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 a....

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 ....

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