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a Geron Corporation, Menlo Park, California, USA;
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b Department of Gene Expression and Development, Roslin Institute, Roslin Midlothian, United Kingdom
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9 {- M: u9 k3 f* g: j( u. AKey Words. Human embryonic stem cells ? Human telomerase reverse transcriptase Telomerase ? Immortalization ? Differentiation- e- a3 A# @" B2 \1 y' l: O
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Correspondence: Chunhui Xu, Ph.D., Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025, USA. Telephone: 650-473-7795; Fax: 650-473-7750; e-mail: cxu@geron.com$ y! [9 k& U. A, J6 c) S ^
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ABSTRACT
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Stem cells may provide the starting material for cell replacement in tissues that are damaged as a result of disease, infection, or congenital abnormalities. Several types of stem cells have the capacity to differentiate into subsets of mature cells that can carry out the unique functions of particular tissues when placed into an appropriate environment. However, many somatic stem cells show limited replicative capacity. In contrast, embryonic stem (ES) cells demonstrate both remarkable proliferative capacity as well as pluripotent differentiative potential and may thus prove to be an optimal source for cell replacement therapy. Human ES cells (hESCs) have been successfully cultured on a layer of mouse embryonic fibroblast (MEF) or human feeders or under feeder-free conditions, in which the cells are maintained on Matrigel or laminin in MEF-conditioned medium (MEF-CM) or are maintained on fibronectin with media supplemented with transforming growth factor-?, leukemia inhibitory factor, and basic fibroblast growth factor . Quantitative analysis of hESCs cultured either on feeders or on Matrigel with MEF-CM demonstrates that hESCs maintained in either condition show similar expression of SSEA-4, TRA-1-81, OCT4, and hTERT and are capable of long-term proliferation in vitro while retaining a normal karyotype . These cells can differentiate into several derivatives, including neural progenitors, cardiomyocytes, trophoblasts, endothelial cells, hematopoietic lineages, hepatocytes, osteoblasts, and insulin-expressing cells, which may have the potential to repair damaged tissues .' @, j0 c/ w8 ~8 D" B- a
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However, differentiated cells derived from hESCs have limited proliferative capacity, as commonly observed for other mammalian somatic cells in culture. Because somatic cell senescence is known to be caused by telomere shortening, it is also possible that senescence of the hESC derivatives is related to reduction of telomere length during each round of cell division. Indeed, significant downregulation of hTERT, the catalytic component of human telomerase, has been observed when hESCs differentiate . Therefore, it is predicted that hESC derivatives may be immortalized through overexpression of hTERT. Ectopic expression of hTERT has previously been demonstrated to extend the lifespan of many somatic cell types in vitro, including fibroblasts, retinal pigment epithelial cells, bone marrow stromal cells, and endothelial cells . Although the hESC derivatives may also be immortalized by other means, such as over-expression of oncogenes, hTERT-mediated immortalization is particularly desirable for cell therapies and drug discovery applications, because it is not associated with neoplastic transformation .( K z, H8 a, H( ?9 ~4 H7 @
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In this report, we demonstrate that telomerase-immortalized cells can be generated from hESCs. Fibroblast-like cells, named HEF1, were obtained by differentiating hESCs in vitro and were subsequently immortalized by infection with a retroviral vector expressing hTERT. These immortal hESC-derived cells produced conditioned medium that supported growth of hESCs under feeder-free conditions. In addition, we found that these cells respond to inductive factors to produce at least one fully differentiated cell type, osteoblasts.
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, Q: U- }7 a* U( MMATERIALS AND METHODS F* U7 y7 C6 R% v3 q
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Derivation of Fibroblast-Like Cells from hESCs
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H1 hESCs were induced to differentiate using embryoid body formation followed by culture on gelatin-coated plates. Initially, the cultures contained a heterogeneous population of cells that became more homogeneous with subsequent passaging (three passages). Most cells showed a fibroblast-like or mesenchymal-like morphology, and the cultures were designated as human embryonic fibroblast-like cells (HEF1 cells) (Fig. 1). At passage 5, the cells were infected with a retrovirus, pBABE-hTERT, containing the hTERT and puromycin resistance genes. Parallel control cultures were infected with a retrovirus pBABE containing only the puromycin resistance gene. Telomerase activity was assessed before and after infection in HEF1-hTERT and control cultures using TRAP analysis. No telomerase activity was detected before infection of the HEF1 cells (Fig. 2A). Twenty days (~ eight population doublings) after infection, the HEF1- hTERT cultures showed strong telomerase activity, whereas the control reference cultures did not (Fig. 2A). The HEF1-hTERT cells retained telomerase activity for at least 65 days (~30 population doublings, the latest time point examined), showing that hTERT was stably introduced into the HEF1 cells.
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+ x7 w3 I: D% a* DFigure 2. Immortalization of HEF1 cells. (A): Telomerase activity of HEF1 cells before and 20 days (~8 population doublings) or 65 days (~30 population doublings) after virus infection, as determined by telomeric repeat amplification protocol assay. An aliquot of each sample equal to approximately 1,000 cells (left lanes under each condition) or 5,000 cells (middle lane under each condition) was assayed. As a control, heat-inactivated samples were also assayed (right lane under each condition). (B): Growth kinetics of HEF1 cells infected with a retrovirus expressing hTERT or a control virus. (C): Analysis of senescence-associated ?-galactosidase in HEF1-hTERT and HEF1-control cells 65 days after virus infection. Abbreviation: HEFI, fibroblast-like cells derived from hESCs; hTERT, human telomerase reverse transcriptase.+ ?# w; A# @' D
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Introduction of hTERT into the HEF1 cells extended the lifespan of the culture. As shown in Figure 2C, HEF1-hTERT cells had very low or undetectable senescence-associated ?-galactosidase activity, an established biomarker associated with cellular aging . In contrast, HEF1 control cells were strongly positive for ?-galactosidase activity (Fig. 2C), indicating that these populations were undergoing senescence. After selection with puromycin, HEF1 cells infected with the hTERT retrovirus (HEF1-hTERT) cells proliferated with an approximate doubling time of 54 hours (Fig. 2B). The HEF1-hTERT cells continued to expand and maintained a consistent morphology and doubling time even after cryopreservation. In contrast, cells infected with the control virus (HEF1-control) stopped proliferating 38 days after infection (Fig. 2A). These data indicate that the introduction of hTERT extended the lifespan of HEF1 cells.
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Feeder-Free hESC Culture Using Medium Conditioned by HEF1-hTERT Cells
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- O: x6 g2 r/ e- @We have previously shown that undifferentiated hESCs can be maintained on Matrigel in media conditioned by cells such as MEF-CM . To examine whether the HEF1-hTERT cells can provide critical factors for hESC growth, hESC medium was conditioned using mitotically inactivated HEF1-hTERT cells. The HEF1-CM was then tested for its ability to support growth of two separate hESC lines (H7 and H9) grown on Matrigel. Similar to parallel cultures maintained in MEF-CM, cells seeded onto Matrigel in HEF1-CM gave rise to many colonies of undifferentiated hESCs with differentiated stroma-like cells between the colonies. Over the next few days, these colonies increased in size and appeared indistinguishable from hESCs maintained on Matrigel in MEF-CM. As shown in Figure 3A, H9 hESCs maintained in these conditions for four passages using standard passaging techniques continued to display undifferentiated hESC morphology. Similar morphology was observed in H7 hESCs after being maintained in HEF1-CM for at least14 passages (98 days). The H7 cells maintained in HEF1-CM retained expression of undifferentiated cell markers, such as OCT-4 and hTERT, as examined by TaqMan real-time reverse transcriptase–PCR analysis (Fig. 3B), and telomerase activity, as measured by the TRAP assay (Fig. 3C). Furthermore, hESCs maintained in HEF1-CM or MEF-CM expressed surface markers for undifferentiated hESCs, such as SSEA-4 (Figs. 3D–3G), TRA-1-60 (data not shown), and TRA-1-81 (Figs. 3H, 3I), predominantly in the colonies but not in the differentiated stroma-like cells. Like the parallel cultures in MEF-CM, the colonies of hESCs maintained in HEF1-CM had alkaline phosphatase activity (Figs. 3J, 3K) and expressed connexin 43, a gap junction protein (Figs. 3H, 3I). Last, after 13 passages in HEF1-CM, H7 hESCs maintained a normal female karyotype, as analyzed by G banding (data not shown).
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$ ?, D ?& T9 G. U: t$ qFigure 3. Cultures of human embryoinic stem cells using CM produced by HEF1-hTERT. (A): Morphology of H9 cells maintained on Matrigel in MEF-CM (passage 29) or HEF1-CM for four passages (passage 25 4). Bar = 300 μm. (B): Real-time reverse transcription–polymerase chain reaction analysis of relative levels of OCT-4 and hTERT expression in H7 cells (passage 29) maintained in HEF1-CM for six passages (passage 29 6) compared with MEF-CM control cells (passage 35). (C): Telomerase activity of the H9 cells (passage 29) maintained in MEF-CM or HEF1-CM for four passages. Three lanes were run for each condition. The left lane represents approximately 1,000 cells, the middle lane represents 5,000 cells, and the right lane is the heat-inactivated control. (D–I): Detection of surface markers in H7 cells maintained in MEF-CM (passage 34) (D, F, H, J) or HEF1-CM for five passages (passage 29 5) (E, G, I, K). SSEA-4 and connexin 43 were detected by fluorescein isothiocyanate, and TRA-1-81 was detected by Texas red. Bar = 200 μm for D, E, J, and K. Bar = 20 μm for F, G, H, and I. Abbreviations:AP, alkaline phosphatase; HEFI-CM, conditioned medium from hESC-derived immortal fibroblast-like cells; hTERT, human telomerase reverse transcriptase; MEF-CM, mouse embryonic fibroblast conditioned medium.
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1 f- K$ X v5 `- ]/ Z, ZTo evaluate the differentiation capacity of the cells maintained in HEF1-CM, H7 cells maintained in the medium for 12 passages were induced to differentiate using EB formation and subsequent culture on a gelatin-coated surface. Like cells cultured in MEF-CM, hESCs maintained in HEF1-CM differentiated into a population of cells with heterogeneous morphologies, including beating cells. We then evaluated the differentiation by immunocytochemical analysis using antibodies specific for cardiac troponin I (cTnI), alpha feto-protein (AFP), and ?-tubulin III. cTnI cells with striations characteristic of the sarcomeric structures of muscle cells, AFP cells with a flat morphology, and ?-tubulin III cells with neuron morphology were detected in these cultures (Fig. 4).0 F' N) w/ i n1 l) N0 T
9 V% ?$ Z8 u8 e5 R. FFigure 4. Differentiation of human embryonic stem cells maintained in HEF1-CM. H7 cells maintained in HEF1-CM for 12 passages (passage 29 12) were induced to differentiate through EB formation. After 4 days in suspension, EBs were plated on gelatin-coated chamber slides. The cultures were fixed after an additional 7 days and subjected to immunocytochemical analysis for expression of AFP, ?-tubulin III, and cTnI. Bar = 50 μm. Abbreviations:AFP, alpha fetoprotein; cTnI, cardiac troponin I; EB, embryoid body; HEFI-CM, conditioned medium from hESC-derived fibroblast-like cells.
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Taken together, these results indicate that cells maintained in HEF1-CM possess characteristics of undifferentiated hESCs, including morphology, surface marker and transcription factor expression, telomerase activity, karyotypic stability, and differentiation capacity.4 y. A- |2 D; t1 J$ G" @/ v
7 b9 u+ g0 l: Y% H& _Characterization of HEF1-hTERT Cells, D6 M6 N, e0 A5 ^: C- ~8 @
4 `3 ^! M1 I- j( V6 e" ~, ABecause the HEF1-hTERT cells are useful for maintaining undifferentiated hESCs, we additionally characterized these cells. Morphologically, HEF1-hTERT cells seem to be similar to hMSCs. Flow cytometry analysis showed that the HEF1-hTERT cells expressed surface markers CD29, CD44, CD71, and CD90 at similar levels to hMSCs. CD106 was expressed at lower levels than observed in hMSC, whereas no CD45 or CD14 expression was observed in either HEF1-hTERT or hMSC (online supplementary Fig. 1). When treated with OS factors, HEF1-hTERT cells and hMSCs differentiated into distinct nodules that stained positive with alizarin red and showed increased calcium deposition and alkaline phosphatase activity (Fig. 5). In contrast, HEK293 cells, a nonosteogenic cell line used as negative control, failed to respond to the same conditions. When treated with an adipogenic or chondrogenic culture conditions promoting hMSC differentiation, HEF1 did not seem to differentiate (online supplementary Fig. 2). These results indicate that HEF1-hTERT cells respond to osteogenic factors in a comparable way to hMSC cells but do not exhibit in vitro adipogenic or chondrogenic potential using the conditions tested.3 M; x7 Y: T, e. Q8 s
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Figure 5. Differentiation of HEF1-hTERT cells. (A): alizarin red staining of cultures after 14 days in control (upper panel) or OS-supplemented medium (lower panel). (B): Time course of calcium deposition (upper panel) and ALP activity (lower panel) after the treatment. Results are presented as mean ± standard error of the mean (n = 4). Bar = 50 μm. Abbreviations: ALP, alkaline phosphatase; hMSC, human mesenchymal stem cell; hTERT, human telomerase reverse transcriptase; OS, osteogenic supplement.) u) ~- Q: H' i3 \; N( Q6 u- |7 g
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DISCUSSION
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We thank Dr. Calvin Harley for insightful discussions and critical review of the manuscript. This work is supported by the Geron Corporation.4 d2 R5 _) O+ r j
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发表于 2009-3-5 10:36
发表于 2015-6-14 17:43
