Embryonic stem cell-based cardiopatches improve cardiac function in infarcted rats.

Pluripotent stem cell-seeded cardiopatches hold promise for in situ regeneration of infarcted hearts. Here, we describe a novel cardiopatch based on bone morphogenetic protein 2-primed cardiac-committed mouse embryonic stem cells, embedded into biodegradable fibrin matrices and engrafted onto infarcted rat hearts. For in vivo tracking of the engrafted cardiac-committed cells, superparamagnetic iron oxide nanoparticles were magnetofected into the cells, thus enabling detection and functional evaluation by high-resolution magnetic resonance imaging. Six weeks after transplantation into infarcted rat hearts, both local (p < .04) and global (p < .015) heart function, as well as the left ventricular dilation (p < .0011), were significantly improved (p < .001) as compared with hearts receiving cardiopatches loaded with iron nanoparticles alone. Histological analysis revealed that the fibrin scaffolds had degraded over time and clusters of myocyte enhancer factor 2-positive cardiac-committed cells had colonized most of the infarcted myocardium, including the fibrotic area. De novo CD31-positive blood vessels were formed in the vicinity of the transplanted cardiopatch. Altogether, our data provide evidence that stem cell-based cardiopatches represent a promising therapeutic strategy to achieve efficient cell implantation and improved global and regional cardiac function after myocardial infarction.

Three-dimensional biomaterials for the study of human pluripotent stem cells.

The self-renewal and differentiation of human pluripotent stem cells (hPSCs) have typically been studied in flat, two-dimensional (2D) environments. In this Perspective, we argue that 3D model systems may be needed in addition, as they mimic the natural 3D tissue organization more closely. We survey methods that have used 3D biomaterials for expansion of undifferentiated hPSCs, directed differentiation of hPSCs and transplantation of differentiated hPSCs in vivo.

Human embryonic stem cell-derived microvascular grafts for cardiac tissue preservation after myocardial infarction.

We present use of a synthetic, injectable matrix metalloproteinase (MMP)-responsive, bioactive hydrogel as an in situ forming scaffold to deliver thymosin ß4 (Tß4), a pro-angiogenic and pro-survival factor, along with vascular cells derived from human embryonic stem cells (hESC) in ischemic injuries to the heart in a rat model. The gel was found to substitute the degrading extracellular matrix in the infarcted myocardium of rats and to promote structural organization of native endothelial cells, while some of the delivered hESC-derived vascular cells formed de novo capillaries in the infarct zone. Magnetic resonance imaging (MRI) revealed that the microvascular grafts effectively preserved contractile performance 3 d and 6 wk after myocardial infarction, attenuated left ventricular dilation, and decreased infarct size as compared to infarcted rats treated with PBS injection as a control (3 d ejection fraction, + ∼7%, P < 0.001; 6 wk ejection faction, + ∼12%, P < 0.001). Elevation in vessel density was observed in response to treatment, which may be due in part to elevations in human (donor)-derived cytokines EGF, VEGF and HGF (1 d). Thus, a clinically relevant matrix for dual delivery of vascular cells and drugs may be useful in engineering sustained tissue preservation and potentially regenerating ischemic cardiac tissue.

Effects of µCT radiation on tissue engineered bone-like constructs.

High-resolution, non-destructive imaging with micro-computed tomography (µCT) enables in situ monitoring of tissue engineered bone constructs. However, it remains controversial, if the locally imposed X-ray dose affects bone development and thus could influence the results. Here, we developed a model system for µCT monitoring of tissue engineered bone-like constructs. We examined the in vitro effects of high-resolution µCT imaging on the cellular level by using pre-osteoblastic MC3T3-E1 cells embedded into three-dimensional collagen type I matrices. We found no significantly reduced cell survival 2 h after irradiation with a dose of 1.9 Gy. However, 24 h post-irradiation, cell survival was significantly decreased by 15% compared to non-irradiated samples. The highest dose of 7.6 Gy decreased survival of the pre-osteoblastic MC3T3-E1 cells by around 40% at 2 days post-irradiation. No significant increase of alkaline phosphatase (ALP) activity at 2 days post-irradiation was found with a dose of 1.9 Gy. However, ALP activity was significantly decreased after 7 days. Using our model system, the results indicate that µCT imaging with doses as low as 1.9 Gy, which is required to obtain a reasonable image quality, can induce irreparable damages on the cellular level.

Isolation, differentiation and characterization of vascular cells derived from human embryonic stem cells.

Herein, we describe a protocol for the isolation of human embryonic stem cells (hESCs)-derived vascular cells at various stages of development. The cells are isolated from 10 to 15-d-old human embryoid bodies (EBs) cultured in suspension. After dissociation, cells are labeled with anti-CD34 or anti-CD31 (PECAM1) antibody and separated from the cell mixture by magnetic-activated cell separation (MACS) or fluorescent-activated cell sorting (FACS). Isolated vascular cells are then cultured in media conditions that support specific differentiation and expansion pathways. The resulting vascular cell populations contain >80% endothelial-like or smooth muscle-like cells. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 1.5 months. Vascular cells isolated and differentiated under the described conditions may constitute a potential cell source for therapeutic application toward repair of ischemic tissues, preparation of tissue-engineered vascular grafts and design of cellular kits for drug screening applications.

Cell-responsive hydrogel for encapsulation of vascular cells.

The in vitro potential of a synthetic matrix metalloproteinase (MMP)-responsive poly(ethylene glycol) (PEG)-based hydrogel as a bioactive co-encapsulation system for vascular cells and a small bioactive peptide, thymosin beta4 (Tbeta4), was examined. We show that the physical incorporation of Tbeta4 in this bioactive matrix creates a three-dimensional (3D) environment conducive for human umbilical vein endothelial cell (HUVEC) adhesion, survival, migration and organization. Gels with entrapped Tbeta4 increased the survival of HUVEC compared to gels without Tbeta4, and significantly up-regulated the endothelial genes vascular endothelial-cadherin and angiopoietin-2, whereas von Willebrand factor was significantly down-regulated. Incorporation of Tbeta4 significantly increased MMP-2 and MMP-9 secretion of encapsulated HUVEC. The gel acts as a controlled Tbeta4-release system, as MMP-2 and MMP-9 enzymes trigger the release. In addition, Tbeta4 facilitated HUVEC attachment and induced vascular-like network formation upon the PEG-hydrogels. These MMP-responsive PEG-hydrogels may thus serve as controlled co-encapsulation system of vascular cells and bioactive factors for in situ regeneration of ischemic tissues.

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

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=4,036+/-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 (alpha(5)beta(1,) alpha(v)beta(3)) 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.