In Vitro Grown Micro-Tissues for Cardiac Cell Replacement Therapy in Vivo.

BACKGROUND/AIMS: Different approaches have been considered to improve heart reconstructive medicine and direct delivery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) appears to be highly promising in this context. However, low cell persistence post-transplantation remains a bottleneck hindering the approach. Here, we present a novel strategy to overcome the low engraftment of PSC-CMs during the early post-transplantation phase into the myocardium of both healthy and cryoinjured syngeneic mice. METHODS: Adult murine bone marrow mesenchymal stem cells (MSCs) and PSC-CMs were co-cultured on thermo-responsive polymers and later detached through temperature reduction, resulting in the protease-free generation of cell clusters (micro-tissues) composed of both cells types. Micro-tissues were transplanted into healthy and cryo-injured murine hearts. Short term cell retention was quantified by real-time-PCR. Longitudinal cell tracking was performed by bioluminescence imaging for four weeks. Transplanted cells were further detected by immunofluorescence staining of tissue sections. RESULTS: We demonstrated that in vitro grown micro-tissues consisting of PSC-CMs and MSCs can increase cardiomyocyte retention by >10fold one day post-transplantation, but could not fully rescue a further cell loss between day 1 and day 2. Neutrophil infiltration into the transplanted area was detected in healthy hearts and could be attributed to the cellular implantation rather than tissue damage exerted by the transplantation cannula. Injected PSC-CMs were tracked and successfully detected for up to four weeks by bioluminescence imaging. CONCLUSION: This approach demonstrated that in vitro grown micro-tissues might contribute to the development of cardiac cell replacement therapies.

Entrapment of embryonic stem cells-derived cardiomyocytes in macroporous biodegradable microspheres: preparation and characterization.

Embryonic Stem (ES) cells-derived cardiomyocytes can possibly be applied for cell therapy of diseases such as heart failure. Biodegradable scaffolds will significantly improve the expansion of sufficient functional ES cell-derived cardiomyocytes and may also increase the survival rate of cardiomyocytes after their transplantation. In the present study, we cultivated cardiomyocytes isolated from a transgenic a-myosin heavy chain (alpha-MHC) ES cell lineage expressing both puromycin resistance and enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter in macroporous gelatine microspheres using small-scale bioreactors and proved that cardiomyocytes function after their cultivation in micropsperes. The average number of cultivated cells per microsphere was optimised once the most suitable agitation conditions and the optimal timeframe of cultivation were identified. Our study shows that 72% of CultiSpher-S beads were colonised by cardiomyocytes under optimal conditions. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) showed that colonization of the beads was not limited to the surface, but that cells also invaded the inner surfaces of the microspheres. Electrophysiological experiments demonstrated that the action potentials (APs) of alpha-MHC(+) cardiomyocytes entrapped in microspheres were identical to action potentials of control cells. This attractive approach for cultivation and expansion of functional cardiomyocytes in biodegradable macroporous may offer a perspective for higher transplantation efficiencies of ES cell-derived cardiomyocytes.

Expansion and differentiation of germline-derived pluripotent stem cells on biomaterials.

Stem cells with broad differentiation potential, such as the recently described germline-derived pluripotent stem cells (gPS cells), are an appealing source for tissue engineering strategies. Biomaterials can inhibit, support, or induce proliferation and differentiation of stem cells. Here we identified (1) polymers that maintain self-renewal and differentiation potential of gPS cells for feeder-free expansion and (2) polymers supporting the cardiomyogenic fate of gPS cells by analyzing a panel of polymers of an established biomaterial bank previously used to assess growth of diverse stem cell types. Identification of cytocompatible gPS cell/biomaterial combinations required analysis of several parameters, including morphology, viability, cytotoxicity, apoptosis, proliferation, and differentiation potential. Pluripotency of gPS cells was visualized by the endogenous Oct4-promoter-driven GFP and by Sox2 and Nanog immunofluorescence. Viability assay, proliferation assay, and flow cytometry showed that gPS cells efficiently adhere and are viable on synthetic polymers, such as Resomer(®) LR704 (poly(L-lactic-D,L-lactic acid), poly(tetrafluor ethylene) (PTFE), poly(vinylidene fluoride) (PVDF), and on gelatine-coated tissue culture polystyrene. Expansion experiments showed that Resomer LR704 is an alternative substrate for feeder-free gPS cell maintenance. Resomer LR704, PTFE, and PVDF were found to be suitable for gPS cell differentiation. Spontaneous beating in embryoid bodies cultured on Resomer LR704 occurred already on day 8 of differentiation, much earlier compared to the other surfaces. This indicates that Resomer LR704 supports spontaneous cardiomyogenic differentiation of gPS cells, which was also confirmed on molecular, protein and functional level.

Microelectrode arrays: a new tool to measure embryonic heart activity.

The analysis of the sequential excitation of cardiac tissue is of high relevance, both for clinical pathophysiological purposes, eg, detection of sustained ventricular arrhythmias, as well as for experimental electrophysiology. Clinically, different technical approaches such as single electrode measurements and bipolar mapping electrode catheters have been used. In experimental setups several techniques to record cardiac activity have been proposed. Beside the well-established intracellular current-clamp recordings of action potentials, recent studies have performed extracellularly activation sequence mapping or simultaneous multichannel action potential electrode array measurements. Measurement of extracellularly recorded field potentials (FPs) hereby especially provides detailed information about the origin and spread of excitation in the heart. A similar analytical approach for cardiac FPs advanced the analysis of excitation spread and arrhythmic activity in multicellular preparations like developmental differentiation tissue of mouse embryonic stem cells, multicellular preparations of isolated native embryonic cardiomyocytes or the embryonic heart in toto. The use of substrate-integrated Microelectrode Arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) with 60 electrodes of 10-30 microm diameters on a 100-200 microm grid, coated with porous titanium nitride to minimize the impedance allows recording of FPs at a high signal to noise ratio. The possibility to electrically stimulate the tissue further expands the range of applications and bioassays. It may thus facilitate the evaluation of drug research providing detailed information about the interplay of the complex cardiac network, and might improve the predictability of physiological and pathophysiological conditions or drug effects in embryonic heart tissue.