Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Summary

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Abstract

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Introduction

Mesoangioblasts (MABs) are vessel-associated muscle progenitors derived from a subset of pericytes^1-3. The main advantage of MABs over canonical muscle progenitors such as satellite cells⁴ resides in their ability to cross the vessel wall when delivered intra-arterial and hence contribute to skeletal muscle regeneration in cell therapy protocols. This feature has been evaluated and confirmed in both murine and canine models of muscular dystrophy^1,5,6. These preclinical studies have built the foundations for a first-in-man phase I/II clinical trial based upon intra-arterial transplantation of donor HLA-identical MABs in children with Du....

Protocol

1. Assessment of Myogenic and Engraftment Potential

1. In vitro: MyoD-induced differentiation
   1. Generate a stable cell line of IDEMs transduced with the tamoxifen-inducible MyoD-ER lentiviral vector, titrating the multiplicity of infection (MOI; e.g. 1, 5 and 50) using the staining described in 1.1.9 (MyHC) as an outcome of the efficiency of the procedure.
   2. Coat a 3.5 cm dish with 1 ml of 1% Matrigel and incubate for 30 min at 37 °C.
   3. Wash the plate twice with medium, seed 1 x 10⁵ MyoD-ER transduced IDEMs in the 3.5 cm tissue culture dish and incubate at 37 °C in growth medium.
   <....

Results

The reported representative results follow the main in vitro/in vivo assays depicted in the workflow in Figure 1. 48 hr after 4OH-tamoxifen administration MyoD-positive nuclei are identifiable within MyoD-ER transduced IDEMs in culture (Figure 2A). The cells then fuse and differentiate into multinucleated myotubes (Figure 2B). When transplanted intramuscularly into a murine model of acute muscle injury, IDEMs contribute to tissue regeneration (Figure 3

Discussion

iPSCs can be expanded indefinitely while preserving their self-renewal potential and thus be directed to differentiate into a broad range of cell lineages¹². For this and other reasons iPSC-derived stem/progenitor cells are considered a promising source for autologous gene and cell therapy approaches¹³. A previously published work from the Authors reported the generation of mouse and human myogenic progenitors from iPSCs with a phenotype similar to that of pericyte-derived MABs (IDEMs) and their eff.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
MegaCell DMEM	Sigma	M3942
DMEM	Sigma	D5671
IMDM	Sigma	I3390
Horse serum	Euroclone	ECS0090L
Foetal Bovine Serum	Lonza	DE14801F
PBS Calcium/Magnesium free	Lonza	BE17-516F
L-Glutammine	Sigma	G7513
Penicilline/Streptomicin	Sigma	P0781
2-Mercaptoethanol	Gibco	31350-010
ITS (Insulin-Transferrin-Selenium)	Gibco	51500-056
Nonessential amino acid solution	Sigma	M7145
Fer-In-Sol	Mead Johnson
Ferlixit	Aventis
Oleic Acid	Sigma	01257-10 mg
Linoleic Acid	Sigma	L5900-10 mg
Human bFGF	Gibco	AA 10-155
Grow factors-reduced Matrigel	Becton Dickinson	356230
Trypsin	Sigma	T3924
Sodium heparin	Mayne Pharma
Trypan blue solution	Sigma	T8154	HARMFUL
Patent blue dye	Sigma	19, 821-8
EDTA	Sigma	E-4884
Paraformaldehyde	TAAB	P001	HARMFUL
Tamoxifen	Sigma	T5648
4-OH Tamoxifen	Sigma	H7904
pLv-CMV-MyoD-ER(T)	Addgene	26809
Cardiotoxin	Sigma	C9759	HARMFUL
Povidone iodine
Tragachant gum	MP Biomedicals	104792
Isopenthane	VWR	24,872,323
Tissue-tek OCT	Sakura	4583
Sucrose	VWR	27,480,294
Polarized glass slides	Thermo	J1800AMNZ
Eosin Y	Sigma	E4382
Hematoxylin	Sigma	HHS32
Masson's trichrome	Bio-Optica	04-010802
Mouse anti Myosin Heavy Chain antibody	DSHB	MF20
Mouse anti Lamin A/C antibody	Novocastra	NLC-LAM-A/C
Hoechst 33342	Sigma Fluka	B2261
Rabbit anti Laminin antibody	Sigma	L9393
MATERIALS AND EQUIPMENT
Adsorbable antibacterial suture 4-0	Ethicon	vcp310h
30 G Needle syringe	BD	324826
Treadmill	Columbus instrument
Steromicroscope	Nikon	SMZ800
Inverted microscope	Leica	DMIL LED
Isoflurane unit	Harvad Apparatus
Fiber optics	Euromecs (Holland)	EK1
Heating pad	Vet Tech	C17A1
Scalpels	Swann-Morton	11REF050
Surgical forceps	Fine Scientific Tools		5/45
High temperature cauteriser	Bovie Medical	AA01
MEDIA COMPOSITION
Media composition is detailed below. HIDEMs growth medium: MegaCell Dulbecco's Modified Eagle Medium (MegaCell DMEM) 5% fetal bovine serum (FBS) 2 mM glutamine 0.1 mM β-mercaptoethanol 1% nonessential amino acids 5 ng/ml human basic fibroblast growth factor (bFGF) 100 IU ml penicillin 100 mg/ml streptomycin Alternatively, if some of the above reagents are not available, HIDEMs can be grown in: Iscove's Modified Dulbecco's Medium (IMDM) 10% FBS 2 mM glutamine 0.1 mM β-mercaptoethanol 1% Nonessential amino acids (NEAA) 1% ITS (insulin/transferrin/selenium) 5 ng/ml human bFGF 100 IU ml penicillin 100 mg / ml streptomycin 0.5 μM oleic and linoleic acids 1.5 μM Fe2+ (Fer-In-Sol) 0.12 μM Fe3+ (Ferlixit) MIDEMs growth medium: DMEM 20% FBS 2 mM glutamine 5 ng/ml human bFGF 100 IU ml penicillin 100 mg/ml streptomycin Differentiation medium: DMEM 2% Horse Serum (HS) 100 IU ml penicillin 100 mg/ml streptomycin 2 mM glutamine
Table 1. List of Reagents, Materials, Equipment, and Media.