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.

Figure 1

Knockdown of GFP expression in GFP-hESC and GFP-hEB seeded scaffolds: A) Bright filed micrographs of GFP-hESC and GFP-hEB following 2 days and 3 days post seeding on scaffolds containing lipidoid- siGFP, lipid-siScrambled (vi) or untreated (iv, v). B) Live cell staining of GFP-hESC seeded scaffolds containing lipidoid-siGFP complexes. Live cells are stained in red, GFP-hESCs in green and colocalized live GFP-hESCs cells in yellow.

Figure 2

Quantitative analysis of GFP knockdown in GFP-hESC and GFP-hEB seeded scaffolds: A) FACS analysis of GFP-hESCs grown for three days on 3D scaffolds containing lipidiod-siGFP and untreated. B) hEB viability measured by PI staining after growing in scaffolds for 2 and 3 days;. C) GFP expression of hESCs and hEBs measured by FACS. D) GFP expression of hESCs and hEBs measured by rt-qPCR normalized to untreated hESCs grown on 3D scaffolds. E) Dose response of hESCs treated with lipidoid-siGFP in 2D and 3D.

Figure 3

RT-qPCR analysis of hESCs differentiation to the three germ layers following 3D delivery of (a) lipidoid-siKDR, and (b) lipidoid-siGAPDH. The siKDR upregulates the mesodermal genes with concomitant downregulation of the endoderm specific genes. Gene expression was normalized to untreated hESCs grown on 3D scaffolds.

Figure 4

A) Protein-protein interaction network that leads to the expression of the gastrulation specific genes during KDR siRNA knockout. We then constructed a KDR reduced network introducing all genes that can preserve the connectivity of the network. These genes were identified as responsible for the expression of the gastrulation specific transcription factors once KDR is absent. B) Role of androgen receptor (AR): RT-qPCR of hESC gene expression following 3D delivery of lipidoid-siAR. C) Role of integrins: RT-qPCR of hESC gene expression following 3D delivery of lipidoid-siKDR in the presence of synthetic RGD peptide blocking integrins.