Abstract
Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of blastocyst staged embryos. Spare blastocyst staged embryos were obtained by in vitro fertilization (IVF) and donated for research purposes. hESCs carrying specific mutations can be used as a powerful cell system in modeling human genetic disorders. We obtained preimplantation genetic diagnosed (PGD) blastocyst staged embryos with genetic mutations that cause human disorders and derived hESCs from these embryos. We applied laser assisted micromanipulation to isolate the inner cell mass from the blastocysts and plated the ICM onto the mouse embryonic fibroblast cells. Two hESC lines with lesions in FOXP3 and NF1 were established. Both lines maintain a typical undifferentiated hESCs phenotype and present a normal karyotype. The two lines express a panel of pluripotency markers and have the potential to differentiate to the three germ layers in vitro and in vivo. The hESC lines with lesions in FOXP3 and NF1 are available for the scientific community and may serve as an important resource for research into these disease states.
Highlights
Human embryonic stem cells carrying genetic mutations offer the potential to model genetic diseases, especially if the affected tissues can be reliably generated by differentiating the cells in vitro [1]
The preimplantation genetic diagnosis (PGD) embryos affected with a FOXP3 (IVS8+5G>A) mutation and the PGD embryos heterozygous for a NF1 (IVS1+1G>C) mutation were used for the derivation of Human embryonic stem cells (hESCs) lines with lesions in FOXP3 and NF1
One inner cell mass (ICM) from the FOXP3 donations survived with a cellular outgrowth appearing as early as day 2 post plating (Fig 1A-c) and small colony appearing at day 15 (Fig 1A-e)
Summary
Human embryonic stem cells (hESCs) carrying genetic mutations offer the potential to model genetic diseases, especially if the affected tissues can be reliably generated by differentiating the cells in vitro [1]. We derive two hESC lines from PGD embryos diagnosed as containing lesions that cause the monogenic diseases IPEX and neurofibromatosis type 1. One day before PGD embryo dissection, feeder cells were thawed and plated in 10% gelatin coated 4-well tissue culture dishes (Thermo Fisher Scientific, Inc., Waltham, MA) in DMEM medium supplemented with 10% FBS.
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