Received date: April 25, 2013; Accepted date: May 15, 2013; Published date: May 20, 2013
Citation: Noguchi H, Kobayashi N (2013) Controlled Expansion of Mammalian Cell Populations by Reversible Immortalization. J Biotechnol Biomater 3:158. doi:10.4172/2155-952X.1000158
Copyright: © 2013 Noguchi H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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In 1996, reversible immortalization using SV40 large T antigens and the Cre/LoxP system was successfully achieved with primary human fibroblasts. The concept of reversible immortalization involves introducing an immortalizing agent, SV40 large T antigens, into primary cells, expanding the cells in the culture, and finally, efficiently removing the immortalizing agent using Cre/LoxP site-specific recombination. The resulting cell population is essentially identical to the initial primary cells, but greatly increased in number. Since this report, reversible immortalization has been realized with hepatocytes, pancreatic β-cells, hepatic stellate cells, endothelial cells, renal epithelial cells and myogenic cells. This method facilitates the study of cell transplantation as well as cell differentiation, the cell cycle and senescence, by allowing one to control cell proliferation.
Reversible immortalization; myogenic cells; β-cells
Various research strategies have been hampered by difficulties in obtaining populations of primary cells that actively divide, while maintaining their stage of differentiation. Transferring specific oncogenes can generate cell lines that propagate in an intermediate stage of differentiation, a process known as cell immortalization. The simian virus 40 gene encoding the large tumor antigen (SV40Tag) is widely used to obtain continuously growing cell lines. Unlike most other oncogene products, SV40Tag alone can immortalize cells in the absence of other oncoproteins, owing to its multiple effects on the cell cycle . SV40Tag is able to induce transformation in cell culture and in vivo transgenic systems, a phenomenon effected through its interaction with at least five cellular targets: hsc70, the three Rb tumor suppressor proteins (pRb, p107, a nd p130), and the tumor suppressor p53 [2,3]. SV40Tag binds p53 through interactions with exposed amino acids on the surface of its ATPase domain . Similarly, the three Rbproteins bind to an LXCXE motif located in the flexible linker between the J domain and the origin binding domain. Finally, the J domain governs recruitment and activation of hsc70, a cellular chaperone [5,6]. SV40Tag has been shown to interact with another three targets, and these interactions could contribute to transformation as well, but they have been less studied. Two of these factors, the checkpoint kinase Bub1 and the cullin Cul7, interact with SV40Tag via the flexible linker near the LXCXE motif [7-10]. Finally, the transcriptional adapter proteins CBP/p300 [11-13], bind SV40Tag through interactions with p53, and could also contribute to transformation.
Recently, the telomerase expression has also been used, either alone or in the company of other immortalizing genes, to create genetically stable, nontumorigenic cell lines capable of apparently indefinite proliferation. The introduction of SV40Tag sometimes does not induce immortalization, but rather extends the in vitro life span of cells. This event reflects the critical attrition of telomere length, and can be overcome by expressing the catalytic component of the enzyme telomerase (human telomerase reverse transcriptase: hTERT) . However, established cell lines frequently exhibit different characteristics from the primary cells, especially in terms of cell growth.
Recently, the Cre/LoxP system has been used to temporarily “immortalize” primary cells, in order to obtain populations of primary cells that actively divide in vitro without entering senescence . Cre recombinase catalyzes site-specific recombination between two specific 34-base-pair direct repeats called LoxP. The binding of Cre to LoxP results in the formation of a synapse between both LoxP sites, in addition to Cre-mediated excision of the intervening sequences, which are permanently removed from the genome. Reversible immortalization involves the introduction of immortalizing genes, such as SV40Tag, and/or hTERT, into primary cells, expanding the cells in culture, and ultimately efficiently removing the immortalizing genes using the Cre/LoxP system (Figure 1). The cells reverted to their preimmortalized state after Cre expression, as indicated by changes in both the growth characteristics and p53 levels, and their fate conformed to the telomere hypothesis of replicative cell senescence . Reversible immortalization has been realized with fibroblasts, hepatocytes, hepatic stellate cells, endothelial cells, renal epithelial cells, myogenic cells and pancreatic beta cells.
Figure 1: Illustration demonstrating reversible cell immortalization. Reversible immortalization involves the introduction of immortalizing genes into primary cells, expanding the cells in culture, and ultimately efficiently removing the immortalizing genes. Immortalizing genes such as SV40Tag, and/or hTERT are expressed in transduced cells in the absence of Cre recombinase. After Cre/LoxP recombination, the intervening DNA segment between the two recombination targets is excised. The cells reverted to their preimmortalized state after Cre expression.
LTR: Long Terminal Repeat; SV40Tag: Simian Virus 40 large T antigen; hTERT: Human Telomerase Reverse Transcriptase.
In this review, we focus on reversible cell immortalization using gene transfer and site-specific recombination.
The transplantation of hepatocytes, which has been proposed as temporary metabolic support in patients awaiting liver transplantation or spontaneous reversion of liver disease, is an attractive alternative to liver transplantation. Our group demonstrated the reversible immortalization of human hepatocytes . A highly-differentiated cell line, NKNT-3, was established via retroviral transfer. We performed intrasplenic transplantation of NKNT-3 cells treated with Cre recombinase (reverted NKNT-3 cells) into a rat model of liver failure induced by 90% hepatectomy. The reverted NKNT-3 cells provided lifesaving metabolic support during acute liver failure.
Our group also showed the reversible immortalization of human hepatocytes using a retroviral vector expressing a catalytic subunit of hTERT flanked by a pair of LoxP recombination targets . One of the 24 clones was further subjected to transfection with the plasmid pCAGMerCreMer/PuroR, which expresses a Cre recombinase protein fused to two mutant estrogen-receptor ligand-binding domains (MerCreMer), under the control of the CAG promoter. Cre/LoxP recombination was performed in the established cells using simple exposure to 500 nM of tamoxifen for one week. Following transfection with the pCAGMerCreMer/PuroR, a resultant clone, 16-T3, was isolated for further studies based on the Cre/LoxP recombination efficiency. 16-T3 cells grew steadily in the culture and doubled in number within approximately 48 hours. The 16-T3 cells that reverted after treatment with tamoxifen showed an increased expression of hepatic markers, in association with enhanced levels of transcription factors. Transplantation of the reverted 16-T3 cells significantly prolonged the survival of pigs with acute liver failure induced by d-galactosamine injection.
The successes achieved over the last few decades with islet transplantation of whole pancreata and isolated islets suggest that diabetes can be cured by replenishing deficient β-cells [20,21]. It is logical that replacing the islet tissue itself is a better approach than simply replacing the lost insulin. However, the clinical benefits of islet transplantation are obtained in only a small minority of patients and are not permanent . Our strategy was to transform human primary β-cells with immortalizing genes of SV40Tag and hTERT, and to screen for clones that are not tumorigenic and that express insulin and β-cellassociated factors. Suitable clones could then be expanded, and Cremediated excision of the immortalizing genes would allow for the removal of tumorigenic potential and recovery of the primary β-cell function. Our reversibly immortalized pancreatic β-cell clone (NAKT- 15) secreted insulin in response to glucose stimulation and non-glucose secretagogues, resulting in the expression of proteins characteristic of β-cells (such as Isl-1, Pax 6, Nkx 6.1, Pdx-1, prohormone convertase (PC) 1/3 and PC 2), and secretory granule proteins (such as chromogranin A and synaptophysin). The NAKT-15 cells did not senesce after more than fifty passages in culture, and were continuously expanded. The transplantation of reverted NAKT-15 cells into streptozotocin (STZ)- induced diabetic severe combined immunodeficiency (SCID) mice resulted in perfect control of blood glucose within two weeks, and the mice remained normoglycemic for more than thirty weeks .
Reversible immortalization of murine pancreatic β-cells has also been realized using regulatory elements of the bacterial tetracycline (tet) operon for the conditional expression of SV40Tag oncoproteins in transgenic murine β-cells . The tet-on regulatory system was used to generate β-cell lines that divide in the presence of the tet derivative doxycycline (dox), and undergo growth arrest in its absence. The cells produce and secrete high amounts of insulin, and can restore and maintain euglycemia in syngeneic STZ-induced diabetic mice in the absence of dox. Moreover, reversible immortalization of rat pancreatic β-cells has also been reported using tricistronic retroviral vectors, in which Cre-ER, SV40Tag or hTERT and a reporter gene are flanked by the same pair of LoxP sites . The Cre-ER protein was induced to translocate from the cytoplasm to the nucleus by 4-hydroxytamoxifen, in order to excise SV40Tag, hTERT and the Cre-ER gene itself without the need for secondary gene transfer. Reversible immortalization of human pancreatic β-cells via the lentivector-mediated transfer of specific genes has also been reported .
The promising results afforded by islet transplantation, coupled with the shortage of cadaver pancreata relative to the potential demand, have provided strong impetus to search for new sources of insulin-producing cells. Alternative sources of islets have been sought in stem cells [27-29], porcine islets [30-33], and β-cell expansion with growth factors [34,35]. However, the differentiation of embryonic and pancreatic stem cells and expansion of differentiated β-cells in vitro is limited . The expansion of primary β-cells by growth factors is also hampered by the senescence of the cells . Establishing reversibly immortalized pancreatic β-cells, is one step toward overcoming the limitations of transplanting primary pancreatic β-cells to control diabetes.
The use of ex vivo gene therapy strategies and cell replacement therapy in the clinical field is often limited by the low number of cells harvested from biopsies, as well as the poor proliferation and premature senescence of these cells in vitro. The proliferative potential of human myogenic precursors declines considerably during early postnatal growth [38,39], in parallel with progressive reduction in the telomere length, which occurs in the first two decades of life . To overcome this problem, Berghella et al.  employed Cre-mediated excision of a LoxP-flanked SV40Tag sequence to establish reversibly immortalized human myogenic cells. The clonal isolates of the SV40Tag-positive myogenic cells exhibited modified growth characteristics, and a significantly extended life span while maintaining full myogenic potential. The transient expression of Cre recombinase allowed for excision of the entire provirus, with up to >90% efficiency, although 10% of the “unexcised” cells may still be cancerous. As a result, it is important to improve the excision efficiency, in order to remove these remaining cells. The reverted cells, which were injected into the regenerating muscle of SCID/bg-immunodeficient mice, underwent terminal differentiation in vivo, giving rise to clusters of hybrid fibers, with an efficiency comparable to that of control untransduced cells. This approach may eventually lead to the ex vivo production of an adequate number of myogenic cells to reconstitute at least a few essential muscles in dystrophic patients via transplantation.
Transplantation of primary adrenal chromaffin cells has been used to deliver functional molecules for a variety of therapeutic indications . However, a serious limitation is the need to harvest fresh cells from donors, requiring safety screening for each batch of cells, and a resultant mixture of cell types that is incompletely characterized and nonhomogeneous. Eaton et al.  described the generation of chromaffin cell lines using the temperature-sensitive allele of SV40Tag. The cells are able to reverse neuropathic pain after being transplanted in the spinal subarachnoid space . Even with 100% disappearance of SV40Tag in the grafts within a few weeks after transplantation, the oncogene expression in vivo remains a potential possibility, and the use of such cells is not an appropriate strategy for safe clinical application in humans. The same group developed a strategy based on reversible immortalization of primary adrenal chromaffin cells, with a retroviral vector expressing the temperature-sensitive allele of SV40Tag, excisable by means of the Cre/LoxP recombination system . The immortalized cells expressed immunoreactivity for all catecholamine enzymes, including tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH) and phenylethanolamine-N-methyltransferase (PNMT). When chromaffin cells reverted by the Cre expression were transplanted into a model of neuropathic pain and partial nerve injury, the grafts were equally able to reverse the behavioral hypersensitivity induced by the injury. The use of Cre/Lox site-directed recombination of SV40Tag in chromaffin cells that are able to deliver neuroactive molecules may overcome the limitations of these cells for transplantation.
These cell lines are useful in the development of bioartificial organs, as well as cell transplantation and ex vivo gene therapy. One of our long-term goals is to develop bioartificial organ systems that closely mimic the function of normal organs in vivo. Recently, heterotypic cell interactions between parenchymal cells and nonparenchymal neighbors have been recognized to be central to the function of many organ systems. Pure cultures of hepatocytes recapitulate several key liver functions, but fail to provide adequate levels of a few important detoxifying enzymes, including cytochrome p450-associated enzymes (CYPs). Hepatic stellate cells are believed to play an essential role in the known crosstalk between hepatocytes and other liver cells, such as endothelial cells [46,47]. Extending the reversible immortalization system to other cell types present in the human liver would allow for the study of cell-cell interactions, and further contribute to the development of bioartificial livers. We established reversibly immortalized human hepatic stellate cells via the retroviral transfer of hTERT flanked by a pair of LoxP sequences . TWNT-1, an immortalized human stellate cell line, is highly differentiated and exhibits the functions of human stellate cells, including the uptake of acetylated low-density lipoprotein,s and synthesis of collagen type I and hepatocyte growth factor. The efficient excision of retrovirally transferred hTERT cDNAs was achieved via the TAT-mediated transduction of Cre recombinase, a new technology for transducing proteins into cells [49-51]. When cocultured with TWNT- 1 cells, NKNT-3 increases the protein expressions of the detoxifying cytochrome P450-associated protein isoenzymes 3A4 and 2C9, in addition to urea synthesis. This finding supports the contention that heterotypic cell interactions are important for enhancing the production of liver-specific enzymes by hepatocytes in vitro [52,53].
In both the developing and mature adult liver, hepatocyte-toendothelial cell interactions are imperative for the coordination of the sophisticated liver functions . Therefore, we also applied the Cre/LoxP system to human liver endothelial cells [55,56]. Liver endothelial cells were transfected with a retroviral vector that expresses the SV40Tag, flanked by a pair of LoxP recombination targets. One of the transduced clones, HNNT-2, extended the life span from passages 10 to 40; however, complete immortalization was not achieved . This finding is explained by the absence of spontaneous activation of endogenous telomerase, known to be an essential participant in cellular immortalization processes in SV40Tag-transduced cells [57-59]. To enhance the immortalization potential, we used another retroviral vector expressing hTERT flanked by a pair of LoxB target sequences. One of the clones, TMNK-1, expressed EC markers, including factor VIII, vascular endothelial growth factor receptors (flt-1, KDR/Flk- 1) and CD34. TMNK-1 exhibited uptake of Di-I-acetylated-lowdensity lipoprotein and angiogenic potential in Matrigel assays. Following lipopolysaccharide treatment, TMNK-1 produced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and exhibited an increased expression of intracellular adhesive molecule-1, vascular cellular adhesive molecule-1 and VE-cadherin. Efficient excision of the retrovirally-transferred hTERT and SV40Tag cDNAs was achieved via the TAT-mediated transduction of Cre recombinase .
Recently, much attention has been paid to a novel therapy for liver failure, using a hybrid bioartificial liver support device that incorporates living liver cells. Researchers in various fields have considered the following cells for potential use in bioartificial livers: human embryonic stem (ES) cells, somatic stem cells, differentiated tissue cells and cells derived from tissues of different animal species, particularly pigs. We recommend that researchers adopt a reversible immortalization system that uses the Cre/LoxP site-specific recombination reaction targeting human hepatocytes, and other liver-related cells in their final differentiated state. This system has allowed us to establish a safe human liver cell line for generating bioartificial livers, that is capable of differentiation at a low cost and on a large scale.
Reversibly immortalized human melanocytes created using a retroviral vector expressing SV40Tag-EGFP flanked by a pair of LoxP recombination targets have been reported to provide melanocytes with rapid replicative potential in vitro . Following the transplantation of reverted melanocytes into an established vitiligo animal model, the pigmentation formed black macula within three months without tumorigenicity. The pathological results showed that there was significant melanocyte and melanin accumulation in the epidermis with some hair follicles in the transplanted area, which confirmed that the reverted cells display melanogenesis in vivo. Reversibly immortalized human melanocytes may be useful as a successful repigmentation method for depigmented skin disease therapy.
Reversibly immortalized human renal proximal tubule epithelial cells (RPTECs) have been established using two lentivirus vectors carrying hTERT and SV40Tag flanked by LoxP sites . The transduced RPTEC clones continued to proliferate, while retaining the biochemical and functional characteristics of the primary cells. The clones exhibited contact-inhibited, anchorage- and growth factor-dependent growth, and did not form tumors in nude mice. The transient Cre expression observed in these cells resulted in efficient proviral deletion, the upregulation of some renal-specific activities and decreased growth rates. Ultimately, the cells underwent replicative senescence, indicating intact cell cycle control. These data suggest that reversible immortalization of human RPTECs can lead to the largescale production of RPTECs that retain most tissue-specific properties.
A reversibly immortalized murine dental papilla cell line (mDPCET) has been generated by combining the traditional strategy of “Cre/LoxP-based reversible immortalization”, with a tamoxifenregulated Cre recombination system . Tamoxifen-mediated reversible immortalization allowed for the expansion of primary mDPCs that led to the production of odontoblast-like cells that retained most odontoblast-specific properties, representing a safe and ready-touse method due to its simple manipulation.
Reversible immortalization of cardiomyocytes has also been reported . The immortalized cardiomyocytes exhibited the morphological features of dedifferentiation (an increased expression of vimentin and reduced expressions of troponin I and Nkx2.5), along with the continued expression of cardiac markers (alpha-actin, connexin-43 and calcium transients). After the immortalization was reversed, the cells returned to their differentiated state. This strategy for the controlled expansion of primary cardiomyocytes has the potential to provide large amounts of an individual patient’s own cardiomyocytes for cell therapy, and the cardiomyocytes derived using this method may constitute a useful cellular model for studying cardiogenesis.
We previously reported the in vitro amplification of human umbilical vein endothelial cell (HUVEC) populations during the first phase of reversible immortalization resulting from the retroviral transfer of the SV40Tag gene, which was subsequently excised via Cre/LoxP-mediated site-specific recombination . The transduced HUVECs exhibited the morphological characteristics of endothelial cells, and were maintained in the culture up to passage 40. The cells expressed endothelial cell markers, including factor VIII, VEGF receptors (Flt-1 and KDR/Flk-1) and CD34, and endocytosed acetylated low-density lipoproteins. The formation of capillary-like structures in the cells was observed in a Matrigel assay. The complete elimination of the transferred SV40Tag gene was achieved in virtually 100% of the cells, following infection with a recombinant adenovirus expressing Cre recombinase and subsequent selection. The reverted cells maintained their differentiated endothelial cell phenotype. Qiu et al.  showed similar data. These studies provide a means of expanding primary endothelial cells of various sources for basic studies and possible cell and gene therapy.
Some groups have also reported the reversible immortalization of progenitor cells. Nishioka et al.  and our group collaboratively reported the reversible immortalization of human marrow-derived mesenchymal stem cells (MSCs), with the potential to differentiate into mesenchymal tissues, such as bone, cartilage, adipose tissue and bone marrow stroma, using a retroviral vector carrying SV40Tag, which can be excised via Cre/LoxP site-specific recombination. One of the MSCs cell lines, HMSC-1, retained the original surface characteristics and differentiation potential, and exhibited a higher proliferative capacity than the parental cells. Other groups have demonstrated the reversible immortalization of Nestin-positive progenitor cells (NPPCs) obtained from the murine pancreas, using the tet-on system of the SV40Tag expression [66,67]. The reversibly immortalized NPPCs were efficiently induced to differentiate into insulin-producing cells that contained a combination of glucagon like peptide-1 (GLP-1) and sodium butyrate.
Cre/LoxP site-specific recombination has been used for genetic engineering. Employing this technology with the genes of SV40Tag, and/or hTERT, the transient expression of an immortalizing gene induced via gene transfer is used to generate cell lines that propagate rapidly in cell culture, and display an increased life span without entering senescence. The reversible immortalization system allows for the ex vivo amplification of primary cells in culture, facilitating the study of cell transplantation, as well as cell differentiation, the cell cycle and senescence by allowing one to control cell proliferation. However, some immortalized cell lines have been reported to demonstrate an abnormal karyotype . Therefore, further studies are required before such cells can be used in clinical situations.
This work was supported in part by the Japan Society for the Promotion of Science; and the Ministry of Health, Labour and Welfare.
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