Cell Extract–Derived Differentiation of Embryonic Stem Cells
http://www.100md.com
《干细胞学杂志》
a Tissue Engineering & Regenerative Medicine Centre, Chelsea & Westminster Campus, Imperial College, London, United Kingdom;
b Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
Key Words. Embryonic stem cells ? Cell extract–based reprogramming
Correspondence: Julia M. Polak, D.B.E., M.D., F.R.C.Path., Tissue Engineering & Regenerative Medicine Centre, Imperial College Faculty of Medicine, Chelsea & Westminster Campus, Fulham Road, London SW10 9NH, U.K. Telephone: 44-208-237-2670; Fax: 44-208-746-5619; e-mail: julia.polak@imperial.ac.uk
ABSTRACT
Embryonic stem cells (ESCs) have been shown to differentiate in vitro to cell phenotypes arising from each of the three germ layers . A variety of means has been used to try to direct the differentiation of these cells toward specific cell lineages, in particular for the purposes of tissue engineering . In the distal lung, type II pneumocytes undergo proliferation and differentiation to the type I phenotype following injury and are crucial to the natural regenerative process of the alveoli . Thus, as part of our strategy to produce pulmonary cell types for tissue engineering purposes, we have derived type II pneumocytes from ESCs using soluble factors . However, as this process is lengthy and does not provide high yields of the target phenotype, we have sought a more efficient method.
A method of cell reprogramming, based on the exposure of permeabilized cells to a nuclear and cytoplasmic extract derived from a "target" cell, has been used successfully to derive mature cells from nonrelated phenotypes, including T cells and pancreatic ? (insulin-producing) cells from 293T cells and fibroblasts, and cardiomyocytes from adipose stem cells . We hypothesized, therefore, that this in vitro reprogramming technology could be used to differentiate ESCs to type II pneumocytes. In this study, a murine ESC line (E14tg2) was stably transfected with a construct containing an enhanced green fluorescent protein (eGFP) reporter gene under the control of the surfactant protein C (SPC) promoter, a specific marker of type II pneumocytes. Reversibly permeabilized ESCs were reprogrammed with extracts of "target cells" (transformed murine pneumocytes). The reprogrammed ESCs became GFP-positive cells and coexpressed SPC, specific for type II pneumocytes, and thyroid transcription factor-1 (TTF-1) that is found in immature pulmonary epithelium. In accordance with the well-known, in vitro differentiation of type II pneumocytes to the type I phenotype , we were able to show that SPC gene expression declines over time and is accompanied by an increase in aquaporin 5 mRNA and protein, a marker for type I pneumocytes . These findings demonstrate that differentiation of murine ESCs can be driven by using an extract-based, in vitro reprogramming approach.
MATERIALS AND METHODS
Reversible Permeabilization of EB Cells
In the absence of SLO, few EB cells were labeled with Texas Red (Fig. 1A). With 200–700 ng/ml SLO, the cells were not sufficiently permeabilized, and only approximately 10% of cells were found to be labeled with Texas Red (Fig. 1B). At 800–900 ng/ml SLO, 80% of the cells were labeled with Texas Red (Figs. 1C, 1E, 1F), indicating that a high proportion of EB cells could take up the dextran and be successfully replated. Increasing the SLO concentration to 1000 ng/ml appeared to damage the cells, and fewer were labeled (Fig. 1D).
Figure 1. Optimization of membrane permeabilization by SLO. Embryoid body cells were permeabilized with SLO at concentrations of 0–1000 ng/ml. Texas Red–stained cells treated with (A) 0 ng/ml SLO, (B) 200 ng/ml SLO, (C) 800 ng/ml SLO, and (D) 1000 ng/ml SLO. Original magnification x200. (E–H): Corresponding phase-contrast images of the fields are shown in (A–D). (I, J): Higher magnification (x400) of cells treated with 800 ng/ml SLO, the concentration found to give the greatest uptake of Texas Red–dextran without overt cell damage: (I) uptake of Texas Red–dextran, (J) phase-contrast image of the same cells. Abbreviation: SLO, streptolysin O.
Transfection
An eGFP reporter gene driven by the pneumocyte-specific murine SPC promoter was transfected into undifferentiated ES, MEF, and MLE-12 cells. As expected, no SPC/GFP expression was detected in transfected ESCs (Fig. 2A) or MEFs (Fig. 2B). However, SPC/GFP was expressed in transfected MLE-12 cells (Fig. 2C). All cell types expressed eGFP transfected alone, indicating that transfection efficiency was satisfactory (Figs. 2G–I). These results demonstrate that the SPC/GFP construct is specifically expressed in type II pneumocytes.
Figure 2. Transfection of SPC/GFP. Forty-eight hours after transfection of the SPC/GFP construct, no expression of GFP could be seen in undifferentiated ESCs (A) (fluorescence microscopy) and (D) (same cells seen in phase contrast) or MEFs (B) (fluorescence microscopy) and (E) (same cells seen in phase contrast), but was clearly present in MLE-12 cells (C) (fluorescence microscopy) and (F) (same cells seen in phase contrast). In the cells transfected with pTracer-GFP construct, GFP was found in all three types (all fluorescence microscopy and phase contrast, as before): (G, J) undifferentiated ESCs; (H, K) MEFs; and (I, L) MLE-12 cells. Original magnification x200. Abbreviations: ESC, embryonic stem cell; GFP, green fluorescent protein; MEF, murine embryonic fibroblast; SPC, surfactant protein C.
Induction of SPC/GFP Expression in Differentiated Cells
EB cells reversibly permeabilized with 800 ng/ml SLO were incubated for 1 hour in a nuclear and cytoplasmic extract of MLE-12 pneumocytes. Reprogramming was assessed by the induction of SPC promoter-driven eGFP expression in the EB cells. In cells exposed to MLE-12 extract, SPC/GFP expression was detected as early as day 3 of exposure to the extract, and the proportion of GFP-positive cells increased up to day 7 to 7.53 ± 1.14% (mean + SE) (Fig. 3Ac). No expression of SPC/GFP could be detected in cells reprogrammed with ATP-regenerating buffer (Fig. 3Aa) or MEF extract (Fig. 3Ab). Induction of SPC gene expression was also analyzed by real-time PCR. At day 7 of exposure to MLE-12 extract, SPC mRNA expression was markedly higher than that of control cells incubated in buffer only or MEF extract (Fig. 3B). SPC gene expression in cells exposed to buffer only or MEF extract was also found to be higher (3.28 ± 0.74 fold) than that of undifferentiated ESCs, as some of these cells had probably undergone spontaneous differentiation to pneumocytes. Furthermore, SPC mRNA expression of ESCs treated with 800 ng/ml SLO was higher (p < .005) than that of cells treated with 200 or 1000 ng/ml (Fig. 3C), corroborating our Texas Red–dextran uptake observations. Collectively, these results indicate that the MLE-12 extract is capable of inducing expression of a pneumocyte-specific promoter in ESCs.
Figure 3. SPC/GFP and SPC expression in reprogrammed cells. (A): Seven days after reprogramming, SPC/GFP could not be detected in cells reprogrammed with ATP-regenerating buffer (a) (fluorescence microscopy) and (d) (same cells seen in phase contrast) or MEF extract (b) (fluorescence microscopy) and (e) (same cells seen in phase contrast), but was clearly present in the cells reprogrammed with MLE-12 extract (c) (fluorescence microscopy) and (f) (same cells seen in phase contrast). Original magnification x200. (B): Real-time PCR analysis of SPC mRNA expression at day 7 in cells reprogrammed with ATP-regenerating buffer (No ext), MEF extract (MEF ext), or MLE-12 extract (MLE ext). Data are shown relative to SPC mRNA of the control cells reprogrammed with buffer only (No ext). (C): Real-time PCR analysis of SPC mRNA expression in cells permeabilized with different concentrations of SLO (200, 800, or 1000 mg/ml). Data shown are from three independent experiments; bars = SE of the mean. ** indicates p < .05 as compared with each control. Abbreviations: GFP, green fluorescent protein; MEF, murine embryonic fibroblast; PCR, polymerase chain reaction; SLO, streptolysin O; SPC, surfactant protein C.
The phenotype of SPC/GFP-transfected reprogrammed cells was examined by immunocytochemistry and electron microscopy. Pro-SPC and TTF-1, two markers for type-II pneumocytes, were detected in SPC/GFP-expressing reprogrammed cells (Figs. 4A–C, 4G–I). Both markers were also detected in control MLE-12 cells (tMLE-12) transfected with SPC/GFP, (Figs. 4D–F, 4J–L). In all cells, immunoreactivity of pro-SPC was found in the cytoplasm and that of TTF-1, a transcription factor, was found only in the nucleus. The phenotype of the pneumocytes was confirmed by electron microscopy that revealed the presence of organelles with the appearance of lamellar bodies in the cytoplasm of reprogrammed cells harvested after 7 days of culture (Figs. 4M, 4N).
Figure 4. SPC and TTF-1 expression in SPC/GFP-positive reprogrammed cells. Immunostaining of pro-SPC and TTF-1. (A–C): SPC/GFP-expressing reprogrammed ESCs. (A): Immunostained for pro-SPC; (B) the same cells labeled by transfected SPC/GFP; and (C) nuclei of the same cells stained with DAPI. (D–F): tMLE-12 cells. (D): Immunostained for pro-SPC; (E) the same cells labeled by transfected SPC/GFP; and (F) nuclei. (G–I): SPC/GFP-expressing reprogrammed cells. (G): Immunostained for TTF-1; (H) the same cells labeled by transfected SPC/GFP; and (I) DAPI-stained nuclei. (J–L): tMLE-12 cells. (J): Immunostained for TTF-1; (K) the same cells labeled by transfected SPC/GFP; and (L) DAPI-stained nuclei. Original magnification x200. (M, N): Electron micrograph showing abundant organelles with the appearance of lamellar bodies in reprogrammed cells after 7 days in culture. Original magnification x10,000. (N): A higher magnification of area indicated in (M). Original magnification x40,000. Abbreviations: DAPI, 4,6-diamindino-2-phenylindole-2; GFP, green fluorescent protein; SPC, surfactant protein C; TTF-1, thyroid transcription factor-1.
At day 7, pro-SPC–expressing reprogrammed cells, assessed by GFP fluorescence and immunostaining of pro-SPC, reached a maximal proportion (5%–10%), with some of the cells showing very strong SPC/GFP expression. During the second week of culture, the proportion of SPC/GFP-expressing cells started to decrease, although some cells were still positive at day 14. Real-time PCR analysis showed a concomitant gradual decrease in SPC mRNA expression (Fig. 5B) with a more rapid decrease of GFP expression after day 7 (Fig. 5A). Our contention that this decline in SPC over 14 days of culture was due to differentiation of type II pneumocytes to the type I phenotype, was supported by the finding of an increase in aquaporin 5 mRNA, a marker for type I pneumocytes, during that period (Fig. 5C). The cells also changed their morphology during this time, becoming larger and thinner, an appearance consistent with that of type I pneumocytes, and were found to express aquaporin 5 protein (Figs. 5D and 5E).
Figure 5. SPC, GFP, and aquaporin (AQP) expression in SPC/GFP-positive reprogrammed cells from 7–14 days of culture. Real-time PCR analysis of (A) SPC/GFP, (B) SPC, and (C) AQP-5 gene expression in SPC/GFP-transfected reprogrammed cells at days 7, 11, and 14. Data for SPC and GFP RNA are shown relative to the level of expression of the cells at day 14 of culture. For AQP-5, the data are relative to expression on day 7 of culture. Expression of SPC and GFP RNA decreased from day 7 to day 14, and this was associated with an increase in AQP RNA expression. Data shown are from three independent experiments; bars = SE of the mean. (D): Reprogrammed embryonic stem cells after 14 days of culture immunostained for AQP-5. The staining pattern on the surface of the cells is consistent with the localization of AQP, an integral membrane protein. (E): DAPI-stained nuclei. Original magnification x200. Abbreviations: DAPI, 4,6-diamindino-2-phenylindole-2; GFP, green fluorescent protein; SPC, surfactant protein C.
DISCUSSION
This work was supported by the Medical Research Council (Cooperative Group G9900355 and Component Grant G0300106). The authors thank Prof. Austin G. Smith, Institute of Stem Cell Research, University of Edinburgh, Scotland, U.K., for the E14Tg2 ES cells; Prof. Jeffrey A. Whitsett, The Children’s Hospital, Cincinnati, for the 4.8-kbmurine SPC promoter/GFP construct; Mrs. Margaret Mobberley, Histopathology Department, Charing Cross Campus, Imperial College, for expert electron microscopy, and Prof. Richard L. Lubman, Keck School of Medicine of the University of Southern California, for his advice on the manuscript. Authors Qin and Tai contributed equally to this article.
REFERENCES
Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines. STEM CELLS 2001;19:193–204.
Wobus AM. Potential of embryonic stem cells. Mol Aspects Med 2001;22:149–164.
Bishop AE, Buttery LDK, Polak JM. Embryonic stem cells. J Pathol 2002;197:424–429.
Rippon HJ, Bishop AE. Embryonic stem cells. Cell Prolif 2004;27:23–34.
Otto WR. Lung stem cells. Int J Exp Pathol 1997;78(5):291–310.
Bishop AE. Pulmonary epithelial stem cells. Cell Prolif 2004;37:89–96.
Ali NN, Edgar AJ, Samadikuchaksaraei A et al. Derivation of type II alveolar epithelial cells from murine embryonic stem cells. Tissue Eng 2002;8:541–550.
Rippon HJ, Ali NN, Polak JM et al. Initial observations on the effect of medium composition on the differentiation of murine embryonic stem cells to alveolar type II cells. Cloning Stem Cells 2004;6:49–56.
H?kelien AM, Landsverk HB, Robl JM et al. Reprogramming fibroblasts to express T-cell functions using cell extracts. Nat Biotechnol 2002;20:460–466.
H?kelien AM, Gaustad KG, Collas P. Transient alteration of cell fate using a nuclear and cytoplasmic extract of an insulinoma cell line. Biochem Biophys Res Commun 2004;316:834–841.
Gaustad KG, Boquest AC, Anderson BE et al. Differentiation of human adipose tissue stem cells using extracts of rat cardiomyocytes. Biochem Biophys Res Commun 2004;314:420–427.
Dobbs LG, Williams MC, Gonzalez R. Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells. Biochim Biophys Acta 1988;970:146–156.
Dobbs LG. Isolation and culture of alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 1990;258:134–147.
Nielsen S, King LS, Christensen BM et al. Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat. Am J Physiol 1997;273:C1549–C1561.
Wikenheiser KA, Vorbroker DK, Rice WR et al. Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice. Proc Natl Acad Sci U S A 1993;90:11029–11033.(Mingde Qina, Guangping Ta)
b Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
Key Words. Embryonic stem cells ? Cell extract–based reprogramming
Correspondence: Julia M. Polak, D.B.E., M.D., F.R.C.Path., Tissue Engineering & Regenerative Medicine Centre, Imperial College Faculty of Medicine, Chelsea & Westminster Campus, Fulham Road, London SW10 9NH, U.K. Telephone: 44-208-237-2670; Fax: 44-208-746-5619; e-mail: julia.polak@imperial.ac.uk
ABSTRACT
Embryonic stem cells (ESCs) have been shown to differentiate in vitro to cell phenotypes arising from each of the three germ layers . A variety of means has been used to try to direct the differentiation of these cells toward specific cell lineages, in particular for the purposes of tissue engineering . In the distal lung, type II pneumocytes undergo proliferation and differentiation to the type I phenotype following injury and are crucial to the natural regenerative process of the alveoli . Thus, as part of our strategy to produce pulmonary cell types for tissue engineering purposes, we have derived type II pneumocytes from ESCs using soluble factors . However, as this process is lengthy and does not provide high yields of the target phenotype, we have sought a more efficient method.
A method of cell reprogramming, based on the exposure of permeabilized cells to a nuclear and cytoplasmic extract derived from a "target" cell, has been used successfully to derive mature cells from nonrelated phenotypes, including T cells and pancreatic ? (insulin-producing) cells from 293T cells and fibroblasts, and cardiomyocytes from adipose stem cells . We hypothesized, therefore, that this in vitro reprogramming technology could be used to differentiate ESCs to type II pneumocytes. In this study, a murine ESC line (E14tg2) was stably transfected with a construct containing an enhanced green fluorescent protein (eGFP) reporter gene under the control of the surfactant protein C (SPC) promoter, a specific marker of type II pneumocytes. Reversibly permeabilized ESCs were reprogrammed with extracts of "target cells" (transformed murine pneumocytes). The reprogrammed ESCs became GFP-positive cells and coexpressed SPC, specific for type II pneumocytes, and thyroid transcription factor-1 (TTF-1) that is found in immature pulmonary epithelium. In accordance with the well-known, in vitro differentiation of type II pneumocytes to the type I phenotype , we were able to show that SPC gene expression declines over time and is accompanied by an increase in aquaporin 5 mRNA and protein, a marker for type I pneumocytes . These findings demonstrate that differentiation of murine ESCs can be driven by using an extract-based, in vitro reprogramming approach.
MATERIALS AND METHODS
Reversible Permeabilization of EB Cells
In the absence of SLO, few EB cells were labeled with Texas Red (Fig. 1A). With 200–700 ng/ml SLO, the cells were not sufficiently permeabilized, and only approximately 10% of cells were found to be labeled with Texas Red (Fig. 1B). At 800–900 ng/ml SLO, 80% of the cells were labeled with Texas Red (Figs. 1C, 1E, 1F), indicating that a high proportion of EB cells could take up the dextran and be successfully replated. Increasing the SLO concentration to 1000 ng/ml appeared to damage the cells, and fewer were labeled (Fig. 1D).
Figure 1. Optimization of membrane permeabilization by SLO. Embryoid body cells were permeabilized with SLO at concentrations of 0–1000 ng/ml. Texas Red–stained cells treated with (A) 0 ng/ml SLO, (B) 200 ng/ml SLO, (C) 800 ng/ml SLO, and (D) 1000 ng/ml SLO. Original magnification x200. (E–H): Corresponding phase-contrast images of the fields are shown in (A–D). (I, J): Higher magnification (x400) of cells treated with 800 ng/ml SLO, the concentration found to give the greatest uptake of Texas Red–dextran without overt cell damage: (I) uptake of Texas Red–dextran, (J) phase-contrast image of the same cells. Abbreviation: SLO, streptolysin O.
Transfection
An eGFP reporter gene driven by the pneumocyte-specific murine SPC promoter was transfected into undifferentiated ES, MEF, and MLE-12 cells. As expected, no SPC/GFP expression was detected in transfected ESCs (Fig. 2A) or MEFs (Fig. 2B). However, SPC/GFP was expressed in transfected MLE-12 cells (Fig. 2C). All cell types expressed eGFP transfected alone, indicating that transfection efficiency was satisfactory (Figs. 2G–I). These results demonstrate that the SPC/GFP construct is specifically expressed in type II pneumocytes.
Figure 2. Transfection of SPC/GFP. Forty-eight hours after transfection of the SPC/GFP construct, no expression of GFP could be seen in undifferentiated ESCs (A) (fluorescence microscopy) and (D) (same cells seen in phase contrast) or MEFs (B) (fluorescence microscopy) and (E) (same cells seen in phase contrast), but was clearly present in MLE-12 cells (C) (fluorescence microscopy) and (F) (same cells seen in phase contrast). In the cells transfected with pTracer-GFP construct, GFP was found in all three types (all fluorescence microscopy and phase contrast, as before): (G, J) undifferentiated ESCs; (H, K) MEFs; and (I, L) MLE-12 cells. Original magnification x200. Abbreviations: ESC, embryonic stem cell; GFP, green fluorescent protein; MEF, murine embryonic fibroblast; SPC, surfactant protein C.
Induction of SPC/GFP Expression in Differentiated Cells
EB cells reversibly permeabilized with 800 ng/ml SLO were incubated for 1 hour in a nuclear and cytoplasmic extract of MLE-12 pneumocytes. Reprogramming was assessed by the induction of SPC promoter-driven eGFP expression in the EB cells. In cells exposed to MLE-12 extract, SPC/GFP expression was detected as early as day 3 of exposure to the extract, and the proportion of GFP-positive cells increased up to day 7 to 7.53 ± 1.14% (mean + SE) (Fig. 3Ac). No expression of SPC/GFP could be detected in cells reprogrammed with ATP-regenerating buffer (Fig. 3Aa) or MEF extract (Fig. 3Ab). Induction of SPC gene expression was also analyzed by real-time PCR. At day 7 of exposure to MLE-12 extract, SPC mRNA expression was markedly higher than that of control cells incubated in buffer only or MEF extract (Fig. 3B). SPC gene expression in cells exposed to buffer only or MEF extract was also found to be higher (3.28 ± 0.74 fold) than that of undifferentiated ESCs, as some of these cells had probably undergone spontaneous differentiation to pneumocytes. Furthermore, SPC mRNA expression of ESCs treated with 800 ng/ml SLO was higher (p < .005) than that of cells treated with 200 or 1000 ng/ml (Fig. 3C), corroborating our Texas Red–dextran uptake observations. Collectively, these results indicate that the MLE-12 extract is capable of inducing expression of a pneumocyte-specific promoter in ESCs.
Figure 3. SPC/GFP and SPC expression in reprogrammed cells. (A): Seven days after reprogramming, SPC/GFP could not be detected in cells reprogrammed with ATP-regenerating buffer (a) (fluorescence microscopy) and (d) (same cells seen in phase contrast) or MEF extract (b) (fluorescence microscopy) and (e) (same cells seen in phase contrast), but was clearly present in the cells reprogrammed with MLE-12 extract (c) (fluorescence microscopy) and (f) (same cells seen in phase contrast). Original magnification x200. (B): Real-time PCR analysis of SPC mRNA expression at day 7 in cells reprogrammed with ATP-regenerating buffer (No ext), MEF extract (MEF ext), or MLE-12 extract (MLE ext). Data are shown relative to SPC mRNA of the control cells reprogrammed with buffer only (No ext). (C): Real-time PCR analysis of SPC mRNA expression in cells permeabilized with different concentrations of SLO (200, 800, or 1000 mg/ml). Data shown are from three independent experiments; bars = SE of the mean. ** indicates p < .05 as compared with each control. Abbreviations: GFP, green fluorescent protein; MEF, murine embryonic fibroblast; PCR, polymerase chain reaction; SLO, streptolysin O; SPC, surfactant protein C.
The phenotype of SPC/GFP-transfected reprogrammed cells was examined by immunocytochemistry and electron microscopy. Pro-SPC and TTF-1, two markers for type-II pneumocytes, were detected in SPC/GFP-expressing reprogrammed cells (Figs. 4A–C, 4G–I). Both markers were also detected in control MLE-12 cells (tMLE-12) transfected with SPC/GFP, (Figs. 4D–F, 4J–L). In all cells, immunoreactivity of pro-SPC was found in the cytoplasm and that of TTF-1, a transcription factor, was found only in the nucleus. The phenotype of the pneumocytes was confirmed by electron microscopy that revealed the presence of organelles with the appearance of lamellar bodies in the cytoplasm of reprogrammed cells harvested after 7 days of culture (Figs. 4M, 4N).
Figure 4. SPC and TTF-1 expression in SPC/GFP-positive reprogrammed cells. Immunostaining of pro-SPC and TTF-1. (A–C): SPC/GFP-expressing reprogrammed ESCs. (A): Immunostained for pro-SPC; (B) the same cells labeled by transfected SPC/GFP; and (C) nuclei of the same cells stained with DAPI. (D–F): tMLE-12 cells. (D): Immunostained for pro-SPC; (E) the same cells labeled by transfected SPC/GFP; and (F) nuclei. (G–I): SPC/GFP-expressing reprogrammed cells. (G): Immunostained for TTF-1; (H) the same cells labeled by transfected SPC/GFP; and (I) DAPI-stained nuclei. (J–L): tMLE-12 cells. (J): Immunostained for TTF-1; (K) the same cells labeled by transfected SPC/GFP; and (L) DAPI-stained nuclei. Original magnification x200. (M, N): Electron micrograph showing abundant organelles with the appearance of lamellar bodies in reprogrammed cells after 7 days in culture. Original magnification x10,000. (N): A higher magnification of area indicated in (M). Original magnification x40,000. Abbreviations: DAPI, 4,6-diamindino-2-phenylindole-2; GFP, green fluorescent protein; SPC, surfactant protein C; TTF-1, thyroid transcription factor-1.
At day 7, pro-SPC–expressing reprogrammed cells, assessed by GFP fluorescence and immunostaining of pro-SPC, reached a maximal proportion (5%–10%), with some of the cells showing very strong SPC/GFP expression. During the second week of culture, the proportion of SPC/GFP-expressing cells started to decrease, although some cells were still positive at day 14. Real-time PCR analysis showed a concomitant gradual decrease in SPC mRNA expression (Fig. 5B) with a more rapid decrease of GFP expression after day 7 (Fig. 5A). Our contention that this decline in SPC over 14 days of culture was due to differentiation of type II pneumocytes to the type I phenotype, was supported by the finding of an increase in aquaporin 5 mRNA, a marker for type I pneumocytes, during that period (Fig. 5C). The cells also changed their morphology during this time, becoming larger and thinner, an appearance consistent with that of type I pneumocytes, and were found to express aquaporin 5 protein (Figs. 5D and 5E).
Figure 5. SPC, GFP, and aquaporin (AQP) expression in SPC/GFP-positive reprogrammed cells from 7–14 days of culture. Real-time PCR analysis of (A) SPC/GFP, (B) SPC, and (C) AQP-5 gene expression in SPC/GFP-transfected reprogrammed cells at days 7, 11, and 14. Data for SPC and GFP RNA are shown relative to the level of expression of the cells at day 14 of culture. For AQP-5, the data are relative to expression on day 7 of culture. Expression of SPC and GFP RNA decreased from day 7 to day 14, and this was associated with an increase in AQP RNA expression. Data shown are from three independent experiments; bars = SE of the mean. (D): Reprogrammed embryonic stem cells after 14 days of culture immunostained for AQP-5. The staining pattern on the surface of the cells is consistent with the localization of AQP, an integral membrane protein. (E): DAPI-stained nuclei. Original magnification x200. Abbreviations: DAPI, 4,6-diamindino-2-phenylindole-2; GFP, green fluorescent protein; SPC, surfactant protein C.
DISCUSSION
This work was supported by the Medical Research Council (Cooperative Group G9900355 and Component Grant G0300106). The authors thank Prof. Austin G. Smith, Institute of Stem Cell Research, University of Edinburgh, Scotland, U.K., for the E14Tg2 ES cells; Prof. Jeffrey A. Whitsett, The Children’s Hospital, Cincinnati, for the 4.8-kbmurine SPC promoter/GFP construct; Mrs. Margaret Mobberley, Histopathology Department, Charing Cross Campus, Imperial College, for expert electron microscopy, and Prof. Richard L. Lubman, Keck School of Medicine of the University of Southern California, for his advice on the manuscript. Authors Qin and Tai contributed equally to this article.
REFERENCES
Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines. STEM CELLS 2001;19:193–204.
Wobus AM. Potential of embryonic stem cells. Mol Aspects Med 2001;22:149–164.
Bishop AE, Buttery LDK, Polak JM. Embryonic stem cells. J Pathol 2002;197:424–429.
Rippon HJ, Bishop AE. Embryonic stem cells. Cell Prolif 2004;27:23–34.
Otto WR. Lung stem cells. Int J Exp Pathol 1997;78(5):291–310.
Bishop AE. Pulmonary epithelial stem cells. Cell Prolif 2004;37:89–96.
Ali NN, Edgar AJ, Samadikuchaksaraei A et al. Derivation of type II alveolar epithelial cells from murine embryonic stem cells. Tissue Eng 2002;8:541–550.
Rippon HJ, Ali NN, Polak JM et al. Initial observations on the effect of medium composition on the differentiation of murine embryonic stem cells to alveolar type II cells. Cloning Stem Cells 2004;6:49–56.
H?kelien AM, Landsverk HB, Robl JM et al. Reprogramming fibroblasts to express T-cell functions using cell extracts. Nat Biotechnol 2002;20:460–466.
H?kelien AM, Gaustad KG, Collas P. Transient alteration of cell fate using a nuclear and cytoplasmic extract of an insulinoma cell line. Biochem Biophys Res Commun 2004;316:834–841.
Gaustad KG, Boquest AC, Anderson BE et al. Differentiation of human adipose tissue stem cells using extracts of rat cardiomyocytes. Biochem Biophys Res Commun 2004;314:420–427.
Dobbs LG, Williams MC, Gonzalez R. Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells. Biochim Biophys Acta 1988;970:146–156.
Dobbs LG. Isolation and culture of alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 1990;258:134–147.
Nielsen S, King LS, Christensen BM et al. Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat. Am J Physiol 1997;273:C1549–C1561.
Wikenheiser KA, Vorbroker DK, Rice WR et al. Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice. Proc Natl Acad Sci U S A 1993;90:11029–11033.(Mingde Qina, Guangping Ta)