Bollini, Sveva (2008) Cardiomyogenic Potential of Amniotic Fluid Stem Cells As A New Tool For Cell Based Cardiac Tissue Engineering. [Ph.D. thesis]
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In the last years tissue engineering for cardiac pathologies has been broadly developed with the aim to restore or improve the diseased or damaged heart. Novel cardiac tissue engineering approaches combine the use of biocompatible scaffolds with stem cells to conjugate material science, surgery and cell therapy techniques. So far, different kinds of stem cells have been described and their potential for cardiac regeneration broadly investigated. We have previously described that it is possible to derive lines of broadly multipotent cells from the amniotic fluid (Amniotic Fluid Stem cells; AFS cells). The aim of this study was to characterize more in detail the AFS cells cardiomyogenic potential both in vitro and in vivo.
Neonatal rat cardiomyocyte (rCM) cells were obtained by enzymatic digestion of 2-3-days old rat hearts. GFP-positive rat AFS (gfp+rAFS) cells were obtained from amniotic fluid samples from GFP-positive transgenic pregnant rats. Human AFS (hAFS) cells were obtained from healthy amniotic fluid back up samples from prenatal diagnosis, following informed consent. AFS cells were isolated by immunosorting for the stem marker c-kit.
Before applying a tissue engineering approach, using biocompatible scaffolds, to the AFS and rCM cells coculture, the AFS cells “cardiomyocyte-like” phenotype, acquired in cocolture, had been functionally evaluated by patch-clamp analysis.
In this work two different kinds of bidimensional micropatterned scaffolds were used: hydrogel films and PDMS (silicon) membranes. The scaffolds were obtained by microcontact printing technique and using a mold scratched with the desidered micropattern and their viability was tested using, at first, the rat neonatal primary culture. AFS and rCM cells were seeded together on the micropatterned PDMS membranes and analyzed for the expression of troponin T by immunostaining after 6 and 10 days of culture.
For the in vivo study, immunodeficient nude male rats underwent a cryoinjury on the heart left ventricle with a 3D collagen scaffold implantation and 5x10e6 hAFS cells/animal local or systemic injection after 15 days. hAFS cells were previously labelled with the red intracellular fluorescent dye CMTMR. Animals were sacrificed at 24 hours, 15 and 30 days after cells injection and hearts stained for cardiac and inflammatory markers. For the acute myocardial infarct model, male Wistar rats underwent an ischemic injury by left anterior descendent coronary artery ligation for 30 minutes and then they were reperfused injecting via the external jugular vein 10e7 or 10e6 gfp+rAFS and 10e7 or 5x10e6 hAFS cells/animal for 2 hours; rats were sacrificed afterwards and hearts analyzed for infarct size measurement by Evans blue staining, by 2,3,5-triphenolltetrazolium chloride (TTC) staining and planimetry with the software Image J. Heart, lungs, spleen and liver were analyzed as well by immunostaining for evaluating hAFS cells content. hAFS cells were also analyzed for the presence of a subpopulation of cardiac progenitors, by RT-PCR analysis, for the expression of early cardiac commitment genes as Isl1 and Kdr. The cells were then studied by ELISA essay to speculate if they can secrete in the culture medium the protein thymosin beta 4, paracrine and cardioprotector factor.
Results and Conclusions.
Regarding the in vitro results, AFS cells were demonstrated to express a “pace maker cell-like” action potential, when cocultured with rat neonatal cardiomyocyte cells. Moreover, when cultured on the bidimensional scaffold, AFS cells showed to follow the longitudinal orientation of the microstruttured membrane, expressing beating activity and the cardiac protein troponin T.
Our in vivo data revealed that hAFS cells, injected into the cryoinjured rat heart, survived in the host up to 30 days, moved from the injection site to the lesioned area in the heart and gave rise to new chimeric capillaries in the patch and cryoinjury area. In the acute myocardial infarct model the results obtained suggested that hAFS cells could exert a paracrine effect in vivo, decreasing the infarct size (measured as the ratio between the infarct area and the ischemic area at risk of necrosis) from a 53,9 ± 2,3% (obtained in control animals receiving PBS injection) to 40,0 ± 3,0% of the ischemic area. Furthermore, hAFS cells were also demonstrated to have a subpopulation of cardiac progenitors, positive for the expression of the early cardiac commitment genes Isl1 and Kdr and to to secrete in the culture medium thymosin beta 4, a paracrine factor previously shown to act as cardioprotector and angiogenic agent.
In conclusions, our results are very encouraging and challenging, suggesting that AFS cells can show cardiomyogenic potential and cardioprotective therapeutic application in cell based therapy tissue engineering.
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