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.

Reprogramming somatic cells to become induced pluripotent stem (iPS) cells is important in making regenerative medicine a reality [1]-[3]. The best iPS cells for therapeutic applications are derived from cells harvested from individual patients and the reprogramming should not involve permanent genetic changes because strategies involving insertional modifications of the genome increase the risk of insertional mutagenesis [4] and perturbation of differentiation potential [5]. To avoid permanent, detrimental modification of the host genome while reprogramming somatic cells, several vectors and protocols that exclude permanent transgene integration into the host genome have been developed: the piggyBac transposon [6]-[8], adenovirus vectors [9], Sendai virus vectors [10], EB-derived episomal vectors [11] and iterant administration of non-replicative materials (i.e. plasmid [12], minicircle DNA [13], protein [14], and synthetic modified mRNA [15]). However, these vectors and methods should be scrutinized with regard to quality of individual iPS cells, reprogramming efficiency and genome integrity. In addition, iPS cells should have a safeguard system because iPS cells with teratoma-forming potential can persist even after differentiation, leading to unexpected and undesired events [16].

With respect to the generation of iPS cells, human artificial chromosomes (HACs) have two important and unique characteristics as gene-delivery vectors; effectively unlimited carrying capacity for transgenic material and autonomous maintenance through cell division that is independent of host chromosomes. We have created several HAC vectors from human chromosome 21 using a top down method [17], [18] and have demonstrated that full-length genomic loci, such as DMD [19], HPRT [20] and p53 [20] could be cloned into a defined HAC cloning site. We have also shown that these loci are efficiently transcribed. Moreover, expression in human cells of cDNAs introduced into HACs was more stable and sustained and less subject to position effects [21] than expression of cDNAs from conventional plasmids and viral vectors. In addition, our HAC vectors encode EGFP [18]; therefore, because HACs are lost spontaneously at a low frequency [22] we can isolate HAC-free cells from reprogrammed iPS populations by identifying EGFP-negative cells.

Here, we have taken advantage of these features of HAC vectors to generate vector-free and transgene-free iPS cells. Recent attempts to generate iPS cells using polycistronic vectors to express multiple proteins demonstrated that a significant portion of the iPS clones carried more than two copies of the polycistronic vector [6], [8], [23], [24], suggesting that multiple copies of the polycistronic transgenes were needed to generate iPS cells. Thus, we devised a reprogramming cassette with four defined reprogramming factors and introduced multiple copies of the cassette into the cloning site of a HAC vector. We constructed a closely related cassette by adding a p53 short hairpin RNA (shRNA) expression construct to the four-factor cassette because suppression of the p53 pathway leads to more efficient reprogramming [25]-[29]. Moreover, our HAC vector encodes Herpes Simplex virus thymidine kinase (HSV-TK), and we confirmed that iPS cells and/or their differentiated derivatives carrying our HAC can be killed by ganciclovir (GCV), providing a safeguard system if unexpected events (e.g., tumor formation) occur.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Tottori University (the permit number: 08-Y-69).

We constructed individual expression cassettes for each reprogramming factor in pBSII (Stratagene) and combined all cassettes into a pPAC4 backbone as follows. The pBSII multiple cloning site was replaced with either KpnI-XhoI-AscI-BsiWI-NheI-ClaI-SalI-PstI-AvrII-PmeI-FseI-XbaI-SpeI-SacI or KpnI-XhoI-AscI-BsiWI-NheI-ClaI-SalI-MluI-SphI-SnaBI-NotI-SacII-BamHI-AvrII-PmeI-FseI-XbaI-SpeI-SacI, resulting in the vectors pB3 and pB4, respectively. Two different 1.2 kb fragments of the chicken HS4 insulator were excised by either SacI or XbaI digestion of pCJ5-4 (a gift from Dr. G. Felsenfeld, National Institutes of Health, Bethesda, MD, USA), blunted by KOD polymerase (Toyobo), and cloned into the SmaI site or the blunted HindIII site of pBSII, respectively. The resulting vector, harboring 2 copies of HS4, was called pBSI-I. A ClaI-BamHI fragment of pBSI-I was cloned into (1) a blunted ClaI site of pB3, (2) a blunted ClaI site of pB4, or (3) blunted ClaI and PmeI sites of pB4, resulting in (1) pinsB3, (2) pinsB4 and (3) pB4ins2, all of which retained the BamHI site immediately downstream of the HS4 dimer. All subcloned HS4 insulators had the same orientation.

Mouse Klf-4, c-Myc, Sox2 and Oct4 were PCR-amplified and individually cloned into the EcoRI site of pCAGGS (a gift from Dr. M. Okabe, Osaka University, Japan), resulting in pCX-Klf4, pCX-c-Myc, pCX-Sox2 and pCX-Oct3/4, respectively. SalI-BamHI fragments of pCX-Klf4, pCX-c-Myc and pCX-Sox2 were blunted and cloned into blunted BamHI sites of pinsB4, resulting in pB4K, pB4M and pB4S, respectively; an SspI-BamHI fragment of pCX-Oct3/4 was cloned into a SnaBI site of pB4ins2, resulting in pB4O. To combine four factors in a single vector, AscI-AvrII fragments from pB4K and pB4S were inserted into the AscI-NheI sites of pB4M and pB4O, resulting in pB4KM and pB4SO, respectively. Finally, an AscI-AvrII fragment of pB4KM and an NheI-FseI fragment of pB4SO were ligated into the AscI and FseI sites of pPH3-9, which was generated by modifying pPAC4; specifically we exchanged the region between the pUC link and the CMV promoter with HPRT ex3-ex9 and added an FseI site immediately downstream of HPRT ex9. The resulting vector was designated pPAC-KMSO. This KMSO reprogramming cassette was duplicated by the same strategy, resulting in pPAC-2CAG-KMSO. A fragment of the duplicated pB4O was cloned into the AscI-NheI site of pPAC-2CAG-KMSO, resulting in pPAC-2CAG-O2.

A mouse p53-knockdown construct was generated by annealing two complementary synthetic oligonucleotides with the target sequence GTACATGTGTAATAGCTCC and cloning the product into the BglII-XbaI sites of pENTR4-H1 (a gift from Dr. H. Miyoshi, RIKEN, Japan), resulting in pENTR4-H1-mp53sh. A SalI-XbaI fragment of pENTR4-H1-mp53sh was inserted into the SalI-AvrII site of pinsB3, resulting in pinsB3mp53sh. Finally, an AscI-SpeI fragment of pinsB3mp53sh was inserted into the AscI-NheI site of pPAC-2CAG-O2, resulting in pPAC-2CAG-O2mp53sh.

Hprt-deficient Chinese hamster ovary cells (JCRB0218, JCRB Cell Bank, Japan) each bearing a HAC vector, (CHO(21HAC2), CHO/iHAC1/E15 and CHO/iHAC2/mp25) were maintained at 37C in Hams F-12 nutrient mixture (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 8 /ml Blasticidin S (Funakoshi). Mouse embryonic fibroblasts (MEFs), isolated from 13.5 day post-coitum (d.p.c.) wild-type embryos (C57BL/6-J), were grown in Dulbeccos modified Eagles medium (DMEM) (Sigma) plus 10% FBS. The mouse ES cell lines, TT2 (a gift from Dr. S. Aizawa, RIKEN, Japan) [30] and B6ES (DAINIPPON SUMITOMO PHARMA, Osaka, Japan), and the microcell hybrid clones, were maintained on mitomycin C-treated Jcl:ICR (CLEA Japan) MEF feeder layers in ES medium [DMEM with 18% FBS (Hyclone), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine (Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma), and 1000 U/ml leukemia inhibitory factor (LIF) (Millipore)].

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Integration-Free iPS Cells Engineered Using Human ...

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