Variations in humanized and defined culture conditions supporting derivation of new human embryonic stem cell lines

Fletcher, Judy M., Ferrier, Patricia M., Gardner, John O., Harkness, Linda, Dhanjal, Seema, Serhal, Paul, Harper, Joyce, Delhanty, Joy, Brownstein, David G., Prasad, Yogesh R., Lebkowski, Jane, Mandalam, Ram, Wilmut, Ian and De Sousa, Paul A. (2006) Variations in humanized and defined culture conditions supporting derivation of new human embryonic stem cell lines. Cloning and Stem Cells, 8 4: 319-334. doi:10.1089/clo.2006.8.319

Author Fletcher, Judy M.
Ferrier, Patricia M.
Gardner, John O.
Harkness, Linda
Dhanjal, Seema
Serhal, Paul
Harper, Joyce
Delhanty, Joy
Brownstein, David G.
Prasad, Yogesh R.
Lebkowski, Jane
Mandalam, Ram
Wilmut, Ian
De Sousa, Paul A.
Title Variations in humanized and defined culture conditions supporting derivation of new human embryonic stem cell lines
Journal name Cloning and Stem Cells   Check publisher's open access policy
ISSN 1536-2302
Publication date 2006-12-31
Sub-type Article (original research)
DOI 10.1089/clo.2006.8.319
Open Access Status Not yet assessed
Volume 8
Issue 4
Start page 319
End page 334
Total pages 16
Place of publication New Rochelle, NY, United States
Publisher Mary Ann Liebert
Language eng
Formatted abstract
The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved by integration of a multitude of individual approaches to replace or eliminate specific animal sourced reagents into a single comprehensive protocol. In the present study our objective was to integrate strategies obviating reliance on some of the most poorly defined and path-critical factors associated with hESC derivation, namely the use of animal immune compliment to isolate embryo inner cell mass, and animal sourced serum products and feeder cells to sustain hESC growth and attachment. As a result we report the derivation of six new hESC lines isolated by outgrowth from whole blastocysts on an extracellular matrix substrate of purified human laminin (Ln) with transitional reliance on mitotically inactivated human fibroblast (HDF) feeder cells. With this integrated system hESC lines were isolated using either HDF conditioned medium supplemented with a bovine-sourced serum replacement (bSRM), or a defined serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype and the potential to form cells representative of all three germinal lineages in vitro and in vivo, when transitioned off of feeders onto Laminin or Matrigel™. Our study thus demonstrates the capacity to integrate derivation strategies eliminating a requirement for animal immune compliment and serum products, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Australian Institute for Bioengineering and Nanotechnology Publications
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Citation counts: TR Web of Science Citation Count  Cited 32 times in Thomson Reuters Web of Science Article | Citations
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Created: Wed, 23 Sep 2015, 15:55:29 EST by Linda Harkness on behalf of Learning and Research Services (UQ Library)