Human prenatal progenitors for pediatric cardiovascular tissue engineering

Pediatric cardiovascular tissue engineering is a promising strategy to overcome the lack of autologous, growing replacements for the early repair of congenital malformations in order to prevent secondary damage to the immature heart. Therefore, cells should be harvested during pregnancy as soon as the cardiovascular defect is detected enabling the generation of living autologous implants with the potential of growth, remodeling and regeneration ready to use at or shortly after birth. Furthermore, the ideal cell source should be easily accessible and allow cell harvest without substantial risks for both the mother and the child and without sacrifice of intact infantile donor tissue. In this work, human prenatal progenitor cells obtained from different extra-embryonically situated fetal tissues were investigated with regard to the pediatric cardiovascular tissue engineering concept. In individual studies prenatal progenitor cells were isolated from different fetal tissues including umbilical cord blood and cord tissue, chorionic villi and amniotic fluid. Cells were expanded and differentiated into cell types that are required for cardiovascular replacements in order to match the characteristics of their native counterparts: a myofibroblast-fibroblast-like cell type producing extracellular matrix and an endothelial cell type forming an antithromobogenic and blood-compatible surface. Thereby, cell phenotypes were analyzed by flowcytometry and immunohistochemistry and genotypes were determined. For the fabrication of cardiovascular tissues, biodegradable cardiovascular scaffolds (PGA/P4HB) were seeded with fibroblast-myofibroblast-like cells derived from either umbilical cord tissue, chorionic villi or amniotic fluid. Constructs were implanted in an in vitro pulse duplicator and exposed to biochemical and/or mechanical stimulation. After, in vitro maturation time, the surfaces of cardiovascular constructs were endothelialized with differentiated umbilical cord blood-derived endothelial progenitor cells or amniotic fluid-derived endothelial progenitor cells and conditioned for an additional 7d. Analysis of the neo-tissues comprised histology, immunohistochemistry (vimentin, a- SMA, desmin, Ki-67), biochemistry (extracellular matrix (ECM) - analysis, DNA), mechanical testing and scanning electron microscopy (SEM). Neo-endothelia were analysed by immunohistochemistry (CD31, vWF, thrombomodulin, tissue factor, eNOS). After differentiation, cells demonstrated characteristics of fibroblast-myofibroblast-like cells expressing vimentin, desmin and partly a-SMA independent of the cell source. Furthermore, umbilical cord blood-derived endothelial progenitor cells and amniotic fluid-derived cells expressed typical endothelial cell markers such as CD31, vWF, thrombomodulin, tissue factor, and eNOS, respectively. Genotyping confirmed the fetal origin of the cells without contamination with maternal cells. All cardiovascular constructs showed cellular tissue formation with functional endothelia as indicated by the expression of eNOS. Expression of Ki-67 confirmed proliferation of cells in all parts of the neo-tissues. Matrix analysis (collagen and proteoglycans) and DNA content demonstrated constituents typical of native cardiovascular tissues. Mechanical properties revealed native analogous profiles but did not reach native values. SEM showed cell-ingrowth into the polymer and smooth surfaces covered densely with endothelial cells. Prenatal progenitors from different sources were successfully used for the in vitro fabrication and maturation of living autologous cardiovascular constructs. With regard to clinical application the use of amniotic fluid-derived prenatal progenitor cells represents the most attractive approach as it enables the prenatal fabrication of cardiovascular replacements based on a single cell source ready to use at birth.