Integration-Free iPS Cells Engineered Using Human Artificial Chromosome Vectors

Abstract

Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.

Stable maintenance of de novo assembled human artificial chromosomes in embryonic stem cells and their differentiated progeny in mice

“…3,[6][7][8] This HAC has also been successfully used to deliver reprogramming factors, but its subsequent removal from de novo generated iPS cells is quite problematic and relies on spontaneous loss during mitotic divisions, which is extremely rare. 9 A novel, truly artificial HAC has recently come to the fore as a highly promising vector system. This HAC has been assembled de novo from a synthetic alphoid DNA array with embedded tetracycline operator (tetO) that binds tet-repressor fusion proteins, providing the option to incorporate conditional inhibition of kinetochore assembly, resulting in subsequent loss of the HAC from populations of dividing cells.…”

Generation of induced pluripotent stem cells by using a mammalian artificial chromosome expression system

“…The suppression of the p53 pathway [11], the determination of the optimal protein concentration or optimal ratio of different proteins, or the application of small molecules [11,13,21,24] would also probably improve the reprogramming efficiency. An alternative method of reprogramming mouse embryonic fibroblasts into iPS with engineered human artificial chromosome vector had recently been described [10]. The lack of DNA-based manipulations, which may generate unexpected and uncontrollable insertional mutagenesis of the host genome, is an advantage of our method.…”

Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models

“…HACs/MACs generally harbor loxP sites for gene loading. In system (b), circular vectors such as plasmids, P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) were successfully loaded onto HACs/MACs by the Cre/loxP system for various purposes including functional analysis, monitoring system development, and gene therapy [58][59][60][61][62][63][64] (Fig. 3b).…”