Intramuscular Transplantation of Human Pluripotent Stem Cell-derived Pancreatic Endocrine Cells in Mice

Summary

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.

Abstract

Human pluripotent stem cell (hPSC)-derived pancreatic endocrine cells constitute a promising source for diabetes cell therapy and disease modeling. Although several differentiation protocols have been established, in vivo implantation is commonly required to study maturation and function within a physiological context. Most transplantation approaches rely on sites such as the kidney capsule or subcutaneous space, which may pose technical or physiological limitations. Here, we present a detailed protocol for the intramuscular transplantation of hPSC-derived pancreatic SC-islets into immunodeficient mice. This site allows for straightforward surgical access, reduced invasiveness, and reliable graft retrieval for subsequent analysis. The procedure enables intramuscular implantation of stem cell-derived islets and supports their recovery for downstream assessment of insulin secretion following an oral glucose challenge. The protocol includes steps for cluster preparation and compaction, transplantation surgery, in vivo glucose-stimulated insulin secretion (GSIS), and graft retrieval for histological analysis, providing a reproducible framework for intramuscular transplantation in preclinical mouse models.

Introduction

Diabetes is a heterogeneous metabolic disorder whose prevalence has doubled over the past three decades and is expected to affect 1.3 billion people by 2050¹. While environmental factors, such as diet and lifestyle, play a significant role in the onset of type 2 diabetes (T2D), genetic predisposition also contributes substantially to its pathogenesis². Pancreatic β cells are responsible for insulin synthesis and secretion and play a central role in diabetes pathophysiology³. To understand how genetic risk contributes to β-cell dysfunction, human stem cell-derived β cells organized as three-dimensional endocrine clusters (hereafter referred to as SC-islets) provide a highly informative and relevant model system^4,5. Recent protocols effectively guide the differentiation of human stem cells into insulin-producing β cells by recapitulating key stages of pancreatic development using specific inhibitors and growth factors^6,7.

These systems reliably replicate developmental milestones in a scalable, reproducible manner, overcoming the limitations of donor-derived islets, which do not support developmental modeling and are subject to supply constraints. Murine models have provided valuable insights into pancreatic development, but they often fail to fully reproduce the phenotypic manifestations observed in human patients⁵. Consequently, the use of a stem cell-based system enables the characterization of genetic variants involved in human pancreatic development and β cell dysfunction⁸.

Despite these advances, SC-islets generated in vitro remain transcriptomically and functionally immature compared to native adult β cells. They exhibit suboptimal glucose-stimulated insulin secretion and lack expression of mature β-cell markers such as MAFA and G6PC2^9,10. To bridge this gap, in vivo maturation via transplantation into immunodeficient mice is often employed; this typically improves both insulin secretion dynamics and gene-expression profiles⁶.

Glucose metabolism is tightly regulated and requires the integration of systemic cues. Transplanted SC-islets are able to integrate signals from multiple organs, providing a more physiologically relevant environment crucial for assessing the functional consequences of genetic variation in human β cells¹¹.

SC-islet transplantation in mice has been performed at different anatomical sites. The conventional site is the kidney capsule, effective for promoting β-cell maturation and reversing diabetes in streptozotocin (STZ)-treated rodents¹², but it involves a complex invasive surgery, which limits its implementation across laboratories¹³. Moreover, this method is not translatable to clinical settings to treat patients with type 1 diabetes due to size and vascularization limitations¹⁴. Alternative transplantation sites, such as intramuscular, subcutaneous, and adipose tissue, have been reported, offering less invasive and more clinically applicable routes. Although intramuscular transplantation may present challenges for achieving optimal β-cell maturation and insulin secretion compared with the kidney capsule¹², it can nevertheless achieve effective insulin secretion and maintenance of glucose homeostasis, while offering a simpler and more accessible surgical approach^15,16.

Therefore, in this protocol, we present an intramuscular transplantation approach that addresses several limitations of other transplantation protocols. This method is minimally invasive and provides a reliable in vivo strategy for maturing SC-islets and investigating genetic variants linked to β-cell dysfunction.

Protocol

All experiments involving pluripotent stem cells (hPSCs) and laboratory animals were approved by institutional, regional, and national ethics committees. The hPSC lines used here were originally derived and distributed in accordance with the appropriate ethical approvals and informed consent procedures at the source institutions. Figure 1 shows the overall workflow, from hPSC maintenance through directed pancreatic lineage differentiation and maturation into SC-islets suitable for transplantation.

1.Differentiation of hPSCs to pancreatic progenitors

NOTE: This protocol builds upon two essential preparatory steps: 1) the maintenance and expansion of hPSCs, and 2) their directed differentiation toward the endocrine pancreas lineage. Both steps have been described in detail^7,17. hPSCs are maintained in coated plates with B8 media following the recommendations of Lyria-Leyte et al.¹⁷. Harvest hPSCs for differentiation during exponential growth before they become confluent. At the recommended seeding density, they should reach 90-100% confluence the next day, ready to start differentiation. The differentiation toward the endocrine pancreas lineage is performed according to the procedure described by Barsby et al.⁷. Full details of the media can be found in Table 1.

1. Freshly coat the differentiation plates with a basement membrane-derived extracellular matrix.
2. Seed 0.16-0.19 million cells/cm² on Day 0 (1.7 million cells in a well from a 6-well plate). Use 1.5-2x the volume of B8 + CEPT to prevent nutrient depletion (3-4 mL per well in a 6-well plate).
   NOTE: When passaging hPSCs or seeding differentiation experiments, we preferentially supplement B8 media with CEPT, a cocktail of chroman 1, emricasan, polyamines, and trans-ISRIB, rather than the conventional ROCK inhibitor Y-27632, due to its proven ability to enhance hPSC survival¹⁸. Nonetheless, Y27632 remains a valid alternative for this protocol.
3. Perform media changes using differentiation media S1 through S4 according to the following schedule: S1 (Days 1-4), S2 (Days 4-7), S3 (Days 7-9), and S4 (Days 9-11).
   NOTE: Refer to Table 1 for full media compositions; additional procedural details are described by Barsby et al.⁷.

2. Generation of 3D clusters and differentiation to insulin-producing cells

NOTE: On day 11, differentiated cells should have developed into pancreatic progenitors. To further promote endocrine differentiation at this stage, the cells transition from planar culture to 3D culture.

1. Add 0.5 mL of 5% (w/v) anti-adherence solution (see Table 1 for details) to cover the bottom of a 24-well microwell plate containing inverted pyramidal microwells (400 µm diameter). Centrifuge at 1,300 g for 10 min at room temperature (RT).
   NOTE: The anti-adherence solution creates an anti-adherent surface that prevents cells from sticking to the plastic and promotes uniform aggregation.
2. Examine the microwells under a microscope for air bubbles and re-centrifuge as needed to remove any trapped air (Figure 2A). Aspirate the anti-adherence solution and wash 2x with 0.5 mL of PBS/well. Leave the wells covered in PBS until ready for use.
3. Wash the cells with 1 mL of PBS-0.5 mM EDTA, aspirate the PBS-EDTA, and cover the wells with trypsin (1 mL for a well of a 6-well plate). Incubate at 37 ºC for 8-12 min (until tapping causes noticeable detachment).
4. Do not remove the trypsin; instead, dilute with 2x volumes of DMEM-F12 or PBS, and gently resuspend by pipetting. Place the suspension in a conical tube and immediately proceed to the next step.
   NOTE: It is better to incubate for a longer time in trypsin than to perform a harsh mechanical trituration.
5. Centrifuge the suspension at 200 g for 3 min at RT. Aspirate the supernatant carefully and resuspend the pellet gently in prewarmed S4 medium (1.25 mL for each well of a 24-well microwell).
6. Aspirate the PBS from microwell dishes. Gently, add 1.25 mL of the suspended cells to each well of the microwell plate.
   NOTE: Each well of the 24-microwell plate contains 1,200 microwells. The seeding density required to obtain appropriate-sized clusters is 1,500 cells per microwell, corresponding to 1.8 million cells per well. Alternatively, 6-well microwells can be used, adjusting volumes and number of cells (approximately 8.85 million cells in 5 mL of Stage 4 medium).
7. Centrifuge the plate at 100 g for 3 min at RT to distribute the cells evenly in the microwells. Carefully transfer the microwell plates to a humidified incubator.
8. Perform media changes using differentiation media S5 (D14-D18), S6 (D18-D25), and S7 (D25-D46).
   NOTE: Full composition details are provided in Table 1, with additional procedural details described by Barsby et al.⁷.
   CEPT cocktail can be used during this step instead of the ROCK inhibitor Y-27632, due to its proven ability to enhance cell survival during organoid formation¹⁸. Successful aggregation is indicated by the formation of compact, spherical clusters within 24-48 h (see Figure 2B).

3.Transplantation of SC-islets into the tight muscle of immunodeficient mice

NOTE: SC-islets have been transplanted at various stages of differentiation (from stage 4 to stage 7). While transplantation of differentiated S7 SC-islets represents the optimal strategy for assessing glucose responsiveness⁶, the transplantation of earlier-stage progenitors (S4-S5) or immature SC-islets (S6) may help reveal differentiation or maturation differences that are masked in vitro by the use of defined culture conditions. However, transplanting less differentiated progenitors carries a heightened risk of teratoma formation, primarily due to residual undifferentiated cell populations.

1. Compaction of clusters
   1. Transfer the appropriate quantity of clusters to a 1.5 mL microcentrifuge tube (see note).
      ​NOTE: Cell dose depends on the experimental goal. For histology, 0.5-1 million cells per mouse are sufficient for reliable graft retrieval, while functional assays or diabetes reversal in STZ-treated mice typically require higher doses, up to 2-5 million cells. Prior to compaction, SC-islets should exhibit a uniform spherical morphology with a target diameter of ~150-250 µm (acceptable range: 100-300 µm). This size range corresponds approximately to ~10³-4 × 10³ cells per cluster, oversized aggregates (>300-400 µm) should be avoided, as they are more susceptible to central necrosis. Cluster size and uniformity are verified before compaction by brightfield or stereomicroscope imaging of representative wells, followed by diameter measurement using standard image analysis software.
   2. Connect a 23 G syringe to the needle-tubing assembly and fill it with S7 medium.
   3. Aspirate the clusters into the tubing directly from the microcentrifuge tube. Fold the tubing in half and insert the folded section into a 200 µL pipette tip. Insert the tip-tubing-needle assembly in a 15 mL conical tube and secure the ends to the tube's rim using tape.
   4. Centrifuge the tube at 100 g for 2 min at 4 °C to compact the clusters. Immediately place the tube on ice.
2. Transplantation of clusters into mice
   ​NOTE: All surgical procedures must be performed using appropriate personal protective equipment and in compliance with institutional biosafety and animal welfare regulations. Anesthetic agents and sharps should be handled and disposed of according to approved safety procedures.
   1. Induce anesthesia in NOD-scid-gamma mice using 3-5% isoflurane.
   2. Shave the medial aspect of the right leg below the knee and disinfect with 70% ethanol.
   3. 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.
   4. Position the mouse prone, leaning to the right side, and extend the right leg. Insert the needle 4-5 mm deep into the medial compartment of the right thigh, in the region between the adductor and gracilis muscles.
   5. Inject the clusters, maximum volume of 50 µL, while retracting the needle by ~3 mm. After completing the injection, gently rotate the syringe 180° during needle withdrawal to help release the deposited clusters and minimize reflux of transplanted material.
   6. Monitor the mouse until fully recovered from anesthesia.
      ​NOTE: For functional characterization (i.e., in vivo GSIS, section 4), SC-islets of a single genotype per mouse must be used; a single transplantation is sufficient. However, for histological or transcriptomic characterization of maturation post implantation, both legs can be used (e.g., one leg bearing SC-clusters derived from hPSCs containing a disease-associated genetic variant, and the other leg with the variant corrected). Correct intramuscular delivery cannot be directly confirmed in vivo immediately after injection. Successful implantation is instead verified retrospectively by macroscopic identification of the graft at the time of retrieval (Figure 3) and by histological analysis (Figure 4).

4. In vivo glucose-stimulated insulin secretion (GSIS)

NOTE In vivo GSIS via oral glucose challenge assesses functional insulin secretion in response to a physiological stimulus. All procedures must follow institutional animal care and use guidelines. Functional responses of transplanted β-cells improve with in vivo maturation: human C-peptide can be detected as early as 2 weeks post transplant, while robust glucose-responsive secretion is typically achieved by 4 weeks. These timelines can vary depending on the developmental stage of the SC-islets at transplantation

1. Fast mice for 4-6 h prior to glucose administration, ensuring access to water.
2. Prepare a 20% (w/v) D-(+)-glucose solution in water. Sterilize by passing through a 0.22 µm filter and warm to room temperature before use.
3. Weigh each mouse and calculate the required glucose dose at 3 g/kg body weight.
4. Load the appropriate volume of glucose solution into a 20 G curved gavage needle attached to a 1 mL syringe.
5. Gently restrain the mouse and perform oral gavage, ensuring smooth insertion of the gavage needle to avoid tracheal aspiration.
6. Collect blood samples via submandibular or tail vein puncture at baseline (0 min) and 15 and 30 min post gavage, using capillary tubes and 1.5 mL microcentrifuge tubes. Allow the blood to clot at room temperature for approximately 15 min, then store the tubes on ice.
7. Centrifuge the samples at 1,000 g for 15 min at 4 °C and transfer serum to new tubes. Store at −80 °C until analysis by ELISA.
   NOTE: Time points can be adjusted depending on the experimental model. It is critical to use an assay capable of distinguishing between human and mouse insulin or C-peptide to assess graft-derived insulin secretion in xenotransplant models.

5. Retrieval of grafted clusters

1. Euthanize the mouse following institutional animal ethics guidelines.
2. To minimize tissue contamination with fur, spray ethanol 70% (v/v) over the body.
3. Remove the skin to expose the lower limb muscles and locate the grafted clusters using a magnifying glass. Excise graft-containing tissue using scissors and forceps.
4. Rinse the tissue once with PBS and transfer it to 4% (w/v) paraformaldehyde (PFA) on ice. Fix the tissue in 4% PFA at 4 °C for 4 h with gentle shaking.
5. Wash the sample in PBS for 1 h with gentle shaking. Transfer the tissue to 30% (w/v) sucrose on PBS and incubate overnight at 4 °C.
   NOTE: Biological tissues, blood samples, and chemical waste (including fixatives and anesthetic residues) must be disposed of following institutional biosafety and chemical waste management guidelines.

6. Staining of explants

1. OCT embedding
   1. Embed the tissue in a mold filled with Optimal Cutting Temperature (OCT) compound.
   2. Place the mold in a dry ice-isopropanol bath to flash-freeze the sample. Store at -80 °C until cryosectioning.
   3. Using a cryostat, cut 5-7 µm sections and store the slides at -80 °C.
2. Immunostaining
   1. Equilibrate the slides at RT for 15 min inside a container to avoid condensation.
   2. Rinse the slides with PBS for 3 x 5 min.
   3. Incubate each section with 100 µL of blocking/permeabilization solution for 30 min at RT (0.1% nonionic surfactant in PBS, with 5% serum of the species where the secondary antibody was raised).
   4. Incubate each section with 50 µL of primary antibody diluted in blocking/permeabilization solution overnight at 4 °C.
   5. Rinse the slides with PBS for 3 x 5 min.
   6. Incubate each section with 50 µL of secondary antibody diluted in blocking/permeabilization solution 1 h at RT.
   7. Rinse the slides with PBS for 3 x 5 min.
   8. Mount the slides with 15 µL of mounting solution with DAPI and apply a coverslip. Store at 4 °C in the dark until imaging.

Results

The results shown correspond to intramuscular transplantation of Stage 7 SC-islets into immunodeficient mice and analysis performed 6 weeks post transplantation. A limited number of animals bearing representative grafts were analyzed to illustrate the feasibility of the procedure. In this study, a successful outcome is defined as the macroscopic retrieval of the intramuscular graft followed by histological confirmation of survival and endocrine identity of transplanted SC-islets.

Discussion

In this protocol, we describe intramuscular transplantation of SC-islets as a straightforward, reproducible, and minimally invasive approach for in vivo maturation, with the potential for functional assessment. Although transplantation under the kidney capsule is widely regarded as the "gold standard" for islet or SC-islet engraftment in rodents, it requires greater technical expertise and a longer surgical procedure. In contrast, the intramuscular site offers procedural simplicity and rapid execution, a...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Phospho-L-ascorbic acid trisodium salt	Sigma Aldrich	49752
Activin A	Qkine	Qk001
AggreWell 400 24-well (microwell plate)	Stemcell	34415
Betacellulin	GenScript	Z03102
Bovine Serum Albumin, Fraction V, Fatty Acid Free	Goldbio	A-421
CHIR 99021	LC labs	C-6556
Chroman-1	Medchem Express	HY-15392	CEPT cocktail
CMRL-1066	Pan Biotech	P04-84600
D-(+)-Glucose	Sigma Aldrich	G7021
Dibenzazepine (DBZ)	Syncom	Custom product	Gamma-secretase inhibitor
Dulbecco's Modified Eagle Medium and Ham’s F12 media (DMEM/F12)	Corning	15383541
EGF	Qkine	Qk011
Emricasan	Medchem Express	HY-10396	CEPT cocktail
Ethanolamine	Sigma Aldrich	E9508
FGF2-G3	GenScript	Custom product
FGF7	GenScript	Z03047
Heparin	Sigma Aldrich	H3149
Insulin	Gibco	30284510
L-Alanyl-L-Glutamine	Linus	X0551-100
L-Ascorbic Acid	Santa Cruz	sc-202686
LDN-193189	Sigma Aldrich	SML0559
Lipid Concentrate	Gibco	11548846
Matrigel (basement membrane-derived extracellular matrix)	Falcon	354230
MCDB 131	Corning	15-100-CV
N-acetylcysteine	Thermo Scientific	10521221
NaHCO3	Fisher Scientific	10553325
Nicotinamide	Sigma Aldrich	N0636
NRG-1	Qkine	Qk045
Pluronic F-127 (anti-adherence solution)	Sigma Aldrich	82184	Used at 5% (w/v)
Polyamine Supplement (1000×)	Sigma Aldrich	P8483	CEPT cocktail
RepSox	MedChemExpress	HY-13012	Alk5 Inhibitor
Retinoic Acid	Sigma Aldrich	R2625
SANT-1	Selleckchem	S7092
Sobetirome (GC1)	Sigma Aldrich	SML1900-5MG	Cell-permeable T3 analog
Sodium Pyruvate	Corning	25-000-CI
Sodium Selenite	Sigma Aldrich	S5261
T3 (3,3′,5-Triiodo-L-thyronine sodium salt)	Sigma Aldrich	T6397
TGF-β1	Qkine	Qk010
TPB	Tocris	5343
Trace Elements A	Corning	15333641
Trace Elements B	Corning	15343641
Trans-ISRIB	Medchem Express	HY-12495	CEPT cocktail
Transferrin	InVitria	777TRF029
Triton X-100 (nonionic surfactant)	Thermo Scientific	11488696
TrypLE	Gibco	11538856
Y-27632	LC Laboratories	Y-5301	ROCK inhibitor
ZM-447439	MedChemExpress	HY-10128
ZnSO4	Sigma Aldrich	Z0251
Antibodies
Rat anti-Human C-peptide (insulin)	DSHB	GN-ID4-s	Used at 150 ng/mL
Mouse anti-glucagon	Proteintech	67286-1-Ig	1:2000
Rabbit anti-somatostatin	Millipore	AB5494	1:500
Goat anti-rat AF488	Invitrogen	A-11006	1:500
Donkey anti-mouse AF488	Invitrogen	A-21202	1:500
Donkey anti-rabbit AF488	Invitrogen	A-21206	1:500