Hamilton Thorne - Products - XYClone - Transgenic Applications - apps icm

By: Hamilton Thorne  09-12-2011
Keywords: Assisted Hatching

XYClone is for non-clinical research use only. hESC research using the XYClone should be conducted in accordance with any applicable Federal, State or local laws and regulations.

Objective

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.

Conclusion

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.


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

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 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.

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.

Keywords: Assisted Hatching

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Hamilton Thorne - Products - XYClone - Transgenic Applications - Developmental Biology

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Hamilton Thorne - Products - XYClone - Transgenic Applications - apps lah

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Hamilton Thorne - Products - XYClone - Transgenic Applications - apps transgenic

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