This study describes the production of two new human embryonic stem cell (hESC) lines affected by cystic fibrosis. These cell lines are heterozygous compounds, each a carrier of the DF508 mutations associated either with E585X or with 3849+10 kb C-->T. The derivation process was performed on irradiated human placental mesenchymal stromal cells and designed to minimize contact with xeno-components. This new source of feeder cells is easy to obtain and devoid of ethical concerns. The cells have a great capacity to proliferate which reduces the need for continuous preparation of new feeder cell lines. In addition, three normal hESC lines were obtained in the same conditions. The five stem cell lines retained hESC-specific features, including an unlimited and undifferentiated proliferation capacity, marker expression and the maintenance of stable karyotype. They also demonstrated pluripotency in vitro, forming cell lineages of the three germ layers, as indicated by immunolocalization of beta-tubulin, alpha-fetoprotein and actin. These new genetic cell lines represent an important in-vitro tool to study the physiological processes underlying this genetic disease, drug screening, and tissue engineering.