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In Vitro and In Vivo Cardiomyogenic Differentiation of Amniotic Fluid Stem Cells

Abstract

Cell therapy has developed as a complementary treatment for myocardial regeneration. While both autologous and allogeneic uses have been advocated, the ideal candidate has not been identified yet. Amniotic fluid-derived stem (AFS) cells are potentially a promising resource for cell therapy and tissue engineering of myocardial injuries. However, no information is available regarding their use in an allogeneic context. c-kit-sorted, GFP-positive rat AFS (GFP-rAFS) cells and neonatal rat cardiomyocytes (rCMs) were characterized by cytocentrifugation and flow cytometry for the expression of mesenchymal, embryonic and cell lineage-specific antigens. The activation of the myocardial gene program in GFP-rAFS cells was induced by co-culture with rCMs. The stem cell differentiation was evaluated using immunofluorescence, RT-PCR and single cell electrophysiology. The in vivo potential of Endorem-labeled GFP-rAFS cells for myocardial repair was studied by transplantation in the heart of animals with ischemia/reperfusion injury (I/R), monitored by magnetic resonance imaging (MRI). Three weeks after injection a small number of GFP-rAFS cells acquired an endothelial or smooth muscle phenotype and to a lesser extent CMs. Despite the low GFP-rAFS cells count in the heart, there was still an improvement of ejection fraction as measured by MRI. rAFS cells have the in vitro propensity to acquire a cardiomyogenic phenotype and to preserve cardiac function, even if their potential may be limited by poor survival in an allogeneic setting.

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Acknowledgments

This work was supported by grant # 07/02 from “Città della Speranza”, Malo, Vicenza, Italy (SB, PDC) and by the Wellcome Trust (MN and AT). The authors also acknowledge the support of the Biotechnology and Biological Sciences Research Council, the British Heart Foundation and the Engineering and Physical Sciences Research Council.

Conflict of Interest and Disclosures

None to declare.

Author information

Correspondence to Sveva Bollini.

Electronic supplementary material

Below is the link to the electronic supplementary material.

GFP-rAFS cell with spontaneous contractile activity in co-culture with rCMs. After 4 days of co-culture, some GFP-rAFS cells were detected in CM-enriched beating areas expressing contractile activity as detected by the video recording. (MPG 583 kb)

Supplement Movie 1

GFP-rAFS cell with spontaneous contractile activity in co-culture with rCMs. After 4 days of co-culture, some GFP-rAFS cells were detected in CM-enriched beating areas expressing contractile activity as detected by the video recording. (MPG 583 kb)

Supplement Figure 1

Calibration curve for Endorem particles concentration mg/ml, [c] versus R2 (1/T2), demonstrating a linear relation between iron particles concentration and T2 (as long as the T2 values are between 20 and 90 seconds). (GIF 7.23 kb)

Supplement Figure 2

Analysis of differentiation of GFP-rAFS cells by immunofluorescence and gene expression analysis after 6 and 9 days of indirect co-culture with rCMs with Transwell® Membrane Inserts and after treatment with rCMs-conditioned medium. (ac) GFP-rAFS cells after indirect co-culture with rCMs and (d–f) after rCMs-conditioned medium treatment for 9 days, showing no expression of CM-specific markers as cTnT, bar, 100 μm. (g) Gel electrophoresis of RT-PCR products of control untreated GFP-rAFS cells (control GFP-rAFS cells, lane 1), GFP-rAFS cells co-cultured with rCMs on Transwell® Membrane Inserts for 6 days (lane 2), GFP-rAFS cells treated with rCM-conditioned medium for 6 days (lane 3), GFP-rAFS cells co-cultured with rCMs on Transwell® Membrane Inserts for 9 days (lane 4), GFP-rAFS cells treated with rCM-conditioned medium for 9 days (lane 5), control rCMs (lane 6) and H2O (negative control, lane 7) for the expression of the housekeeping gene β-Actin and the cardiac genes troponin I (cTnI) and sarcomeric α-actinin (cαA). GFP-rAFS cells co-cultured with rCMs with inserts and treated with rCMs-conditioned medium did not show any expression of cardiomyocyte genes (lane 2–5) compared to control undifferentiated GFP-rAFS cells (lane 1). (JPEG 2.09 mb)

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Bollini, S., Pozzobon, M., Nobles, M. et al. In Vitro and In Vivo Cardiomyogenic Differentiation of Amniotic Fluid Stem Cells. Stem Cell Rev and Rep 7, 364–380 (2011) doi:10.1007/s12015-010-9200-z

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Keywords

  • Amniotic fluid
  • Stem cells
  • In vitro differentiation
  • Cardiomyocyte
  • Cell transplantation