Intramuscular Transplantation of Human Pluripotent Stem Cell-derived Pancreatic Endocrine Cells in Mice
April 10th, 2026
We provide a minimally invasive protocol for the intramuscular transplantation of human stem cell-derived pancreatic stem cell islets into immunodeficient mice for in vivo implantation and downstream analysis.
We study human endocrine pancreas development using stem cell-derived eyelets to understand the genetic basis of diabetes. Traditional kidney capsule transplantation is highly invasive. Intramuscular transplantation protocol offer a simpler, reproducible, and minimally invasive alternative.
To begin, add 0.5 milliliters of 5%anti-adherent solution to cover the bottom of a 24-well microwell plate containing inverted pyramidal microwells. Centrifuge the plate at 1, 300 g for 10 minutes at room temperature. Then examine the microwells under a microscope for air bubbles.
After re-centrifuging the plate, to remove any trapped air, aspirate the anti-adherent solution. Then wash each well with 0.5 milliliters of PBS and repeat the washing step once more. Next, wash the cultured pancreatic progenitors developed from hPSCs with one milliliter of PBS with 0.5 millimolar EDTA.
Aspirate the PBS with 0.5 millimolar EDTA and cover the wells with trypsin. Then incubate at 37 degrees Celsius for eight to 12 minutes. Dilute the trypsin with two volumes of DMEM/F12 or PBS and gently re-suspend the cells by pipetting.
Transfer the suspension to a conical tube and immediately proceed to the next step. Centrifuge the suspension at 200 g for three minutes at room temperature. Carefully aspirate the supernatant and gently re-suspend the pellet in pre-warmed S4 medium.
Next, aspirate the PBS from the microwell dishes. Gently add 1.25 milliliters of the suspended cells to each well of the microwell plate. Centrifuge the plate at 100 g for three minutes at room temperature to distribute the cells evenly in the microwells.
Carefully transfer the microwell plate to a humidified incubator. To perform media changes with differentiation media, gently aspirate and dispense the media along the well walls to prevent turbulence and displacing cell clusters from microwells. After transferring the appropriate quantity of generated clusters to a 1.5-milliliter microcentrifuge tube, connect the syringe to the needle tubing assembly and fill it with S7 medium.
Aspirate the clusters into the tubing directly from the microcentrifuge tube. Fold the tubing in half and insert the folded section into a 200-microliter pipette tip. Then insert the tip tubing needle assembly into a 15-milliliter conical tube and secure the ends to the tube rim using tape.
Centrifuge the tube at 100 g for two minutes at 4 degrees Celsius to compact the clusters and immediately place the tube on ice. To transplant the clusters into the anesthetized NOD scid gamma mice, shave the medial aspect of the right leg below the knee and disinfect with 70%ethanol. Then detach the tubing from the pipette tip and connect it to the syringe.
Cut the tubing close to the compacted clusters and attach it to the blunt end of the needle. Push the saline until the first cluster reaches the tip of the needle. Then position the mouse prone, leaning to the right side and extend the right leg.
Insert the needle four to five millimeters deep into the medial compartment of the right thigh between the adductor and gracilis muscles. Then inject the clusters while retracting the needle by approximately three millimeters. Rotate the syringe, withdraw the needle, and allow the mouse to recover.
Following graft recovery six weeks post-transplantation and tissue sectioning, perform immunostaining to enable subsequent imaging analyses. Single cell suspensions of stage-four pancreatic progenitors were seated into microwell plates to generate size-controlled three-dimensional aggregates. Within 24 to 48 hours, compact spherical clusters formed.
Fluorescence microscopy confirmed the presence of PDX1 and insulin-expressing cells in reporter hPSC lines. After six weeks, the graft site was visualized macroscopically with eyelet-like masses identifiable within the muscle. Following excision, explants were identifiable under a stereo microscope as pale spherical tie structures.
Robust detection of C-peptide positive cells was observed in transplanted animals. Insulin staining confirmed the presence of human beta-like cells within the graft. Glucagon and somatostatin-positive cells were detected, indicating preservation of alpha and delta cell populations.
This protocol facilitates in vivo functional assays and evaluation of survival and maturation of transplanted human grafts. The most important consideration is ensuring high-quality stem cell differentiation and gentle cell handling during cluster formation to maximize viability and ensure good cell survival. These models facilitates the interrogation of genetic mechanisms underlying monogenic diabetes and beta cell dysfunction.
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Overview
This JoVE article presents a detailed protocol for intramuscular transplantation of human pluripotent stem cell (hPSC)-derived pancreatic SC-islets into immunodeficient mice. The method offers a less invasive alternative to kidney capsule or subcutaneous transplantation, enabling straightforward surgical access and reliable graft retrieval. The protocol supports assessment of islet function via in vivo glucose-stimulated insulin secretion (GSIS) following an oral glucose challenge and facilitates downstream histological analysis.
Key Study Components
Area of Science
- Regenerative medicine
- Stem cell therapy
- Diabetes research
Background
- hPSC-derived pancreatic endocrine cells hold promise for diabetes cell therapy and disease modeling.
- Current transplantation sites like kidney capsule or subcutaneous space present technical or physiological limitations.
- In vivo implantation is often required to study maturation and function of stem cell-derived islets in a physiological context.
- There is a need for reproducible, accessible transplantation sites that allow for graft retrieval and functional assessment.
Purpose of Study
- To provide a detailed protocol for intramuscular transplantation of hPSC-derived SC-islets in immunodeficient mice.
- To enable evaluation of islet engraftment, survival, and function in a minimally invasive site.
- To support functional assessment via in vivo GSIS and histological analysis post-transplantation.
- To establish a reproducible framework for preclinical diabetes research using stem cell-derived islets.
Methods Used
- Preparation and compaction of hPSC-derived pancreatic SC-islet clusters.
- Intramuscular transplantation surgery into immunodeficient mice.
- In vivo glucose-stimulated insulin secretion (GSIS) assessment following oral glucose challenge.
- Graft retrieval and histological analysis for post-transplantation evaluation.
Main Results
- Successful engraftment and survival of hPSC-derived islets in the intramuscular transplant site.
- Detection of functional insulin secretion in response to glucose challenge via in vivo GSIS.
- Reliable retrieval of grafts for histological analysis confirming islet structure and marker expression.
- Demonstration of a reproducible, minimally invasive method for preclinical islet transplantation studies.
Conclusions
- Intramuscular transplantation is a viable, accessible site for hPSC-derived islet engraftment in mice.
- The procedure supports functional evaluation of stem cell-derived islets through in vivo GSIS.
- Graft retrieval enables comprehensive histological and molecular analysis post-transplantation.
- This protocol provides a reproducible preclinical model for advancing diabetes cell therapy research.
Frequently Asked Questions
Executive Industry Relevance
Intramuscular transplantation of human pluripotent stem cell-derived pancreatic endocrine cells provides a minimally invasive, reproducible preclinical model for diabetes cell therapy development. This approach enables functional assessment of stem cell-derived islets via in vivo glucose-stimulated insulin secretion and supports reliable graft retrieval for downstream analysis, addressing key limitations of traditional transplantation sites. The method enhances predictive confidence in preclinical evaluation by offering physiological context for islet maturation and function while improving surgical accessibility and reducing animal invasiveness.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by assessing functional maturation of stem cell-derived islets in a physiological context.
- Operational Value: Supports biological de-risking through reliable engraftment and glucose-responsive insulin secretion in immunodeficient mice.
- Strategic Value: Facilitates predictive confidence in target validation by providing reproducible functional readouts prior to lead optimization.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream compound screening by establishing functional islet engraftment.
- Operational Value: Standardizes transplantation procedures to ensure reproducibility and scalability across preclinical studies.
- Strategic Value: Enhances assay readiness by enabling quantitative functional outputs via in vivo GSIS for compound effect evaluation.
Translational & Preclinical Research
- Scientific Value: Maintains disease relevance by modeling human islet function in vivo, supporting translational biomarker alignment.
- Operational Value: Ensures continuity from discovery through preclinical validation with consistent graft retrieval and histological analysis.
- Strategic Value: Supports risk-adjusted advancement decisions by providing functional and structural data on islet engraftment and maturation.
Pipeline & Workflow Integration
This method integrates into the discovery continuum by supporting hypothesis testing in early discovery, enabling assay standardization for screening, and providing functional readouts that inform lead identification and preclinical advancement decisions.
- Discovery Biology: Supports hypothesis testing and pathway clarification by evaluating islet function in a physiological transplant site.
- Screening: Enhances assay readiness through standardized transplantation and reproducible glucose-stimulated insulin secretion measurements.
- Analytics: Delivers quantitative dependent variable measurements via in vivo GSIS to compare graft function across conditions.
- Translational Research: Connects to preclinical continuity by enabling histological and molecular analysis of retrieved grafts for maturation assessment.
- Enterprise Reuse: Establishes a reusable preclinical platform for iterative design-test cycles in stem cell-based diabetes therapy development.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by reducing mechanistic ambiguity in stem cell-derived islet function and maturation.
- Operational Value: Improves standardization, reproducibility, and scalability of transplantation procedures across research sites.
- Strategic Value: Enhances go/no-go decisions by providing reliable functional data, reducing late-stage biological risk in diabetes programs.
- Portfolio Impact: Informs risk-adjusted prioritization through reproducible engraftment and functional validation of stem cell-derived islets.
Implementation Considerations
- Requires expertise in stem cell differentiation, microsurgical transplantation techniques, and in vivo metabolic testing.
- Dependent on sterile surgical infrastructure, glucose challenge systems, and insulin detection assays for functional readouts.
- Necessitates cross-team standardization between cell processing, surgical, and analytical teams to ensure consistent outcomes.
- Involves adaptation considerations for different immunodeficient mouse strains and islet maturation timelines.
- Limited by the need for postoperative monitoring and graft retrieval procedures, which may affect throughput in large-scale studies.
