This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.
Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.
Type 1 diabetes (T1D) is caused by the autoimmune destruction of the insulin-producing beta cells of the pancreas. The loss of beta cell mass results in insulin deficiency and hyperglycemia. T1D patients rely on multiple daily injections of exogenous insulin and experience episodes of severe hyperglycemia and hypoglycemia throughout their lives. The complications related to these episodes include diabetic retinopathy, decreased kidney function, and neuropathy1.
Insulin injections are a treatment but not a cure for T1D. Replacing the lost beta cell mass, however, has the potential to reverse the disease by enabling patients to produce their own insulin. However, the supply of cadaveric donor islets is limited2. Stem cell-derived islets (SC-islets) may provide a virtually unlimited supply of beta cells for transplant. Several groups have demonstrated that human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be differentiated to generate functional beta-like cells3,4,5. Promising early clinical trial data indicate that these cells maintain their function following transplant and may enable patients to become insulin-independent6. Chronic immunosuppression is required, however, thus increasing their susceptibility to cancer and infection. In addition, immunosuppressive agents may be cytotoxic to grafts in the long term7. To eliminate the need for immunosuppression, SC-islets may be genetically modified to protect them from recurrent autoimmunity as well as alloimmunity after transplant.
Stem cell research is highly demanding in costs and labor. Mouse cell lines and animal models are useful tools for the initial identification and experimental validation of strategies to protect transplanted cells from autoimmunity. The NOD mouse develops spontaneous autoimmune diabetes with many similarities to human T1D8, and the NIT-1 insulinoma cell line shares a genetic background with this mouse strain9. Diabetes can be adoptively transferred to the related immunodeficient NOD-scid mouse strain via the injection of diabetic splenocytes from NOD mice in order to temporally synchronize the onset of diabetes in replicate experimental mice10. This model can be used to identify genetic targets relatively quickly and inexpensively for further validation in SC-islets. Recently, the method was applied to identify and validate RNLS, a target that was found to protect primary human islets from autoimmunity in vivo and iPSC-derived islets from beta cell stress in vitro11. Described here is a straightforward protocol to transplant genetically engineered NIT-1 cells and non-invasively monitor their survival in an adoptive transfer model of autoimmune diabetes in mice.
Figure 1: The workflow for transplanting and imaging grafts in an adoptive transfer model of diabetes in mice. NIT-1 cells expressing the firefly transgene luciferase (luc2) are transplanted subcutaneously into NOD-scid mice. The mice are simultaneously injected with autoreactive splenocytes isolated from a spontaneously diabetic NOD mouse. The grafts are imaged at regular intervals by non-invasive bioluminescent imaging. Figure created by BioRender.com. Abbreviations: NOD = non-obese diabetic; scid = severe combined immunodeficient. Please click here to view a larger version of this figure.
All animal care and study protocols were approved by and performed in accordance with the Institutional Animal Care and Use Committee (IACUC) at the Joslin Diabetes Center. NOD and NOD-scid mice may be readily obtained from commercial sources. All mice in this study are maintained in a sentinal-monitored facility. See the Table of Materials for details related to all the materials, animals, instruments, and software used in this protocol.
1. Engineering and maintenance of NIT-1 cell lines
2. Preparation of NIT-1 cells for transplant
3. Transplantation of NIT-1 cells into NOD-scid mice
4. Isolation and purification of autoreactive splenocytes
5. Intravenous injection of diabetic splenocytes via the lateral tail vein
6. In vivo bioluminescent imaging of NIT-1 grafts
NOTE: Image the grafts one to two times per week. On the day of the transplant, wait at least 2 h after the transplant to allow the grafts to settle and ensure stable luciferase expression. If time is a limiting factor, the initial measurement may be taken on Day 1 instead. A recommended initial imaging schedule is Day 0 or Day 1 post injection, Day 5, Day 10, Day 14, Day 18, and Day 25. Adjust the schedule, however, based on the progress of autoimmunity as judged by the loss of bioluminescent signal.
Figure 2: Screenshots of the software commands for imaging bioluminescent grafts. (A) Prior to imaging, select Initialize to prepare the instrument. The images may be auto-saved to a folder of choice by selecting Acquisition | Auto-save to… (B) Overview of the imaging parameters. Once the mouse has been positioned in the instrument, select Acquire. (C) Screenshot of the dialog box that pops up during imaging. Details such as the time point and mouse strain may be entered here. Please click here to view a larger version of this figure.
7. Data analysis
An overview of the protocol is outlined in Figure 1. The survival of two cell lines, such as a mutant and a non-targeting control, may be compared, or the survival of one cell line may be measured in multiple groups of mice, such as drug-treated mice versus vehicle-treated controls. Figure 3A shows three 8-week-old female NOD-scid mice transplanted with a non-targeting control (left) and a mutant (right) cell line. The mice were also injected intravenously with autoreactive splenocytes to transfer diabetes.
The mice were imaged on Day 0, Day 5, Day 10, Day 14, and Day 18 post injection. In two out of the three mice, the control graft was destroyed by Day 18, as evidenced by the loss of bioluminescent signal, while the mutant graft was protected. In one mouse, the mutant graft but not the control graft was destroyed, highlighting the biological variation between animals. The bioluminescent signal was quantified as described in Figure 3B. The graft survival is reported as the percentage of residual bioluminescent signal compared to the first time point (Figure 3C). It is also useful to calculate the ratio of mutant to control luminescent signals for each mouse to visualize the variation between animals (Figure 3D). At the end of the data collection period, the mice were euthanized by CO2 inhalation followed by cervical dislocation.
Prior to disease transfer, the transplanted cells may replicate, resulting in expanded grafts that are visible through the skin (Figure 4A). These grafts will have saturated bioluminescent signals when imaged (Figure 4B), which may not be accurately quantified.
Figure 3: Representative images and quantification of the bioluminescent signal. (A) Three 8-week-old female NOD-scid mice were transplanted with non-targeting control (right) and mutant cells (left) and intravenously injected with autoreactive splenocytes to induce diabetes. The mice were non-invasively imaged on Day 0, Day 5, Day 10, Day 14, and Day 18 post injection. The bioluminescent signal is illustrated by a color spectrum ranging from low intensity (blue) to high intensity (red). (B) Instructions for quantifying the bioluminescent signal. (C) Average percentage of graft luminescence remaining over time. (D) Ratio of the percentages of graft luminescence over time for mutant versus non-targeting control grafts for each mouse. Ratio = 1 indicates time points at which the percentage of signal remaining is equal for both grafts. Abbreviations: NOD = non-obese diabetic; scid = severe combined immunodeficient; NTC = non-targeting control; Mut = mutant. Please click here to view a larger version of this figure.
Figure 4: Examples of grafts that are excessively expanded. (A) Expanded grafts may bulge through the skin of the mouse (indicated by the red arrow). (B) Expanded grafts may yield saturated bioluminescent signals. The bioluminescent signal is illustrated by a color spectrum ranging from low intensity (blue) to high intensity (red). Please click here to view a larger version of this figure.
Supplemental File 1: pLenti-luciferase-blast sequence. Please click here to download this File.
T1D is a devastating disease for which no cure currently exists. Beta cell replacement therapy offers a promising treatment for patients with this disease, but the critical barrier to this strategy is the potential for recurrent autoimmune attack against the transplanted beta cells. The genetic engineering of SC-beta cells to reduce their immune visibility or susceptibility is one potential solution to this problem. Described here is a protocol for non-invasively imaging transplanted beta cells to measure their survival in an adoptive transfer model of autoimmune diabetes in mice.
A key advantage of this method is the adaptability of the protocol. The method is applied here to compare a mutant cell line to a non-targeting control (Figure 3), but the protocol can accommodate a variety of experimental questions. The protective effects of a wide range of genetic modifications, drugs, or other treatments against autoimmune attack could be explored. When evaluating the efficacy of a drug or other treatment in protecting against autoimmunity, only cells expressing luciferase with no additional genetic modifications are needed. While syngeneic transplants to test autoimmune protection are used in this paper, the method may be also be adapted to evaluate alloimmune rejection by using a combination of a cell line and a mouse strain that are not syngeneic, such as NIT-1 cells and C57BL/6J mice. The protocol may also be modified to explore immune infiltration into the grafts. Additional mice from which grafts are retrieved at various time points may be included, and the infiltrating immune cells may be profiled by immunohistochemistry or flow cytometry.
A major limitation of the application of this method is the failure of the results obtained in mice to translate to human cells and patients. Over 500 strategies for autoimmune protection have been implemented with success in diabetic mouse models, yet the majority have failed to demonstrate clinical benefit7. Primary human islets are a limited resource, however, and stem cell work has high financial and labor costs. The protocol described here is a tool for the preliminary identification of potential therapeutic targets utilizing a relatively inexpensive and simple mouse technique. Subsequent experiments should be performed with primary and/or SC-islets, which are typically transplanted under the kidney capsule in mice. The method described here can be adapted to evaluate the efficacy of therapeutic strategies in human cells. Both primary and SC-islets can be engineered to express luciferase, and their survival can be measured by bioluminescent imaging following transplant under the kidney capsule into humanized mouse models12,13. Methods for non-invasively monitoring the survival of islet grafts transplanted onto the iris of the eye by confocal microscopy have been also been reported14.
An additional limitation associated with the method described in this paper is the variation in the autoreactive potential of isolated splenocytes. Although the rate of disease transfer is high10, the timeline of diabetes onset post splenocyte injection can vary from 2 weeks to 4 weeks. During this time, the transplanted cells can expand (Figure 4), resulting in hyperinsulinemia and severe hypoglycemia. Combining splenocytes from multiple diabetic donor mice can reduce the variation between experiments.
Despite these limitations, the method is a straightforward and informative tool for testing strategies for autoimmune protection. The protocol utilizes basic cloning and cell culture techniques, a simple and minimally invasive mouse surgery, and non-invasive imaging, allowing the user to maximize the data collection while minimizing the number of animals needed per experiment. As an alternative to in vivo bioluminescent imaging, grafts may be recovered at various time points, weighed, photographed, and further processed for immune cell characterization. This strategy requires larger numbers of mice, as graft recovery requires euthanasia, and it is desirable to have replicates at each time point. In addition, the graft retrieval method allows for only one size measurement to be taken per graft. As the timeline of beta cell killing by autoreactive splenocytes varies across individuals, it can be difficult to determine the optimal time point for graft recovery. The approach described in this paper allows the researcher to monitor the progress of autoimmunity in live mice and obtain a complete timeline of graft destruction. As long as mice maintain euglycemia, protected grafts can be left in the mice and imaged over long periods of time to elucidate the degree of protection.
Described here is a straightforward protocol for transplanting and non-invasively imaging beta cell grafts in an adoptive transfer mouse model of autoimmune diabetes. This method enables a minimal number of animals to be used and allows flexibility in the timing of graft analysis, which is critical given the variation in the progress of autoimmunity. The protocol can also be readily adapted to address a wide range of experimental questions. Mouse models are critical in the discovery and validation of new therapeutic strategies, and this method is a valuable tool for advancing the beta cell replacement therapy field.
The authors have nothing to disclose.
We thank Dr. Erica P. Cai and Dr. Yuki Ishikawa for developing the method described in this protocol (see ref. 11). Research in S.K. and P.Y.'s laboratories is supported by grants from the National Institutes of Health (NIH) (R01DK120445, P30DK036836), JDRF, the Harvard Stem Cell Institute, and the Beatson Foundation. T.S. was supported by a postdoctoral fellowship from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (T32 DK007260-45), and K.B. was supported in part by a fellowship from the Mary K. Iacocca Foundation.
0.05% Trypsin, 0.53 mM EDTA | Corning | 25-052-CI | |
293FT | Invitrogen | R70007 | Fast-growing, highly transfectable clonal isolate derived from human embryonal kidney cells transformed with the SV40 large T antigen |
ACK Lysing Buffer | Gibco | A10492-01 | |
Alcohol prep pads, 70% Isopropyl alcohol | Amazon/Ever Ready First Aid | B08NWF31DX | |
BD 5ml Syringe Luer-Lok Tip | BD | 309646 | |
BD PrecisionGlide Needle 26G x 5/8 (0.45 mm x 16 mm) Sub-Q | BD | 305115 | |
BD 1 mL TB Syringe Slip Tip | BD | 309659 | |
Blasticidin S HCl | Corning | 30-100-RB | |
Cell strainer premium SureStrain, 70 µm, sterile | Southern Labware | C4070 | Or use similar sterile strainer with 40-70um pore size |
CellDrop automated cell counter | Denovix | CellDrop BF-PAYG | Or use similar cell counter device |
Corning 100 mL Penicillin-Streptomycin Solution, 100x | Corning | 30-002-CI | |
Disposable Aspirating Pipets, Polystyrene, Sterile, Capacity=2 mL | VWR | 414004-265 | Or use similar aspirating pipette |
D-Luciferin, Potassium Salt , Molecular Biology Grade, Powder, >99% | Goldbio | LUCK-100 | |
DMEM, high glucose, pyruvate, no glutamine | Gibco | 10313039 | |
Falcon BD tubes, 50 mL | Fisher Scientific | 14-959-49A | |
Fetal Bovine Serum | Gibco | 10437-028 | |
Forceps premium for tissues, 1 x 2 teeth 5 in, German Steel | Fisher Scientific | 13-820-074 | |
Glucose urine test strip | California Pet Pharmacy | u-tsg100 | Or use similar test strip for glucose measurments in urine/blood |
GlutaMAX–1 (100x) | Gibco | 35050-061 | |
Infrared heating lamp | Cole Parmer | 03057-00 | Or use similar infrared lamp |
Insulin syringe 0.5 mL, U-100 29 G 0.5 in | Becton Dickinson | 309306 | |
Isoflurane, USP | Piramal Critical Care | 6679401725 | |
IVIS Spectrum in vivo imaging system | Perkin Elmer | 124262 | Instrument for non-invasively collecting bioluminescent images of transplanted cells |
Living Image Analysis Software | Perkin Elmer | 128113 | Software for collecting and quantifying bioluminescent signal |
Microcentrifuge tubes seal-rite, 1.5 mL | USA Scientific | 1615-5510 | Or use similar sterile microcentrifuge tubes |
NIT-1 | ATCC | CRL-2055 | Pancreatic beta-celll line derived from NOD/Lt mice |
NOD.Cg-Prkdcscid/J | The Jackson Laboratory | 001303 | Mice homozygous for the severe combined immune deficiency spontaneous mutation Prkdcscid, commonly referred to as scid, are characterized by an absence of functional T cells and B cells, lymphopenia, hypogammaglobulinemia, and a normal hematopoietic microenvironment. |
NOD/ShiLtJ | The Jackson Laboratory | 001976 | The NOD/ShiLtJ strain of mice (commonly called NOD) is a polygenic model for autoimmune type 1 diabetes |
PBS, pH 7.4 | Thermo Fisher Scientific | 10010031 | No calcium, no magnesium, no phenol red |
pCMV-VSV-G | Addgene | 8454 | |
pLenti-luciferase-blast | Made in-house | Plasmid available upon request | See Supplemental File 1 |
pMD2.G | Addgene | 12259 | |
pMDLg/pRRE | Addgene | 12251 | |
Polyethylenimine, Linear, MW 25,000, Transfection Grade (PEI 25K) | Fisher Scientific | NC1014320 | |
pRSV-Rev | Addgene | 12253 | |
Restrainer for rodents, broome-style round 1 in | Fisher Scientific | 01-288-32A | |
Scissors, sharp-pointed | Fisher Scientific | 08-940 | Or use other scissors made of surgical-grade stainless steel |
Tissue-culture treated culture dishes | Millipore Sigma | CLS430167-20EA | Or use other sterile cell culture-treated Petri dishes |
Tweezers/Forceps, fine precision medium tipped | Fisher Scientific | 12-000-157 |