Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans
Histological analysis of cell survival and differentiation
Francis D. Pagani, MD, PhD, FACC*,*,
Harout DerSimonian, PhD¶,
Agatha Zawadzka, MS¶,
Kristie Wetzel, BS¶,
Albert S. B. Edge, PhD¶,
Douglas B. Jacoby, PhD¶,
Jonathan H. Dinsmore, PhD¶,
Susan Wright, BS, RN*,
Tom H. Aretz, MD ,
Howard J. Eisen, MD and
Keith D. Aaronson, MD, MPH
* Section of Cardiac Surgery, University of Michigan, Ann Arbor, MI, USA
Division of Cardiology, University of Michigan, Ann Arbor, Michigan, USA
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA
Temple University, Philadelphia, Pennsylvania, USA
¶ Diacrin, Inc., Charlestown, Massachusetts, USA
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Figure 1 Human skeletal myoblast expansion and purity. A shows the anti-CD56 staining profile of the final myoblast product implanted to Patient 2 using flow cytometry. B (10x) and C (4x) show the myoblast cells in culture during expansion. D (10x) and E (4x) show the potential of these cells to fuse and form multinucleated myotubes in an in vitro assay. Some of these multinucleated myotubes are highlighted with arrows in D.
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Figure 2 Representative trichrome and MY-32 stain of grafted skeletal myoblasts. A shows an area of the graft in a section stained with trichrome. B shows an adjacent section that was stained with the skeletal myosin specific MY-32 antibody. The transplant derived myofibers can be identified by the red staining in trichrome and the dark blue staining in the MY-32 stain. Asterisks (*) mark areas of host myocardial fibers. Scale bar = 50 µm.
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Figure 3 Representative trichrome and MY-32 stain of grafted skeletal myoblasts. A and B show an area of the graft in a section stained with trichrome at two magnifications. In A, the myotubes can be clearly identified, and in B the higher magnification shows the presence of striated myofibers in some myotubes. C shows an adjacent section that was stained with the skeletal myosin specific MY-32 antibody.
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Figure 4 Skeletal fast and slow twitch myosin. Shown are surviving skeletal myofibers in the heart from Patient 5 stained with trichrome (A and B), skeletal-specific fast myosin MY-32 (identification of fast twitch skeletal muscle myosin isoforms only) (C) and myosin heavy chain beta slow (D). A, C, and D show adjacent sections from the same graft site, and B shows a different graft site from the same patient, but where the myofibers can be seen in a longitudinal profile. Grafted skeletal myofibers have a distinct stain by trichrome that are marked by stars in B, and the alignment between grafted myofibers and host myofibers is striking. The area of the graft is marked by the dotted line in A, C, and D and individual skeletal myofibers that stained with slow myosin and not fast myosin (MY-32, C) are marked by arrowheads. All images were shot at the same magnification.
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Figure 5 Representative CD-3 staining of a graft site. The micrograph shows a graft site stained with an antibody to CD-3 to stain immune cells. Some scattered macrophages can be identified in and around the graft site. Some of the macrophages can be seen in association with an area of the containing giant cells. There is no collection of infiltrating immune cells at the graft site to indicate any ongoing immune reaction. The dotted line demarcates an area of viable myocardium. The stars highlight the area with giant cells. Arrows mark the surviving transplanted muscle cells, and the arrowheads mark the CD-3 positive macrophages.
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Figure 6 Trichrome staining of a graft site demonstrating the presence of giant cells. A and B show an area adjacent to the grafted cells where giant cells have formed. The boxed area in A is shown at higher magnification in B. Multinucleated giant cells are associated with refractile material (arrows), probably plastic that was introduced with the cells. Note that the area of engrafted cells is free from any giant cells.
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Figure 7 Representative CD-31 staining of a graft site. An antibody to human CD-31 was used to stain graft sections. A shows a representative micrograph in the area of the graft. The dotted line demarcates the border area between the transplant and the adjacent scar. Note the enhanced area of CD-31 staining within the engrafted section. B shows the results from quantitative counts to compare the number of CD-31 vessels at the graft (white bar) and in the adjacent scar (gray bar). Scale bar = 100 µm.
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