Elsevier

Biomaterials

Volume 29, Issue 18, June 2008, Pages 2757-2766
Biomaterials

Three-dimensional extracellular matrix-directed cardioprogenitor differentiation: Systematic modulation of a synthetic cell-responsive PEG-hydrogel

https://doi.org/10.1016/j.biomaterials.2008.03.016Get rights and content

Abstract

We show that synthetic three-dimensional (3D) matrix metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG)-based hydrogels can direct differentiation of pluripotent cardioprogenitors, using P19 embryonal carcinoma (EC) cells as a model, along a cardiac lineage in vitro. In order to systematically probe 3D matrix effects on P19 EC differentiation, matrix elasticity, MMP-sensitivity and the concentration of a matrix-bound RGDSP peptide were modulated. Soft matrices (E = 322 ± 64.2 Pa, stoichiometric ratio: 0.8), mimicking the elasticity of embryonic cardiac tissue, increased the fraction of cells expressing the early cardiac transcription factor Nkx2.5 around 2-fold compared to embryoid bodies (EB) in suspension. In contrast, stiffer matrices (E = 4036 ± 419.6 Pa, stoichiometric ratio: 1.2) decreased the number of Nkx2.5-positive cells significantly. Further indicators of cardiac maturation were promoted by ligation of integrins relevant in early cardiac development (α5β1, αvβ3) by the RGDSP ligand in combination with the MMP-sensitivity of the matrix, with a 6-fold increased amount of myosin heavy chain (MHC)-positive cells as compared to EB in suspension. This precisely controlled 3D culture system thus may serve as a potential alternative to natural matrices for engineering cardiac tissue structures for cell culture and potentially therapeutic applications.

Introduction

Nearly eight million persons in the United States suffer from myocardial infarction, with an estimated 800,000 new incidences every year [1]. Current therapies are limited by the restricted intrinsic regeneration capacity of the myocardium and by the lack of organs for transplantation. In recent attempts to regenerate functional heart muscle, adult cells such as bone marrow-derived cells have been injected into the ventricular wall or coronary vessels in animal models with myocardial infarction, as well as in human patients in first clinical trials [2], [3]. Although improved cardiac performance has been reported, it remains unclear if paracrine signaling effects of the implanted cells, endogenous stem cell recruitment or even trans-differentiation are responsible for the improvement of cardiac function [4], [5]. Furthermore, efficacy of the cell engraftment is very low: less than 10% of the injected cells typically engraft, mainly due to cell death, and less than 2% appear to take on the in vivo fate of cardiomyocytes [6], [7]. To better retain the cells at the site of infarction and to better control growth and differentiation, cardiac grafts have been engineered in vitro employing biodegradable materials as cell carriers or as cell ingrowth matrices. Both naturally derived [8], [9], [10] and synthetic materials [11], [12] have been demonstrated to support embryonic and adult cell-derived cardiac tissue development containing contracting cardiomyocytes. However, mechanisms controlling 3D cardiac development are still poorly understood.

We have recently developed a 3D PEG-based synthetic hydrogel material mimicking key biochemical characteristics of natural collagenous matrices [13], the major constituent of cardiac extracellular matrices [14]. This artificial extracellular matrix system allows the nearly independent control of matrix elasticity, integrin-stimulating ligands and protease-sensitivity and is thus a potentially powerful tool to direct 3D cardiac development. We have previously reported that matrix metalloproteinase (MMP)-sensitive peptides crosslinking branched PEG chains enable cell-mediated proteolytic matrix degradation and remodeling [15]. These characteristics are also important in the heart, both under physiological and pathological conditions, where increased MMP-expression and activation has been observed [16], [17]. Furthermore, matrix-bound RGDSP peptide promotes cell adhesion and stimulation of integrins relevant in early cardiac development (α5β1, αVβ3) [18], [19], [20].

Here, we examined the ability of these cell-responsive PEG-based hydrogels for directing cardiac differentiation of pluripotent P19 embryonal carcinoma cells, a well-accepted model system for embryonic stem cell-based cardiac differentiation [21], [22]. We systematically modulated matrix elasticity, the concentration of the matrix-bound RGDSP oligopeptide and MMP-sensitivity to dissect biophysical and biochemical matrix parameters potentially involved in cardiac differentiation.

Section snippets

Synthesis of poly(ethylene glycol)–vinylsulfone and peptide precursors (RGDSP, MMP-substrate)

PEG-vinylsulfone was synthesized adapting a previous protocol [23]. Branched 4-arm PEG-OH (Mw = 20,000 g/mol) (Shearwater Polymers, Huntsville, AL) was dried by azeotropic distillation in toluene (VWR, Nyon, Switzerland) for 4 h using a Dean–Stark trap. Toluene was distilled off and the residue dissolved in dichloromethane (Fisher Scientific, Wohlen, Switzerland). To the clear solution, sodium hydride (Sigma–Aldrich, Buchs, Switzerland) was added at 20-fold molar excess over OH-groups. After

Physicochemical characteristics of PEG-based hydrogels

To modulate the biophysical matrix characteristics in a range possibly relevant for cardiac differentiation, the stoichiometric ratio between PEG and MMP-sensitive linkers was systematically altered (Fig. 1A). Cardiac cell differentiation was examined in a soft matrix (E = 322 ± 64.2 Pa) and in a stiffer one (E = 4036 ± 64.2 Pa) obtained at stoichiometric ratio of 0.8 and 1.2, respectively. The minimum value of the Young's modulus (E = 322 ± 64.2 Pa) corresponds to the maximum value of the swelling ratio (Q = 40

Discussion

Here, we explore the utility of a material system [13] to support and direct differentiation of a stem cell model along a targeted lineage, employing the pluripotent P19 EC model and targeting cardiomyocytes. Our data indicates the necessity to control several biophysical and biochemical features of the extracellular matrix to drive P19 EC cells, and presumably stem cells as cardioprogenitors, along the cardiac differentiation pathway. Compared to common matrices used in cardiac tissue

Conclusions

We present here a method for engineering 3D cardiac tissue clusters using a precisely controlled artificial extracellular matrix system that may serve as an alternative to natural biopolymer matrices such as collagen. We show that soft PEG-based matrices mimicking the elasticity of embryonic cardiac tissue can direct cardiac commitment of pluripotent P19 EC cells. However, further cardiac maturation was promoted by the MMP-sensitivity of the matrix, allowing cell-triggered matrix remodeling,

Acknowledgement

We thank Dr. Mayumi Mochizuki and Conlin P. O'Neil for help with peptide synthesis, Dr. Mathieu Hauwel for support with RT-PCR, Miriella Pasquier for assistance in tissue embedding and processing, Olivier Brun for supporting with confocal imaging and Sergei Startchik for helping with confocal image processing. We also thank Prof. Ilona Skerjanc, University of Western Ontario, Canada, and Dr. Marcel Van der Heyden, University Medical Center Utrecht, The Netherlands, for helpful discussions. This

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