Name: Intramuscular Transplantation of Human Pluripotent Stem Cell-derived Pancreatic Endocrine Cells in Mice (Video) | JoVE Journal
Uploaded: 2026-04-10T12:40:00.000+00:00
Duration: 6 min 57 s
Description: Learn intramuscular transplantation of hPSC-derived pancreatic islets in immunodeficient mice. This protocol enables functional assessment via glucose-stimulated insulin secretion and supports graft retrieval for histological analysis.

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5 minutes

We thank Dr. Danwei Huangfu (Memorial Sloan Kettering Cancer Center, New York, USA) for generously providing the hPSC lines used in this study. This work was supported by grants PID2021-122284NA-I00 and CNS2023-145179 from the Spanish Ministry of Science, Innovation and Universities MICIU/AEI/10.13039/501100011033 and Next GenerationEU/PRTR (A.B.), and by the CIBER-Consortium for Biomedical Research in Network, CB07/08/0029 and PID2023-150719OB-I00 (M.V.), Instituto de Salud Carlos III (ISCIII). The Center for Biomedical Research in Diabetes and Associated Metabolic Disorders (CIBERDEM) is an initiative of ISCIII and partially supported by the European Regional Development Fund (FEDER).

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 detail7,17. 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.
<ol>
	<li>Freshly coat the differentiation plates with a basement membrane-derived extracellular matrix.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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 &#181;m 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.</li>
	<li>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.</li>
	<li>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 &#186;C for 8-12 min (until tapping causes noticeable detachment).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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.</li>
	<li>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).</li>
</ol>
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.
<ol>
	<li>Compaction of clusters
	<ol>
		<li>Transfer the appropriate quantity of clusters to a 1.5 mL microcentrifuge tube (see note). 
		&#8203;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 &#181;m (acceptable range: 100-300 &#181;m). This size range corresponds approximately to ~10&#179;-4 &#215; 10&#179; cells per cluster, oversized aggregates (&#62;300-400 &#181;m) 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.</li>
		<li>Connect a 23 G syringe to the needle-tubing assembly and fill it with S7 medium.</li>
		<li>Aspirate the clusters into the tubing directly from the microcentrifuge tube. Fold the tubing in half and insert the folded section into a 200 &#181;L pipette tip. Insert the tip-tubing-needle assembly in a 15 mL conical tube and secure the ends to the tube&#39;s rim using tape.</li>
		<li>Centrifuge the tube at 100 g for 2 min at 4 &#176;C to compact the clusters. Immediately place the tube on ice.</li>
	</ol>
	</li>
	<li>Transplantation of clusters into mice 
	&#8203;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.
	<ol>
		<li>Induce anesthesia in NOD-scid-gamma mice using 3-5% isoflurane.</li>
		<li>Shave the medial aspect of the right leg below the knee and disinfect with 70% ethanol.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Inject the clusters, maximum volume of 50 &#181;L, while retracting the needle&#160;by ~3 mm. After completing the injection, gently rotate the syringe 180&#176; during needle withdrawal to help release the deposited clusters and minimize reflux of transplanted material.</li>
		<li>Monitor the mouse until fully recovered from anesthesia. 
		&#8203;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).</li>
	</ol>
	</li>
</ol>
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 &#946;-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
<ol>
	<li>Fast mice for 4-6 h prior to glucose administration, ensuring access to water.</li>
	<li>Prepare a 20% (w/v) D-(+)-glucose solution in water. Sterilize by passing through a 0.22 &#181;m filter and warm to room temperature before use.</li>
	<li>Weigh each mouse and calculate the required glucose dose at 3 g/kg body weight.</li>
	<li>Load the appropriate volume of glucose solution into a 20 G curved gavage needle attached to a 1 mL syringe.</li>
	<li>Gently restrain the mouse and perform oral gavage, ensuring smooth insertion of the gavage needle to avoid tracheal aspiration.</li>
	<li>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.</li>
	<li>Centrifuge the samples at 1,000 g for 15 min at 4 &#176;C and transfer serum to new tubes. Store at &#8722;80 &#176;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.</li>
</ol>
5. Retrieval of grafted clusters
<ol>
	<li>Euthanize the mouse following institutional animal ethics guidelines.</li>
	<li>To minimize tissue contamination with fur, spray ethanol 70% (v/v) over the body.</li>
	<li>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.</li>
	<li>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 &#176;C for 4 h with gentle shaking.</li>
	<li>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 &#176;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.</li>
</ol>
6. Staining of explants
<ol>
	<li>OCT embedding
	<ol>
		<li>Embed the tissue in a mold filled with Optimal Cutting Temperature (OCT) compound.</li>
		<li>Place the mold in a dry ice-isopropanol bath to flash-freeze the sample. Store at -80 &#176;C until cryosectioning.</li>
		<li>Using a cryostat, cut 5-7 &#181;m sections and store the slides at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>Equilibrate the slides at RT for 15 min inside a container to avoid condensation.</li>
		<li>Rinse the slides with PBS for 3 x 5 min.</li>
		<li>Incubate each section with 100 &#181;L 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).</li>
		<li>Incubate each section with 50 &#181;L of primary antibody diluted in blocking/permeabilization solution overnight at 4 &#176;C.</li>
		<li>Rinse the slides with PBS for 3 x 5 min.</li>
		<li>Incubate each section with 50 &#181;L of secondary antibody diluted in blocking/permeabilization solution 1 h at RT.</li>
		<li>Rinse the slides with PBS for 3 x 5 min.</li>
		<li>Mount the slides with 15 &#181;L of mounting solution with DAPI and apply a coverslip. Store at 4 &#176;C in the dark until imaging.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to declare.

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 &#34;gold standard&#34; 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...

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 &#946; cells are responsible for insulin synthesis and secretion and play a central role in diabetes pathophysiology3. To understand how genetic risk contributes to &#946;-cell dysfunction, human stem cell-derived &#946; cells organized as three-dimensional endocrine clusters (hereafter referred to as SC-islets) provide a highly informative and relevant model system4,5. Recent protocols effectively guide the differentiation of human stem cells into insulin-producing &#946; cells by recapitulating key stages of pancreatic development using specific inhibitors and growth factors6,7.
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 &#946; cell dysfunction8.
Despite these advances, SC-islets generated in vitro remain transcriptomically and functionally immature compared to native adult &#946; cells. They exhibit suboptimal glucose-stimulated insulin secretion and lack expression of mature &#946;-cell markers such as MAFA and G6PC29,10. 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 &#946; cells11.
SC-islet transplantation in mice has been performed at different anatomical sites. The conventional site is the kidney capsule, effective for promoting &#946;-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 &#946;-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 approach15,16.
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 &#946;-cell dysfunction.

<table><tbody><tr><td>2-Phospho-L-ascorbic acid trisodium salt</td><td>Sigma Aldrich</td><td>49752</td><td></td></tr><tr><td>Activin A</td><td>Qkine</td><td>Qk001</td><td></td></tr><tr><td>AggreWell 400 24-well (microwell plate)</td><td>Stemcell</td><td>34415</td><td></td></tr><tr><td>Betacellulin</td><td>GenScript</td><td>Z03102</td><td></td></tr><tr><td>Bovine Serum Albumin, Fraction V, Fatty Acid Free</td><td>Goldbio</td><td>A-421</td><td></td></tr><tr><td>CHIR 99021</td><td>LC labs</td><td>C-6556&amp;nbsp;</td><td></td></tr><tr><td>Chroman-1</td><td>Medchem Express</td><td>HY-15392</td><td>CEPT cocktail</td></tr><tr><td>CMRL-1066</td><td>Pan Biotech</td><td>P04-84600</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma Aldrich</td><td>G7021</td><td></td></tr><tr><td>Dibenzazepine (DBZ)</td><td>Syncom</td><td>Custom product</td><td>Gamma-secretase inhibitor</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium and Ham&amp;rsquo;s F12 media (DMEM/F12)</td><td>Corning</td><td>15383541</td><td></td></tr><tr><td>EGF</td><td>Qkine</td><td>Qk011</td><td></td></tr><tr><td>Emricasan</td><td>Medchem Express</td><td>HY-10396</td><td>CEPT cocktail</td></tr><tr><td>Ethanolamine</td><td>Sigma Aldrich</td><td>E9508</td><td></td></tr><tr><td>FGF2-G3</td><td>GenScript</td><td>Custom product</td><td></td></tr><tr><td>FGF7</td><td>GenScript</td><td>Z03047</td><td></td></tr><tr><td>Heparin</td><td>Sigma Aldrich</td><td>H3149</td><td></td></tr><tr><td>Insulin</td><td>Gibco</td><td>30284510</td><td></td></tr><tr><td>L-Alanyl-L-Glutamine</td><td>Linus</td><td>X0551-100</td><td></td></tr><tr><td>L-Ascorbic Acid</td><td>Santa Cruz</td><td>sc-202686</td><td></td></tr><tr><td>LDN-193189&amp;nbsp;</td><td>Sigma Aldrich</td><td>SML0559</td><td></td></tr><tr><td>Lipid Concentrate</td><td>Gibco</td><td>11548846</td><td></td></tr><tr><td>Matrigel (basement membrane-derived extracellular matrix)</td><td>Falcon</td><td>354230</td><td></td></tr><tr><td>MCDB 131</td><td>Corning</td><td>15-100-CV</td><td></td></tr><tr><td>N-acetylcysteine</td><td>Thermo Scientific</td><td>10521221</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>10553325</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma Aldrich</td><td>N0636</td><td></td></tr><tr><td>NRG-1</td><td>Qkine</td><td>Qk045</td><td></td></tr><tr><td>Pluronic F-127 (anti-adherence solution)</td><td>Sigma Aldrich</td><td>82184</td><td>Used at 5% (w/v)</td></tr><tr><td>Polyamine Supplement (1000&amp;times;)</td><td>Sigma Aldrich</td><td>P8483</td><td>CEPT cocktail</td></tr><tr><td>RepSox</td><td>MedChemExpress</td><td>HY-13012</td><td>Alk5 Inhibitor</td></tr><tr><td>Retinoic Acid</td><td>Sigma Aldrich</td><td>R2625</td><td></td></tr><tr><td>SANT-1</td><td>Selleckchem</td><td>S7092</td><td></td></tr><tr><td>Sobetirome (GC1)</td><td>Sigma Aldrich</td><td>SML1900-5MG</td><td>Cell-permeable T3 analog</td></tr><tr><td>Sodium Pyruvate</td><td>Corning</td><td>25-000-CI</td><td></td></tr><tr><td>Sodium Selenite</td><td>Sigma Aldrich</td><td>S5261</td><td></td></tr><tr><td>T3 (3,3&amp;prime;,5-Triiodo-L-thyronine sodium salt)</td><td>Sigma Aldrich</td><td>T6397</td><td></td></tr><tr><td>TGF-&amp;beta;1&amp;nbsp;</td><td>Qkine</td><td>Qk010</td><td></td></tr><tr><td>TPB</td><td>Tocris</td><td>5343</td><td></td></tr><tr><td>Trace Elements A</td><td>Corning</td><td>15333641</td><td></td></tr><tr><td>Trace Elements B</td><td>Corning</td><td>15343641</td><td></td></tr><tr><td>Trans-ISRIB</td><td>Medchem Express</td><td>HY-12495</td><td>CEPT cocktail</td></tr><tr><td>Transferrin</td><td>InVitria</td><td>777TRF029</td><td></td></tr><tr><td>Triton X-100 (nonionic surfactant)</td><td>Thermo Scientific</td><td>11488696</td><td></td></tr><tr><td>TrypLE</td><td>Gibco</td><td>11538856</td><td></td></tr><tr><td>Y-27632</td><td>LC Laboratories</td><td>Y-5301</td><td>ROCK inhibitor</td></tr><tr><td>ZM-447439&amp;nbsp;</td><td>MedChemExpress</td><td>HY-10128</td><td></td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>Z0251</td><td></td></tr><tr><td>Antibodies</td><td></td><td></td><td></td></tr><tr><td>Rat anti-Human C-peptide (insulin)</td><td>DSHB</td><td>GN-ID4-s</td><td>Used at 150 ng/mL</td></tr><tr><td>Mouse anti-glucagon</td><td>Proteintech</td><td>67286-1-Ig</td><td>1:2000</td></tr><tr><td>Rabbit anti-somatostatin</td><td>Millipore</td><td>AB5494</td><td>1:500</td></tr><tr><td>Goat anti-rat AF488</td><td>Invitrogen</td><td>A-11006</td><td>1:500</td></tr><tr><td>Donkey anti-mouse AF488</td><td>Invitrogen</td><td>A-21202</td><td>1:500</td></tr><tr><td>Donkey anti-rabbit AF488</td><td>Invitrogen</td><td>A-21206</td><td>1:500</td></tr></tbody></table>

intramuscular transplantation of human pluripotent stem cell-derived pancreatic endocrine cells in mice

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

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.

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. <strong ...

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.

Human pluripotent stem cell (hPSC)-derived pancreatic endocrine cells constitute a promising source for diabetes cell therapy and disease modeling. ...

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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Research

JoVE Journal

Developmental Biology

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice.  Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

This video describes the protocol for grafting human pancreatic islets into the anterior chamber of the mouse’s eye to monitor revascularization of human islet grafts in vivo. It also helps to understand the contribution of recipient versus donor cells in promoting the encapsulation and vascularization of the graft.

Grafting Pancreatic Islets: A Method to Implant Pancreatic Islets into the Anterior Chamber of the Mouse Eye for In Vivo Imaging

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

A Method for Islet Transplantation to the Omentum in Mouse

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal Transplantation of Pancreatic Islets in Mouse Model

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

This article presents a protocol for directed differentiation and functional analysis of β-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional β-cell like cells secreting insulin in response to glucose.

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Learn intramuscular transplantation of hPSC-derived pancreatic islets in immunodeficient mice. This protocol enables functional assessment via glucose-stimulated insulin secretion and supports graft retrieval for histological analysis.

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

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