(317f) Light-Controlled Enhancement of Glucose-Stimulated Insulin Secretion of Optogenetically Engineered Human Pancreatic Beta-Cells

Diabetes is a group of disorders linked to the lack of insulin-producing pancreatic β-cells or insulin resistance exhibited by glucose-absorbing tissues. The transplantation of β-cells or islets is a promising avenue for restoring normal blood glucose homeostasis in diabetes patients. Yet, the number of cells required to reconstitute glucose-stimulated insulin secretion (GSIS) – whether by directly transplanting islet cells or employing a bioartificial pancreas – poses significant challenges on maintaining optimal cell viability and function. To mitigate this issue, optogenetic tools can be applied to lower the oxygen and nutrient demand as fewer cells are required with insulin production upregulated by 2- to 3-fold in rodent β-cells, as we reported [1][2]. Optogenetics allow modulation of cellular processes in a non-invasive and reversible manner with high specificity and fewer side effects in comparison to pertinent pharmacological treatments. The secondary messenger cyclic adenosine monophosphate (cAMP) plays a central role in the regulation of GSIS in pancreatic β-cells. The increased cAMP activates the protein kinase A (PKA), which phosphorylates and enhances the function of voltage-dependent calcium channels, allowing for greater Ca²⁺influx and therefore stimulation of membrane depolarization for subsequent release of insulin granules. We engineered human β-cells to express a blue light photoactivatable adenylyl cyclase (bPAC) for photoinduced augmentation of insulin secretion at various extracellular glucose levels. Alginate hydrogels were adopted to encapsulate and facilitate the delivery of the optogenetically modified β-cells.

We have demonstrated that murine insulinoma cells can form PIs in stirred-suspension spinner flasks and in low-adhesion plates under orbital shaking [3]. To this end, human EndoC-βH3 cells were seeded in 50-100 mL culture medium in spinner flasks and cultured at 37 °C and 5% CO₂ with agitation speed set to 70 rpm. For up to nine days, cells formed aggregates and were maintained with half-volume media changes every 3 days. The average aggregate size increased from 67.8 ± 7.8 mm on day 3 to 94. 8 ± 19.9 mm on day 9 as determined from the quantification of PI images acquired by bright field microscopy. A significant increase in size was noted on days 7 and 9 of the cultures by 7% and 40% respectively. The PIs were infected with an adenoviral vector, AdbPAC, carrying the bPAC-cMyc-IRES-mCherry cassette (cMyc tag for immunodetection of bPAC; mCherry: fluorescent reporter coexpressed with bPAC; IRES: internal ribosomal entry site) under the CMV promoter. The expression of mCherry was detected by fluorescence microscopy in about 50% of cells in monolayers and PIs. Given the similarities in the molecular circuitries governing GSIS in rodent and human β-cells, we hypothesized that expression of the PAC to human β-cells will provide a similar handle for controlling insulin function. Insulin secretion and intracellular cAMP were measured under different glucose conditions. Insulin release was upregulated by 2.5-fold stimulated by the addition of glucose at 20 mM compared to 0.5 mM. A 2-fold and 1.5-fold increase of secreted insulin was observed by raising [cAMP]i with forskolin at 20 mM glucose, and 0.5 mM glucose level, respectively.

For the delivery of the optogenetically engineered β-cells, we set out to develop a scaffold featuring wireless circuitry and an LED for optostimulation in a closed-loop system. Alginate, which exhibits excellent biocompatibility, minimal cytotoxicity and benign gelation conditions, has been used successfully for encapsulating islet cells as reported by numerous groups. The scaffold is designed to harbor PIs and an LED with a magnetic reed for remote control during in vitro testing. Alginate polymers can be crosslinked with calcium ions. Using CaCl₂, the gelation rate of alginate is rapid, but the polymer gels are opaque rendering them undesirable for optogenetic applications, which rely on light transmissibility. To control the gelation rate of 1.5% (w/v) alginate and obtain homogeneous hydrogels, we used CaCO₃ and 50-200 mM glucono-δ-lactone (GDL) (1:2) as the crosslinking agents, which resulted in a slower, more controlled gelation process leading to structural uniformity and notably higher transparency. An increase in hydrogel opacity was observed along with a faster gelation time with higher Ca²⁺ concentration. Gelation with CaCO₃ and GDL improved scaffold transparency by 80% compared to CaCl₂-crosslinked alginate gels at higher Ca²⁺ concentration. The Young modulus of the hydrogels was determined by a dynamic mechanical analyzer (RSA3, TA Instruments, New Castle, DE) with a 35-N loading cell at room temperature. Scaffolds formed using CaCl₂ showed concentration-dependent mechanical properties. Young’s modulus ranged between 4 kPa – 25 kPa for 25 mM – 100 mM CaCl₂. For reference, the Young modulus of murine pancreas is 4.2 kPa and of human normal pancreatic tissue is 1 kPa, whereas higher modulus values are reported for pancreas under pathological conditions [4]. Our premise is that hydrogels with mechanical properties resembling those of the native pancreatic tissue will provide an environment that is more akin to the physiological milieu potentially contributing to enhanced functionality of the optogenetically engineered cells. Moreover, a blue microLED light that can be activated remotely with a radio-frequency identification (RFID) reader was also encapsulated in the alginate disk scaffold. Viability of encapsulated EndoC-βH3 and incubated with Calcein AM dye was 80% when determined by confocal microscopy an hour after the gelation. Ongoing experiments aim to determine the glucose stimulation and release rate of insulin from bPAC-expressing cells in hydrogels.

Next-generation technologies for diabetic management are expected to incorporate glucose-sensing, insulin-releasing cells. The transplantation of β-cells is shown to restore normal blood glucose level autonomously unlike treatments entailing exogenous insulin injections. Through the expression of optogenetic switches like bPAC, GSIS can be enhanced without the use of pharmacological agents with potentially adverse side effects. Molecular customization of human β-cells with optogenetic moieties, scalable cultivation of the engineered islet cells as PIs, and encapsulation within an LED-harboring hydrogel are key elements in the design and development of bioartificial pancreas devices. Such technologies will be a significant step toward translating optogenetic control-based solutions to therapeutic modalities for diabetes patients.

References:

[1] F. Zhang, E. S. Tzanakakis, "Amelioration of diabetes in a murine model upon transplantation of pancreatic β-cells with optogenetic control of cyclic adenosine monophosphate." ACS Synth. Biol. 8(10): 2248-2255, 2019.

[2] F. Zhang, E. S. Tzanakakis, "Optogenetic regulation of insulin secretion in pancreatic β-cells." Sci. Rep. 7(1): 1-10, 2017.

[3] L.T. Lock, S. G. Laychock, E.S. Tzanakakis, “Pseudoislets in stirred-suspension culture exhibit enhanced cell survival propagation and insulin secretion”, J. Biotechnol. 151(3): 278-86, 2011.

[4] A. Nabavizadeh et al. “Noninvasive Young’s modules visualization of fibrosis progression and delineation of pancreatic ductal adenocarcinoma (PDAC) tumors using harmonic motion elastography in vivo”, Theranostics10(10):4614-26, 2020.