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Department of Reproductive Biology and Pathology, National Research Institute for Child Health and Development, Tokyo, Japan,1 Department of Pathology, Keio University School of Medicine, Tokyo, Japan,2 Virology Division, National Cancer Center Research Institute, Tokyo, Japan,3 Department of Orthopedic Surgery, Keio University School of Medicine, Tokyo, Japan,4 Cardiopulmonary Division, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan,5 Department of General Medicine and Clinical Investigation, Nara Medical University, Nara, Japan,6 Department of Allergy and Immunology, National Research Institute for Child Health and Development, Tokyo, Japan7
Received 11 January 2005/ Returned for modification 13 February 2005/ Accepted 14 March 2005
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ABSTRACT |
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INTRODUCTION |
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Large numbers of cells may be necessary for repairing damaged human tissues to restore function. However, there have been no reports of a sufficient number of differentiated neurons ever having been obtained from human marrow stromal cells. One reason is that normal human cells undergo a limited number of divisions in culture and then enter a nondividing state referred to as "senescence." Senescence is classified into two categories: "stress-induced premature senescence," or "telomere-independent senescence," and "replicative senescence," or "telomere-dependent senescence" (3, 5, 38). p16Ink4a (p16), a cyclin-dependent kinase (CDK) inhibitor, is induced by certain oncogenes and other damage or stress signals and is required for "premature senescence" in human mammary epithelial cells and keratinocytes. p16 inhibits dephosphorylation of pRb by Cdk4/6-cyclin D, and hypophosphorylated pRb actively represses the genes required for the S phase by sequestering the E2F transcription factors. "Replicative senescence" is caused by telomere size reduction during successive cell divisions because of the chromosome end replication problem. Ectopic expression of telomerase alone bypasses replicative senescence in certain cell types, such as human foreskin fibroblasts.
To obtain enough human cells to restore the function of failing organs and to establish a model of cell therapy, the life span of human marrow stromal cells was extended by infecting them with retrovirus encoding human telomerase reverse transcriptase (hTERT) and the human papillomavirus E6 and E7 genes. Both p16/Rb inactivation by E7 and telomerase activation by E6 are required to extend the life span of human mammary epithelial cells (21) and umbilical cord blood-derived cells (36). E6 also accelerates the degradation of p53, which induces the CDK inhibitor p21 (35). In contrast to foreskin fibroblasts (5), however, the increase in telomerase activity as a result of the introduction of hTERT is insufficient to prolong the life span of marrow stromal cells (27).
The protocols for in vitro differentiation into neuronal cells include the use of a demethylating agent and/or the neurotrophic cytokines, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3. Basic fibroblast growth factor (bFGF) activates essential neuronal transcription factors, such as Hes-1 (10), in neural precursor cells and embryonic stem cells (19, 31). The Notch-Hes1 pathway plays an essential role in inhibition of neuronal differentiation (17), and the B27 supplement is effective in achieving long-term viability of primary neurons in culture (7). Taking these findings into consideration, a simple protocol for neural transdifferentiation was developed in this study.
The first purpose of this study was to determine whether prolonging the cell life span with cell cycle-associated molecules would inhibit neurogenic differentiation of marrow stromal cells in vitro. The second purpose was to determine if transdifferentiation of marrow cells of mesodermal origin to neurogenic cells was accompanied by global changes in gene expression or only leaky expression of some neurogenic markers. The in vitro differentiation process appears to be highly specific, and the life span of marrow-derived stromal cells can be extended by retrovirus-mediated transfer of the bmi-1 gene, which reduces expression of p16 (14, 15, 37), stimulates cell proliferation (9), and is required for maintenance of self-renewing hematopoietic stem cells (29).
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MATERIALS AND METHODS |
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Infection with recombinant retroviruses. The cells were prepared for infection with recombinant retroviruses carrying the bmi-1, E6, E7, and hTERT genes, as previously described (1). Stably transduced cells with an expanded life span were designated UBT-5, UBE6T-6, UBE6T-7, UE7T-9, UE6E7T-11, UE6E7T-12, UE7T-13, UBT-15, and UE6E7-16 cells.
Neuronal differentiation of bone marrow stromal cells. Cells removed from the flask bottom were replated onto a coverslip coated with laminin-polylysine (no. 354455; Becton Dickinson BioScience) in MSCGM. One day after passage, the medium was replaced with B27-supplemented Dulbecco's modified Eagle's medium-F12 (Gibco, BRL) containing 20 ng/ml of BDNF (R&D), 10 ng/ml of bFGF (R&D), and 50 ng/ml of NGF (Invitrogen) for neuroectodermal differentiation. Bone marrow stem cells were processed for immunocytochemistry and reverse transcription (RT)-PCR 7 to 21 days after induction.
RT-PCR. Total RNA was prepared from cultured cells with Isogen (Nippon Gene, Tokyo, Japan). Human neuronal RNA was purchased (human total brain RNA; lot 2110667; Becton Dickinson BioScience). RNA for RT-PCR was converted to cDNA with a First-strand cDNA Synthesis kit (Amersham Pharmacia Biotech) according to the manufacturer's recommendations. The following PCR primer sets were used for neuron-associated genes: nestin mRNA, sense (5'-AGAGGGGAATTCCTGGAG-3') and antisense (5'-CTGAGGACCAGGACTCTCTA-3'); NF-M mRNA, sense (5'-TGAGCTACACGTTGGACTCG-3') and antisense (5'-TCTCCGCCTCAATCTCCTTA-3'); notch-I mRNA, sense (5'-TCACGCTGACGGAGTACAAG-3') and antisense (5'-CCACACTCGTTGACATCCTG-3'); Sox-2 mRNA, sense (5'-CACAACTCGGAGATCAGCAA-3') and antisense (5'-GTTCATGTGCGCGTAACTGT-3'); neuronal cell adhesive molecule (N-CAM) mRNA, sense (5'-TCCATCACCTGGAGGACTTC-3') and antisense (5'-CTCCAGATAGCTGGCAGAGG-3'); MAP-2 mRNA, sense (5'-GGATTCTGGCAGCAGTTCTC-3') and antisense (5'-TCCTTGCAGACACCTCCTCT-3'); tubulin-beta III mRNA, sense (5'-ACCTCAACCACCTGGTATCG-3') and antisense (5'-TGCTGTTCTTGCTCTGGATG-3'); and Nur-related factor-1 (Nurr1) mRNA, sense (5'-TTTCTGCCTTCTCCTGCATT-3') and antisense (5'-GTGGCACCAAGTCTTCCAA-3'); 18S mRNA sense (5'-GTGGAGCGATTTGTCTGGTT-3') and antisense (5'-CGCTGAGCCAGTCAGTGTAG-3') were used as a positive control. PCR was performed with TaKaRa Z-Taq (TAKARA SHUZO Co., Ltd.) for 30 cycles, with each cycle consisting of 98°C for 5 s, 68°C or 60°C for 1 s, and 72°C for 10 s, with an additional 30-s incubation at 72°C after completion of the final cycle.
Western blot analysis. To detect p16, p53, p21, p27, Rb, Bmi-1, and actin, immunoblotting was performed as previously described (30) with antibodies against p16 (G3-245; BD Pharmingen, San Diego, CA), Rb (G174-405; BD Pharmingen), p53 (DO-1), p21 (Ab-1; Oncogene Science, Boston, MA), Bmi-1 (monoclonal antibody generated by T. Kiyono), p27 (BD Pharmingen), and actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA).
Telomere length assay. Genomic DNA was extracted from cultured cells. Restriction enzyme digestion of genomic DNA was carried out with HinfI and RsaI. The fragments obtained were resolved on 0.7% agarose gels, transferred to a Hybond N membrane (Amersham, United Kingdom), and hybridized with digoxigenin-labeled (TTAGGG)3 probe. The membrane was then incubated with anti-digoxigenin alkaline phosphatase, and detection was performed with a chemiluminescence solution. The size range and intensity were determined with X-ray film.
Telomerase activity analysis. Telomerase activity was determined with a telomere repeat amplification protocol (TRAP) assay kit, Telo TAGGG telomerase PCR ELISA plus (Roche, Indianapolis, IN), according to the manufacturer's instructions.
G-banding karyotypic analysis. Metaphase spreads were prepared from cells treated with Colcemid (Karyo Max; Gibco BRL; 100 ng/ml for 6 h). We performed a standard G-banding karyotypic analysis on at least 50 metaphase spreads for each population.
SKY analysis. Spectral karyotyping (SKY) was performed on metaphase-transduced cells (UBE6T-7, UE6E7T12, and UE7T13) in 80 population doublings (PDs) according to the kit manufacturer's instructions (ASI, Carlsbad, CA) and a previously published method (34).
Immunocytochemical analysis. Immunocytochemical analysis was performed as previously described (30) with antibodies to MAP-2 (Zymed, San Francisco, CA), tubulin 3 (Sigma, St. Louis, Missouri), GFAP (DAKO, Denmark), Nurr1 (N-20; Santa Cruz), and nestin (Biogenesis, United Kingdom) in phosphate-buffered saline containing 1% bovine serum albumin. As a methodological control, the primary antibody was omitted. After being washed in phosphate-buffered saline, the slides were incubated with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin antibody or phycoerythrin (PE)-conjugated anti-rabbit immunoglobulin antibody (DAKO, Denmark).
GeneChip expression analysis. Human genomewide gene expression was examined with the Human Genome U133A Probe array (GeneChip; Affymetrix), which contains the oligonucleotide probe set for approximately 23,000 full-length genes and expressed sequence tags, according to the manufacturer's protocol (Expression Analysis technical manual and GeneChip Small Sample Target Labeling Assay version 2 technical note [http://www.affymetrix.com/support/technical/index.affx]). Total RNA was isolated with an RNeasy minikit (QIAGEN, Chatsworth, CA). Double-stranded cDNA was synthesized, and the cDNA was subjected to in vitro transcription in the presence of biotinylated nucleoside triphosphates. The biotinylated cRNA was hybridized with a probe array for 16 h at 45°C, and the hybridized biotinylated cRNA was stained with streptavidin-PE and scanned with a Hewlett-Packard Gene Array Scanner. The fluorescence intensity of each probe was quantified by using the GeneChip Analysis Suite 5.0 computer program (Affymetrix). The expression level of a single mRNA was determined as the average fluorescence intensity among the intensities obtained with 11 paired (perfectly matched and single-nucleotide-mismatched) probes consisting of 25-mer oligonucleotides. If the intensities of mismatched probes were very high, gene expression was judged to be absent, even if high average fluorescence was obtained with the GeneChip Analysis Suite 5.0 program. The level of gene expression was determined with the GeneChip software as the average difference (AD). Specific AD levels were then calculated as percentages of the mean AD level of six probe sets for housekeeping genes (actin and GAPDH [glyceraldehyde-3-phosphate dehydrogenase] genes). Further data analysis was performed with Genespring software version 5 (Silicon Genetics, San Carlos, CA). To normalize the staining intensity variations among chips, the AD values for all genes on a given chip were divided by the median of all measurements on that chip. To eliminate changes within the range of background noise and to select the most differentially expressed genes, data were used only if the raw data values were less than 100 AD and gene expression was judged to be present by the Affymetrix data analysis. Hierarchical clustering analysis with standard correlation was used to identify gene clusters. The separation ratio was set at 0.5. Normalization values were considered significant when (i) expression changed by at least twofold (activation program) and (ii) increased gene expression included at least one present absolute call (Affymetrix algorithm). Normalized values were averaged for two donors and used for the data analysis, and the normalized values were used to classify the genes into up-regulated or down-regulated. There was a statistically significant correlation between the expression levels of genes of the same cells analyzed twice (r = 0.997). These criteria confirmed the reproducibility of the differences that were seen between different cells under different conditions.
Calcium imaging. The intracellular Ca2+ concentration was monitored by means of the fluorescent Ca2+ indicator Fluo-3. The cultured cells were exposed to 10 µmol/liter Fluo-3 acetoxymethylester (Molecular Probes, Eugene, Oregon) at 37°C for 30 min and then washed in Tyrode's solution containing (in mmol/liter) NaCl, 140; KCl, 4; MgCl2, 0.5; CaCl2, 1.8; HEPES, 5; and D-glucose, 55 (pH adjusted to 7.4 with NaOH). The signal from the cell was monitored with a fluorescence microscope (BX50WI; Olympus, Tokyo, Japan) and a high-resolution optical mapping system (MiCAM-01; SciMedia Ltd. Tokyo, Japan) at a wavelength of 530 nm and an excitation wavelength of 488 nm. The cells were challenged with a depolarization stimulus in the form of high-potassium (144 mmol/liter) Tyrode's solution (KCl substituted for NaCl) applied with a rapid solution changer (RSC-160; Molecular Kinetics, Indianapolis, Indiana) at a flow rate of 100 µl/s at a distance of 1 mm from the cell at a room temperature of 25°C. The image analysis was performed with Igor Pro software (Wavemetrics, Lake Oswego, OR) and by customized procedures. The background fluorescence intensity was subtracted from the data, and the areas were normalized to the control response to normal Tyrode's solution and shown on a color scale.
Quantitative RT-PCR. RNA was extracted from cells using the RNeasy kit (QIAGEN, Valencia, CA). Contaminating DNA was eliminated by two sequential DNase (Invitrogen) treatments. An aliquot (1 µg) of total RNA was reverse transcribed by using an oligo(dT) primer. For the thermal cycle reactions, cDNA was amplified (ABI PRISM 7000; Perkin-Elmer Applied Biosystems) using the SYBR RT-PCR kit (Takara Bio, Japan) under the following reaction conditions: 40 cycles of PCR (95°C for 15 seconds and 60°C for 1 min) after an initial denaturation (95°C for 10 min). The controls consisted of amplifications without reverse transcription and reactions without the addition of a cDNA template. The authenticity and sizes of the PCR products were confirmed using a melting curve analysis (using software provided by Perkin-Elmer) and a gel analysis. mRNA levels were normalized using the GAPDH gene as a housekeeping gene. The following primer sets were used: hTERT mRNA, sense (5'-CGGTGTGCACCAACATCTACAAG-3') and antisense (5'-TCAGAGATGACGCGCAGGA-3'); Bmi-1 mRNA, sense (5'-CGCTTGGCTCGCATTC-3') and antisense (5'-AGCTCAGTGATCTTGATTCTCGTTG-3'); E6 mRNA, sense (5'-GCACAGAGCTGCAAACAACT-3') and antisense (5'-CTCACGTCGCAGTAACTGTTG-3'); E7 mRNA, sense (5'-ATGACAGCTCAGAGGAGGAG-3') and antisense (5'-TCCTAGTGTGCCCATTAACAG-3'); NR4A2/Nurr1 mRNA, sense (5'-TTCGGCAGAGTTGAATGAATG-3') and antisense (5'-GAAATTAAAGGTGGACAGTGTCGTA-3'); and GAPDH mRNA, sense (5'-CCAGCCGAGCCACATCGCTC-3') and antisense (5'-ATGAGCCCCAGCCTTCTCCAT-3').
Nucleotide sequence accession numbers. The gene chip datasets have been deposited in the GEO database with accession number GSE2110 (GSM38114 to GSM38116).
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RESULTS |
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None of the cells exhibited malignant transformation activity: they did not form a focus after confluence in vitro; cells grafted into subcutaneous tissue of immunodeficient mice (nonobese diabetic [NOD]-SCID-interleukin 2 receptor knockout mice) did not form tumors, at least during the observation period (more than 30 days); and the morphologies of all clones remained unchanged for 40 to 200 PDs.
Expression of Bmi-1, Rb, p53, p27, and p21 proteins in transduced cells. The expression of cell cycle-associated proteins was analyzed in cells transduced with the hTERT and bmi-1, E6, or E7 genes and in parental H4-1 cells (Fig. 2). Human dermal keratinocytes at senescence (PD 27) served as a control for expression of p16Ink4a, hypophosphorylated RB, and p53 proteins at a high level.
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Increase in telomerase activity and maintenance of telomere length in cells transduced with the hTERT gene. No telomerase activity was detected by the TRAP assay in the parental H4-1 cells at PDs 20 and 40, but telomerase activity was detected in UBT-5, UBE6T-7, UE6E7T-12, and UE7T-13 cells transduced with the hTERT gene at all of the PDs tested. UE6E7-16 cells not transduced with the hTERT gene did not exhibit any telomerase activity at PDs 40 to 120 (Fig. 3A). Likewise, the telomere length of the parental H4-1 cells decreased with the number of PDs, whereas the telomere length of UBT-5, UBE6T-7, UE6E7T-12, and UE7T-13 cells was maintained irrespective of the PD number (Fig. 3B, C, and D).
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Gene chip analysis during neuroectodermal differentiation. To clarify the specific gene expression profile of human marrow stromal cells, we compared the expression levels of approximately 23,000 genes by neuronally differentiated UE7T-13 cells, undifferentiated UE7T-13 cells, and parental H4-1 cells (GEO accession number GSE2110 [http://1954.jukuin.keio.ac.jp/umezawa/chip/mori]) using the Affymetrix gene chip oligonucleotide arrays. Of the approximately 23,000 genes represented on the gene chip, 2,123 genes were up-regulated to more than twice their expression level in undifferentiated cells. Genes whose expression increased or decreased are shown in Table 2. The Nurr1, kynureninase, apolipoprotein, RARRES1, ABC1, rab27B, STC1, WISP2, rhoGAP6, SLC2A5, and palmderphin genes were among the top 0.1% in terms of increase in expression. Surprisingly, expression of Nurr1/NR4A2, which is essential for differentiation of the nigral dopaminergic neurons (23, 32), was up-regulated 26.1-fold. Wnt-5a promotes the acquisition of dopaminergic neurons (8), and its expression was increased 7.1-fold. These findings imply that the characteristics of these cells are similar to those of midbrain neurons. The dendrogram analysis clearly showed that the change in gene expression pattern is dramatic and neuron specific during neuroectodermal differentiation rather than being leaky, subtle, and nonspecific (Fig. 6).
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DISCUSSION |
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The lack of change in phenotypes of bmi-1-transduced cells, for example, in their differentiation abilities, growth rates, cell surface markers, and gene expression profiles, is rather surprising, because bmi-1 suppresses p16 protein and has been identified as a c-Myc-cooperating gene in murine B- and T-cell lymphomas (15, 37). A critical target of bmi-1 is the Ink4a locus, which encodes p16 and p19 (p14 in humans) (14). Interestingly, bmi-1 is required for maintenance of adult self-renewing hematopoietic stem cells (29) and neural stem cells (25), whereas the simian virus 40 large T antigen oncogene, which prolongs the cell life span, interferes with the differentiation program and transforms cells. The human marrow-derived stromal cells transfected with simian virus 40 large T antigen (12) did not exhibit contact inhibition in vitro and formed tumors within a month when implanted into nude mice (data not shown).
Transdifferentiation of human mesoderm-derived cells into a functional neuronal lineage. This study was also conducted to determine whether prolongation of the cell life span by hTERT and bmi-1, E6, or E7 would predominate over neurogenic differentiation of marrow stromal cells in vitro. In contrast to our previous study of neurogenic differentiation of immortalized murine marrow-derived stromal cells (22) by demethylating agents, the transdifferentiation of the human stromal cells was limited to neurons, but not astrocytes or oligodendrocytes. This was probably due to the developmentally logical protocol we employed in this study: use of bFGF, NGF, BDNF, and laminin-ornithine coating. The idea for the neuronal differentiation protocol without 5-azacytidine arose from reports of the following: NGF and BDNF support neuron survival and growth (4), and bFGF induces initial differentiation of neural precursor cells and activates transcription factors related to the differentiation of neural precursor cells and embryonic stem cells (19, 31).
The extremely high level of expression of dopaminergic neuron-associated genes, such as nurr1 and wnt5a, in the neuronally differentiated stromal cells, which we accidentally found by the GeneChip analysis and confirmed by RT-PCR, is surprising. wnt5a and nurr1 are involved in the differentiation of midbrain precursors into dopaminergic neurons (20, 39). Although further analysis of functionally differentiated cells is beyond the scope of this study, it is quite interesting that dopaminergic neurons can be generated from marrow-derived stromal cells, since one of the target cells for regenerative medicine is dopaminergic neurons.
Are marrow stromal cells traced back to their default state, i.e., neural lineage, by neurotrophic factors? The mechanism of the transdifferentiation from marrow stromal cells to neuronal cells remains unresolved. It must be emphasized that the GeneChip analysis showed that the change in gene expression during differentiation is global and drastic: the differentiated cells no longer exhibited the profile of mesenchymal cells or the biphenotypic pattern of neuronal and mesenchymal cells. Contamination of human stromal cell cultures by neural precursor cells is inconceivable, because the cells were subcloned several times after gene transduction and exhibited mesenchymal phenotypes after subcloning. Our previous study of murine stromal cells clearly showed that osteoblasts capable of membranous ossification are likely to differentiate into neuronal lineages but that adipocytes do not (22). The craniofacial membranous bones develop from the neural crest, which is of ectodermal origin. Our finding of in vitro differentiation from mesoderm- to ectoderm-derived cells in this study may be the opposite of the developmental process, i.e., from ectoderm- to mesoderm-derived cells. Development naturally progresses from neural crest cells to terminally differentiated osteoblasts (28). Converting differentiated osteoblasts or marrow stromal cells to neuronal cells, a key future task for any cell-based therapy, would thus oppose the usual direction of cell differentiation. This can now be achieved by exposing stromal cells to neurotrophic factors, at least in vitro.
Are human marrow stromal cells with an extended life span available for cell-based therapy? Human stromal cells transduced with hTERT and bmi-1, E6, or E7 did not transform according to the classical criteria: they did not generate tumors in immunosuppressed NOD-SCID-interleukin 2 receptor knockout mice, they did not form foci in vitro, and they stopped dividing after confluence (data not shown). However, we cannot rule out the possibility that gene-transduced stromal cells might become tumorigenic in patients several decades after cell therapy. We believe that these gene-modified stromal cells may be used to supply defective enzymes to patients with genetic metabolic diseases, such as neuro-Gaucher disease, Fabry disease, and mucopolysaccharidosis, which have a poor prognosis and are sometimes lethal. The "risk-versus-benefit" balance is essential when applying these gene-modified cells clinically, and the "risk" or "drawback" in this case is transformation of implanted cells.
hTERT is normally expressed in stem cells and in over 90% of human cancers. Ectopic expression of telomerase is sufficient to prevent telomere shortening and can thereby promote indefinite proliferation in human foreskin fibroblasts (5). Several stromal cell strains with extended life spans and maintenance of differentiation capability, such as UBT5, UBE6T7, UE6E7T-12, UE7T-13, and UE6E7-16 cells, were developed based on the above notion. UE6E7-16 cells not transduced with hTERT have a longer life span but enter a "crisis" period in culture at 67 PDs. "Crisis" is the stage in which widespread death occurs in a population of cultured cells or when karyotypic instability develops as a result of fusion of telomere ends, after the cells manage to circumvent senescence or initial blockades. Human cells transduced with bmi-1 or E6 and E7 alone enter a "crisis," and the cells that spontaneously circumvent premature senescence without gene induction enter replicative senescence. Replicative senescence or crisis may be a tumor suppressor mechanism that avoids the risk of cell transformation after implantation of cells as a source for cell-based therapy.
Even when cells transduced with nononcogenic genes are used for cell-based therapy, the cases of leukemia in SCID patients treated with gene-modified lymphocytes must be taken into account (3). The infected virus genome was integrated into the LMO2 locus in the leukemia cases and resulted in induction of the oncogenic LMO2 gene. Because of these failures, it will take time before gene-modified cells can be used for regenerative medicine. Inhibition of the p16/Rb pathway is sufficient to prolong the life span of cells in cultures of marrow-derived stroma, as shown in this study. The p16/Rb pathway was activated in marrow-derived stromal cells in vitro, the same as in mammary epithelial cells and hepatocytes, but not in foreskin fibroblasts (21). The development of an appropriate culture system without gene transduction to neutralize the p16/Rb braking system, i.e., nonstress medium, will be essential for cell therapy and further experiments.
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ACKNOWLEDGMENTS |
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This work was supported in part by a special grant for Advanced Research on Cancer from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan to T.K. and A.U.; a grant from MEXT to A.U.; Health and Labour Sciences Research Grants to A.U.; and a grant from the Organization for Pharmaceutical Safety and Research to A.U.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. Allan, E. H., P. W. Ho, A. Umezawa, J. Hata, F. Makishima, M. T. Gillespie, and T. J. Martin. 2003. Differentiation potential of a mouse bone marrow stromal cell line. J. Cell Biochem. 90:158-169.[CrossRef][Medline]
3. Allsopp, R. C., H. Vaziri, C. Patterson, S. Goldstein, E. V. Younglai, A. B. Futcher, C. W. Greider, and C. B. Harley. 1992. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA 89:10114-10118.
4. Batchelor, P. E., G. T. Liberatore, M. J. Porritt, G. A. Donnan, and D. W. Howells. 2000. Inhibition of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression reduces dopaminergic sprouting in the injured striatum. Eur. J. Neurosci. 12:3462-3468.[CrossRef][Medline]
5. Bodnar, A. G., M. Ouellette, M. Frolkis, S. E. Holt, C. P. Chiu, G. B. Morin, C. B. Harley, J. W. Shay, S. Lichtsteiner, and W. E. Wright. 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279:349-352.
6. Brazelton, T. R., F. M. Rossi, G. I. Keshet, and H. M. Blau. 2000. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 290:1775-1779.
7. Brewer, G. J., J. R. Torricelli, E. K. Evege, and P. J. Price. 1993. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci Res. 35:567-576.[CrossRef][Medline]
8. Castelo-Branco, G., J. Wagner, F. J. Rodriguez, J. Kele, K. Sousa, N. Rawal, H. A. Pasolli, E. Fuchs, J. Kitajewski, and E. Arenas. 2003. Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc. Natl. Acad. Sci. USA 100:12747-12752.
9. Dimri, G. P., J. L. Martinez, J. J. Jacobs, P. Keblusek, K. Itahana, M. Van Lohuizen, J. Campisi, D. E. Wazer, and V. Band. 2002. The bmi-1 oncogene induces telomerase activity and immortalizes human mammary epithelial cells. Cancer Res. 62:4736-4745.
10. Faux, C. H., A. M. Turnley, R. Epa, R. Cappai, and P. F. Bartlett. 2001. Interactions between fibroblast growth factors and Notch regulate neuronal differentiation. J. Neurosci. 21:5587-5596.
11. Gojo, S., N. Gojo, Y. Takeda, T. Mori, H. Abe, S. Kyo, J. Hata, and A. Umezawa. 2003. In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells. Exp. Cell Res. 288:51-59.[CrossRef][Medline]
12. Hacein-Bey-Abina, S., C. Von Kalle, M. Schmidt, M. P. McCormack, N. Wulffraat, P. Leboulch, A. Lim, C. S. Osborne, R. Pawliuk, E. Morillon, R. Sorensen, A. Forster, P. Fraser, J. I. Cohen, G. de Saint Basile, I. Alexander, U. Wintergerst, T. Frebourg, A. Aurias, D. Stoppa-Lyonnet, S. Romana, I. Radford-Weiss, F. Gross, F. Valensi, E. Delabesse, E. Macintyre, F. Sigaux, J. Soulier, L. E. Leiva, M. Wissler, C. Prinz, T. H. Rabbitts, F. Le Deist, A. Fischer, and M. Cavazzana-Calvo. 2003. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302:415-419.
13. Imabayashi, H., T. Mori, S. Gojo, T. Kiyono, T. Sugiyama, R. Irie, T. Isogai, J. Hata, Y. Toyama, and A. Umezawa. 2003. Redifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis. Exp. Cell Res. 288:35-50.[CrossRef][Medline]
14. Jacobs, J. J., K. Kieboom, S. Marino, R. A. DePinho, and M. van Lohuizen. 1999. The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature 397:164-168.[CrossRef][Medline]
15. Jacobs, J. J., B. Scheijen, J. W. Voncken, K. Kieboom, A. Berns, and M. van Lohuizen. 1999. bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-induced apoptosis via INK4a/ARF. Genes Dev. 13:2678-2690.
16. Jiang, Y., D. Henderson, M. Blackstad, A. Chen, R. F. Miller, and C. M. Verfaillie. 2003. Neuroectodermal differentiation from mouse multipotent adult progenitor cells. Proc. Natl. Acad. Sci. USA 100(Suppl. 1):1854-1860.
17. Kageyama, R., and T. Ohtsuka. 1999. The Notch-Hes pathway in mammalian neural development. Cell Res. 9:179-188.[CrossRef][Medline]
18. Kageyama, R., T. Ohtsuka, and K. Tomita. 2000. The bHLH gene Hes1 regulates differentiation of multiple cell types. Mol. Cell 10:1-7.
19. Kalyani, A. J., T. Mujtaba, and M. S. Rao. 1999. Expression of EGF receptor and FGF receptor isoforms during neuroepithelial stem cell differentiation. J. Neurobiol. 38:207-224.[CrossRef][Medline]
20. Kim, J. H., J. M. Auerbach, J. A. Rodriguez-Gomez, I. Velasco, D. Gavin, N. Lumelsky, S. H. Lee, J. Nguyen, R. Sanchez-Pernaute, K. Bankiewicz, and R. McKay. 2002. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 418:50-56.[CrossRef][Medline]
21. Kiyono, T., S. A. Foster, J. I. Koop, J. K. McDougall, D. A. Galloway, and A. J. Klingelhutz. 1998. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396:84-88.[CrossRef][Medline]
22. Kohyama, J., H. Abe, T. Shimazaki, A. Koizumi, K. Nakashima, S. Gojo, T. Taga, H. Okano, J. Hata, and A. Umezawa. 2001. Brain from bone: efficient "meta-differentiation" of marrow stroma-derived mature osteoblasts to neurons with Noggin or a demethylating agent. Differentiation 68:235-244.[CrossRef][Medline]
23. Le, W. D., P. Xu, J. Jankovic, H. Jiang, S. H. Appel, R. G. Smith, and D. K. Vassilatis. 2003. Mutations in NR4A2 associated with familial Parkinson disease. Nat. Genet. 33:85-89.[CrossRef][Medline]
24. Makino, S., K. Fukuda, S. Miyoshi, F. Konishi, H. Kodama, J. Pan, M. Sano, T. Takahashi, S. Hori, H. Abe, J. Hata, A. Umezawa, and S. Ogawa. 1999. Cardiomyocytes can be generated from marrow stromal cells in vitro. J. Clin. Investig. 103:697-705.[Medline]
25. Molofsky, A. V., R. Pardal, T. Iwashita, I. K. Park, M. F. Clarke, and S. J. Morrison. 2003. bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425:962-967.[CrossRef][Medline]
26. Ochi, K., G. Chen, T. Ushida, S. Gojo, K. Segawa, H. Tai, K. Ueno, H. Ohkawa, T. Mori, A. Yamaguchi, Y. Toyama, J. Hata, and A. Umezawa. 2003. Use of isolated mature osteoblasts in abundance acts as desired-shaped bone regeneration in combination with a modified poly-DL-lactic-co-glycolic acid (PLGA)-collagen sponge. J. Cell Physiol. 194:45-53.[CrossRef][Medline]
27. Okamoto, T., T. Aoyama, T. Nakayama, T. Nakamata, T. Hosaka, K. Nishijo, T. Nakamura, T. Kiyono, and J. Toguchida. 2002. Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells. Biochem. Biophys. Res. Commun. 295:354-361.[CrossRef][Medline]
28. Olsen, B. R., A. M. Reginato, and W. Wang. 2000. Bone development. Annu. Rev. Cell Dev. Biol. 16:191-220.[CrossRef][Medline]
29. Park, I. K., D. Qian, M. Kiel, M. W. Becker, M. Pihalja, I. L. Weissman, S. J. Morrison, and M. F. Clarke. 2003. bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 423:302-305.[CrossRef][Medline]
30. Sano, M., A. Umezawa, H. Abe, A. Akatsuka, S. Nonaka, H. Shimizu, M. Fukuma, and J. Hata. 2001. EAT/mcl-1 expression in the human embryonal carcinoma cells undergoing differentiation or apoptosis. Exp. Cell Res. 266:114-125.[CrossRef][Medline]
31. Santa-Olalla, J., and L. Covarrubias. 1995. Epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and basic fibroblast growth factor (bFGF) differentially influence neural precursor cells of mouse embryonic mesencephalon. J. Neurosci. Res. 42:172-183.[CrossRef][Medline]
32. Satoh, J., and Y. Kuroda. 2002. The constitutive and inducible expression of Nurr1, a key regulator of dopaminergic neuronal differentiation, in human neural and non-neural cell lines. Neuropathology 22:219-232.[CrossRef][Medline]
33. Scheffner, M., K. Munger, J. M. Huibregtse, and P. M. Howley. 1992. Targeted degradation of the retinoblastoma protein by human papillomavirus E7-E6 fusion proteins. EMBO J. 11:2425-2431.[Medline]
34. Schrock, E., T. Veldman, H. Padilla-Nash, Y. Ning, J. Spurbeck, S. Jalal, L. G. Shaffer, P. Papenhausen, C. Kozma, M. C. Phelan, E. Kjeldsen, S. A. Schonberg, P. O'Brien, L. Biesecker, S. du Manoir, and T. Ried. 1997. Spectral karyotyping refines cytogenetic diagnostics of constitutional chromosomal abnormalities. Hum. Genet. 101:255-262.[CrossRef][Medline]
35. Sekiguchi, T., and T. Hunter. 1998. Induction of growth arrest and cell death by overexpression of the cyclin-Cdk inhibitor p21 in hamster BHK21 cells. Oncogene 16:369-380.[CrossRef][Medline]
36. Terai, M., T. Uyama, T. Sugiki, X. K. Li, A. Umezawa, and T. Kiyono. 2005. Immortalization of human fetal cells: the life span of umbilical cord blood-derived cells can be prolonged without manipulating p16INK4a/RB braking pathway. Mol. Biol. Cell 16:1491-1499.
37. van Lohuizen, M., S. Verbeek, B. Scheijen, E. Wientjens, H. van der Gulden, and A. Berns. 1991. Identification of cooperating oncogenes in E mu-myc transgenic mice by provirus tagging. Cell 65:737-752.[CrossRef][Medline]
38. Wright, W. E., and J. W. Shay. 1992. The two-stage mechanism controlling cellular senescence and immortalization. Exp. Gerontol. 27:383-389.[CrossRef][Medline]
39. Zetterstrom, R. H., L. Solomin, L. Jansson, B. J. Hoffer, L. Olson, and T. Perlmann. 1997. Dopamine neuron agenesis in Nurr1-deficient mice. Science 276:248-250.
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