Human Adult Bone Marrow Mesenchymal Stem Cells Repair Experimental Conduction Block in Rat Cardiomyocyte Cultures
Saskia L.M.A. Beeres, MD*,
Douwe E. Atsma, MD, PhD*,*,
Arnoud van der Laarse, PhD*,
Daniël A. Pijnappels, MSc*,
John van Tuyn, MSc*, ,
Willem E. Fibbe, MD, PhD ,
Antoine A.F. de Vries, PhD,
Dirk L. Ypey, PhD ,
Ernst E. van der Wall, MD, PhD* and
Martin J. Schalij, MD, PhD*
* Department of Cardiology, Leiden University Medical Center, Leiden, the Netherlands
Department of Molecular Cell Biology Section Gene Therapy, Leiden University Medical Center, Leiden, the Netherlands
Department of Hematology, Leiden University Medical Center, Leiden, the Netherlands
Department of Physiology, Leiden University Medical Center, Leiden, the Netherlands
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Figure 1 Light microscopy of micro-electrode array (MEA) cultures (A) before and (B) immediately after channel abrasion. (C) Fluorescence microscopy of MEA cultures 24 h after application of eGFP-labelled human mesenchymal stem cells.
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Figure 2 Before channel abrasion, there is a consistent time interval between local activation times (LATs) in the upper and lower field of the culture (all points are on a straight line) and the culture is considered to be activated synchronously with a similar excitation spread in consecutive excitation waves (A). After abrasion, the two cardiomyocyte fields beat independently (correlation between LATs on either side of the channel is lost) (B). Twenty-four hours after human mesenchymal stem cell (hMSC) application, the correlation between LATs is restored, indicating resynchronization. Because of the conduction delay within the channel, the lower field is now activated with an 80-ms delay (C).
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Figure 3 Color-coded activation maps before channel abrasion (A), immediately after channel abrasion (B), and 24 h after human mesenchymal stem cell application (C). See text for explanation.
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Figure 4 Electrograms recorded at eight adjacent electrodes in a synchronized culture before channel abrasion (A), immediately after channel abrasion (B) (gray squares indicate electrodes located at the channel level), and 24 h after human mesenchymal stem cell (hMSC) seeding (C). After abrasion no electrical activity is present at the level of the channel (B, E). Resynchronization of the cardiomyocyte fields and the presence of electrical activity in the channel is shown in F. Panels D and F give typical recordings of an electrode underneath a cardiomyocyte and an hMSC.
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Figure 5 Action potential recordings in a resynchronized culture 24 h after seeding human mesenchymal stem cells (hMSCs) in a channel-crossing pattern. Recordings on the left side are membrane potentials of cardiomyocytes; recordings on the right are derived from hMSCs within the channel.
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Figure 6 Connexin (Cx)43-positive gap junctions along a region of intimate cell-cell contact between a DsRed-labeled human mesenchymal stem cell (hMSC) and a cardiomyocyte (A, white arrowhead). Cx43 is also present in the cytoplasm of the DsRed-labeled hMSC (A, black arrowhead, B, black arrowhead), between adjacent cardiomyocytes (A, white arrowhead) and between adjacent hMSC (B, white arrowhead).
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Figure 7 Light microscopy of a mixed culture of DsRed-labeled human mesenchymal stem cell (hMSCs) and calcein-loaded cardiomyocytes (cm) (A). Fluorescence in red channel (B), fluorescence in green channel (C). The hMSCs in contact with calcein-loaded cardiomyocytes display green fluorescence indicative of dye transfer (C), whereas hMSCs that have no contact with calcein-loaded cardiomyocytes do not fluoresce green (A through C, white arrowhead).
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