Patterning human stem cells and endothelial cells with laser printing for cardiac regeneration
Introduction
A cardiac patch can provide a support matrix which allows stem/progenitor cell adhesion and proliferation in a damaged heart [1], [2], [3]. It is a possible alternative to the current approach of direct cell injection for cell-based therapy. However, inadequate neovascularization remains the major limitation in clinical application of cardiac patch. Due to the impaired nutrients and oxygenation perfusion after myocardial infarction (MI), cardiac tissue formation will be confined to limited area with only marginal functional improvement.
Laser-Induced-Forward-Transfer (LIFT) derived from industrial electronic manufacturing demonstrates advantages for controlled transfer of inorganic and biological materials in conjunction with proteins, peptides, DNA, RNA and cells [4]. A precise arrangement of cells could be obtained in three-dimensional (3D) patterns [5]. Refinement of this methodical approach achieved survival rates of printed cells of nearly 100%. There were no signs of DNA-damage, increased apoptosis rates or any effect on the proliferation [4]. The advantage of LIFT is the potential to form a precise multi-layered 3D construct in a single step. The technique allows integration of an endothelial cell pattern for vascular network formation to construct a myocardium analog with precise 3D organization.
Mesenchymal stem cells or multipotent stromal cells (MSC) are capable of self-renewing and have the potential to differentiate into multiple lineage [6]. MSC were tested in animal models for a number of diseases [7], [8] and in clinical trials for myocardial ischemia with promising angiogenic outcome [9]. MSC have profound impacts on the local cell survival, cardiac remodeling and regeneration [10]. Their regenerative effect was attributed mainly to endocrine or paracrine factors [11]. The paracrine factors of MSC could activate the PI3 K-Akt pathway in hypoxic endothelial cells to inhibit apoptosis, increase survival, and stimulate angiogenesis [12]. Further LIFT process could retain the phenotype of MSC as well as their differential potential [13], and MSC which were printed in close proximity to EC by LIFT could stabilize the neovasculature [14]. To mimic the natural vascular structure and favor the close cell–cell interaction [15] we printed human MSC (hMSC) and EC with a defined manner on a PEUU cardiac patch by LIFT and assessed the therapeutic potential of the patch in cardiac regeneration in an immunodeficient Rowett Nude (RNU) rats left anterior descending (LAD) ligation model with respect to cell survival, infarct wall thickness, angiogenesis, cardiac remodeling and functional improvement.
Section snippets
Material and methods
The study conforms to the Declaration of Helsinki and cell donors gave their informed written consent to use their umbilical cord or bone marrow for experimental purposes.
Cell isolation and characterization
Cell isolation, expansion, characterization and differentiation of human bone marrow MSC (hMSC) have been established according to previous reports [21]. The morphology of hMSC from bone marrow displayed a homogenous spindle-shaped population and maintained a similar morphology during the subsequent passages. FACS analysis was employed to identify the expression of specific cell surface markers at passage 3 (Table 1, Fig. 2).
LIFT printing of cells on the cardiac patch
Human MSC and HUVEC were transferred by LIFT on the PEUU cardiac patch
Discussion
This study presents a method (LIFT) to arrange EC together with hMSC in a defined manner to produce functional PEUU cardiac patch. Human MSC and EC were isolated, cultured and seeded onto PEUU cardiac patch carefully in a defined manner. The cardiac patch with printed cells could attenuate LV remodeling and dysfunction in RNU rats that underwent MI. The patterned human stem cells and endothelial cells on PEUU patch could enhance the angiogenesis in the border zone of infarction and form
Conclusions
In this study, we successfully patterned human mesenchymal stem cells and endothelial cells on a PEUU cardiac patch with laser printing that enhanced angiogenesis in the border zone of infarction and preserved cardiac functions after acute myocardial infarction. Transplantation of LIFT-based tissue engineering of cardiac patches may provide an effective approach to mediate substantial functional recovery of the infarcted heart.
Disclosures
The authors confirm that there are no known conflicts of interest associated with this publication.
Acknowledgments
This work was supported by Standardization for Regenerative Therapy - Mesenchymal Stem Cells (START-MSC); Sonderforschungsbereich/Transregio 37 (B5, B2 and A4); German Federal Ministry of Education and Research, BioChancePlus program (0313191); The German Helmholtz Association, Mecklenburg-Vorpommern, German Federal Ministry of Education and Research, German Research Foundation (Nachwuchsgruppe Regenerative Medizin Regulation der Stammzellmigration 0402710); REBIRTH Cluster of Excellence
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- 1
Authors contributed equally to this work.
- 2
Present address: BCRT & CBD, Institute of Polymer Research, HZG Germany.