Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture.

Zoldan J, Lytton-Jean AK, Karagiannis ED, Deiorio-Haggar K, Bellan LM, Langer R, Anderson DG
Biomaterials. 2011 32 (31): 7793-800

PMID: 21835461 · PMCID: PMC3313656 · DOI:10.1016/j.biomaterials.2011.06.057

Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60-90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27-90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.

Copyright © 2011 Elsevier Ltd. All rights reserved.

MeSH Terms (15)

Animals Cell Culture Techniques Cell Differentiation Cells, Cultured Embryoid Bodies Embryonic Stem Cells Gene Knockdown Techniques Gene Transfer Techniques Green Fluorescent Proteins Humans Mice Protein Interaction Maps Reverse Transcriptase Polymerase Chain Reaction RNA, Small Interfering Viruses

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