Myospheres are composed of two cell types: one that is myogenic and a second that is mesenchymal.

Previously, in an attempt to isolate stem cells that would be capable of regenerating injured skeletal muscle, we cultured cells derived from muscle, non-adherently, in serum-free media. As a result of the culture conditions used, these cells formed spheres, and thus were referred to as myospheres. It was found that myosphere-derived cells expressed Sca-1, a marker that is not typically associated with myogenic cells, and as a result has generated some questions as to the origin of these cells. The goal of this study was to clearly determine the origin of myosphere-derived cells, and in particular to answer the question of whether myospheres contain myogenic cells. To determine if myospheres were composed of myogenic cells without altering the structure of myospheres or the culture conditions used to maintain myospheres, I isolated these cells from yellow fluorescent protein (YFP)-Myf5, YFP-MyoD, and ZsGreen-Pax7 lineage-tracing mice and monitored their growth over time. I found that myospheres do contain myogenic cells, but that these cells are gradually lost over time (within 2 months). Additionally, the use of the lineage-tracing mice gave an interesting perspective into the composition of myospheres. I found that myospheres were composed of two distinct cell types, one that is myogenic (α7 integrin+) and contains cells expressing Myf5, MyoD, and Pax7, and a second that is non-myogenic (α7 integrin-) expressing platelet-derived growth factor receptor alpha (PDGFRα) and Sca-1, both of which have been associated with fibro/adipocyte mesenchymal cells.

Sca-1 is involved in the adhesion of myosphere cells to αVß3 integrin.

A myosphere cell is a unique type of muscle stem cell that is able to maintain its pre-myogenic state in culture over time. These cells are propagated in culture as free-floating, non-adherent spheres. We believe that the 3-dimensional adhesive cell-cell interactions involved in maintaining the sphere-like myosphere structures are also involved in maintaining their longevity in culture. We found that Sca-1, which is highly expressed by myosphere cells, plays a role in the growth and the formation of the myospheres. In comparing adhesion molecules expressed by 3-dimensionally grown myosphere cells to those expressed by 2-dimensionally grown primary myoblasts, we found that there was a distinct difference in the expression of ß3 integrin. Upon further investigation we discovered that there is an adhesive interaction between Sca-1(+) cells and αVß3 integrin. Here we show that Sca-1(+) cells (myosphere cells and NIH3T3 cells) adhere to αVß3 integrin and that Sca-1(-) cells (primary myoblasts) do not adhere. The interaction between Sca-1 and αVß3 integrin was confirmed using antibody blocking, shRNA knockdown of Sca-1 in Sca-1(+) cells, and by expressing Sca-1 cDNA in Sca-1(-) cells, which demonstrated that the level of adhesion of these cells to αVß3 integrin was dependent on the presence of Sca-1. Additionally, we found that the co-expression of Sca-1 and ß3 resulted in significantly greater adhesion of Sca-1(+) cells to αVß3 integrin. In conclusion, our data indicate that Sca-1 is involved in maintaining the 3-dimensional myosphere cell-cell contacts and that Sca-1 is involved in the binding of cells to αVß3 integrin.

Adult muscle 'stem' cells can be sustained in culture as free-floating myospheres.

The effectiveness of cell-based therapy to treat muscle disease has been hampered by difficulties in isolating, maintaining and propagating the stem cells that are needed for treatment. Here we report the isolation of muscle-derived stem cells from both young and old mice and their propagation over extended periods of time in culture as "free-floating" myospheres. Analysis of these sphere-forming cells showed that they express stem cell antigen-1 (Sca-1), beta1 integrin (CD29), Thy-1 (CD90), and CD34, but did not express CD45, CD31, or myogenic markers (Pax7, Myf5, and MyoD). We found that cells derived from myospheres and then grown adherently (MDACs) behaved similar to primary myoblasts, in that these cells expressed myogenic markers and were able to easily form multinucleated myotubes. Unlike the parental myospheres but analogous to primary myoblasts, MDACs expressed Pax7, Myf5, and MyoD, indicating that the parent myosphere cells were a more primitive type of cell. In support of this we demonstrated that myospheres were also able to differentiate into adipogenic and osteogenic cells in culture, as well as being able to contribute to injured muscle in vivo. In summary, we report that primitive adult muscle stem cells can be easily isolated and sustained in culture as myospheres.

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.

Design of a trans protease lentiviral packaging system that produces high titer virus.

BACKGROUND: The structural and enzymatic proteins of the human immunodeficiency virus (HIV) are initially generated as two long polyproteins encoded from overlapping reading frames, one producing the structural proteins (Gag) and the second producing both structural and enzymatic proteins (Gag-Pol). The Gag to Gag-Pol ratio is critical for the proper assembly and maturation of viral particles. To minimize the risk of producing a replication competent lentivirus (RCL), we developed a "super-split" lentiviral packaging system in which Gag was separated from Pol with minimal loss of transducibility by supplying protease (PR) in trans independently of both Gag and Pol. RESULTS: In developing this "super-split" packaging system, we incorporated several new safety features that include removing the Gag/Gag-Pol frameshift, splitting the Gag, PR, and reverse transcriptase/integrase (RT/IN) functions onto separate plasmids, and greatly reducing the nucleotide sequence overlap between vector and Gag and between Gag and Pol. As part of the construction of this novel system, we used a truncated form of the accessory protein Vpr, which binds the P6 region of Gag, as a vehicle to deliver both PR and RT/IN as fusion proteins to the site of viral assembly and budding. We also replaced wt PR with a slightly less active T26S PR mutant in an effort to prevent premature processing and cytoxicity associated with wt PR. This novel "super-split" packaging system yielded lentiviral titers comparable to those generated by conventional lentiviral packaging where Gag-Pol is supplied intact (1.0 x 106 TU/ml, unconcentrated). CONCLUSION: Here, we were able to create a true "split-function" lentiviral packaging system that has the potential to be used for gene therapy applications. This novel system incorporates many new safety features while maintaining high titers. In addition, because PR is supplied in trans, this unique system may also provide opportunities to examine viral protein processing and maturation.

HIF-dependent induction of adenosine A2B receptor in hypoxia.

Adenosine has been widely associated with hypoxia of many origins, including those associated with inflammation and tumorogenesis. A number of recent studies have implicated metabolic control of adenosine generation at sites of tissue hypoxia. Here, we examine adenosine receptor control and amplification of signaling through transcriptional regulation of endothelial and epithelial adenosine receptors. Initial studies confirmed previous findings indicating selective induction of human adenosine A2B receptor (A2BR) by hypoxia. Analysis of the cloned human A2BR promoter identified a functional hypoxia-responsive region, including a functional binding site for hypoxia-inducible factor (HIF) within the A2BR promoter. Further studies examining HIF-1alpha DNA binding and HIF-1alpha gain and loss of function confirmed strong dependence of A2BR induction by HIF-1alpha in vitro and in vivo mouse models. Additional studies in endothelia overexpressing full-length A2BR revealed functional phenotypes of increased barrier function and enhanced angiogenesis. Taken together, these results demonstrate transcriptional coordination of A2BR by HIF-1alpha and amplified adenosine signaling during hypoxia. These findings may provide an important link between hypoxia and metabolic conditions associated with inflammation and angiogenesis.

Postnatal neovascularization by endothelial progenitor cells immortalized with the simian virus 40T antigen gene.

Endothelial progenitor cells (EPCs) contribute to blood vessel formation in ischemic and tumorous tissues, but comprise only a small population in circulation. We attempted to immortalize putative EPCs from human cord blood. Human CD34+ cord blood cells were cultured in the presence of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF), and transfected with a retroviral vector encoding the simian virus 40 large T (SV40T) antigen. This resulted in the immortalization of cord blood cells, leading to the establishment of several cell lines. One of these lines, HYCEC-1, exhibited a phenotype characteristic of the endothelial lineage, including expression of von Willebrand factor and VEGF receptor-2 (VEGFR-2/KDR/Flk-1) and uptake of acetylated-low density lipoprotein. Flow cytometric analysis revealed that HYCEC-1 cells were strongly positive for CD31 and CD146, moderately positive for CD144, weakly positive for CD133 and CD34, and negative for CD14 and CD45. HYCEC-1 cells formed capillary-like structures on basement matrix gel in vitro. Upon transplantation into the ischemic hind limb of nude rats, HYCEC-1 cells efficiently participated in neovascularization and augmented blood flow. The immortalized HYCEC-1 cells are suggested to be a class of EPCs that can efficiently participate in postnatal neovasculogenesis in the ischemic hind limb, and may also be a useful tool for studying tumor vessel formation.

HIF-1-dependent repression of equilibrative nucleoside transporter (ENT) in hypoxia.

Extracellular adenosine (Ado) has been implicated as central signaling molecule during conditions of limited oxygen availability (hypoxia), regulating physiologic outcomes as diverse as vascular leak, leukocyte activation, and accumulation. Presently, the molecular mechanisms that elevate extracellular Ado during hypoxia are unclear. In the present study, we pursued the hypothesis that diminished uptake of Ado effectively enhances extracellular Ado signaling. Initial studies indicated that the half-life of Ado was increased by as much as fivefold after exposure of endothelia to hypoxia. Examination of expressional levels of the equilibrative nucleoside transporter (ENT)1 and ENT2 revealed a transcriptionally dependent decrease in mRNA, protein, and function in endothelia and epithelia. Examination of the ENT1 promoter identified a hypoxia inducible factor 1 (HIF-1)-dependent repression of ENT1 during hypoxia. Using in vitro and in vivo models of Ado signaling, we revealed that decreased Ado uptake promotes vascular barrier and dampens neutrophil tissue accumulation during hypoxia. Moreover, epithelial Hif1alpha mutant animals displayed increased epithelial ENT1 expression. Together, these results identify transcriptional repression of ENT as an innate mechanism to elevate extracellular Ado during hypoxia.

A human beta-cell line for transplantation therapy to control type 1 diabetes.

A human pancreatic beta-cell line that is functionally equivalent to primary beta-cells has not been available. We established a reversibly immortalized human beta-cell clone (NAKT-15) by transfection of primary human beta-cells with a retroviral vector containing simian virus 40 large T-antigen (SV40T) and human telomerase reverse transcriptase (hTERT) cDNAs flanked by paired loxP recombination targets, which allow deletion of SV40T and TERT by Cre recombinase. Reverted NAKT-15 cells expressed beta-cell transcription factors (Isl-1, Pax 6, Nkx 6.1, Pdx-1), prohormone convertases 1/3 and 2, and secretory granule proteins, and secreted insulin in response to glucose, similar to normal human islets. Transplantation of NAKT-15 cells into streptozotocin-induced diabetic severe combined immunodeficiency mice resulted in perfect control of blood glucose within 2 weeks; mice remained normoglycemic for longer than 30 weeks. The establishment of this cell line is one step toward a potential cure of diabetes by transplantation.

High-level beta-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells.

Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and beta-thalassemia. Here, we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling betaA-T87Q-globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scid mice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene, and erythroid cells derived in vitro from these in vivo-regenerated cells produced high levels of betaA-T87Q-globin protein. Linker-mediated PCR analysis identified multiple transgene-positive clones in all mice analyzed with 2.1 +/- 0.1 integrated proviral copies per cell. Genomic sequencing of vector-containing fragments showed that 86% of the proviral inserts had occurred within genes, including several genes implicated in human leukemia. These findings indicate effective transduction of very primitive human cord blood cells with a candidate therapeutic lentiviral vector resulting in the long-term and robust, erythroid-specific production of therapeutically relevant levels of beta-globin protein. However, the frequency of proviral integration within genes that regulate hematopoiesis points to a need for additional safety modifications.

Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT.

BACKGROUND AND AIMS: Liver endothelial cells (LECs) perform an essential role in important pathophysiologic functions in the liver. Establishment of a human LEC line facilitates advances in LEC research. Here, we present immortalization of human LECs using retroviral gene transfer of simian virus 40 large T antigen (SV40T) and human telomerase reverse transcriptase (hTERT). We also demonstrate excision of SV40T and hTERT with TAT-mediated Cre/loxP recombination and subsequent cell sorting. METHODS: First, human LECs were transduced with a retroviral vector somatostatin receptor (SSR)#69 expressing SV40T and hygromycin-resistance genes flanked by a pair of loxA recombination targets. Then, cells were retrovirally superinfected with SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs that were intervened by two loxBs. One SV40T-and hTERT-immortalized LEC clone, TMNK-1, was established and analyzed for its biologic characteristics. RESULTS: The cells were hygromycin-resistant and uniformly positive for GFP expression. TMNK-1 expressed EC markers, including factor VIII, vascular endothelial growth factor receptors (flt-1, KDR/Flk-1), and CD34, showed uptake of Di-I-acetylated-low-density lipoprotein and angiogenic potential in Matrigel assays. After lipopolysaccharide treatment, TMNK-1 produced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and exhibited increased expression of intra-cellular adhesive molecule-1, vascular cellular adhesive molecule-1, and VE-cadherin. After treatment with TAT-Cre recombinase fusion protein, approximately 60% of TMNK-1 was negative for GFP expression, and subsequent cell sorting of this population for GFP allowed for collection of the reverted form of TMNK-1. CONCLUSIONS: This study demonstrates the utility and efficiency of the reversible immortalization procedure to expand primary human LECs for basic studies.

Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT.

BACKGROUND: Cholangiocytes perform an essential role in important pathophysiologic functions in the liver. Establishment of a human cholangiocyte line facilitates advances in cholangiocyte research and clinical applications for cell therapies. Here, we describe the immortalization of human cholangiocytes using serial transfection of simian virus 40 large T (SV40T) followed by human telomerase reverse transcriptase (hTERT). METHODS: SV40T-transduced human liver OUMS-21 cells were superinfected with a retroviral vector SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs. Resulting cell lines were evaluated for gene expression, functional cholangiogenic characteristics in vitro and in vivo, and response to lipopolysaccharide (LPS). RESULTS: One of the SV40T- and hTERT-immortalized cholangiocyte clones, MMNK-1, was established. MMNK-1 expressed cholangiocyte markers, including cytokeratin (CK)-7 and -19 and exhibited cholangiogenic tubule formation in a Matrigel assay. When transplanted into the immunodeficient mice, MMNK-1 cells developed bile duct-like structures in the spleen. After LPS treatment, MMNK-1 cells produced interleukin-6 and failed to form well-developed tubular structures in Matrigel. CONCLUSION: We have established an immortalized cholangiocyte cell line, MMNK-1, using SV40T and hTERT transduction.

Transplantation of reversibly immortalized insulin-secreting human hepatocytes controls diabetes in pancreatectomized pigs.

Type 1 diabetes results from the destruction of insulin-producing pancreatic beta-cells by a beta-cell-specific autoimmune process. Although converting other cell types into insulin-producing cells may compensate for the loss of the beta-cell mass while evading beta-cell-specific T-cell responses, proof-of-principle of this approach in large animal models is lacking. This investigation was initiated to determine whether an insulin-producing human hepatocyte line can control diabetes when transplanted into totally pancreatectomized diabetic pigs. We established a reversibly immortalized human hepatocyte line, YOCK-13, by transferring a human telomerase reverse transcriptase cDNA and a drug-inducible Cre recombinase cassette, followed by cDNA for a modified insulin under the control of the L-type pyruvate kinase (L-PK) promoter. YOCK-13 cells produced small amounts of modified insulin and no detectable endogenous L-PK at low glucose concentrations, whereas they produced large amounts of both modified insulin and L-PK in response to high glucose concentrations. Xenotransplantation of YOCK-13 cells via the portal vein into immunosuppressed, totally pancreatectomized pigs decreased hyperglycemia and prolonged survival without adverse effects such as portal thrombosis, liver necrosis, pulmonary embolism, and tumor development. We suggest that this reversibly immortalized, insulin-secreting human hepatocyte line may overcome the shortage of donor pancreata for islet transplantation into patients with type 1 diabetes.

Immortalization of bone marrow-derived human mesenchymal stem cells by removable simian virus 40T antigen gene: analysis of the ability to support expansion of cord blood hematopoietic progenitor cells.

Human marrow-derived mesenchymal stem cells (MSC), which have the potential to differentiate into mesenchymal tissues, such as bone, cartilage, adipose and bone marrow stroma, were transduced with a retroviral vector carrying the simian virus 40 large T antigen, hygromycin-resistant gene and herpes simplex virus thymidine kinase gene, that can be excised by Cre/loxP site-specific recombination. This resulted in establishment of an MSC cell line, HMSC-1, which retained original surface characteristics and differentiation potential, and exhibited a higher proliferative capacity than parental cells. HMSC-1 expressed mRNAs of BMP-4, Jagged-1, and SCF that are known to promote hematopoiesis. Human CB CD34+ hematopoietic progenitor cells (HPC) cultured on a layer of HMSC-1 cells showed high expansion of CD34+CD38- immature HPC, capable of reconstituting human hematopoiesis in non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. This cell line may be of value for developing strategies for ex vivo expansion of human HPC.

Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers.

BACKGROUND: Maintenance of liver-specific functions has been shown to be stabilized by co-cultivation of hepatocytes with hepatic stellate cells (HSC). Because the limited lifespan of human HSC is a major hurdle to their use, the authors report here the amplification of human HSC populations in vitro by retroviral transfer of human telomerase reverse transcriptase (hTERT). METHODS: Human HSC strain LI 90 cells were transduced with a retroviral vector SSR#197 expressing hTERT and green fluorescent protein (GFP) cDNA flanked by a pair of loxP. TWNT-1, one of SSR#197-immortalized HSC, was characterized. Differentiated liver functions were evaluated in an immortalized human hepatocyte NKNT-3-TWNT-1 co-culture system. RESULTS: TWNT-1 cells showed differential functions of HSC, including uptake of acetylated low-density lipoproteins and synthesis of collagen type I and hepatocyte growth factor. Efficient excision of the retrovirally transferred hTERT and GFP cDNAs was achieved by TAT-mediated expression of the Cre recombinase and subsequent GFP-negative cell sorting. When co-cultured with TWNT-1 cells, NKNT-3 increased protein expression of the detoxifying cytochrome P450-associated protein isoenzymes 3A4 and 2C9 and urea synthesis. CONCLUSIONS: TWNT-1 cells could be valuable in the study of integrated liver functions and contribute to the optimization of liver cell therapies and bioartificial livers.

Permanent and panerythroid correction of murine beta thalassemia by multiple lentiviral integration in hematopoietic stem cells.

Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid beta globin gene expression has not yet been achieved in beta thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid correction of severe beta thalassemia in mice, resulting from a homozygous deletion of the beta major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-1-derived [beta globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time approximately 95% of the red blood cells in all mice contained human beta globin contributing to 32 +/- 4% of all beta-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free alpha globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in beta thalassemia patients.

Treatment of liver failure in rats with end-stage cirrhosis by transplantation of immortalized hepatocytes.

The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, 35 immortalized rat hepatocyte clones were developed by transduction with the gene encoding the simian virus 40 T antigen (SV40Tag). The SV40Tag sequence and a suicide gene, herpes simplex virus thymidine kinase (HSV-tk), were flanked by loxP sequences so that they could be excised by Cre/lox recombination. When transplanted into the spleens of portacaval-shunted rats, 3 of the 35 immortalized hepatocyte clones prevented the development of hyperammonemia-induced hepatic encephalopathy. The protection was reversed by treatment with ganciclovir, which kills HSV-tk-expressing cells. Transplantation of alginate-encapsulated, immortalized hepatocytes into the spleens of cirrhotic rats resulted in significant improvement in prothrombin time, serum albumin and bilirubin levels, hepatic encephalopathy score, and duration of survival. The metabolic support provided by the immortalized cells equaled that observed after transplantation of primary rat hepatocytes. In conclusion, immortalized hepatocytes can function as well as primary hepatocytes following transplantation and can be engineered to contain safeguards that could make them clinically useful. Further investigation is warranted regarding the mechanisms of loss of mass or function of the transplanted hepatocytes over time and how the relatively few engrafted hepatocytes can ameliorate liver decompensation in cirrhosis.

Controlled expansion of human endothelial cell populations by Cre-loxP-based reversible immortalization.

Endothelial cells (ECs) play multiple physiological functions and are central to many pathological processes. Various biological studies as well as cell and gene therapy applications would benefit substantially from a procedure that would result in the expansion in culture of large numbers of highly differentiated human ECs. Here, we report the amplification in vitro of human EC populations, which occurred during the first phase of reversible immortalization resulting from the retroviral transfer of an oncogene that was subsequently excised by Cre-loxP-mediated site-specific recombination. Human umbilical vein endothelial cells (HUVECs) and human liver sinusoidal endothelial cells (HLSECs) were transduced with a retroviral vector that expresses the simian virus 40 large T (SV40T) gene flanked by positive and negative selectable markers and a pair of loxP recombination targets. Transduced HUVECs and HLSECs yielded clones with greatly extended life spans, referred to as HNNT-1 and HNNT-2 cells, respectively. HNNT-1 and HNNT-2 cells showed morphological characteristics of ECs and were maintained in culture up to population doubling level (PDL) 80 for HNNT-1 and PDL 65 for HNNT-2 cells. HNNT-1 and HNNT-2 cells were not tumorigenic when transplanted into severe combined immunodeficiency mice and were sensitive to ganciclovir as well as G418. Both cell clones expressed EC markers, which include factor VIII, VEGF receptors (Flt-1 and KDR/Flk-1), and CD34, and endocytosed acetylated low-density lipoproteins. Formation of capillary-like structures in a Matrigel assay was observed with HNNT-1 and HNNT-2 cells until at least PDL 50. Complete elimination of the transferred SV40T gene was achieved in virtually 100% of HNNT-1 and HNNT-2 cells after infection with a recombinant adenovirus expressing the Cre recombinase fused to a nuclear localization signal and subsequent selection with G418. Reverted cells maintained their differentiated EC phenotype. This study extends the utility of the reversible immortalization procedure and provides a means to expand primary human ECs of various sources for basic studies and possible cell and gene therapies.

A reversibly immortalized human hepatocyte cell line as a source of hepatocyte-based biological support.

The application of hepatocyte transplantation (HTX) is increasingly envisioned for temporary metabolic support during acute liver failure and provision of specific liver functions in inherited liver-based metabolic diseases. Compared with whole liver transplantation, HTX is a technically simple procedure and hepatocytes can be cryopreserved for future use. A major limitation of this form of therapy in humans is the worldwide shortage of human livers for isolating an adequate number of transplantable human hepatocyes when needed. Furthermore, the numbers of donor livers available for hepatocyte isolation is limited by competition for their use in whole organ transplantation. Considering the cost of hepatocyte isolation and the need for immediate preparation of consistent and functional cells, it is unlikely that human hepatocytes can be obtained on such a scale to treat a large number of patients with falling liver functions. The utilization of xenogenic hepatocytes will result in additional concerns regarding transmission of infectious pathogens and immunological and physiological incompatibilities between animals and humans. An attractive alternative to primary human hepatocytes is the use of tightly regulated human hepatocyte cell lines. Such cell lines can provide the advantages of unlimited availability, sterility and uniformity. We describe here methods for creating transplantable human hepatocyte cell lines using currently available cell cultures and gene transfer technology.

Cre/loxP-Based Reversible Immortalization of Human Hepatocytes 1.

An ideal alternative to the primary human hepatocytes for hepatocyte transplantation would be to use a clonal cell line that grows economically in culture and exhibits the characteristics of differentiated, nontransformed hepatocytes following transplantation. The purpose of the present studies was to establish a reversibly immortalized human hepatocyte cell line. Human hepatocytes were immortalized with a retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40Tag) gene flanked by a pair of loxP recombination targets. One of the resulting clones, NKNT-3, showed morphological characteristics of liver parenchymal cells and expressed the genes of differentiated liver functions. NKNT-3 cells offered unlimited availability. After an adenoviral delivery of Cre recombinase and subsequent differential selection, efficient removal of SV40Tag from NKNT-3 cells was performed. Here we represent that elimination of the retrovirally transferred SV40Tag gene can be excised by adenovirus-mediated site-specific recombination.