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First published online September 1, 2005
Stem Cells Vol. 23 No. 10 November 2005, pp. 1608 -1616
doi:10.1634/stemcells.2005-0021; www.StemCells.com
© 2005 AlphaMed Press

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A Novel Human Artificial Chromosome Vector Provides Effective Cell Lineage–Specific Transgene Expression in Human Mesenchymal Stem Cells

Xianying Rena, Motonobu Katohb, Hidetoshi Hoshiyac, Akihiro Kurimasac, Toshiaki Inoueb, Fumiaki Ayabec, Kotaro Shibatad, Junya Toguchidad, Mitsuo Oshimuraa,b,c

a Departments of Molecular and Cell Genetics,
b Human Genome Sciences (Kirin Brewery), and
c Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medicine, Tottori University, Yonago, Tottori, Japan;
d Department of Tissue Regeneration, Institute for Frontier Medical Science, Kyoto University, Sakyo-ku, Kyoto, Japan

Key Words. Human artificial chromosome vector • Insulator • Mesenchymal stem cells • Cell lineage–specific transgene expression • Microcell-mediated chromosome transfer • Differentiation

Correspondence: Mitsuo Oshimura, Ph.D., Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan. Telephone: 81-859-348261; Fax: 81-859-348134; e-mail: oshimura{at}grape.med.tottori-u.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mesenchymal stem cells (MSCs) hold promise for use in adult stem cell–mediated gene therapy. One of the major aims of stem cell–mediated gene therapy is to develop vectors that will allow appropriate levels of expression of therapeutic genes along differentiation under physiological regulation of the specialized cells. Human artificial chromosomes (HACs) are stably maintained as independent chromosomes in host cells and should be free from potential insertional mutagenesis problems of conventional transgenes. Therefore, HACs have been proposed as alternative implements to cell-mediated gene therapy. Previously, we constructed a novel HAC, termed 21 {Delta}pq HAC, with a loxP site in which circular DNA can be reproducibly inserted by the Cre/loxP system. We here assessed the feasibility of lineage-specific transgene expression by the 21{Delta}pq HAC vector using an in vitro differentiation system with an MSC cell line, hiMSCs, which has potential for osteogenic, chondrogenic, and adipogenic differentiation. An enhanced green fluorescent protein (EGFP) gene driven by a promoter for osteogenic lineage-specific osteopontin (OPN) gene was inserted onto the 21 {Delta}pq HAC and then transferred into hiMSC. The expression cassette was flanked by the chicken HS4 insulators to block promoter interference from adjacent drug-resistant genes. The EGFP gene was specifically expressed in the hiMSC that differentiated into osteocytes in coordination with the transcription of endogenous OPN gene but was not expressed after adipogenic differentiation induction or in noninduction culture. These results suggest that use of the HAC vector is suitable for regulated expression of transgenes in stem cell–mediated gene therapy.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In cell-based gene therapy, human bone marrow–derived mesenchymal stem cells (MSCs) draw attention as a potential progenitor cell source for repair and regeneration of diverse adult tissues, including fat, cartilage, bone, marrow stroma, and skeletal muscle [1–3]. Recent studies have shown that the MSCs can regenerate myocardium, liver, neural tissue, and hepatocytes by cell fusion [4–10]. Their accessibility, ease of expansion, and manipulation in vitro has made MSCs an attractive delivery vehicle for applications of cell-based gene therapy.

Numerous studies have been performed using MSCs as platforms for the systemic delivery of therapeutic genes in vivo by viral vector systems [11–17]. Transgene production mainly achieved by the use of viral promoters evokes high-level gene expression in all cell types transduced. However, achieving high expression is not the only goal of gene therapy, including stem cell–based gene therapy. Because inappropriate doses, timing, or localization of transgene expression can result in potentially harmful effects, providing appropriate levels of expression of therapeutic genes along differentiation under physiological regulation in the specialized cells is particularly important. One of the major challenges is to develop vectors that will allow appropriate levels of spatial and temporal expression of therapeutic genes.

Human artificial chromosomes (HACs) that rely on the mechanisms governing replication and accurate segregation of natural chromosomes offer a new approach for creating gene delivery vectors with potential therapeutic applications [18–20]. Because they are maintained as independent chromosomes in host cells, they should be free from potential random transgene integration into the host genome and silencing of transgene expression caused by chromosomal position effect. In a previous study, we constructed a novel HAC from human chromosome 21 by telomere-directed chromosome breakage [21]. It contains a single loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. The HAC, designated 21{Delta}pq HAC vector, was mitotically stable in human cells and achieved reproducible introduction and expression of enhanced green fluorescent protein (EGFP) gene driven by human cytomegalovirus (hCMV) promoter. It was proven that introduction of transgenes into specific, single, defined sites within the 21{Delta}pq HAC vector provides competence to create a genetic environment for reproducible transgene expression. From the features mentioned above, the 21{Delta}pq HAC vector may be feasible to be developed for a transgene regulation system.

Here we addressed whether the 21{Delta}pq HAC vector can provide inducible transgene expression using an in vitro differentiation system with a human bone marrow–derived MSC cell line. To this end, we made reporter gene constructs with EGFP genes driven by the promoter from osteopontin (OPN) gene [22], whose expression is upregulated along differentiation of MSCs into osteoblastic lineage [23]. Two types of 21{Delta}pq HAC vectors, with or without insulators in both sides of OPN-EGFP expression units, were constructed and transferred into an MSC cell line: hiMSCs [24] that are capable of in vitro tridirectional differentiation into chondrocytes, adipocytes, and osteocytes in response to appropriate culture stimuli. Expression status of the reporter gene in the hiMSC hybrids containing these HAC vectors was tested before and after induction of osteogenic and adipogenic differentiation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
A Chinese hamster ovary (CHO) hybrid cell line containing the 21{Delta}pq HAC vector [21] H8 was maintained in Ham’s F-12 nutrient mixture (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 10% calf serum (HyClone, Logan, UT, http://www.hyclone.com) and 8 µg/ml blasticid in Shydro chloride (Funakoshi, Tokyo, http://www.funakoshi.co.jp). A human immortalized mesenchymal stem cell line (hiMSC) [24] was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) supplemented with 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS, www.jrhbio.com) and incubated at 37°C with 5% CO2

Construction and Transfection of OPN-EGFP Reporter Construct
The plasmid pSVmCAT-124, which contains the OPN gene promoter [22], was kindly provided by S. Yamamoto (Oita Medical University, Oita, Japan), and pBS226 vector containing hCMV-loxP-5'neo sequence [25] was purchased from Invitrogen.

The 250-bp insulator core of chicken ß-globin 5'cHS4 region containing a CTCF binding site was obtained from DT40 genomic DNA by a two-step polymerase chain reaction (PCR), and three copies of the insulator core were ligated and cloned into pCR4-TOPO (Invitrogen) to generate pIN-J2 [26]. pBS226/In-OPN-EGFP reporter construct with insulator was constructed as follows. A 750-bp EcoRI fragment of pIN-J2 carrying the insulator was inserted into the EcoRI site of pBS226, followed by changing the BamHI site into NheI site to generate pBS226/In. A XhoI/SalI fragment carrying the 750-bp insulator from pIN-J2 was inserted into the XhoI site of pEGFP-N1 (Clontech, Palo Alto, CA, http://www.clontech.com), followed by changing the DraIII site into the SalI site to generate pEGFP-N1/I. Then a SalI/NheI fragment of pEGFP-N1/I was inserted into SalI/NheI-digested pBS226/In to generate pBS226/In-EGFP. A 214-bp fragment containing the OPN promoter was amplified from pSVmCAT-124 by PCR with the primer pair 5'-ATACCGCGGGGGGAAGTGTGGGAGCAG and 5'-AAAGATCTCCTTGGTCGGCGTTTGGC containing SacII and BglII restriction sites. The PCR products were then digested with these enzymes and cloned into SacII/BamHI-digested pBS226/EGFP-In plasmid to generate pBS226/In-OPN-EGFP, which contains an OPN-EGFP expression cassette flanked by three copies of the 250-bp core element from cHS4 region at 5' upstream of the chicken ß-globin gene.

pBS226/OPN-EGFP reporter construct without insulator was constructed as follows. A 1.2-kb fragment containing OPN-EGFP sequence was amplified from pBS226/In-OPN-EGFP plasmid by PCR with the primer pair 5'-aaaggatccggggaagtgtgggagcaggt and 3'-tttgaattccctgatagacggtttttcgc containing EcoRI and BamHI restriction sites. The PCR products were then digested with these enzymes and cloned into EcoRI/BamHI-digested pBS226 plasmid.

A total of 4 x 105 H8 cells were cotransfected with 2 µg of reporter construct plasmids and 1 µg of Cre-expression vector pBS185 plasmid using 7.5 µl of Lipofectamine 2000 reagent, according to the supplier’s protocol (Invitrogen). After 24 hours of culture in basic medium, cells were trypsinized and cultured in the presence of G418 (800 µg/ml). Drug-resistant colonies were isolated and expanded for further analysis.

Microcell-Mediated Chromosome Transfer
Introduction of empty 21{Delta}pq HAC/OPN-EGFP or 21{Delta}pq HAC/In-OPN-EGFP into hiMSCs was performed by microcell-mediated chromosome transfer (MMCT) as described [27], with the following modifications. Briefly, CHO hybrid cells containing 21{Delta}pq HAC vector were treated with 0.1 µg/ml colcemid to induce the formation of micronuclei and centrifuged in culture flasks filled with medium containing cytochalasin B (10 µg/ml). Recovered microcells were loaded on hiMSCs and treated with 47% polyethylene glycol (MW 1,000) for 1 minute, followed by extensive washing in serum-free DMEM. After 24 hours of culture in DMEM supplemented with 10% FBS, cells were trypsinized, and 4 x 106 cells were plated into 10 90-mm dishes with selection medium containing blasticidin S hydrochloride (4 µg/ml).

Fluorescence In Situ Hybridization
Fluorescence in situ hybridization (FISH) was carried out according to standard protocol [27]. In single-color FISH, hChr21-derived alphoid DNA p11-4 [28] was labeled with digoxigenin-dUTP for detection of the 21{Delta}pq HAC vector in CHO or hiMSC hybrids. In two-color FISH, in addition to digoxigenin-dUTP–labeled p11-4 probe (gift from H. Masumoto, Nagoya University, Nagoya City, Japan), pBS226/In-OPN-EFGP probe was labeled with biotin-16-dUTP for detection of the OPN-EGFP sequence on 21{Delta}pq HAC vector in CHO or hiMSC hybrids. Images were captured using a fluorescence microscope (Nikon, Tokyo, http://www.nikon.co.jp) equipped with a photometric CCD camera and digitally visualized with an Argus system (Hamamatsu Photonics, Hamamatsu City, Japan, http://www.hamamatsu.com/).

Mitotic Stability Assay
To test the mitotic stability of 21{Delta}pq HAC vector, the hiMSC hybrid cells were divided into two sublines, which were maintained independently either with blasticidin S hydrochloride or without selective agent. Cells were plated 1:8 every 3 days up to population-doubling levels (PDLs) of 100. At various time points, a portion of the culture was harvested for FISH and analyzed for the presence of the 21{Delta}pq HAC vector. Fifty metaphases were scored at each time point.

Induction of Differentiation and Histochemical Staining
The multidirectional differentiation experiments and histochemical staining for osteocytes, chondrocytes, and adipocytes were carried out as described [24] according to the supplier’s protocols (Cambrex Corporation, Walkersville, MD, http://www.cambrex.com).

Osteogenic Differentiation   A total of 3 x 104 cells were seeded in a six-well plate and cultured with 2 ml osteogenesis induction medium. The cultures were maintained for 3 weeks, and the culture medium was replaced every 3 days.

Adipogenic Differentiation   A total of 2 x 105 cells were seeded in 2 ml medium in a six-well plate. When the cells reached confluence, three cycles of induction/maintenance culture stimulated optimal adipogenic differentiation. Each cycle consisted of 3 days of adipogenesis induction culture, followed by 1–3 days of culture in maintenance medium. After three complete cycles of induction/maintenance, the cells were cultured for 7 days more in adipogenic maintenance medium.

Chondrogenic Differentiation   Cells (2.5 x 105) were placed in a 15-ml polypropylene tube, centrifuged at 800 rpm for 5 minutes at room temperature, and resuspended in 0.5 ml of chondrogenic differentiation medium. Cells were recentrifuged and maintained as a small pellet for 21 days. The chondrogenesis induction medium was replaced every 2 days.

After 3 weeks of osteogenic, chondrogenic, or adipogenic induction culture, cells were rinsed twice with phosphate-buffered saline and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Fixed cells were treated with alkaline phosphatase substrate (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) for osteogenic differentiation assay or stained with 0.3% oil red O (Nakalai Tesque, Kyoto, Japan, http://www.nacalai.co.jp/en/) for the adipogenic differentiation assay. In the chondrogenic differentiation assay, the fixed pellet was dehydrated with a series of graded ethanol, cleaned by treatment with xylene, and infiltrated with paraffin. Paraffin-embedded sections were stained with Alcian blue.

Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted by Isogen reagent (NipponGene, Tokyo, http://www.nippongene.com). Reverse transcription (RT)–PCR for detection of OPN and peroxisome proliferator-activated receptor gamma (PPAR{gamma}) was carried out according to a standard protocol using the following primers: sense: 5'-ACCTGCCAGCAACCGAAGTT, antisense: 5'-TGGCTGTGGGTTTCAGCACT; sense: 5'-GAGCCCAAGTTTGAGTTTGC, and anti-sense: 5'-CTGTGAGGACTCAGGGTGGT.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
21{Delta}pq HAC Is Stably Maintained in hiMSC Hybrid Cells
The 21{Delta}pq HAC vector is maintained in CHO hybrids (Fig. 1AGo) [21]. To determine whether the 21{Delta}pq HAC can be stably maintained in hiMSCs throughout mitotic divisions, we transferred the HAC from the CHO hybrids into the hiMSCs by means of MMCT. Four drug-resistant clones (M8 #8-1, M8 #1, M8 #3, and M8 #4) were obtained by selection with blasticidin S hydrochloride. In these clones, retention of the 21{Delta}pq HAC was confirmed by PCR amplifying the blasticidin-resistant gene (data not shown). FISH analysis was performed to test the transfer of the HAC. A single copy of 21{Delta}pq HAC was detected in all metaphases observed (Fig. 1BGo). Neither insertion into host chromosome nor apparent amplification of the 21{Delta}pq HAC was observed. In addition, these clones showed normal karyotype. These data suggested that 21{Delta}pq HAC vector had been transferred into hiMSCs without introducing lesions in the host cells’ chromosomes.



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  		Figure 1. FISH analysis of CHO and hiMSC hybrid cells carrying the 21{Delta}pq HAC vectors or 21{Delta}pq HAC/In-OPN-EGFP vector. Human chromosome 21–derived alphoid DNA probe was hybridized to the 21{Delta}pq HAC vector in (A) H8 and (B) hiMSC hybrid cell-M8#1(red signals with arrows). In the hiMSC hybrid cell, 21{Delta}pq HAC vector (red signal with arrow) was identified as a chromosome fragment whose size was reduced compared with intact chromosome 21. Additional red signals were detected on the centromere region of endogenous human chromosome 21 and chromosome 13, which possess high homology to alphoid satellite on chromosome 21. pBS226/In-OPN-EFGP plasmid probe (green signal) was hybridized to 21{Delta}pq HAC/In-OPN-EGFP vector in (C) CHO hybrid cell H8-In-OPN#9 and (D) hiMSC hybrid cell M8-In-OPN#B72. Chromosomal DNA was counterstained with DAPI (blue). The inset showed enlargement of the 21{Delta}pq HAC vector (C, D) with or (A, B) without the OPN-EGFP insert. Note that the single copy of 21{Delta}pq HAC vector was maintained independently in CHO hybrid cells and hiMSC hybrid cells, without any translocation or insertion to host chromosomes. Background karyotype of the host hiMSC was apparently normal. Images are representative of the results from the other hiMSC hybrid cell lines containing 21{Delta}pq HAC vector. Original magnification x 1,600. Abbreviations: CHO, Chinese hamster ovary; EGFP, enhanced green fluorescent protein; FISH, fluorescence in situ hybridization; HAC, human artificial chromosome; hiMSC, human immortalized mesenchymal stem cell; OPN, osteopontin.

 
To further investigate mitotic stability of the 21{Delta}pq HAC vector in hiMSCs, two sublines from four hybrid clones were independently maintained either in the presence or absence of blasticidin up to PDLs of 100. Metaphase chromosomes were prepared at PDLs of 10, 23, 49, and 100. Retention rate of the HAC vector was then analyzed by FISH. The results are summarized in Table 1Go. A single copy of the 21{Delta}pq HAC vector was constantly observed in most metaphase spreads. Loss rate of the 21{Delta}pq HAC vector was very low at any time point, regardless of the absence or presence of selective pressure. These results suggested that the 21{Delta}pq HAC vector was mitotically stable in hiMSCs.


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  		Table 1. 21{Delta}pq HAC stability in the absence of selective pressure in human bone marrow–derived mesenchymal stem cells
 
21{Delta}pq HAC Vector Does Not Affect Multipotent Differentiation Ability of hiMSCs
The original hiMSC cell line had the ability to differentiate into adipocytes, chondrocytes, and osteocytes [24], but there was a possibility that in vitro serial culture and subcloning procedures by MMCT could prevent differentiation ability of the hiMSCs. To evaluate whether the hiMSC hybrids containing the 21{Delta}pq HAC vector still maintained its differentiation potential, four hybrid clones (M8 #8-1, M8 #1, M8 #3, and M8 #4) at PDLs of 10 were cultured in differentiation induction medium. Histochemical staining assays revealed that these clones differentiated into adipocytes, chondrocytes, and osteocytes (Fig. 2Go), indicating that the presence of 21{Delta}pq HAC vector did not affect the tridirectional differentiation potential of the hiMSCs.



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  		Figure 2. Differentiation of hiMSC hybrids containing 21{Delta}pq HAC vector. The cells were induced to differentiate into adipocytes, chondrocytes, and osteocytes. Adipogenesis (A), chondrogenesis (B), and (C) osteogenesis were indicated by the histochemical staining with oil red O and alcian blue and by the detection of alkaline phosphatase activity, respectively. It was suggested that the introduction of the 21{Delta}pq HAC vector did not affect the pluripotent stem cell phenotype of hiMSC. The images show details of histochemical staining from M8#1, representative of the results from three other hiMSC hybrid cell lines containing 21{Delta}pq HAC vector. Original magnification x200. Abbreviations: HAC, human artificial chromosome; hiMSCs, human immortalized mesenchymal stem cells.

 
Site-Specific Insertion of the OPN-EGFP Reporter Gene into the 21{Delta}pq HAC and Transfer into hiMSCs
To investigate whether the 21{Delta}pq HAC vector implements regulation of a transgene expression in differentiated hiMSCs, we constructed an EGFP reporter gene under the control of lineage-specific transcriptional regulatory elements. For this purpose, we chose a 0.2-kb fragment from the upstream region of the human OPN gene, which showed promoter activity [22].

On the 21{Delta}pq HAC vector, acceptor loxP site is surrounded by viral promoters for driving drug-resistant genes (Fig. 3Go). Although the EGFP reporter gene was efficiently expressed from the 21{Delta}pq HAC vector housed in an HT1080 hybrid [21], there was still a possibility that surrounding sequences may interfere with lineage-specific transcriptional regulation [29]. To prevent such interferences, in reporter construct pBS226/In-OPN-EGFP, three copies of 250-bp insulator sequences from cHS4 region at 5'-upstream of chicken ß-globin gene [29] were positioned at both sides of the transcriptional units of OPN-EGFP. Another reporter construct, pBS226/OPN-EGFP without insulator, was used as a control.



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  		Figure 3. Site-specific insertion of pBS226/OPN-EGFP reporter construct or pBS226/In-OPN-EGFP reporter construct into the loxP site on the 21{Delta}pq HAC vector. In pBS226/OPN-EGFP, a circular targeting construct carries EGFP gene driven by OPN promoter; in pBS226/In-OPN-EGFP, the OPN-EGFP expression unit was flanked on both sides with the insulators, hCMV promoter for neo gene, and the loxP sequence (top). Cre-recombinase–mediated site-specific integration of the targeting construct into the loxP site on the HAC vector (middle) regenerates a functional neo gene on the HAC vector. The resulting inserted allele is shown at the bottom. Cotransfection of CHO hybrids harboring 21{Delta}pq HAC vector with the reporter construct and Cre recombinase expression vector yielded G418-resistant transfectants. Abbreviations: CHO, Chinese hamster ovary; EGFP, enhanced green fluorescent protein; HAC, human artificial chromosome; hCMV, human cytomegalovirus; OPN, osteopontin.

 
Because the hiMSCs were tagged with a neo gene during the immortalization process [24], G418 resistance was not applicable as a selection marker for hiMSCs that underwent site-directed insertion of the gene of interest into the 21{Delta}pq HAC vector. We therefore inserted the pBS226/OPN-EGFP or pBS226/In-OPN-EGFP reporter constructs into the 21{Delta}pq HAC vector first in CHO hybrid clone H8 and then transferred these HAC vectors into hiMSCs by MMCT. The H8 cells were cotransfected with these reporter constructs and Cre-recombinase expression vector (Fig. 3Go).

When the H8 cells were transfected by pBS226/In-OPN-EGFP vector, eight G418-resistant hybrids (H8-In-OPN#1, #3, #4, #5, #6, #7, #8, and #9) were obtained. PCR amplifying the OPN-EGFP expression unit was performed for identifying the insertion event on the 21{Delta}pq HAC vector. Three of eight clones (H8-In-OPN#3, #7, and #9) showed the expected size bands (data not shown). Southern blot using a GFP probe revealed correct insertion in these three clones (Fig. 4AGo). In two-color FISH analysis, a single independent 21{Delta}pq HAC/In-OPN-EGFP vector was observed (Fig. 1CGo), suggesting that the 21{Delta}pq HAC was successfully transferred.



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  		Figure 4. Southern analysis for site-specific insertion of pBS226/In-OPN-EGFP constructs into the loxP site on the 21{Delta}pq HAC in H8 cells (A) and detection of the 21{Delta}pq HAC/In-OPN-EGFP vector in hiMSC hybrid cells after MMCT (B). A neo probe was hybridized to BamHI-digested genomic DNA. A 3.3-kb fragment from the wild-type allele was replaced with a 9.6-kb fragment in successfully targeted transfectants H8-In-OPN#3, #7, and #9 (A). An EGFP probe was hybridized to Bst XI-digested genomic DNA. A 9.7 kb fragment is detected in all successfully transferred M8-In-OPN clones except M8-In-OPN#E-4(b). Abbreviations: EGFP, enhanced green fluorescent protein; HAC, human artificial chromosome; hiMSC, human immortalized mesenchymal stem cell; MMCT, microcell-mediated chromosome transfer; OPN, osteopontin.

 
Among these three clones, we arbitrarily chose clone H8-In-OPN#9 as the donor of the 21{Delta}pq HAC/In-OPN-EGFP vector and transferred this vector to hiMSC. Sixteen resistant clones (M8-In-OPN#B-11, #B-12, #B-13, #B-42, #B-43, #B-51, #B-52, #B-61, #B-71, #B-72, #B-92, #B-101, #C-11, #E-3, #E-4, and #E-6) were obtained by selection with blasticidin S hydrochloride, and no clones expressed EGFP in maintenance culture. PCR was performed to verify intact OPN-EGFP gene expression unit. PCR results revealed that the OPN-EGFP expression unit was retained in all clones (data not shown). Southern blot with the EGFP probe confirmed the presence of the In-OPN-EGFP construct in all but one clone (Fig. 4BGo). FISH analysis showed the presence of a single, independent 21{Delta}pq HAC vector in the hiMSC hybrid cells (Fig. 1DGo). These data indicate that the intact 21{Delta}pq HAC/In-OPN-EGFP vector was successfully transferred into the hiMSC. It was noted that EGFP expression was not observed in maintenance culture (data not shown).

On the other hand, we prepared the HAC vector without insulator. Transfection of the H8 cells with pBS226/OPN-EGFP vector yielded eight G418-resistant clones. Presence of OPN-EGFP expression unit was confirmed by PCR (data not shown). One of these clones was arbitrarily chosen as the donor of the 21{Delta}pq HAC/OPN-EGFP vector, and MMCT was performed. Four drug-resistant hiMSC clones were obtained (M8-OPN#A7, #C1, #C2, and #C4). Among them, two clones (M8-OPN#C1 and #C4) expressed EGFP, even in noninduction culture (supplemental online Fig. 1Go). The EGFP expression in clone M8-OPN#C1 and #C4 may result from interference of hCMV in adjacent genes on the same HAC vector. Thus, we used only the hiMSC hybrids carrying the 21{Delta}pq HAC/In-OPN-EGFP for the following studies.

Osteocyte-Specific Transgene Expression After Differentiation of hiMSC Hybrids
As described above, the hiMSC hybrids containing 21{Delta}pq HAC vector retained the ability to differentiate into multiple lineages. To investigate whether 21{Delta}pq HAC vector can mediate cell lineage–specific transgene expression, the hiMSC hybrids containing 21{Delta}pq HAC/In-OPN-EGFP vector were induced to differentiate into osteocytes and adipocytes. Four hiMSC hybrid clones (M8-In-OPN#B-51, #B-61, #B-72, and #B-92) were arbitrarily chosen, split into three sublines, and cultured independently in osteogenic induction, adipogenic induction, or noninduction medium. Evidence for osteogenic and adipogenic differentiation was obtained by lineage-specific histochemical staining for alkaline phosphatase activity and oil red O staining, respectively. Notably, EGFP fluorescence was observed only in the cells cultured in osteogenic induction culture (Fig. 5AiiGo). The EGFP expression level increased in a time-dependent manner, and EGFP fluorescence was observed in the cell fraction expressing alkaline phosphatase (Figs. 5Ai, 5AiiGo). At 3 weeks after induction, more than 90% of the cells expressed EGFP (Fig. 5AiiGo). For adipogenic differentiation, the accumulation of oil red O–positive lipid vesicles was used as a marker for adipogenic differentiation (Fig. 5CiiiGo). In contrast to osteogenic induction culture, no expression of EGFP was observed in the cells cultured with either adipogenic induction medium (Fig. 5CiiGo) or noninduction medium (Fig. 5BiiGo). This indicated that the EGFP reporter gene in the 21{Delta}pq HAC vector exhibits lineage-specific regulation.



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  		Figure 5. Lineage-specific EGFP expression in hiMSC hybrids containing 21{Delta}pq HAC/In-OPN-EGFP vector after osteogenic differentiation. Representative fluorescence and phase-contrast microscopic view of hiMSC hybrids. Cross panel (A) after osteogenic differentiation, (B) after adipogenic differentiation, and (C) control culture without differentiation induction. Vertical panel (i): Detection of red fluorescence produced by alkaline phosphatase activity; (ii): detection of green fluorescence of EGFP; (iii): phase-contrast microscopic view of the identical field as depicted in (i) and (ii). (Ai): Osteogenic differentiation was indicated by the alkaline phosphatase activity stained by alkaline phosphatase substrate, and (Biii): adipogenic differentiation was indicated by the accumulation of lipid vacuoles stained by Oil Red. EGFP was exclusively expressed (Aii) in hiMSC hybrid cells at 3 weeks postinduction of osteogenic differentiation but not in (Cii) undifferentiated cells or (Bii) cells at postinduction of adipogenic differentiation. The images show details of histochemical staining from M8-In-OPN#B-72, representative of the results from other hiMSC hybrid cell lines (M8-In-OPN#B-51, B-61, and B-92) containing 21{Delta}pq HAC/In-OPN-EGFP vector. Original magnification x200. Abbreviations: EGFP, enhanced green fluorescent protein; HAC, human artificial chromosome; hiMSC, human immortalized mesenchymal stem cell; OPN, osteopontin.

 
We next investigated expression status of the endogenous OPN gene in differentiation induction culture. RT-PCR detected OPN transcripts exclusively in osteogenic induced cells, in which the EGFP reporter gene was driven by an OPN promoter (Fig. 6Go). Taken together, our results indicate that the 21{Delta}pq HAC vector is capable of mediating lineage-specific transgene expression in hiMSCs after in vitro differentiation.



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  		Figure 6. Detection of lineage-specific transcription of endogenous marker genes in hiMSC hybrids by reverse transcription–polymerase chain reaction. OPN and PPAR{gamma} were tested as markers for osteogenic and adipogenic differentiation, respectively. Three weeks after induction of osteogenic and adipogenic differentiation, total RNA was extracted and analyzed for OPN, PPAR{gamma}, and GAPDH expression. Lineage-specific marker genes were upregulated at transcriptional level in the hiMSC hybrids along differentiation induction. Lanes 1–4 (M8-In-OPN#B-51, #B-61, #B-72, and #B-92, respectively), are representative of the hiMSC hybrid cells containing 21{Delta}pq HAC/In-OPN-EGFP vector. Abbreviations: D, differentiated; EGFP, enhanced green fluorescent protein; HAC, human artificial chromosome; hiMSC, human immortalized mesenchymal stem cell; OPN, osteopontin, PPAR{gamma}, peroxisome proliferator-activated receptor gamma; U, undifferentiated.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One of the major goals of stem cell–based gene therapy is to develop vectors that will allow appropriate levels of expression of therapeutic genes in defined target cells. In the present study, we aimed to address whether the 21{Delta}pq HAC vector could provide lineage-specific expression of a transgene using an in vitro differentiation system in an MSC cell line. Our results demonstrated that the 21{Delta}pq HAC vector allowed inducible expression of EGFP gene driven by OPN promoter in hiMSCs differentiated into osteogenic lineage.

The hiMSCs can differentiate into osteoblasts or osteocytes without transfer of the HAC vector in osteogenic induction culture conditions because the hiMSC is an established mesenchymal stem cell line that can differentiate into osteoblasts, chondrocytes, or adipocytes [24]. As shown in Figure 5Go, almost all cells were alkaline phosphatase–positive and EGFP-positive, suggesting that osteogenic differentiation was induced at a high rate. However, targets in the stem cell–based ex vivo gene therapy will be the primary culture of bone marrow stromal-derived MSCs, which are a heterogeneous population constituted by a group of cells with non differentiation or unidirectional, bidirectional, or tridirectional differentiation potential [24]. When a gene of interest is transduced into the primary culture MSCs, there is a possibility that the transgene does not contribute to lineage-specific expression because limited cell population can differentiate into osteoblasts. Thus, in this case, we can easily concentrate precursor cells that differentiate into a desired direction using the EGFP tag.

When considering the stem cell–based ex vivo gene therapy, the recipient stem cells transduced with therapeutic genes ex vivo should be propagated before autologous transplantation into the patient. Stable maintenance of the HAC in undifferentiated MSCs during long-term culture could promise substantial supply of treated cells that are competent to express therapeutic genes after induction of appropriate differentiation. In future studies, our aim is to harbor a tissue-specific therapeutic gene into HAC vector to achieve a more safe and effective therapy.

Recently, Stewart et al. [30] reported transfection of a mammalian artificial chromosome (ACE) derived from mouse satellite DNA carrying multiple copies of the red fluorescent protein (RFP) reporter gene into MSCs [31]. The ACE was maintained as a single chromosome in MSCs and provided stable expression of the RFP transgene along differentiation into adipogenic or osteogenic lineages. These results supported the possible application of artificial chromosomes for ex vivo stem cell therapy. However, the lineage-specific transgene expression along with differentiation was not intended in their study. In this context, our results are the first demonstration of lineage-specific expression induction of transgene by an artificial chromosome vector in MSCs.

The 21{Delta}pq HAC vector could be transduced from CHO donor hybrids into hiMSCs by MMCT. MMCT is a powerful tool for transferring HACs into recipient cells, but there is also a possibility that the nonhuman genomic DNA derived from CHO cells is concomitantly transferred into hiMSCs. One supposed case is an introduction of HAC-integrated hamster chromosome; the other is carry over of an independent hamster chromosome with HAC. To address these issue, we first analyzed the CHO hybrid by FISH and confirmed that the HAC remained an intact extra chromosome and that no translocation took place. After transfer of HAC into hiMSCs, obtained hybrid cells were analyzed by FISH again and it was confirmed that the hiMSCs showed normal karyotype (46, x y) plus an extra HAC. These results indicated that the carry over of hamster chromosomes was not a concern.

In transgenesis, it is known that two transgenes in a single construct often interfere mutually when they are driven by different transcriptional elements [29]. Insulators are endogenous cis-acting boundary elements that can protect chromatin domains from the nonspecific effects of the surrounding chromatin by blocking the passage of regulatory signals from adjacent loci and chromatin domains [32–35]. Hasegawa and Nakatsuji tested the effect of cHS4 insulator to prevent transcriptional interference between ubiquitous and tissue-specific transcriptional regulatory elements in the transgene study [29]. Another study using the 21{Delta}q HAC vector showed that highly reproducible tetracycline-regulating system was established with insulator [26]. In our study, the hiMSC hybrids containing 21{Delta}pq HAC/OPN-EGFP expressed EGFP in noninduction culture, indicating that transcriptional interference caused leaky transcription from the promoters that should not be activated. In contrast, the hiMSC hybrid containing the HAC vector with insulator achieved strict cell lineage–specific expression along with MSC differentiation. Our results support the significance of placing insulators on the HAC vector for the precise regulation of transgene expression.

For future application of the 21{Delta}pq HAC vector for stem cell–based gene therapy, an issue to be addressed is improvement in efficiency of transferring the HAC. The MMCT has been adopted to transfer a single, intact chromosome from donor to recipient cells, because the risk of truncation or rearrangement of the transferred chromosome was relatively low compared with other methods [36, 37], such as transfection of flow-sorted chromosomes [31]. Most of the chromosome transfer experiments so far have been applied for cell lines with unlimited replication potential. It is more recently that chromosome transfer has begun to be applied for primary cells with limited lifespan. Therefore, experimental protocol has not been fully optimized. In our previous study, the 21{Delta}pq HAC vector carrying human erythropoietin gene has been successfully transferred into primary human fibroblast [38]. Modification of microcell fusion protocol achieved transfer efficiency up to approximately 10–4 order. Further efforts have been made to optimize yet-to-be-examined factors.

In summary, we have demonstrated the successful transcriptional regulation by the 21{Delta}pq HAC vector in hiMSCs along differentiation. A remaining challenge is developing methods that will allow efficient delivery of these very large molecules into primary human cells with limited lifespan. Although substantial improvement in delivery method is indispensable, the demonstration of HAC-mediated cell lineage–specific transgene expression is the first step toward the prospective use of the HAC vector in stem cell–based gene therapy.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Hiroyuki Kugoh and Yasuaki Shirayoshi for valuable discussions. We also thank Hidetoshi Yamazaki for technical advice, Satoko Norikane for technical support, and Candice Ginn T. Tahimic for critical reading of the manuscript. This study was supported in part by a Health and Labour Sciences Research Grant for Research on Human Genome, Tissue Engineering from the Ministry of Health, Labour and Welfare, Japan, and by the 21st Century Program: The Research Core for Chromosome Engineering Technology.

DISCLOSURES
The authors indicate no potential conflicts of interest.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received January 15, 2005; accepted for publication June 7, 2005.



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