Engineering of human pluripotent stem cells by AAV-mediated gene targeting

Abstract

Precise genetic manipulation of human pluripotent stem cells will be required to realize their scientific and therapeutic potential. Here, we show that adeno-associated virus (AAV) gene targeting vectors can be used to genetically engineer human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Different types of sequence-specific changes, including the creation and correction of mutations, were introduced into the human HPRT1 and HMGA1 genes (HPRT1 mutations being responsible for Lesch-Nyhan syndrome). Gene targeting occurred at high frequencies in both ESCs and iPSCs, with over 1% of all colony-forming units (CFUs) undergoing targeting in some experiments. AAV vectors could also be used to target genes in human fibroblasts that were subsequently used to derive iPSCs. Accurate and efficient targeting took place with minimal or no cytotoxicity, and most of the gene-targeted stem cells produced were euploid and pluripotent.

Figure 1

HPRT1 targeting in ESCs and iPSCs. (a) The structures of the AAV2-HPe3PNA targeting vector and human HPRT1 gene with Southern blot probes and enzymes are shown. (b) G418-resistant CFU obtained after infection with AAV2-HPe3PNA at the indicated MOIs, with portion due to targeted and random integration shown. (c) Southern blot performed on transduced, G418-resistant clones and parental BG01 ESCs, probed with HPRT1 or neo sequences. Clones with random (6TG-sensitive) and targeted (6TG-resistant) integration events are shown. CFU, colony-forming unit; hESC, human embryonic stem cell; iPSC, induced pluripotent stem cell; kb, kilobase; MOI, multiplicity of infection; PGK, phosphoglycerate kinase.

Figure 2

HMGA1 gene targeting. (a) The structure of the AAV2-HMGA1-HyTKpA targeting vector and human HMGA1 gene are shown with homologies indicated. (b) Hygromycin-resistant colony-forming unit obtained after infecting the indicated hESC line with AAV2-HMGA1-HyTKpA at a multiplicity of infection of 20,000. Number of independent experiments is indicated. (c) Examples of Southern blots are performed on transduced, hygromycin-resistant clones and parental H1 cells. Kpn I restriction sites and genomic HMGA1 probe location are shown in a. CFU, colony-forming unit; ESC, embryonic stem cell; kb, kilobase.

Figure 3

Targeting in fibroblasts and mesenchymal stem cells used to derive iPSCs. (a) The structures of the AAV2-HPe3(+4) targeting vector and human HPRT1 gene with Southern blot probes and enzymes are shown. (b) Southern blot of six iPSC clones derived from gene-targeted MHF2 fibroblasts with control of nontargeted iPSC-MHF2 c2 DNA and MHF2 DNA. iPSC, induced pluripotent stem cell; kb, kilobase.

Figure 4

Pluripotency of gene-targeted iPSCs. (a) RT-PCR amplification of pluripotency-specific markers, lineage-specific markers, and GAPDH controls in RNA from the indicated iPSC clones and control cells. For lineage-specific markers, iPSC and ESC RNA were isolated from undifferentiated and embryoid body–differentiated (U and D, respectively) cells. (b) Phase-contrast images of embryoid bodies derived from the indicated cell lines. (c,d) Histological sections from teratomas formed by gene-targeted iPSC-HP(+4) c1 (c) and c2 (d), stained with antibodies against the indicated proteins and DAPI, with serial sections stained with hematoxylin and eosin (H&E). Bar = 60 microns. AFP, α-fetoprotein; ESC, embryonic stem cell; iPSC, induced pluripotent stem cell; MSC, mesenchymal stem cell; SMA, smooth muscle actin.

Figure 5

HPRT1 gene correction. (a) Structures of the AAV2-HPe3 targeting vector, and the mutant HPRT1 loci in iPSC-HP(+4) and BG01/HPe3PNA cells are shown with Southern blot probes and enzymes used. (b) HAT-resistant CFU obtained after infection of the indicated cell lines with AAV2-HPe3 at an MOI of 60,000 genome-containing particles/cell. (c) Southern blot of two HAT-resistant gene-targeted iPSC subclones and their parental iPSCs showing corrected HPRT1 loci in subclones after BsoBI digestion and probing as shown in a. (d) HPRT1 exon 3 sequencing of the parental iPSC-HP(+4) c1 and the two HAT-resistant transduced iPSC subclones in c showing corrected wild-type sequence at BsoBI sites. bp, base pair; CFU, colony-forming unit; iPSC, induced pluripotent stem cell; kb, kilobase; PGK, phosphoglycerate kinase.

Figure 6

Karyotypes of ESCs and iPSCs. Flowchart indicates derivation of different cell lines, including reprogramming and gene-targeting steps. Karyotypes are printed in cell with bracketed numbers indicating the number of mitoses observed. OSNL signifies OCT4, SOX2, NANOG, and LIN28. hESC, human embryonic stem cell; iPSC, induced pluripotent stem cell; MSC, mesenchymal stem cell.