Stem Cell Populations in Human Heart Valves: Identification, Isolation and Characterization in Valve Homografts and Surgical Specimens.

This study aims to identify valve stem cells and assess their contribution to postnatal tissue regeneration/homeostasis in inflammatory-associated remodeling. Valve homografts (n=28) have been classified in five age groups: excised aortic and mitral leaflets have been analyzed by histology (H&E, Masson trichrome), histochemistry (von Kossa, Oil O Red) and immunohistochemistry for inflammation (CD45, CD14, CD68, Mast cells, S100, granulocytes) and differentiated cells (vimentin, non-muscle myosin, SM-actin, SM-myosin, von Willebrand factor, CD31). Expression of stem markers from different lineages has been evaluated: hematopoietic (CD34, CD133, CD117), neuronal (NGFr, Nestin, GFAP, beta-catenin), embryonic (SSEA4, OCT4) and mesenchymal (CD90, CD29, CXCR4, CD105). Valvular interstitial primary cultures have been obtained from aortic and/or mitral leaflets (n=10), surgically removed during valve substitutions. Cells have been analyzed by cytocentrifuges for above markers and cytofluorimetry (CD29, CD31, CD34, CD44, CD73, CD105) and tested for transdifferentiation. The detection of lipid deposits tends to be more frequent with aging, from simple fat vesicles to organized cholesterol crystals in the last age group. Rarely calcifications are found. For each age group, an inflammatory cell component (CD45, CD68, Mast cell) is present. Fibroblast population is vimentin- and non-muscle myosin-positive, seldom with SM-actin activated phenotype. Stem markers are variously expressed by valve cells: the hematopoietic CD34, the neuronal GFAP, the mesenchymal CD90 and CD29 are present at high percentages. In cusp tissues more free of inflammatory contaminants (frequently 15-20 year-old group), a topographic stem cell expression could suggest the presence of progenitors with regional specialization. Cultured interstitial cells from surgical samples exhibit spindled shaped morphology, prominent nuclei, long cytoplasmic extroflessions and fast multilayer growth. The expression of CD29, CD90 and CD105 implies a mesenchymal stem profile, with low positivity for Nestin, GFAP and CD34. To date, tested endothelial and smooth muscle conversions have been successful. Responsiveness to in vitro calcification stimuli is under evaluation. Ongoing studies are verifying stem regenerative potential and implication in valve pathophysiology.