Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Summary

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Abstract

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Introduction

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. To date, graft materials used in surgery include biocompatible synthetic polymers (polytetrafluoroethylene [Teflon], expanded polytetrafluoroethylene [ePTFE; Gore-Tex] or polyethylene terephthalate [Dacron]), allografts, autologous tissue (pericardium or saphenous vein) and xenografts¹. Whilst artificial grafts (e.g., Gore-Tex and Dacron) are most commonly used, these materials likely cause numerous short- and long-term complications that include stenosis, calcium deposition, thrombo-embolization....

Protocol

Perform all animal experiments according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals.

1. Preparation of Culture Media

1. Make culture media for human fibroblast cell line CCL-153: F-12K Medium, 10% fetal bovine serum (FBS) and 100 U/ml penicillin and streptomycin.
2. Make Reprogramming Media for PiPSC generation: Knockout Dulbecco’s modified eagle’s medium (DMEM) containing 20% Knockout Serum Replacement, 0.1 mM β-mercaptoethanol, 0.1 mM Minimum Essential Medium (MEM) Non-Essential Amino Acids, 10 ng/ml basic Fibroblast Growth Factor 2 (bFGF-2) and 100 U/ml penicillin and streptomy....

Results

The successful generation of PiPSC was confirmed 4 days after nucleofecting human fibroblasts with a linearized pCAG2LMKOSimO plasmid carrying 4 transcription factors, OCT4, SOX2, KLF4 and c-MYC (OSKM). PiPSC displayed a markedly distinct phenotype when compared to fibroblasts (Figure 2A) and expressed the 4 reprogramming factors at mRNA (Figure 2B) and protein (Figure 2C) levels¹⁰. The efficacy of a PiPSC-based vascular graft is highly dependent on the abilit.......

Discussion

The current protocol indicates a sound, fast, simple, efficient and reproducible strategy in which functional tissue engineered vessels can be generated using PiPSC from human fibroblasts. This technique represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell therapy in the near future. Critical steps to ensure the efficacy of the protocol include the preparation of PiPSC, preparation of sterile and fully decellularized aortic grafts, successful seeding and different.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Human Fibroblasts CCL-153	ATCC	CCL-153	Prenatal human embryonic fibroblasts
ATCC F-12K Medium (Kaighn's Modification of Ham's F-12 Medium)	ATCC	30-2004
Fetal Bovine Serum	ATCC	30-2020
Knockout DMEM medium optimized for embryonic stem cells	Life technologies (Gibco)	12660-012
Knockout Serum Replacement	Life technologies (Invitrogen)	10828-028
Human Basic FGF-2	Miltenyi Biotech	130-093-837
alpha-MEM medium	Life technologies (Invitrogen)	32571093
Human PDGF	R&D System	120-HD-001
Gelatin Solution 2%	Sigma	G1393
Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC)	Addgene	20866
PvuI Restriction Enzyme	New England Biolabs	RO150S
SureClean Plus	Bioline	BIO-37047
Nucelofection Kit (NHDF Kit)	LONZA	VPD-1001
Neomycin	SIGMA	G418
KL 1500 LCD, Illumination for Stereo Microscopy	SCHOTT	KL 1500 LCD	Cold light illumination for stereo microscopy
Nikon Zoom Steromicroscope SMZ800	Nikon	SMZ800
Heparin sodium salt	Sigma	H3393
10% SDS Stock Solution Molecular Biology Reagent	Severn Biotech	CAS 151-21-3
Dulbecco's Phosphate Buffered Saline	Sigma	D8537
Matrigel (10mg/ml)	BD	A6661
Shaker IKA Vibrax with Shaking platform VX 7	Jepson Bolton's, Janke&Kunkel	S32-102
Masterflex L/S Digital Pump Drive	Cole-Parmer	WZ-07523-80
Masterflex L/S 6-channel, 6-roller cartridge pump head	Cole-Parmer	EW-07519-15
Masterflex L/S large cartridges for pump head	Cole-Parmer	EW-07519-75
Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft	Cole-Parmer	WZ-96410-14	Tubing goes through the peristaltic pump
0.5mm ID, 0.8 mm OD Silicone Tubing	SILEX	N/A	Tubings connect incubation chamber, media reservoir and compliance chamber
Fitting Reducer 0.5 to 1.6, natural Polypropyline	Ibidi	10829	Adapter connect above two types of tubings
1/32" Tubing, ID 0.01" (250µm) Material: PEEK	LabSmith	T-132-010P	Tubing through the incubation chamber wall which connects the graft with outside tubing
One-Piece Fittings	LabSmith	T-132-100	Fix the above tubings through the incubation chamber wall
Nylon tubes (OD 0.9mm, ID 0.75mm)	Smiths Medical	N/A	Tubings insert into two ends of the aorta graft
NOD.CB17-Prkdcscid/NcrCrl mouse	Charles River
Surgical sutures, 8-0  silk	ETHICON	W819
Hypnorm	Vetapharm	Vm21757/4000	Neuroleptanalgesic for use in mice
Hypnovel (Midazolam)	Roche	59467-70-8	Induction of anaesthesia
Dissecting microscope	Carl Zeiss	Stemi 2000
Nylon Tubing	Portex LTD	800/200/100/200	0.65 mm in diameter and 1 mm in length; to make artery cuff
Electrocoagulator	Martin	SN 54.131	Ligation of artery branches on aorta
Bipolar micro hemostat forceps	Martin	80-91-12-04	Fixation of vessel ends
Vessel Dilator	S&T	JFX-7
Vessel Dilator	S&T	JFL-3dZ
Vessel Dilator	S&T	D-5aZ
Mini applier	AESCULAP	FE572K
Micro hemostats clips	AESCULAP	FE720K
Surgical sutures, 6-0 VICRYL	ETHICON	V489