Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.
BACKGROUND
Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products.


METHODS
Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines.


RESULTS
eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers.


CONCLUSIONS
These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.
Turetsky
T
T
The Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Medical Center, Ein Kerem, PO BOX 12000, Jerusalem 91120, Israel.
Aizenman
E
E
Gil
Y
Y
Weinberg
N
N
Shufaro
Y
Y
Revel
A
A
Laufer
N
N
Simon
A
A
Abeliovich
D
D
Reubinoff
B E
BE
D015415:Biomarkers / Q000378:metabolism; D053624:Blastocyst Inner Cell Mass / Q000473:pathology; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000196:embryology / Q000235:genetics / Q000473:pathology; D004210:Dissection / Q000379:methods*; D004622:Embryo, Mammalian / Q000473:pathology*; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000473:pathology*; D005307:Fertilization in Vitro; D005600:Fragile X Syndrome / Q000175:diagnosis / Q000196:embryology / Q000473:pathology; D006467:Hemophilia A / Q000175:diagnosis / Q000196:embryology / Q000473:pathology; D006579:Heterozygote; D006801:Humans; D007834:Lasers; D009223:Myotonic Dystrophy / Q000175:diagnosis / Q000196:embryology / Q000473:pathology; D039904:Pluripotent Stem Cells / Q000378:metabolism; D019836:Preimplantation Diagnosis
D015415:Biomarkers