Inner Cell Mass Isolation
Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model.
Noriko Tanaka, Takumi Takeuchi, Queenie V Neri, Eric Scott Sills and Gianpiero D Palermo. Journal of Translational Medicine 2006, 4 : 20 doi:10.1186/1479-5876-4-20
The key role of mouse embryonic fibroblasts (MEF) is recognized as the feeder cell layer for establishing ESCs. However, feeder cell culture is technically involving and more importantly is a source of contamination with xenogenic by-products. Recently, it has been identified that human ESCs are coated with immunogenic sialic acid. Moreover, the adoption of methods aimed at directly isolating inner cell masses (ICM) by immunosurgery, entails exposure to allogenic antibodies and complement. Therefore, the avoidance of feeder cells and serum-free media as well as the utilization of mechanical ICM dissection would be ideal. In this study, we attempted mouse ESC derivation without feeder cells and sera. To facilitate ICM isolation, laser-assisted blastocyst dissection was performed.
With laser-assisted ICM dissection, we were able to obtain in standard culture conditions, supported by feeder cells, ESC colonies at a similar rate than intact blastocysts. With the same method, we were able to generate an ESC line in feeder cell- and serum-free culture system. This preliminary data represents a feasible model to establish ESC lines in a controlled and xenogenic by-product free environment.
Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.
T. Turetsky, E. Aizenman, Y. Gil, N. Weinberg, Y. Shufaro, A. Revel, N. Laufer, A. Simon, D. Abeliovich and B.E. Reubinoff. Human Reproduction. Advanced Access, November 2007doi:10.1093/humrep/dem351
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.
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.
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 40511G>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.
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.