2 clinical trial by Viacyte (ClinicalTrials.gov identifier NCT02239354). Therefore, although newer differentiation protocols have been reported (Pagliuca et al., 2014; Rezania et al., 2014; Russ et al., 2015), it remains important to understand the development of pancreatic progenitor cells in vivo because clinical trials are underway in patients with diabetes. There are several obvious differences between the pre-clinical transplant recipients tested to date (immunodeficient mice) and the target patient population, including the species, distinct metabolic profiles, and large size difference. Although rats are not directly comparable with humans, their physiology is reportedly more similar to humans than mice, particularly in terms of cardiovascular parameters (Davies and Morris, 1993). We have demonstrated previously that hESC-derived grafts were capable of robust glucose-stimulated insulin secretion (GSIS) after just 14 weeks in nude rats, whereas GSIS was not observed until after 30 weeks in similar studies with severe combined immunodeficiency (SCID)-beige mice (Rezania et al., 2012). However, these studies were performed at different facilities and with different batches of cells, so we could not make direct comparisons between species. Interestingly, others have reported that hESC-derived pancreatic progenitor cells did not efficiently differentiate into pancreatic endocrine tissue following transplantation in nude rats (Matveyenko et al., 2010). The authors speculated that the nude rat may be a less accommodating host environment compared with immunodeficient mice (Matveyenko et al., 2010). To address these conflicting observations, we performed a carefully controlled study within a single research facility to directly compare the in vivo development of hESC-derived pancreatic progenitor cells from the same preparation and transplanted in parallel into either immunodeficient nude rats or SCID-beige mice.