Derivation of Human Embryonic Stem Cell Line From Preimplantation Genetic Diagnosed (PGD) Fragile X-Affected Embryos for the Study of Fragile X Syndrome

Abstract

To produce a heretofore nonexistent cellular model for in-depth study of the molecular mechanism of fragile X syndrome. hES cells may be easily obtained as a by-product of the PGD program in which embryos are genetically analyzed at the 6-8-cell stage, and only disease-free embryos are transferred, while the affected ones are discarded. The latter may be utilized for the establishment of hES cell lines that express the molecular defect underlying fragile X syndrome. Since ES cells can be kept and propagated in culture in an undifferentiated state, they can serve as an ideal research model to study the detailed molecular mechanism for the disease. The potential of these ES cells to develop into many different cell types may also provide us with an unlimited source of differentiated cells for researching the cellular phenotype of the syndrome. Such cell lines should be used for studying the molecular mechanism of the disease at the DNA, RNA and proteins level. PGD-diagnosed fragile X-affected embryos were grown to late-blastocyst stage and used for isolation and propagation of their inner cell mass (ICM) cells. The resulting fragile X-affected hES cell line was further expanded and examined for karyotype, self-renewal ability, differentiation potential and the expression of pluripotent-associated genes. The genetic diagnosis was confirmed by PCR analysis. In addition, the genetic lesion (a triplet expanding repeat) was determined by southern blot analysis. Differentiation potential was examined in-vivo by injecting the cells under the kidney capsule of immunocompromised mice to induce teratomas. Isolation of ICM from a PGD-diagnosed fragile X-affected blastocyst yielded one cell line with ES cells morphological characteristics consistent with previously characterized pluripotent hES cell lines. The line was named Human Embryonic Fragile X SC line #1 (HEFX1). The cells were able to self-renew without restriction while remaining undifferentiated. In addition, they had a normal karyotype and were male in origin (XY). The HEFX1 cells expressed the typical markers of pluripotent stem cells, including OCT4, alkaline phosphatase and several others. PCR analysis for 3 polymorphic markers and SRY performed on DNA extracted from the HEFX1 cells confirmed the results of the PGD diagnosis. Southern blot analysis demonstrated that the HEFX1 cell line we had established indeed displays the genetic lesion that characterizes fragile X-affected patients, having a full mutation (at least 285 CGG repeats). When injected into immunocompromised mice, various types of differentiated cells were found in the teratomas. The successful derivation of HEFX1 cell line opens a new avenue for the scientific study of the molecular basis of fragile X syndrome. This work represents the feasibility and importance of deriving human ES cell lines from genetically abnormal pre-embryos, especially in cases where no suitable cellular and/or animal models are available.