We propose the following array of multiple assays with their associated rationale, as well as a decision tree/order of the proposed assays.
Protocol 1 – Testing Physiological Function and Viability by Pulsed Glucose and KCI Stimulation
We first test the functionality of the cell sample by simple pulse stimulation with a high glucose concentration, followed by 30 mM KCI. From this protocol, we can characterize the physiological similarities or differences of cell-biologics compared to human islets. Furthermore, it acts as an initial test criteria to determine if further testing is needed. If the cell-biologic passes the initial test, the following systematic protocols may follow to further interrogate their insulin secretory pathway.
Protocol 2 – Determination of Optimal Glucose Sensitivity using Incremental Step-Up Glucose Pulse Stimulation and/or Glucose Ramp Stimulation
It is known that some cell derivatives respond to glucose differently when compared to human islets, for example glucose sensitivity shift. This shift in glucose sensitivity is evidence that the cells are responsive to glucose either at lower or higher glucose concentrations when compared to human islets. It is expected that the iBCs derived from pluripotent stem cells might initially form cells that display a variation in glucose sensitivity, due in part to its position as a developmental intermediate. It is important not only to address how this varying threshold may change during maturation, but also to prevent potential complications such as hypoglycemia, which will have profound effects on clinical outcomes.
Step-up glucose pulse stimulation provides useful information on whether a shift in glucose sensitivity occurs. However, a detailed change in threshold/sensitivity can be further verified by a glucose ramp over a narrowed range of glucose concentrations determined by step-up glucose pulse experiments.
Protocol 3. Dissection of Insulin Stimulator-Secretion Coupling Factors
Glucose-induced beta-cell insulin secretion is a cascade that is mainly mediated through the KATP-dependent pathway. This pathway involves several key stimulator-secretion coupling factors. Any defect or change in these coupling factors will significantly impact insulin secretion and, subsequently, in vivo function. Many iBCs are acknowledged to respond to glucose differently when compared to normal human islets. This difference may result from a complete absence of or immaturity of stimulator-secretion coupling factors through the differentiation protocols employed. Additionally, glucose can increase cytoplasmic calcium concentrations by inducing calcium release from the endoplasmic reticulum (ER) calcium stores without biphasic and pulsatile signatures. Often, the ER-induced calcium stimulates small quantities of insulin release in a non-controlled manner. Additionally, this can provide misleading information on iBCs glucose-responsiveness and functional maturity.
Protocol 3.1 Verification of KATP Channel Activity
Since both plasma KATP channel existence and activity play an important role in glucose-induced insulin secretion, the KATP channel activity is assessed with dose responses of KATP channel closers (Tolbutamide and/or glyburide). Additionally, KATP channel activity can be further verified by KATP channel openers (Diazoxide) in response to either glucose or KATP channel closers.
Protocol 3.2 Verification of VDCCs Activity
Glucose-induced calcium influx mainly occurs through the voltage-dependent calcium channels (VDCCs). We have observed that glucose-induced calcium may be independent of VDCCs in some iBCs. This may explain why iBCs often have abnormal insulin secretion patterns and lower insulin release in response to the glucose challenge. Here, we apply Nifedipine (L-type Ca2+ channel closer) pre or post glucose administration to determine the spatiotemporal relationship between [Ca2+]I and insulin secretion in cells to be tested. Additionally, we can determine the origin of cytoplasm calcium by using a calcium-free Krebs-Ringer buffer and assessing [Ca2+]i in response to glucose.
Protocol 3.3 Determination of Intracellular Calcium Source
In addition to activation through the VDCCs, cytoplasm calcium can also be induced by glucose through calcium release from the ER. This in turn then triggers insulin secretion; however, such calcium signaling often does not have normal biphasic and oscillatory patterns, similar to VDCC-regulated calcium signaling. In this protocol, thapsigargin (an ER calcium ATPase pump blocker) is applied to deplete ER calcium before glucose stimulation in order to determine the role, if any, that ER calcium plays in insulin secretion and intracellular signaling of test cell derivatives.
Protocol 4. Determination of Maximal Insulin Secretion Capability and Exhaustion
Beta-cell insulin granules populate two different pools: the readily releasable pool (RRP) and the reserved pool (RP). The RRP pools are associated with phase I secretion. In contrast, phase II secretion requires the trafficking of the RP to the plasma membrane. Normal beta-cells contain excess amounts of insulin granules and only a small percentage (1-5%) is utilized for insulin secretion under each stimulation. We apply repetitive glucose stimulation to mimic in situ glucose homeostasis to determine maximal insulin secretion capabilities and identify insulin secretion defects in phase I/II or beta-cell exhaustion. This pulse stimulation protocol may provide useful information on insulin granule distribution and release capability (RRP pools vs. reserved pools) by analyzing phase I vs. phase II or phase I or II vs. total insulin release. If repeated stimulation elicits a significant reduction of acute insulin release with each pulse then the beta-cells are considered exhausted. This in turn indicates that the cell derivative may not provide long-term in vivo function.
Protocol 5. Determination of the Impact of Microencapsulation
Microencapsulation is a promising approach to prevent islet graft immunorejection. It is important to understand the impact of the encapsulation process, capsule material, and capsule size on the nutrient/insulin diffusion, glucose metabolism, and function of encapsulated cells. Additionally, hypoxia is considered another important factor responsible for the functional loss of encapsulated islets. Encapsulation aggravates islet hypoxia by preventing revascularization and increases the oxygen diffusion distance. Hypoxia may also facilitate the attraction of macrophages and, subsequently, causes cell overgrowth on the microcapsule.
The function of microencapsulated islets, impact of capsule materials, and capsule sizes are assessed using previously described protocols (1-4). For the hypoxia study, microencapsulated islets or islet-like cells are assessed using the perifusion device integrated with an oxygen controller shown in Figure 1C.
If required by interested researchers, we can further provide new experimental protocols focused on the following areas: (please note that all protocols are well established and fully verified).
- Simultaneous measurement of [Ca2+]i/NAD(P)H and insulin.
- Simultaneous measurement of ROS using DCFDA/MitoSox, [Ca2+]i, and insulin.
- Investigation of insulin secretion independent of KATP channels and amplification of KATP-dependent pathways through the application of GLP-1, carbachol, glucose + arginine, or glucose + amino acid mix.
- Investigation of long-term dynamic and dose-response of pharmaceutical agents on islet survival and function using the biochip shown in Figure 1C.
- High-throughput, high-content imaging, and screening of iBCs and pharmaceutical agents using the array biochip (Fig. 1D and E) that can hold up to 200 islets or encapsulated islets in an array.
The UIC Human Islet Transplant Program has a certified cGMP isolation facility and has conducted more than 600 human islet isolations. The program is one of only six centers funded through the IIDP for the distribution of human islets. Additionally, we have archived data from over 150 human islet preparations using this microfluidic assay, as well as in vivo animal transplant data, histological imaging/analysis, and other in vitro data from each preparation that is useful as an historical control. Since we isolate human islets on a weekly basis, freshly isolated human islets can be used as a suitable control whenever required by cell providers.