title: Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture.
abstract: 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.
authors.affiliation: David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
authors.forename: Janet
authors.initials: J
authors.lastname: Zoldan
authors.forename: Abigail K R
authors.initials: AK
authors.lastname: Lytton-Jean
authors.forename: Emmanouil D
authors.initials: ED
authors.lastname: Karagiannis
authors.forename: Kaila
authors.initials: K
authors.lastname: Deiorio-Haggar
authors.forename: Leon M
authors.initials: LM
authors.lastname: Bellan
authors.forename: Robert
authors.initials: R
authors.lastname: Langer
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Anderson
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D058732:Embryoid Bodies / Q000166:cytology / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D055785:Gene Knockdown Techniques; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D060066:Protein Interaction Maps; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014780:Viruses / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins