The Tumor-Immune Response Is Not Compromised by Mesenchymal Stromal Cells in Humanized Mice.

Therapeutic uses of mesenchymal stromal cells (MSCs) have emerged over the past decade. Yet, their effect on tumor growth remains highly debated, particularly in an immune competent environment. In this study, we wanted to investigate the impact of human umbilical cord-derived MSCs (hUC-MSCs) on tumor growth in humanized mice generated by the human adoptive transfer of PBMCs or the cotransplantation of hematopoietic stem cells and human thymic tissue (human BLT [Hu-BLT]). Our results showed that the growth and immune rejection of engineered human fibroblastic tumors was not altered by the injection of hUC-MSCs in immune-deficient or humanized mice, respectively. This was observed whether tumor cells were injected s.c. or i.v. and independently of the injection route of the hUC-MSCs. Moreover, only in Hu-BLT mice did hUC-MSCs have some effects on the tumor-immune infiltrate, yet without altering tumor growth. These results demonstrate that hUC-MSCs do not promote fibroblastic tumor growth and neither do they prevent tumor infiltration and rejection by immune cells in humanized mice.

Andrographolide enhances the anti-metastatic effect of radiation in Ras-transformed cells via suppression of ERK-mediated MMP-2 activity.

BACKGROUND: Activation of Ras oncogene in human tumors is associated with radiation-associated metastatic potential. Although ionizing radiation is one important method of cancer treatments, it has been shown to enhance matrix metalloproteinases (MMPs) activity and facilitates a more aggressive cancer phenotype. Our previous studies showed that andrographolide with lower dose rates of radiation could inhibit RAS-transformed cancer metastasis in vivo; however, the molecular mechanisms are not yet clear. In this study, we aimed to explore the anti-metastatic effect of andrographolide combined with radiation on Ras-transformed cells. METHODS: RAS-transformed cells were treated with andrographolide in the presence or absence of irradiation (2-4 Gy) or angiotensin II to examine cell invasion. In vivo tumorigenesis assays were also performed. The MMP-2 activity was detected by using Gelatin zymography. Signal transduction of NF-κB subunit, p65 and phosphor-ERK 1/2, were examined by using Western blotting analysis. RESULTS: Treatment with andrographolide inhibited migration of Ras-transformed cells. Andrographolide treatment with radiation significantly inhibited cancer metastasis in vivo. We found that andrographolide exhibited anti-migration and anti-invasive ability against cancer metastasis via inhibition of MMP2 activity rather than affected MMP-9 and EMT. In addition, combined andrographolide with radiation appeared to be more effective in reducing MMP-2 expression, and this effect was accompanied by suppression of ERK activation that inhibits cancer cell migration and invasion. CONCLUSIONS: These findings suggest that andrographolide enhances the anti-metastatic effect of radiation in Ras-transformed cells via suppression of ERK-mediated MMP-2 activity.

Honeybee venom possesses anticancer and antiviral effects by differential inhibition of HPV E6 and E7 expression on cervical cancer cell line.

Bee venom (BV) therapy is a type of alternative medical treatment used to treat various diseases in oriental medicine. The mechanisms underlying the effects of BV remain poorly understood. In the present study, we evaluated the antiviral effect of BV on cervical carcinoma cell lines (CaSki, HeLa, C33A and TC-1). BV treatments resulted in a more significant suppression of cell growth in HPV 16-infected cells (CaSki) and a lesser suppression in HPV 18-infected cells (HeLa). However, less suppression was observed in HPV-negative C33A cells. In 10 µg/ml BV-treated CaSki cells, the mRNA expression and protein levels of HPV16 E6 and E7 were significantly decreased by BV, while HPV18 E6 and E7 mRNA expression levels were not significantly altered by 10 µg/ml BV-treated HeLa cells. The antitumor effects of BV were in accordance with in vitro data, in restricting tumor growth in vivo and were much more effective on the suppression of tumor growth. Furthermore, the mRNA and protein expression levels of HPV16 E6 and E7 were decreased by BV in TC-1 tumors. These findings demonstrated the antiviral effects of BV in HPV-infected cervical cancer cells and the anticancer effects of BV in HPV16 E6/E7-expressed TC-1 tumors. Collectively, BV plays a differential role in suppressing HPV16-infected cells (CaSki cells) and HPV18-infected cells (HeLa cells) by the downregulation of E6/E7 protein of HPV16/18.

EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors.

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell transformation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immune-evading monomorphic DLBCL-like tumors in NOD/SCID/γc-/- mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell-chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell-mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional EBNA3B mutations. Thus, EBNA3B is a virus-encoded tumor suppressor whose inactivation promotes immune evasion and virus-driven lymphomagenesis.

Unexpected properties of endostatin-producing mouse BCR-ABL-transformed cells.

We investigated whether a genetic modification of BCR-ABL-transformed mouse cells that resulted in endostatin (ES) production altered their oncogenic potential. Mouse B210 cells, which express p210bcr-abl fusion protein and induce leukemia-like disease and extremely rarely solid tumors after intravenous (i.v.) administration, were used. The cells were transfected with a plasmid carrying genes for mouse ES and resistance to blasticidine. Transduced cells were isolated in media supplemented with blasticidine. Production of ES was determined by Western blotting. For further tests, two clones were selected, and their pathogenicity after i.v. inoculation was tested. Compared with the parental B210 cells, the capability of both gene-modified cell clones to induce lethal leukemia was reduced. However, mice that did not succumb to leukemia subsequently developed solid tumors. They were composed of poorly differentiated cells with irregular nuclei and roughly granular chromatin and were well vascularized. FISH revealed the presence of the BCR-ABL fusion gene both in tumors and spleens. Immunohistological investigation of the tumors demonstrated the production of ES in vivo and the cell lines derived from the tumors produced detectable amounts of ES, this demonstrating that the formation of solid tumors was not associated with the loss or silencing of the ES gene.

Antitumor therapeutic and antimetastatic activity of electroporation-delivered human papillomavirus 16 E7 DNA vaccines: a possible mechanism for enhanced tumor control.

DNA vaccines are known to be lacking in immunogenicity in humans. Presently, electroporation (EP) is thought to overcome this limitation. Here, we investigate whether human papillomavirus 16 E7 DNA vaccines delivered by EP might elicit potent antitumor activity in animal cervical cancer models, with a focus on the underlying mechanism(s). Intramuscular (IM)-EP delivery of E7 DNA vaccines induced more potent antitumor therapeutic and antimetastatic activity compared with IM delivery. Moreover, the tumor-controlled animals by IM-EP possessed long-term memory responses to parental tumor cells. This improved antitumor effect was concomitant with augmented Ag-specific CTL activities. IM-EP also induced IgG and Th-cell responses higher than IM delivery. Finally, IM-EP resulted in more antigen production in and more attraction of immune cells into the site of DNA injection, suggesting that these biological and immunological changes made by IM-EP might be responsible for enhanced CTL activities and antitumor resistance. Thus, this study shows that IM-EP can induce more potent antitumor activity by augmenting CTL responses possibly through more antigen production in and more attraction of immune cells into the muscle sites. This study also suggests that IM-EP of E7 DNA vaccines might be a potential approach toward treating patients with cervical cancer.

Overexpression of a novel gene gankyrin correlates with the malignant phenotype of colorectal cancer.

Gankyrin, a small and highly conserved protein which is identical to the p28 gene product, was found to be related with the malignant phenotypes in liver and esophageal carcinoma. However, the roles of gankyrin in colorectal carcinoma (CRC) are still unknown. In the present study, the gankyrin mRNA and protein expression in human CRC cell lines and clinical tissue samples were evaluated and correlated with clinicopathological features. Possible mechanisms by which gankyrin regulates the malignant phenotype of CRC cells were also investigated. The results demonstrated that gankyrin was obviously overexpressed in CRC tissues and cell lines compared to controls, and gankyrin expression was correlated with TNM stages and metastasis of CRC. Overexpression of gankyrin by PhkitNeo-hGankyrin plasmid transfected into Lovo cells could promote the cell proliferation and tumorigenicity. This finding was further strengthened by experiments that suppressing gankyrin expression by siRNA exerted the opposite effects on CRC cells SW620. In addition, our present study showed that the co-expression of cyclinD1 and beta-catenin were positive correlation with the alteration of gankyrin expression. This data suggested that gankyrin played significant roles in the pathogenesis of human CRC, and might be an important therapeutic target for CRC.

Effects of Schwann cell transplants in an experimental nerve amputee model.

PURPOSE: By using a nerve amputee model of the rat sciatic nerve (Lago and Navarro, 2007), we have tested a strategy for the long-term maintenance of regenerated axons without distal target reinnervation, by grafting Schwann cells (SCs) into a capped silicone chamber containing the ending nerve stump. METHODS: The sciatic nerve of rats was transected and repaired with a silicone tube, the distal nerve was again cut at 10 mm and inserted in a capped tube that was filled with saline or with a suspension of cultured SCs. Transplants of SCs obtained from primary cultures have been compared with those of an immortalized SC line (SCTM41) or the same line overexpressing GDNF. RESULTS: The histological results show that nerve fibers were able to regenerate through a short distal nerve segment ending into the capped chamber, and sustain distal branches without degenerating for several months. There was abundant axonal sprouting forming an ending neuroma, and the caliber of myelinated fibers remained far thinner than normal during the 9 months investigated. With a distal transplant of primary SCs there were significantly more regenerated myelinated fibers than in the control group at 9 months, indicating that the grafted cells stimulated the axonal growth response and helped to maintain survival of axon branches. In contrast, axonal regeneration was significantly reduced with grafts of SCTM41 cells, probably due to physical competition between cell proliferation and axonal growth. SCTM41 cells overexpressing GDNF improved the regenerative response with respect to the parent SCTM41 cells, although not to the same extent as the primary SCs. CONCLUSION: A graft of primary SCs in the capped chamber stimulated axonal growth response and/or maintained survival of axonal branches on the long term in the nerve amputee model.

Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts.

Current therapeutic approaches to treatment of patients with bulky cervical cancer are based on conventional in situ ablative modalities including cisplatin-based chemotherapy and radiation therapy. The 5-year survival of patients with nonresectable disease is dismal. Because over 99% of squamous cervical cancer is caused by persistent infection with an oncogenic strain of human papillomavirus (HPV), particularly type 16 and viral oncoproteins E6 and E7 are functionally required for disease initiation and persistence, HPV-targeted immune strategies present a compelling opportunity in which to demonstrate proof of principle. Sublethal doses of radiation and chemotherapeutic agents have been shown to have synergistic effect in combination with either vaccination against cancer-specific antigens, or with passive transfer of tumor-specific cytotoxic T lymphocytes (CTLs). Here, we explored the combination of low-dose radiation therapy with DNA vaccination with calreticulin (CRT) linked to the mutated form of HPV-16 E7 antigen (E7(detox)), CRT/E7(detox) in the treatment of E7-expressing TC-1 tumors. We observed that TC-1 tumor-bearing mice treated with radiotherapy combined with CRT/E7(detox) DNA vaccination generated significant therapeutic antitumor effects and the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of treated mice. Furthermore, treatment with radiotherapy was shown to render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. In addition, we observed that treatment with radiotherapy during the second DNA vaccination generated the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of TC-1 tumor-bearing mice. Finally, TC-1 tumor-bearing mice treated with the chemotherapy in combination with radiation and CRT/E7(detox) DNA vaccination generate significantly enhanced therapeutic antitumor effects. The clinical implications of the study are discussed.

Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination.

BACKGROUND/AIMS: Hepatocyte transplantation and bioartificial liver treatment are attractive alternatives to liver transplantation. The availability of well-characterized human hepatocyte lines facilitates such cell therapies. METHODS: Human hepatocytes were immortalized with a retroviral vector SSR#197 expressing catalytic subunit of human telomerase reverse transcriptase (hTERT) and enhanced green fluorescent protein (EGFP) cDNAs flanked by a pair of loxP recombination targets. Then, Tamoxifen-dependent Cre recombinase was expressed in SSR#197-immortalized hepatocytes. Cre/LoxP recombination was performed in the established cells by simple exposure to 500 nM Tamoxifen for a week. Then, the reverted population of the cells was recovered by EGFP-negative cell sorting and characterized in vitro and in vivo using a pig model of acute liver failure (ALF) induced by d-galactosamine (0.5 g/kg) injection. RESULTS: A human hepatocyte cell line 16T-3 was established. Reverted 16-T3 cells showed the increased expression of hepatic markers in association with enhanced levels of transcriptional factors. Compared to normal human hepatocytes, albumin production and lidocaine-metabolizing activities of reverted 16-T3 cells were 0.32 and 0.50-fold, respectively. Transplantation of reverted 16T-3 cells significantly prolonged the survival of ALF pigs. CONCLUSIONS: Here we demonstrate the usefulness of Cre/LoxP -mediated reversible immortalization of human hepatocytes with Tamoxifen-mediated self-recombination.

PPARalpha deficiency in inflammatory cells suppresses tumor growth.

Inflammation in the tumor bed can either promote or inhibit tumor growth. Peroxisome proliferator-activated receptor (PPAR)alpha is a central transcriptional suppressor of inflammation, and may therefore modulate tumor growth. Here we show that PPARalpha deficiency in the host leads to overt inflammation that suppresses angiogenesis via excess production of the endogenous angiogenesis inhibitor thrombospondin-1 and prevents tumor growth. Bone marrow transplantation and granulocyte depletion show that PPARalpha expressing granulocytes are necessary for tumor growth. Neutralization of thrombospondin-1 restores tumor growth in PPARalpha-deficient mice. These findings suggest that the absence of PPARalpha activity renders inflammatory infiltrates tumor suppressive and, thus, may provide a target for inhibiting tumor growth by modulating stromal processes, such as angiogenesis.

Melanocyte transformation associated with substrate adhesion impediment.

Exclude experimental models of malignant transformation employ chemical and physical carcinogens or genetic manipulations to study tumor progression. In this work, different melanoma cell lines were established after submitting a nontumorigenic melanocyte lineage (melan-a) to sequential cycles of forced anchorage impediment. The great majority of these cells underwent anoikis when maintained in suspension. After one deadhesion cycle, phenotypic alterations were noticeable in the few surviving cells, which became more numerous and showed progressive alterations after each adhesion impediment step. No significant differences in cell surface expression of integrins were detected, but a clear electrophoretic migration shift, compatible with an altered glycosylation pattern, was observed for beta1 chain in transformed cell lines. In parallel, a progressive enrichment of tri- and tetra-antennary N-glycans was apparent, suggesting increased N-acetylglucosaminyltransferase V activity. Alterations both in proteoglycan glycosylation pattern and core protein expression were detected during the transformation process. In conclusion, this model corroborates the role of adhesion state as a promoting agent in transformation process and demonstrates that cell adhesion disturbances may act as carcinogenic stimuli, at least for a nontumorigenic immortalized melanocyte lineage. These findings have intriguing implications for in vivo carcinogenesis, suggesting that anchorage independence may precede, and contribute to, neoplastic conversion.

Rapid induction of malignant tumor in Sprague-Dawley rats by injection of RK3E-ras cells.

Several tumor animal models have been provided as a tool for developing cancer therapy. Here, we developed rapid, easy-to use, and cost-effective new rat animal model for invasion and metastasis of cancer using genetically k-ras-induced rat kidney cells (RK3E-ras). We observed tumor as early as 3 days after injection of RK3E-ras cells in subcutaneous of Sprague-Dawley rats. Tumor size and volume were increased exponentially for 2 weeks. The tail vein injected rats obtained the lethal infiltration in the lung within 2 weeks. This tumor animal model has great potential for studying cancer processes and short-term screening of variable cancer therapy strategy.

Noninvasive method of immortalized neural stem-like cell transplantation in an experimental model of Huntington's disease.

A loss of neostriatal neurons is a characteristic of Huntington's disease (HD), and neural tissue transplantation has been performed directly into the striatum. Since the neural stem cells have ability to migrate into the lesion site, we administered immortalized neural stem-like cells (NSC) into the ventricle or via a tail vein following unilateral intrastriatal quinolinic acid lesioning in Sprague-Dawley rats. To identify transplanted NSC, cells were encoded with lac Z and beta-galactosidase histochemistry was performed. lac Z+ cells were detected in the lesioned striatum but tissue damage or tumor formation was not observed. This study shows that NSC migrate into the striatum, either from ventricle or from the systemic circulation, providing less invasive routes for stem cell application in HD.

A virosomal immunization strategy against cervical cancer and pre-malignant cervical disease.

In this study, we demonstrate that fusion-active virosomes, containing recombinant human papillomavirus type 16 (HPV16) E7 protein antigen, are capable of inducing a robust class I MHC-restricted cytotoxic T-lymphocyte (CTL) response against HPV-transformed tumour cells in a murine model system. Virosomes are reconstituted viral envelopes, which do not contain the genetic material of the native virus. During the reconstitution process, protein antigens can be encapsulated within the virosomes. In the present study, we used virosomes derived from influenza virus. These virosomes retain the cell binding and membrane fusion characteristics of native influenza virus, and have the capacity to deliver encapsulated antigens to the cytosol of antigen-presenting cells through fusion from within acidic endosomes. After immunization of mice with virosomes containing encapsulated HPV16 E7 protein, the animals developed a strong E7-specific CTL response as assessed by 51Cr release measurements and MHC tetramer staining of spleen cells. Immunization with E7-containing virosomes also resulted in E7-specific antibody responses. In tumour challenge experiments, immunization of mice with E7-containing virosomes prevented tumour outgrowth in >70% of the animals. Thus, influenza-derived virosomes with encapsulated HPV E7 protein antigen act as an excellent vaccine delivery system for induction of cellular immunity against HPV-transformed cells and represent a promising immunotherapeutic vaccine for the treatment of (precursor lesions of) cervical cancer.

Characteristic of two mouse bcr-abl-transformed cell lines: I. General properties of the cells.

In an effort to develop an experimental system suitable for immunological studies in which Bcr-Abl-positive cells are to be used as antigens, we examined the properties of two mouse (Balb/c) established cell lines that express the Bcr-Abl protein and are oncogenic for syngeneic animals. Under standard conditions the two cell lines, viz. Ba-p210 (B210) and 12B1, expressed comparable amounts of the Bcr-Abl protein. However, they differed in a number of characteristics. From the morphological point of view, B210 cells were the more homogeneous, being mainly represented by leukaemic blastic cells with a large number of AgNORs as markers indicating a high proliferative activity. 12B1 cells were more polymorphic and giant cells were detected within their populations. Many 12B1 cells exhibited nuclear segmentation and "band-like" structures. Markers of proliferation were less frequent in 12B1 and the tendency for aging was more pronounced in these cells. The 12B1 cells were slightly more sensitive to imatinib mesylate than B210 cells. In B210 cells, the expression of MHC class I was downregulated, which was not the case with 12B1 cells. Both cell lines induced leukaemia-like disease in mice after intravenous application but, as compared with B210, 12B1 cells were about 100 times more oncogenic and the disease they induced was more aggressive. Moreover, 12B1, but not B210, induced tumours after subcutaneous or intraperitoneal inoculation.

The promoters of human cell cycle genes integrate signals from two tumor suppressive pathways during cellular transformation.

Deciphering regulatory events that drive malignant transformation represents a major challenge for systems biology. Here, we analyzed genome-wide transcription profiling of an in vitro cancerous transformation process. We focused on a cluster of genes whose expression levels increased as a function of p53 and p16(INK4A) tumor suppressors inactivation. This cluster predominantly consists of cell cycle genes and constitutes a signature of a diversity of cancers. By linking expression profiles of the genes in the cluster with the dynamic behavior of p53 and p16(INK4A), we identified a promoter architecture that integrates signals from the two tumor suppressive channels and that maps their activity onto distinct levels of expression of the cell cycle genes, which, in turn, correspond to different cellular proliferation rates. Taking components of the mitotic spindle as an example, we experimentally verified our predictions that p53-mediated transcriptional repression of several of these novel targets is dependent on the activities of p21, NFY, and E2F. Our study demonstrates how a well-controlled transformation process allows linking between gene expression, promoter architecture, and activity of upstream signaling molecules.

Gene therapy for chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a congenital immune deficiency that is a promising therapeutic target for gene replacement into haematopoietic stem cells (HSCs). CGD results from mutations in any one of four genes encoding subunits of the superoxide-generating NADPH oxidase of phagocytes. Life-threatening, recurrent bacterial and fungal infections, as well as inflammatory granulomas, are the hallmarks of the disease. NADPH oxidase activity can be reconstituted by retroviral- or lentiviral-mediated gene transfer to human CGD marrow in vitro and in xenograft transplant models. Gene transfer studies in knockout mouse models that resemble the human disease suggest that correction of oxidase activity in a minority of phagocytes will be of clinical benefit. Phase I clinical studies in unconditioned CGD patients showed transient expression of small numbers of gene-corrected neutrophils. Areas of research at present include efforts to enhance gene transfer rates into repopulating HSCs using vectors that transduce quiescent cells, and to increase the engraftment of genetically corrected HSCs using non-myeloablative conditioning and drug resistance genes for selection.

Immortalization of human nucleus pulposus cells by a recombinant SV40 adenovirus vector: establishment of a novel cell line for the study of human nucleus pulposus cells.

STUDY DESIGN: Establishment and characterization of a de novo cell line derived from human nucleus pulposus cells using a recombinant simian virus 40 (SV40) adenovirus vector. OBJECTIVES: To assess the feasibility of human nucleus pulposus cell line procurement and to evaluate the character of the resultant outcome to better understand the nature of human nucleus pulposus cells. SUMMARY OF BACKGROUND DATA: Despite recent advances in disc cell biologic research, the fundamental nature of nucleus pulposus cells, especially in the context of human cell lines, is still not well understood. Therefore, a broad-based analysis of these cells is of significant necessity. Because of the limited amount of existing human cells, establishment of an immortal cell line would greatly facilitate resource supply. METHODS: After release of informed consent, tissue samples of nucleus pulposus were obtained from the lumbar intervertebral disc of a 19-year-old man undergoing anterior fusion for burst fracture. Samples with no apparent damage were selected and digested enzymatically for primary culture and then were infected with recombinant SV40 adenovirus vector (Ad/SV40). The infected cells were maintained in culture for more than 40 population doublings, after which they were considered immortalized. Next, confirmation of expression of T antigen was performed and resultant immortalized cell lines were designated and classified as human nucleus pulposus cell line derived from Ad/SV40 infection-1 (HNPSV-1). HNPSV-1 cells were characterized and compared with their mother cells under two designated culture conditions: monolayer and three-dimensional. Morphologic and immunocytochemical analyses were performed at various intervals. Cell proliferation, DNA synthesis, proteoglycan synthesis, gene expression profiling, and karyotypic analyses were also performed. Moreover, HNPSV-1 cells were injected into rabbit discs to assess the presence of tumorigenesis. RESULTS: Recombinant SV40 adenovirus vector infected nucleus pulposus cells with relatively high efficiency (90%> at multiplicity of infection 100). HNPSV-1 demonstrated marked prolongation of cell life with continuous cell doublings for over 5 months (60-100 cell population doublings). Despite significant increase in cell proliferation and DNA synthesis when compared with its mother cells, resultant cell lines expressed strikingly similar cell morphology and functional characteristics. Atypical karyotypes were noted; however, no apparent tumorigenesis was seen in rabbit discs 24 weeks after injection of HNPSV-1. CONCLUSIONS: HNPSV-1 was successfully established using recombinant SV40 adenovirus vector. Results showed that human nucleus pulposus cells are capable of immortalization with maintenance of original cell characteristics. It is anticipated that these cells will be useful for in vitro studies of the biologic nature of human nucleus pulposus cells.

Immortalization of hepatic progenitor cells.

Development of cell therapy-based strategies for the treatment of liver failures and of inherited metabolic diseases has become a necessity because of the limitations of orthotopic liver transplantation, including shortage of donor livers. This shortage limits also the availability for hepatocytes and these terminally differentiated cells cannot be expanded in vitro. Thus, other alternative sources of hepatocytes have to be explored such as hepatic stem cells. Foetal hepatic cells have specific intrinsic properties compared to adult hepatocytes that should overcome some of their limitations. Thus, the availability of in vitro expandable progenitor cells by means of immortalization and without inducing a transformed phenotype and disrupting their differentiation potential would facilitate studies on cell engraftment and differentiation within the hepatic parenchyma. A temporally controlled expression of the immortalizing transgene would also permit to revert the immortalized phenotype prior to cell transplantation. Since characteristics of murine stem cells cannot readily be extrapolated to their human or other primate counterparts, we have immortalized one clone of primate hepatic progenitor cells using a retroviral vector expressing SV40 Large T flanked by lox P sites. These hepatic cells were bipotent, expressing markers of both hepatocytic and biliary lineages. After transplantation into athymic mice, approximately 50% of immortalized cells engrafted, stopped proliferating after a few days and differentiated in adult hepatocytes, suggesting that the hepatic microenvironment plays an important role in such regulations. Upon infection with a retrovirus expressing the CRE recombinase, immortalized cells stopped growing and died, showing that immortalization was dependant on SV40 Large T. These studies suggest new approaches to expand hepatic progenitor cells, analyse their fate in animal models aiming at cell therapy of hepatic diseases.