Intracameral Microimaging of Maturation of Human iPSC Derivatives into Islet Endocrine Cells

Abstract

We exploited the anterior chamber of the eye (ACE) of immunodeficient mice as an ectopic site for both transplantation and microimaging of engineered surrogate islets from human induced pluripotent stem cells (hiPSC-SIs). These islets contained a majority of insulin-expressing cells, positive or negative for PDX1 and NKX6.1, and a minority of glucagon- or somatostatin-positive cells. Single, non-aggregated hiPSC-SIs were satisfactorily engrafted onto the iris. They underwent gradual vascularization and progressively increased their light scattering signals, reflecting the abundance of zinc-insulin crystal packaged inside mature insulin secretory granules. Intracameral hiPSC-SIs retrieved from recipients showed enhanced insulin immunofluorescence in correlation with the parallel increase in overall vascularization and light backscattering during the post-transplantation period. This approach enables longitudinal, nondestructive and intravital microimaging of cell fates, engraftment, vascularization and mature insulin secretory granules of single hiPSC-SI grafts, and may offer a feasible and reliable means to screen compounds for promoting in vivo hiPSC-SI maturation.

In Vivo CaV3 Channel Inhibition Promotes Maturation of Glucose-Dependent Ca2+ Signaling in Human iPSC-Islets

“…The procedure has been described elsewhere [ 5 , 19 ]. In brief, the undifferentiated hiPSC line NCRM-1 (Lonza/NIH, Walkersville, MD, USA) was cultured in mTeSR1 medium (Stem Cell Technologies, Vancouver, BC, Canada) on Growth Factor Reduced Matrigel (Corning, Corning, NY, USA) for 3–4 days at 37 °C in a humidified 5% CO 2 incubator.…”

“…At the end of the six-stage induction, cells were removed from the Transwell culture plates, resized and placed into Ultra Low Attachment flasks (Corning) in suspension as stage seven for eight days. Upon completion of stage seven, the resulting hiPSC-islets were obtained for in vitro quality testing and intraocular transplantation [ 5 , 19 ]. All reagents were purchased from R&D Systems (Minneapolis, MN, USA) unless otherwise stated.…”

“…Human-induced pluripotent stem cells (hiPSCs) have successfully been used to produce spherical aggregates of islet hormone-producing cells, referred to here as hiPSC-islets, through in vitro cultivation [ 1 , 2 , 3 , 4 , 5 , 6 ]. However, clinical hiPSC-islet transplantation as a curative treatment for diabetes still remains challenging because hiPSC-islets cannot fully mature in vitro into glucose-responsive insulin-secreting islets and may reliably achieve functional maturation post-transplantation [ 1 , 3 , 6 , 7 , 8 , 9 , 10 , 11 ].…”

“…However, it has not been possible to retrieve single, intact hiPSC-islet grafts, and thus, [Ca 2+ ] i dynamics could not be directly measured in these grafts without losing their in vivo gained phenotypes. This inspired us to exploit the anterior chamber of the eye (ACE) of immunodeficient mice, which has been used as a reliable transplantation site for hiPSCs and their derivative [ 5 , 19 , 20 , 21 , 22 ]. The ACE is filled with aqueous humor endowed with high oxygen and less stress and may serve as a unique niche for the in vivo maturation of single, non-aggregated hiPSC-islet grafts.…”

Mouse models and human islet transplantation sites for intravital imaging

“…For example, an adenoviral calcium biosensor, GCaMP6m, has been used to infect human islets transplanted into the ACE of immunocompromised mice, demonstrating that islet calcium dynamics were functionally conserved ( 20 ). Also, once the animal is being imaged, injection of various treatments and probes may be employed to study beta cell biology during imaging, such as injection of a vasculature dye or cell death marker ( 21 ). Aside from using islets isolated from cadaveric donors, human induced pluripotent stem cells (hiPSCs) can be differentiated in vitro into islet-like structures for transplantation purposes, potentially reducing the burden to obtain human tissue for future studies ( 21 ), making exciting future discoveries using these cells possible.…”