title: Human stem cell-based three-dimensional microtissues for advanced cardiac cell therapies.
abstract: Cardiac stem cell therapy has been proposed as a therapy option to treat the diseased myocardium. However, the low retention rate of transplanted single-cell suspensions remains a major issue of current therapy strategies. Therefore, the concept of scaffold-free cellular self-assembly into three-dimensional microtissues (3D-MTs) prior to transplantation may be beneficial to enhance retention and survival. We compared clinically relevant, human stem cell sources for their ability to generate 3D-MTs with particular regards to formation characteristics, proliferation-activity, viability and extracellular-matrix production. Single-cell suspensions of human bone marrow- and adipose tissue-derived mesenchymal stem cells (hBMMSCs and hATMSCs), Isl1(+) cardiac progenitors derived from human embryonic stem cells (hESC-Isl1(+) cells), and undifferentiated human induced pluripotent cells (hiPSCs) were characterized before to generate 3D-MTs using a hanging-drop culture. Besides the principal feasibility of cell-specific 3D-MT formation, a detailed head-to-head comparison between cell sources was performed using histology, immunocyto- and histo-chemistry as well as flow cytometry. Round-oval shaped and uniform 3D-MTs could be successfully generated from all cell types starting with a loose formation within the first 24 h that fully stabilized after 3 days and resulting in a mean 3D-MT diameter of 194.56 ± 18.01 μm (hBMMSCs), 194.56 ± 16.30 μm (hATMSCs), 159.73 ± 19.20 μm (hESC-Isl1(+) cells) and 120.95 ± 7.97 μm (hiPSCs). While all 3D-MTs showed a homogenous cell distribution, hiPSC-derived 3D-MTs displayed a compact cell formation primarily located at the outer margin. hESC-Isl1(+) and hiPSC-derived 3D-MTs maintained their proliferation-activity which was rather limited in the MSC-based 3D-MTs. All four 3D-MT types revealed a comparable viability in excess of 70% and showed a cell-specific expression profile being comparable to their single-cell counterparts. Extracellular matrix (ECM) production during 3D-MT formation was observed for all cell-specific 3D-MTs, with hiPSC-derived 3D-MTs being the fastest one. Interestingly, ECM distribution was homogenous for hATMSC- and hiPSC-based 3D-MTs, while it appeared to be primarily concentrated within in the center of hESC-Isl1(+) and hBMMSC-based 3D-MTs. The results of this head-to-head comparative study indicated that 3D-MTs can be successfully generated from hESC-derived Isl1(+) cells, hiPSCs and MSC lines upon hanging drop culture. Cell-specific 3D-MTs displayed sufficient viability and instant ECM formation. The concept of 3D-MT in vitro generation prior to cell transplantation may represent a promising delivery format for future strategies to enhance cellular engraftment and survival.
authors.affiliation: Swiss Centre for Regenerative Medicine, University of Zurich, Switzerland. maximilian.emmert@usz.ch
authors.forename: Maximilian Y
authors.initials: MY
authors.lastname: Emmert
authors.forename: Petra
authors.initials: P
authors.lastname: Wolint
authors.forename: Nadine
authors.initials: N
authors.lastname: Wickboldt
authors.forename: Gino
authors.initials: G
authors.lastname: Gemayel
authors.forename: Benedikt
authors.initials: B
authors.lastname: Weber
authors.forename: Chad E
authors.initials: CE
authors.lastname: Brokopp
authors.forename: Alessandro
authors.initials: A
authors.lastname: Boni
authors.forename: Volkmar
authors.initials: V
authors.lastname: Falk
authors.forename: Alexis
authors.initials: A
authors.lastname: Bosman
authors.forename: Marisa E
authors.initials: ME
authors.lastname: Jaconi
authors.forename: Simon P
authors.initials: SP
authors.lastname: Hoerstrup
mesh_terms: D002454:Cell Differentiation; D049109:Cell Proliferation; D064987:Cell- and Tissue-Based Therapy; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D009206:Myocardium / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods*