Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells

Abstract

High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. Disclosure of potential conflicts of interest is found at the end of this article.

Figure 1

Optimization of nucleofection in hES cells. (A): The mean percentage of colonies (±SEM; n = 3) that were GFP-positive after nucleofection of collagenase-dissociated hES cells in V solution (blue bars) or mES solution (green bars). (B): The percentage of colonies (±SEM; n = 3) that were GFP-positive after nucleofection of trypsin-dissociated hES cells in V solution (yellow bars) or mES solution (red bars). (C): The mean percentage of GFP-positive cells (n = 5) in each colony after nucleofection of collagenase-derived (blue and green bars) and trypsin-derived (yellow and red bars) hES cells using program A-23 in either V solution (blue and yellow bars) or mES solution (green and red bars). (D): BF (top) and GFP expression (bottom) of transfected collagenase-dissociated hES cells and trypsin-dissociated hES cells 4 days after nucleofection. Abbreviations: BF, bright-field; GFP, green fluorescent protein; hES, human embryonic stem; mES, mouse embryonic stem.

Figure 2

Nucleofection of hES cells with DNA, shRNA vectors, or siRNAs alters gene expression. (A): Flow cytometric analysis of GFP and SSEA-4 expression in H1 and H9 hES cells 4 days after transfection with the GFP vector (middle and right) or no DNA (left). (B): BF (top) and GFP expression (bottom) of hES cells 4 days after co-transfection of the GFP vector and either β2M siRNA or Oct4 siRNA. GFP expression was used to identify transfected cells. (C): BF (top) and SSEA-4 (bottom) immunostaining of β2M KD and Oct4 KD hES cells. (D): AP staining of β2M KD and Oct4 KD hES cells. (E): Reverse transcription-polymerase chain reaction analysis of hES cells transfected with either shRNA vectors or siRNAs. β-Actin was used as a template loading control. Abbreviations: AP, alkaline phosphatase; BF, bright-field; DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; hES, human embryonic stem; KD, knockdown.

Figure 3

GFP expression in stable GFP-neo cell lines. (A): BF (left) and GFP (right) expression of hES cells stably transfected with the GFP-neo vector (GFP-neo cells). (B): Clonal cell lines derived from GFP-neo cells grown with or without G418. (C): Polymerase chain reaction analysis of genomic DNA isolated from clonal cell lines. Both GFP-positive and GFP-negative clones contain the CMV promoter and hrGFP transgene contiguously in their genomes. Untransfected hES cells served as a control, and β-actin was used a template loading control. Abbreviations: BF, bright-field; CMV, cytomegalovirus; GFP, green fluorescent protein; hES, human embryonic stem; NT, neurotrophin.

Figure 4

Stably transfected clonal hES cell lines retain stem cell characteristics, including expression of stem cell markers, normal karyotype, and the ability to differentiate in vitro and in vivo. (A): DAPI (top), GFP (middle), Oct4 (bottom left), and SSEA-4 (bottom right) staining of GFP-neo hES cells. (B): AP staining (left) and karyotype (right) of GFP-neo hES cells after 25 passages. (C): BF (left) and GFP expression (right) of EBs derived from GFP-neo cells. (D): Hematoxylin and eosin (H&E; top panels), GFP, and DAPI staining (bottom panels) of sectioned GFP-neo teratomas. (E): Gross image of teratomas formed from GFP-neo hES cell clones. Abbreviations: AP, alkaline phosphatase; BF, bright-field; DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein.

Figure 5

shRNA-mediated knockdown of AP does not affect the expression of other stem cell genes. (A): Reverse transcription (RT)-polymerase chain reaction (PCR) analysis of AP KD and β2M KD hES cells. GFP-neo and untransfected hES cells were used as controls. β-Actin was used a template loading control. (B): BF (top), GFP (middle), and AP (bottom) staining of AP KD and β2M KD hES cells. (C): RT-PCR analysis of AP KD and β2M KD hES cells and EBs. GFP-neo cells served as a control, and β-actin was used a template loading control. Abbreviations: αMHC, alpha myosin heavy chain; AFP, alpha fetoprotein; AP, alkaline phosphatase; BF, bright-field; GCM1, glial cells missing-1; GFP, green fluorescent protein; hES, human embryonic stem; KD, knockdown.