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JSRM Code: 017010300003    

Neuregulin-1, in a Conducive Milieu with Wnt/BMP/Retinoic Acid, Prolongs the Epicardial-Mediated Cardiac Regeneration Capacity of Neonatal Heart Explants
Arora H1,2#, Lavin AC1#, Balkan W1,3, Hare JM1,3, White IA1,4

Author Names in full: Himanshu Arora1,2#, Alessia C. Lavin1#, Wayne Balkan1,3, Joshua M. Hare1,3 and Ian A. White1,4


Interdisciplinary Stem Cell Institute and Departments of 2Urology and 3Medicine, University of Miami Miller School of Medicine, Miami FL, 33136, USA. 4Neobiosis, LLC, 12085 Research Dr, Alachua, FL 32615, USA

#These authors contributed equally

Supplementary Information

Supplemental Figure 1. During wound healing in cultured explants TBX18 is re-expressed within and adjacent to sites of injury. The cardiac‑specific transcription factor T‑Box protein 18 (TBX18) is repressed at the site of injury by day 12 (red), indicative of immature cardiomyoblast formation. At this stage no mature cardiomyocytes expressing cTnT can be seen within the reparative site (green). Cell nuclei stained with DAPI (blue).









Supplemental Figure 2. Following a first wave of epicardial cells into the injury site repopulation may be achieved by the migration of adjacent cardiomyocytes. Some mature cardiomyocytes adjacent to the injury appear to disassemble their sarcomeres and migrate into and populate the injury from the surrounding myocardium beginning 3 days post injury, as shown by immunofluorescence for cardiac troponin T (red), cell nuclei are stained with DAPI (blue). Bottom panel, top inset, shows mature cardiomyocytes with intact sarcomeres. Bottom panel, bottom inset, shows a cardiomyocyte with disassembled sarcomere at junction of injury.
































Supplemental Figure 3. FUCCI reporter mice reveal a population of G1, pre-mitotic cells within the sub-epicardium and surrounding the site of injury. By using the FUCCI reporter system we observe cells in the G1 phase of cell cycle expresses the red nuclear reporter, which can be visualized by epifluorescence histology. By day 21 following injury the site (*) is resolved and closely resembles the surrounding myocardium, based on DAPI distribution (blue). However, by using FUCCI mice we see that the entire sub epicardium is populated with cardiomyocytes in a pre-mitotic state in G1 (red). These cells are excluded from the epicardium itself (arrowhead) and the site of injury (*).







Supplemental Figure 4: In the absence of epicardial-derived cells the site of injury repair can be identified by autofluorescence and differences in cardiomyocyte sarcomere structure. EdU+ epicardial-derived cells, which first populate the injury site (A), start to disappear from the injury site after 10 days post injury in culture and cannot be located by day 21. However, a vestige of injury can be identified by non-specific autofluorescence (green channel) (B). The previously injured site (arrowhead) is populated with cardiac troponin T (cTnT+) cells (red) which lack the defined sarcomeric structure of adjacent cardiomyocytes (C). Site of injury demarcated by dotted line. Inset, mature adjacent cardiomycytes (left panel) compared to group of cardiomycytes with disassembled sarcomeres within the injury (right panel). Keratin-5. E) Representative image showing staining of the A/B colonies with the markers of ciliated and basal cells, acetylated a-tubulin and Keratin-5. Scale bars A, B = 50 m, C-E: 100 m.


































Supplemental Video 1. Long-term mechanical function (beating) can be retained in murine cardiac explants. Two-day old neonatal mouse heart explant cultured in maintenance media (Base+P+N) for 14 days still exhibits non-coordinated, punctate beating, indicative of viable, functioning cardiomyocytes. Hearts maintained in canonical media for culturing cardiomyocyte monolayers rapidly undergo coagulative necrosis (cell death due to a lack of nutrients) by day 3 (data not shown). Atria are removed for reasons of experimental consistency.