3D Bioprinting the Cardiac Purkinje System Using Human Adipogenic Mesenchymal Stem Cell Derived Purkinje Cells
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Cardiovascular Engineering and Technology (Ó 2020) https://doi.org/10.1007/s13239-020-00478-8
Original Article
3D Bioprinting the Cardiac Purkinje System Using Human Adipogenic Mesenchymal Stem Cell Derived Purkinje Cells EVAN P. TRACY,1 BRIAN C. GETTLER,1 JOSEPH S. ZAKHARI,1 ROBERT J. SCHWARTZ,2,4 STUART K. WILLIAMS,1 and RAVI K. BIRLA3 1
Department of Physiology, Cardiovascular Innovation Institute, University of Louisville, 302 E. Muhammad Ali Blvd, Louisville, KY 40202, USA; 2Stem Cell Engineering, Texas Heart Institute, 6770 Bertner Avenue, Houston, TX 77225-0345, USA; 3Department of Biomedical Engineering, Science and Engineering Research Center, (SERC-Building 445), 3605 Cullen Blvd, Room 2005, Houston, TX 77204, USA; and 4Department of Biology and Biochemistry, Science and Engineering Research Center, (SERC-Building 445), 3605 Cullen Blvd, Room 5004, Houston, TX 77204-5060, USA (Received 3 November 2019; accepted 9 July 2020) Associate Editor Stephen Hilbert oversaw the review of this article.
Abstract Purpose—The objective of this study was to reprogram human adipogenic mesenchymal stem cells (hADMSCs) to form Purkinje cells and to use the reprogrammed Purkinje cells to bioprint Purkinje networks. Methods—hADMSCs were reprogrammed to form Purkinje cells using a multi-step process using transcription factors ETS2 and MESP1 to first form cardiac progenitor stem cells followed by SHOX2 and TBX3 to form Purkinje cells. A novel bioprinting method was developed based on Pluronic acid as the sacrificial material and type I collagen as the structural material. The reprogrammed Purkinje cells were used in conjunction with the novel bioprinting method to bioprint Purkinje networks. Printed constructs were evaluated for retention of functional protein connexin 40 (Cx40) and ability to undergo membrane potential changes in response to physiologic stimulus. Results—hADMSCs were successfully reprogrammed to form Purkinje cells based on the expression pattern of IRX3, IRX5, SEMA and SCN10. Reprogrammed purkinje cells were incorporated into a collagen type-1 bioink and the left ventricular Purkinje network was printed using anatomical images of the bovine Purkinje system as reference. Optimization studies demonstrated that 1.8 mg/mL type-I collagen at a seeding density of 300,000 cells per 200 lL resulted in the most functional bioprinted Purkinje networks. Furthermore, bioprinted Purkinje networks formed continuous syncytium, retained expression of vital functional gap junction protein Cx40 post-print, and exhibited membrane potential changes in response to electric stimulation and
Address correspondence to Ravi K. Birla, Department of Biomedical Engineering, Science and Engineering Research Center, (SERCBuilding 445), 3605 Cullen Blvd, Room 2005, Houston, TX 77204, USA. Electronic mail: [email protected] Stuart K. Williams, Ravi K. Birla are Joint Senior Authors.
acetylcholine evaluated by DiBAC4(5), an electrically responsive dye. Conclusion—Based on the results of this study, hADMSCs were successfully reprogramme
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