A Novel Human Artificial Chromosome Vector Provides Effective Cell Lineage–Specific Transgene Expression in Human Mesenchymal Stem Cells
http://www.100md.com
《干细胞学杂志》
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@grape.med.tottori-u.ac.jp
ABSTRACT
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 . Recent studies have shown that the MSCs can regenerate myocardium, liver, neural tissue, and hepatocytes by cell fusion . 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 . 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 . 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 . It contains a single loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. The HAC, designated 21pq 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 21pq HAC vector provides competence to create a genetic environment for reproducible transgene expression. From the features mentioned above, the 21pq HAC vector may be feasible to be developed for a transgene regulation system.
Here we addressed whether the 21pq 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 , whose expression is upregulated along differentiation of MSCs into osteoblastic lineage . Two types of 21pq HAC vectors, with or without insulators in both sides of OPN-EGFP expression units, were constructed and transferred into an MSC cell line: hiMSCs 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
21pq HAC Is Stably Maintained in hiMSC Hybrid Cells
The 21pq HAC vector is maintained in CHO hybrids (Fig. 1A) . To determine whether the 21pq 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 21pq 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 21pq HAC was detected in all metaphases observed (Fig. 1B). Neither insertion into host chromosome nor apparent amplification of the 21pq HAC was observed. In addition, these clones showed normal karyotype. These data suggested that 21pq HAC vector had been transferred into hiMSCs without introducing lesions in the host cells’ chromosomes.
Figure 1. FISH analysis of CHO and hiMSC hybrid cells carrying the 21pq HAC vectors or 21pq HAC/In-OPN-EGFP vector. Human chromosome 21–derived alphoid DNA probe was hybridized to the 21pq HAC vector in (A) H8 and (B) hiMSC hybrid cell-M8#1(red signals with arrows). In the hiMSC hybrid cell, 21pq 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 21pq 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 21pq HAC vector (C, D) with or (A, B) without the OPN-EGFP insert. Note that the single copy of 21pq 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 21pq 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 21pq 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 1. A single copy of the 21pq HAC vector was constantly observed in most metaphase spreads. Loss rate of the 21pq HAC vector was very low at any time point, regardless of the absence or presence of selective pressure. These results suggested that the 21pq HAC vector was mitotically stable in hiMSCs.
Table 1. 21pq HAC stability in the absence of selective pressure in human bone marrow–derived mesenchymal stem cells
21pq 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 , 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 21pq 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. 2), indicating that the presence of 21pq HAC vector did not affect the tridirectional differentiation potential of the hiMSCs.
Figure 2. Differentiation of hiMSC hybrids containing 21pq 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 21pq 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 21pq 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 21pq HAC and Transfer into hiMSCs
To investigate whether the 21pq 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 .
On the 21pq HAC vector, acceptor loxP site is surrounded by viral promoters for driving drug-resistant genes (Fig. 3). Although the EGFP reporter gene was efficiently expressed from the 21pq HAC vector housed in an HT1080 hybrid , there was still a possibility that surrounding sequences may interfere with lineage-specific transcriptional regulation . 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 ?-globin gene 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.
Figure 3. Site-specific insertion of pBS226/OPN-EGFP reporter construct or pBS226/In-OPN-EGFP reporter construct into the loxP site on the 21pq 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 21pq 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 , G418 resistance was not applicable as a selection marker for hiMSCs that underwent site-directed insertion of the gene of interest into the 21pq HAC vector. We therefore inserted the pBS226/OPN-EGFP or pBS226/In-OPN-EGFP reporter constructs into the 21pq 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. 3).
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 21pq 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. 4A). In two-color FISH analysis, a single independent 21pq HAC/In-OPN-EGFP vector was observed (Fig. 1C), suggesting that the 21pq HAC was successfully transferred.
Figure 4. Southern analysis for site-specific insertion of pBS226/In-OPN-EGFP constructs into the loxP site on the 21pq HAC in H8 cells (A) and detection of the 21pq 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 21pq 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. 4B). FISH analysis showed the presence of a single, independent 21pq HAC vector in the hiMSC hybrid cells (Fig. 1D). These data indicate that the intact 21pq 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 21pq 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. 1). 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 21pq HAC/In-OPN-EGFP for the following studies.
Osteocyte-Specific Transgene Expression After Differentiation of hiMSC Hybrids
As described above, the hiMSC hybrids containing 21pq HAC vector retained the ability to differentiate into multiple lineages. To investigate whether 21pq HAC vector can mediate cell lineage–specific transgene expression, the hiMSC hybrids containing 21pq 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. 5Aii). The EGFP expression level increased in a time-dependent manner, and EGFP fluorescence was observed in the cell fraction expressing alkaline phosphatase (Figs. 5Ai, 5Aii). At 3 weeks after induction, more than 90% of the cells expressed EGFP (Fig. 5Aii). For adipogenic differentiation, the accumulation of oil red O–positive lipid vesicles was used as a marker for adipogenic differentiation (Fig. 5Ciii). In contrast to osteogenic induction culture, no expression of EGFP was observed in the cells cultured with either adipogenic induction medium (Fig. 5Cii) or noninduction medium (Fig. 5Bii). This indicated that the EGFP reporter gene in the 21pq HAC vector exhibits lineage-specific regulation.
Figure 5. Lineage-specific EGFP expression in hiMSC hybrids containing 21pq 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 21pq 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. 6). Taken together, our results indicate that the 21pq HAC vector is capable of mediating lineage-specific transgene expression in hiMSCs after in vitro differentiation.
Figure 6. Detection of lineage-specific transcription of endogenous marker genes in hiMSC hybrids by reverse transcription–polymerase chain reaction. OPN and PPAR 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, 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 21pq 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, peroxisome proliferator-activated receptor gamma; U, undifferentiated.
DISCUSSION
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.
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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@grape.med.tottori-u.ac.jp
ABSTRACT
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 . Recent studies have shown that the MSCs can regenerate myocardium, liver, neural tissue, and hepatocytes by cell fusion . 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 . 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 . 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 . It contains a single loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. The HAC, designated 21pq 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 21pq HAC vector provides competence to create a genetic environment for reproducible transgene expression. From the features mentioned above, the 21pq HAC vector may be feasible to be developed for a transgene regulation system.
Here we addressed whether the 21pq 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 , whose expression is upregulated along differentiation of MSCs into osteoblastic lineage . Two types of 21pq HAC vectors, with or without insulators in both sides of OPN-EGFP expression units, were constructed and transferred into an MSC cell line: hiMSCs 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
21pq HAC Is Stably Maintained in hiMSC Hybrid Cells
The 21pq HAC vector is maintained in CHO hybrids (Fig. 1A) . To determine whether the 21pq 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 21pq 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 21pq HAC was detected in all metaphases observed (Fig. 1B). Neither insertion into host chromosome nor apparent amplification of the 21pq HAC was observed. In addition, these clones showed normal karyotype. These data suggested that 21pq HAC vector had been transferred into hiMSCs without introducing lesions in the host cells’ chromosomes.
Figure 1. FISH analysis of CHO and hiMSC hybrid cells carrying the 21pq HAC vectors or 21pq HAC/In-OPN-EGFP vector. Human chromosome 21–derived alphoid DNA probe was hybridized to the 21pq HAC vector in (A) H8 and (B) hiMSC hybrid cell-M8#1(red signals with arrows). In the hiMSC hybrid cell, 21pq 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 21pq 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 21pq HAC vector (C, D) with or (A, B) without the OPN-EGFP insert. Note that the single copy of 21pq 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 21pq 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 21pq 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 1. A single copy of the 21pq HAC vector was constantly observed in most metaphase spreads. Loss rate of the 21pq HAC vector was very low at any time point, regardless of the absence or presence of selective pressure. These results suggested that the 21pq HAC vector was mitotically stable in hiMSCs.
Table 1. 21pq HAC stability in the absence of selective pressure in human bone marrow–derived mesenchymal stem cells
21pq 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 , 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 21pq 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. 2), indicating that the presence of 21pq HAC vector did not affect the tridirectional differentiation potential of the hiMSCs.
Figure 2. Differentiation of hiMSC hybrids containing 21pq 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 21pq 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 21pq 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 21pq HAC and Transfer into hiMSCs
To investigate whether the 21pq 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 .
On the 21pq HAC vector, acceptor loxP site is surrounded by viral promoters for driving drug-resistant genes (Fig. 3). Although the EGFP reporter gene was efficiently expressed from the 21pq HAC vector housed in an HT1080 hybrid , there was still a possibility that surrounding sequences may interfere with lineage-specific transcriptional regulation . 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 ?-globin gene 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.
Figure 3. Site-specific insertion of pBS226/OPN-EGFP reporter construct or pBS226/In-OPN-EGFP reporter construct into the loxP site on the 21pq 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 21pq 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 , G418 resistance was not applicable as a selection marker for hiMSCs that underwent site-directed insertion of the gene of interest into the 21pq HAC vector. We therefore inserted the pBS226/OPN-EGFP or pBS226/In-OPN-EGFP reporter constructs into the 21pq 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. 3).
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 21pq 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. 4A). In two-color FISH analysis, a single independent 21pq HAC/In-OPN-EGFP vector was observed (Fig. 1C), suggesting that the 21pq HAC was successfully transferred.
Figure 4. Southern analysis for site-specific insertion of pBS226/In-OPN-EGFP constructs into the loxP site on the 21pq HAC in H8 cells (A) and detection of the 21pq 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 21pq 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. 4B). FISH analysis showed the presence of a single, independent 21pq HAC vector in the hiMSC hybrid cells (Fig. 1D). These data indicate that the intact 21pq 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 21pq 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. 1). 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 21pq HAC/In-OPN-EGFP for the following studies.
Osteocyte-Specific Transgene Expression After Differentiation of hiMSC Hybrids
As described above, the hiMSC hybrids containing 21pq HAC vector retained the ability to differentiate into multiple lineages. To investigate whether 21pq HAC vector can mediate cell lineage–specific transgene expression, the hiMSC hybrids containing 21pq 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. 5Aii). The EGFP expression level increased in a time-dependent manner, and EGFP fluorescence was observed in the cell fraction expressing alkaline phosphatase (Figs. 5Ai, 5Aii). At 3 weeks after induction, more than 90% of the cells expressed EGFP (Fig. 5Aii). For adipogenic differentiation, the accumulation of oil red O–positive lipid vesicles was used as a marker for adipogenic differentiation (Fig. 5Ciii). In contrast to osteogenic induction culture, no expression of EGFP was observed in the cells cultured with either adipogenic induction medium (Fig. 5Cii) or noninduction medium (Fig. 5Bii). This indicated that the EGFP reporter gene in the 21pq HAC vector exhibits lineage-specific regulation.
Figure 5. Lineage-specific EGFP expression in hiMSC hybrids containing 21pq 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 21pq 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. 6). Taken together, our results indicate that the 21pq HAC vector is capable of mediating lineage-specific transgene expression in hiMSCs after in vitro differentiation.
Figure 6. Detection of lineage-specific transcription of endogenous marker genes in hiMSC hybrids by reverse transcription–polymerase chain reaction. OPN and PPAR 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, 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 21pq 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, peroxisome proliferator-activated receptor gamma; U, undifferentiated.
DISCUSSION
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.
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