Engineered human mesenchymal stem cells: a novel platform for skeletal cell mediated gene therapy.

BACKGROUND: Human mesenchymal stem cells (hMSCs) are pluripotent cells that can differentiate to various mesenchymal cell types. Recently, a method to isolate hMSCs from bone marrow and expand them in culture was described. Here we report on the use of hMSCs as a platform for gene therapy aimed at bone lesions. METHODS: Bone marrow derived hMSCs were expanded in culture and infected with recombinant adenoviral vector encoding the osteogenic factor, human BMP-2. The osteogenic potential of genetically engineered hMSCs was assessed in vitro and in vivo. RESULTS: Genetically engineered hMSCs displayed enhanced proliferation and osteogenic differentiation in culture. In vivo, transplanted genetically engineered hMSCs were able to engraft and form bone and cartilage in ectopic sites, and regenerate bone defects (non-union fractures) in mice radius bone. Importantly, the same results were obtained with hMSCs isolated from a patient suffering from osteoporosis. CONCLUSIONS: hMSCs represent a novel platform for skeletal gene therapy and the present results suggest that they can be genetically engineered to express desired therapeutic proteins inducing specific differentiation pathways. Moreover, hMSCs obtained from osteoporotic patients can restore their osteogenic activity following human BMP-2 gene transduction, an important finding in the future planning of gene therapy treatment for osteoporosis.

Exogenously regulated stem cell-mediated gene therapy for bone regeneration.

Regulated expression of transgene production and function is of great importance for gene therapy. Such regulation can potentially be used to monitor and control complex biological processes. We report here a regulated stem cell-based system for controlling bone regeneration, utilizing genetically engineered mesenchymal stem cells (MSCs) harboring a tetracycline-regulated expression vector encoding the osteogenic growth factor human BMP-2. We show that doxycycline (a tetracycline analogue) is able to control hBMP-2 expression and thus control MSC osteogenic differentiation both in vitro and in vivo. Following in vivo transplantation of genetically engineered MSCs, doxycycline administration controlled both bone formation and bone regeneration. Moreover, our findings showed increased angiogenesis accompanied by bone formation whenever genetically engineered MSCs were induced to express hBMP-2 in vivo. Thus, our results demonstrate that regulated gene expression in mesenchymal stem cells can be used as a means to control bone healing.

Expression and characterization of bone morphogenetic protein-2 in Chinese hamster ovary cells.

Bone is a dynamic tissue that responds to many factors including vitamin D, parathyroid hormone, estrogen, calcitonin, and bone morphogenetic proteins (BMPs). The ability to stimulate new bone growth would permit novel therapies for situations where bone mass has been lost due to accident or disease. Purified BMP-2, in conjunction with a suitable matrix, is sufficient to stimulate the synthesis of new bone (Wang et al., 1990). We have expressed recombinant human BMP-2 at high levels in Chinese hamster ovary cells using methotrexate-mediated gene amplification. Several forms of BMP-2 are secreted from CHO cells: (1) an amino-terminal propeptide of 40-45 kDa, (23) a mature active 30 kDa homodimer consisting of 18-22 kDa subunits, and (3) a small amount of uncleaved 60 kDa precursor protein. The mature, active protein is predominantly a 30 kDa homodimer consisting of subspecies of 18 and 22 kDa which differ by proteolytic processing at their amino termini. Mature BMP-2 and propeptide contain high mannose and complex N-linked oligosaccharides, respectively. The molar amount of secreted, processed propeptide is approximately 5-fold higher than mature BMP-2 in conditioned medium. BMP-2 associates with both the extracellular matrix and the surface of CHO cells, which may in part account for the unequal levels of extracellular propeptide and mature forms of the molecule in the conditioned medium. Recombinant BMP-2 can be expressed in sufficient quantities to assess its therapeutic potential for bone regeneration.

Isolation and characterization of the rat chromosomal gene for a polypeptide (pS1) antigenically related to statin.

Increasing evidence shows the existence of nonproliferation-specific gene(s) whose expression is mostly present in growth-arrested cells. One member of this gene family has been identified by previous work as a nuclear protein of 57,000 Da, termed statin. Logical extensions of statin research are to identify the genomic and cDNA clones encoding for statin and to study the regulation of statin gene expression. During the search for the statin gene, we have identified a cDNA clone and a genomic clone named S1 and S10, respectively, by screening a rat brain lambda gt11 expression library with the statin antibody and subsequently using S1 cDNA as a probe to screen a rat genomic cosmid library. Here, we report the cloning and sequencing of the S1 cDNA and S10 genomic clones. Primary sequence analyses indicate that the derived amino acid sequence of S1 shares high homology (greater than 92.6%) with human elongation factor 1 alpha (EF-1 alpha), whereas the 5'- and 3'-untranslated regions are less than 20% homologous. Despite the unusually high degree of similarity between S1 and human EF-1 alpha at the amino acid sequence level, their protein products are different and immunologically distinct. The in vitro transcription and translation product of S1 (pS1), a 49,000-Da polypeptide, reacts only with the monoclonal antibody against statin; this antibody exhibits no antigenic reaction to the EF-1 alpha protein. Northern blot analysis shows that the S1 message is most abundant in G0 phase of 3T3 mouse fibroblasts, but becomes significantly reduced in G1 and S phase cells. EF-1 alpha messages do not show such dramatic changes during cell cycle phase transition. These findings suggest that the expression of the identified S1 cDNA clone is specific for nonproliferating cells and that the in vitro translation product of the S1 cDNA is recognized by the statin antibody. Genomic Southern blots indicate that S1 cDNA is encoded by a single copy gene in the rat genome and is a unique member of the EF-1 alpha/S1 supermultigene family. DNA sequence analysis demonstrates that the rat S1 transcription unit is 12 kilobase pairs in length and contains seven introns. The organization of exons is virtually identical between S1 and human EF-1 alpha. In contrast, neither a TATA box nor a CAAT box is located in the proximal 5'-flanking regions from positions -1 to -1359 of the S1 gene, where we could expect to find the regulatory region containing the elements controlling gene expression; no evident sequence homology to the human EF-1 alpha gene is detected in this region.(ABSTRACT TRUNCATED AT 400 WORDS)

A rat liver 57-kDa protein is identified to share antigenic determinants with statin, a marker for nonproliferating cells.

The nuclear protein statin, found uniquely in nonproliferating cells, has been previously identified by mouse monoclonal antibodies designated as S-30 and S-44. We report here the screening of various rat tissues for proteins cross-reacting with these antibodies. As revealed by immunoblotting, two polypeptides (mol wt 80 and 57 kDa) and a group of lower-molecular-weight proteins migrating between 34 and 38 kDa were found to react with the anti-statin antibodies. The most prominent immunoreactivity was observed with a 57-kDa protein present in rat liver. Upon further fractionation of the liver protein extract with ammonium sulfate [(NH4)2SO4] the 57-kDa protein, designated as rat liver protein 57 or RLp57, was detected independently with both anti-statin antibodies in the 30 to 60% (NH4)2SO4 fraction. In order to determine whether rat liver protein 57 is indeed specifically recognized by anti-statin antibody S-44, we used RLp57 transferred onto nitrocellulose paper as a specific substrate for the adsorption of the S-44 antibody from ascites fluid. As shown by indirect immunofluorescence microscopy the solution remaining after adsorption failed to stain human fibroblasts. The adsorbed immunoglobulin, however, upon elution revealed statin-specific nuclear staining activity on senescent fibroblasts. These findings suggest that rat liver protein 57 and the human fibroblast statin share similar antigenic determinants recognized by the statin-specific S-44 antibody. Our results indicate furthermore that the statin previously identified in fibroblasts may represent one member of a group of several antigenically related proteins detectable with specific anti-statin antibodies.

Expression of carbohydrate binding protein 35 in human fibroblasts: comparisons between cells with different proliferative capacities.

Carbohydrate Binding Protein 35 (CBP35) is a galactose-specific lectin found in the nucleus and cytoplasm of mouse 3T3 fibroblasts. In these cultures, the level of expression and nuclear localization of CBP35 was correlated with the proliferative state of the cells. CBP35 is also found in human fibroblasts. We have compared the expression and localization of CBP35 in human fibroblasts of different replicative capacities: young (passage 11), intermediate (passage 19), and old (passage 33) SL66 cells, and fibroblasts derived from a patient with Werner's syndrome. The results indicate that the expression of CBP35 in cells with either age-acquired or congenital replicative deficiencies was unresponsive to serum stimulation, in contrast with that found in young normal human fibroblasts and in 3T3 cells.

Endogenous lectins from cultured cells: nuclear localization of carbohydrate-binding protein 35 in proliferating 3T3 fibroblasts.

Proliferating 3T3 mouse fibroblasts contain higher levels of the lectin carbohydrate-binding protein 35 (CBP35) than do quiescent cultures of the same cells. An immunofluorescence study was carried out with a rabbit antiserum directed against CBP35 to map the cellular fluorescence distribution in a large population of cells under different growth conditions. This cytometric analysis showed that the lectin is predominantly localized in the nucleus of the proliferating cells. In quiescent 3T3 cultures, the majority of the cells lost their nuclear staining and underwent a general decrease in the overall fluorescence intensity. Stimulation of serum-starved quiescent 3T3 cells by the addition of serum resulted in an increase in the level of CBP35. The percentage of cells showing distinct punctate intranuclear staining reached a maximum at about the same time as the onset of the first S-phase of the cell cycle. All of these results suggest that CBP35 may be a protein whose presence in the nucleus, in discrete punctate distribution, is coordinated with the proliferation state of the cell.

Carbohydrate-binding protein 35: molecular cloning and expression of a recombinant polypeptide with lectin activity in Escherichia coli.

Affinity-purified antibodies directed against carbohydrate-binding protein 35 (CBP35), a galactose-specific lectin, were used to screen a lambda gt 11 expression library derived from mRNA of 3T3 fibroblasts. This screening yielded several putative clones containing cDNA for CBP35, one of which was characterized in terms of its expression of a fusion protein containing beta-galactosidase and CBP35 sequences. Limited proteolysis of lysates containing the fusion protein, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-CBP35, yielded a peptide mapping pattern comparable to that obtained from parallel treatment of authentic CBP35. Such a limited proteolysis followed by affinity chromatography on a Sepharose column coupled with galactose also yielded a 30-kDa polypeptide that exhibited carbohydrate-binding activity. This polypeptide can be immunoblotted with anti-CBP35, but not with antibodies directed against beta-galactosidase. These results indicate that we have identified a cDNA clone for CBP35 that yields a recombinant polypeptide with lectin activity produced in Escherichia coli. Using this cDNA clone as a probe, Northern-blot analysis showed an increased expression of the CBP35 gene when quiescent 3T3 cells were activated by the addition of serum growth factors.

Endogenous lectins from cultured cells: subcellular localization of carbohydrate-binding protein 35 in 3T3 fibroblasts.

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured 3T3 fibroblasts. In the present study, rabbit antibodies directed against CBP35 were used to analyze the subcellular distribution of CBP35 in 3T3 cells. Several lines of evidence indicate that CBP35 is found externally exposed at the cell surface: immunofluorescent staining of live 3T3 cells; agglutination of suspension of 3T3 fibroblasts by specific antibodies; and isolation, by immunoaffinity chromatography, of a Mr 35,000 component from cells surface-labeled with 125I. In addition to the plasma membrane, CBP35 could also be found intracellularly, as revealed by immunofluorescence studies of fixed and permeabilized 3T3 cells. The staining pattern showed the presence of CBP35 on the nucleus and in the cytoplasm. These results are consistent with the finding that among several subcellular fractions, CBP35 can be found by immunoblotting procedures in the nuclear pellet, the soluble fraction, and the plasma membrane fraction of the postnuclear supernatant.

A "hanging" coverslip method for cell surface iodination of monolayer cultures.

A procedure is described by which the use of a 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenyl glycoluril (iodogen)-coated coverslip to iodinate the cell surface proteins of monolayer cultures has been improved by "hanging" the coverslip at a defined distance from the cells. This method allows gentle manipulation of the cell culture, resulting in retention of high cell viability and in recovery of the cell monolayer with a minimum of mechanical damage. In addition, it allows the safe disposal of the radioactive coverslip upon completion of the reaction. Finally, the labeling is surface specific. The application of this procedure to 3T3 fibroblasts results in labeling of proteins comparable to lactoperoxidase-catalyzed iodinations.