DOI 10.1007/s10517-015-2916-7 Cell Technologies in Biology and Medicine, No. 4, February, 2015
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Functionally Active Gap Junctions between Connexin 43-Positive Mesenchymal Stem Cells and Glioma Cells A. N. Gabashvili*, V. P. Baklaushev*, N. F. Grinenko**, A. B. Levinskii*, P. A. Mel’nikov*, S. A. Cherepanov*, and V. P. Chekhonin*,** Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 61-68, January, 2015 Original article submitted November 13, 2014 The formation of functional gap junctions between mesenchymal stem cells and cells of lowgrade rat glioma C6 cells was studied in in vitro experiments. Immunocytochemical analysis with antibodies to connexin 43 extracellular loop 2 showed that mesenchymal stem cells as well as C6 glioma cells express the main astroglial gap junction protein connexin 43. Analysis of migration activity showed that mesenchymal stem cells actively migrate towards C6 glioma cells. During co-culturing, mesenchymal stem cells and glioma C6 form functionally active gap junctions mediating the transport of cytoplasmic dye from glioma cells to mesenchymal stem cells in the opposite direction. Fluorometry showed that the intensity of transport of low-molecular substances through heterologous gap junctions between mesenchymal stem cells and glioma cells is similar to that through homologous gap junctions between glioma cells. This phenomenon can be used for the development of new methods of cell therapy of high-grade gliomas. Key Words: mesenchymal stem cells; glioblastoma multiforme; C6 glioma; connexin 43; gap junctions Human glioblastoma multiforme is the most invasive low-differentiated glial tumor characterized by rapid growth and practically 100% relapse rate after surgical removal. All known methods of combined therapy (surgical resection with intraoperative navigation, chemotherapy, radiotherapy) are low effective [24,31], therefore, the search for new treatment approaches to the therapy of low-differentiated gliomas remains a pressing problem. In early 2000, the data appeared that various stem cells, including mesenchymal stem cells (MSC), can inhibit tumor growth [2,5,10,25,26]. These cells produce factors regulating, along with cell differentiation and chemotaxis, the balance of pro- and antiapoptotic signals. The important role in transmission of the inhibitory signals from neural progenitor cells to the *Department of Medical Nanobiotechnologies, Medico-Biological Faculty, N. I. Pirogov Russian National Research Medical University; **Department of Fundamental and Applied Neurobiology, V. P. Serbsky State Research Center of Forensic and Social Psychiatry, Moscow, Russia. Address for correspondence: [email protected]. A. N. Gabashvili
tumor cells is thought to be played by various secreted factors, in particular, bone marrow protein BMP7 [7] and IL-18 [32]. BMP family proteins are of particular interest in this respect, because they trigger cell differentiation under normal conditions [8]. The role of stem and progenitor cells in the pathogenesis of gliomas is ambiguous. According to one of the modern concept, astroglial progenitor cells are the source of low-differentiated gliomas [9,19,20,23]. On the other hand, normal stem and progenitor cells can migrate to the pathological focus characterized by overexpression CXC- and CC-chemokines and their receptors (CXCR, CCR, CX3C – CX3CR) [34]. Stem cells introduced into tumor focus interact with glioma cells [25] and migrate into the peritumoral area of glioma invasion [3]. The tropism of these cells to the tumor focus allows considering stem cells as a potential vector for targeted therapy of gliomas [5,15,27,29]. The mechanisms mediating the inhibitory effect of stem cells on tumor cells are poorly studied. Until now, no direct evidence of cell–cell interaction be-
0007-4888/15/15910173 © 2015 Springer Science+Business Media New York
174 tween MSC and tumor cells in co-culture or after MSC implantation in the tumor was found. A variant of this cooperation can be the formation of heterologous gap junctions (GJ) between these cells. The formation of GJ is described for the majority of mammalian cells. Every contact (connexon) consists of 6 integral protein subunits, connexins, arranged in a ring-shaped structure and forming a pore in the plasmalemma [14]. Connexon can exist as a hemichannel maintaining water homeostasis of the cell and mediating exchange of low-molecular-weight substances between the cytoplasm and interstitial fluid. Hemichannels of neighboring cells can interact to form true GJ allowing exchange of ions and small molecules (0.5 reflects positive cell migration.
RESULTS Analysis of rat bone marrow stem cells showed the following phenotype: CD34–, CD44+, CD45–, CD90+, CD105+, and CD117–. Expression of CD90, CD105, and CD44 and the absence of CD45, CD34, and CD117 suggest that the cell preparation consisted of MSC and was not contaminated with hemopoietic stem cells (Fig. 1). Immunocytochemical analysis revealed intensive expression of GJ Cx43 in both C6 glioma (Fig. 2, a) and MSC (Fig. 2, b). In both cell preparations, Cx43 was visualized in the cytoplasm and cell membrane in the form of typical placoids. The detected expression of Cx43 does not definitely indicate the formation of functional hemichannels and true GJ between the cells in Cx43+ cultures. To confirm cell–cell communication via GJ, experi-
Fig. 1. Immunophenotyping of MSC. a) FITC-labeled unspecific antibodies (negative control); b) FITC-labeled antibodies to CD90; c) PElabeled unspecific antibodies ; d, e) PE-labeled antibodies to CD34 and CD117, respectively; f) FITC-labeled antibodies to CD45.
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Fig. 2. Cx43 expression in fixed C6 glioma and MSC. Immunocytochemical analysis with primary monoclonal antibodies to extracellular loop 2 of Cx43 (MabCx43) and second Alexa Fluor 488-labeled anti-mouse immunoglobulin antibodies. Nuclei were poststained with DAPI.
ments with fluorescent dye transfer were carried out. Donor cells (C6 or MSC) were loaded with Calcein AM (green fluorescence) and stained with DiI (red fluorescence) that binds to the cell membrane. Dye transfer between glioma C6 cells served as the control (Fig. 3, a). The formation of GJ was followed by Calcein AM transfer between labeled and unlabeled cells and cells containing Calcein AM and not containing DiI appeared around labeled cells, because this membrane dye cannot be transported from one cell to another. For semiquantitative comparison of the dye transfer intensity, a histogram of fluorescence intensity profile was constructed (Fig. 3, b, c). Calcein AM fluorescence (curve 1) considerably increased in the zone of the recipient cell in comparison with fluorescence in point 0 (baseline), while DiI fluorescence (curve 2) remained unchanged. This confirms that unlabeled recipient cells received the dye from the donor cell. Intensive fluorescence from two labels, Calcein AM and DiI, was recorded in sites where scanning vector passed through the location of the donor cells (curves 1 and 2). In experiments with human glioma cells, dye transfer was recorded between MSC and human U87 and U373 glioma cells; however, dye transfer between
SVG p12 astrocytes and tumor cells served as the control in these experiments [36]. The objective was to prove that MSC and rat C6 glioma cells can form functional heterologous GJ characterized by the same activity as homologous GJ between tumor cells. In this case, Calcein AM is transported through heterologous GJ between these two cell types similar as in the control (Fig. 4, a). To compare the intensity of dye transfer between the two types of GJ, the same fluorescence intensity profile histogram for heterologous GJ was constructed (Fig. 4, b, c) The mean fluorescence intensity in the recipient cells (3500±150 arb. units) determined by dye transfer through heterologous GJ corresponded to the mean fluorescence intensity resultant from dye transfer through homologous GJ (4000±180 arb. units). This confirms functional activity of heterologous GJ between MSC and glioma C6 cells. In turn, glioma C6 cells can receive intracellular messengers Ca2+, cAMP, and cGMP from MSC, which can modulate proliferative activity of these cells and their invasive potential. As was already noted, MSC due to their high tropism to glioma cells are promising vectors for targeted drug delivery to tumor focus. Cell migration is usually evaluated using wound healing assay or Transwell migration assay [17,28,30,33]. In the first case, it is very
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Fig. 3. Analysis of the function of homologous GJ in culture of C6 glioma cells. a) Visualization of Calcein AM transfer through GJ. Left: image superposition; center: Calcein AM green fluorescence; right: DiI red fluorescence. 1) Donor cell; 2) Recipient cell. b) Combined microphotograph of fluorescence intensity measurement vector (arrow). Red mark shows the spot where fluorescence intensity is taken as zero (baseline). Then, the vector passes through the location of recipient cell. c) Histogram of Calcein AM (1) and DiI (2) fluorescence intensity in the donor cell.
difficult to create a gradient of chemotactic factors modulating migration, and in the second, cell migration cannot be measured in real time. In our study, we tracked MSC migration towards C6 glioma cells under conditions of indirect co-culture providing a gradient of chemotactic factors using xCELLigence RTCA DP cell analyzer, which allowed us to evaluate the number of migrating MSC in real-time mode at 20-min intervals. We found that the process of MSC migration towards C6 glioma cells started as soon as in 5 h of indirect co-culturing (Fig. 5, curves 1 and 2) and active migration lasted for 25-30 h. In control
wells, no cell migration was recorded throughout the observation period. It has been experimentally proven that IL-8, SDF1α (stromal factor), HGH (human growth hormone), SCF (stem cell factor), uPa (urokinase plasminogen activator), and VEGF are involved in MSC tropism to glioma cells [13,16,21]. However, the mechanisms mediating MSC migration to glioma focus require further investigations. The authors are grateful to Alamed Company (Moscow) for presented xCELLigence RTCA DP demo version.
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Fig. 4. Analysis of the function of heterologous GJ between MSC and C6 glioma cells. a) Superposition of images of green and red fluorescent channels and differential interference contrast microscopy. b) Calcein AM fluorescence. 1) Donor cell; 2) recipient cell. c) DiI fluorescence (shows only primarily labeled cells). d) Combined microphotograph with fluorescence intensity measurement vector (arrow). Asterisk shows recipient cell. Red mark: point on the vector corresponding to donor cell. e) Histogram of Calcein AM (1) and DiI (2) fluorescence intensity in the donor cell. Asterisk shows the zone corresponding to recipient cell fluorescence.
Fig. 5. Analysis of migration activity of MSC towards glioma cells. Curves 1 and 2: MSC migration towards wells with C6 glioma cells. (1 – 20,000, 2 – 2000 MSC per well. Curves 3 and 4: migration of MSC towards wells with culture medium and HEK293 cells, respectively (control, no migration).
A. N. Gabashvili, V. P. Baklaushev, et al.
The study was supported by the Russian Foundation for Basic Research (grant No. 3-04-40202 NKOMPhI).
REFERENCES 1. G. M. Yusubalieva, V. P. Baklaushev, O. I. Gurina, et al., Bull. Exp. Biol. Med., 157, No. 4, 510-515 (2014). 2. K. S. Aboody, A. Brown, N. G. Rainov, et al., Proc. Natl Acad. Sci. USA, 97, No. 23, 12,846-12,851 (2000). 3. V. P. Baklaushev, N. F. Grinenko, E. A. Savchenko, et al., Bull. Exp. Biol. Med., 152, No. 4, 497-503 (2012). 4. V. P. Baklaushev, O. I. Gurina, G. M. Yusubalieva, et al., Bull. Exp. Biol. Med., 148, No. 4, 725-730 (2009). 5. V. Barresi, N. Belluardo, S. Sipione, et al., Cancer Gene Ther., 10, No. 5, 396-402 (2003). 6. R. Caltabiano, A. Torrisi, D. Condorelli, et al., Acta Histochem., 112, No. 6, 529-535 (2010). 7. S. R. Chirasani, A. Sternjak, P. Wend, et al., Brain, 133, Pt. 7, 1961-1972 (2010). 8. P. B. Dirks, Mol. Oncol., 4, No. 5, 420-430 (2010). 9. D. Friedmann-Morvinski, E. Bushong, E. Ke, et al., Science, 338, 1080-1084 (2012). 10. R. Glass, M. Synowitz, G. Kronenberg, et al., J. Neurosci., 25, No. 10, 2637-2646 (2005). 11. M. Gnecchi and L. G. Melo, Methods Mol. Biol., 482, 281-294 (2009). 12. G. S. Goldberg, J. F. Bechberger, and C. C. Naus, Biotechniques, 18, No. 3, 490-497 (1995). 13. M. Gutova, J. Najbauer, R. T. Frank, et al., Stem Cells, 26, No. 6, 1406-1413 (2008). 14. J. C. Hervé, N. Bourmeyster, D. Sarrouilhe, and H. S. Duffy, Prog. Biophys. Mol. Biol., 94, No. 1, 29-65 (2007). 15. B. E. Huber, E. A. Austin, C. A. Richards, et al., Proc. Natl Acad. Sci. USA, 91, No. 17, 8302-8306 (1994). 16. S. E. Kendall, J. Najbauer, H. F. Johnston, et al., Stem Cells, 26, No. 6, 1575-1586 (2008). 17. P. Kovaříková, E. Michalova, L. Knopfová, and P. Bouchal, Klin. Onkol., 27, Suppl. 1, S22-S27 (2014).
179 18. J. H. Lin, T. Takano, M. L. Cotrina, et al., J. Neurosci., 22, No. 11, 4302-4311 (2002). 19. N. Lindberg, M. Kastemar, T. Olofsson, et al., Oncogene, 28, No. 23, 2266-2275 (2009). 20. D. M. Muñoz, T. Tung, S. Agnihotri, et al., Glia, 61, No. 11, 1862-1872 (2013). 21. J. Najbauer, M. K. Danks, N. O. Schmidt, et al., Progress in Gene Therapy, Autologous and Cancer Stem Cell Gene Therapy, Eds. R. Bertolotti, K. Ozawa, Singapore (2007), pp. 335-372. 22. R. Oliveira, C. Christov, J. S. Guillamo, et al., BMC Cell Biol., 6, No. 1, 7 (2005). 23. N. Sanai, A. Alvarez-Buylla, and M. S. Berger, N. Engl. J. Med. 353, No. 8, 811-822 (2005). 24. S. Sathornsumetee and J. N. Rich, Ann. N. Y. Acad. Sci., No. 1142, 108-132 (2008). 25. C. Schichor, V. Albrecht, B. Korte, et al., Exp. Neurol., 234, No. 1, 208-219 (2012). 26. S. Ströjby, S. Eberstal, A. Svensson, et al., J. Neuroimmunol., 274, Nos. 1-2 240-243 (2014). 27. B. Thaci, A. U. Ahmed, I. V. Ulasov, et al., Cancer Gene Ther., 19, No. 6, 431-442 (2012). 28. S. L. Tomchuck, K. J. Zwezdaryk, S. B. Coffelt, et al., Stem Cells, 26, No. 1, 99-107 (2008). 29. M. A. Tyler, I. V. Ulasov, A. M. Sonabend, et al., Gene Ther., 16, No. 2, 262-278 (2009). 30. M. N. Walter, K. T. Wright, H. R. Fuller, et al., Exp. Cell Res., 316, No. 7, 1271-1281 (2010). 31. P. Y. Wen and S. Kesari, N. Engl. J. Med. 359, No. 5, 492-507 (2008). 32. G. Xu, X. D. Jiang, Y. Xu, et al., Cell Biol. Int., 33, No. 4, 466-474 (2009). 33. L. Xue, J. Wang, W. Wang, et al., Cell Biochem. Biophys., 70, No. 3, 1609-1616 (2014). 34. S. Yip, R. Sabetrasekh, R. L. Sidman, and E. Y. Snyder, Eur. J. Cancer., 42, No. 9, 1298-1308 (2006). 35. W. Zhang, C. Nwagwu, D. M. Le, et al., J. Neurosurg., 99, No. 6, 1039-1046 (2003). 36. Z. H. Zhang, Y. Sum, Z. Y. Wang, et al., Zhonghua Yi Xue Za Zhi, 43, No. 9, 788-793 (2013).
173
Functionally Active Gap Junctions between Connexin 43-Positive Mesenchymal Stem Cells and Glioma Cells A. N. Gabashvili*, V. P. Baklaushev*, N. F. Grinenko**, A. B. Levinskii*, P. A. Mel’nikov*, S. A. Cherepanov*, and V. P. Chekhonin*,** Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 61-68, January, 2015 Original article submitted November 13, 2014 The formation of functional gap junctions between mesenchymal stem cells and cells of lowgrade rat glioma C6 cells was studied in in vitro experiments. Immunocytochemical analysis with antibodies to connexin 43 extracellular loop 2 showed that mesenchymal stem cells as well as C6 glioma cells express the main astroglial gap junction protein connexin 43. Analysis of migration activity showed that mesenchymal stem cells actively migrate towards C6 glioma cells. During co-culturing, mesenchymal stem cells and glioma C6 form functionally active gap junctions mediating the transport of cytoplasmic dye from glioma cells to mesenchymal stem cells in the opposite direction. Fluorometry showed that the intensity of transport of low-molecular substances through heterologous gap junctions between mesenchymal stem cells and glioma cells is similar to that through homologous gap junctions between glioma cells. This phenomenon can be used for the development of new methods of cell therapy of high-grade gliomas. Key Words: mesenchymal stem cells; glioblastoma multiforme; C6 glioma; connexin 43; gap junctions Human glioblastoma multiforme is the most invasive low-differentiated glial tumor characterized by rapid growth and practically 100% relapse rate after surgical removal. All known methods of combined therapy (surgical resection with intraoperative navigation, chemotherapy, radiotherapy) are low effective [24,31], therefore, the search for new treatment approaches to the therapy of low-differentiated gliomas remains a pressing problem. In early 2000, the data appeared that various stem cells, including mesenchymal stem cells (MSC), can inhibit tumor growth [2,5,10,25,26]. These cells produce factors regulating, along with cell differentiation and chemotaxis, the balance of pro- and antiapoptotic signals. The important role in transmission of the inhibitory signals from neural progenitor cells to the *Department of Medical Nanobiotechnologies, Medico-Biological Faculty, N. I. Pirogov Russian National Research Medical University; **Department of Fundamental and Applied Neurobiology, V. P. Serbsky State Research Center of Forensic and Social Psychiatry, Moscow, Russia. Address for correspondence: [email protected]. A. N. Gabashvili
tumor cells is thought to be played by various secreted factors, in particular, bone marrow protein BMP7 [7] and IL-18 [32]. BMP family proteins are of particular interest in this respect, because they trigger cell differentiation under normal conditions [8]. The role of stem and progenitor cells in the pathogenesis of gliomas is ambiguous. According to one of the modern concept, astroglial progenitor cells are the source of low-differentiated gliomas [9,19,20,23]. On the other hand, normal stem and progenitor cells can migrate to the pathological focus characterized by overexpression CXC- and CC-chemokines and their receptors (CXCR, CCR, CX3C – CX3CR) [34]. Stem cells introduced into tumor focus interact with glioma cells [25] and migrate into the peritumoral area of glioma invasion [3]. The tropism of these cells to the tumor focus allows considering stem cells as a potential vector for targeted therapy of gliomas [5,15,27,29]. The mechanisms mediating the inhibitory effect of stem cells on tumor cells are poorly studied. Until now, no direct evidence of cell–cell interaction be-
0007-4888/15/15910173 © 2015 Springer Science+Business Media New York
174 tween MSC and tumor cells in co-culture or after MSC implantation in the tumor was found. A variant of this cooperation can be the formation of heterologous gap junctions (GJ) between these cells. The formation of GJ is described for the majority of mammalian cells. Every contact (connexon) consists of 6 integral protein subunits, connexins, arranged in a ring-shaped structure and forming a pore in the plasmalemma [14]. Connexon can exist as a hemichannel maintaining water homeostasis of the cell and mediating exchange of low-molecular-weight substances between the cytoplasm and interstitial fluid. Hemichannels of neighboring cells can interact to form true GJ allowing exchange of ions and small molecules (0.5 reflects positive cell migration.
RESULTS Analysis of rat bone marrow stem cells showed the following phenotype: CD34–, CD44+, CD45–, CD90+, CD105+, and CD117–. Expression of CD90, CD105, and CD44 and the absence of CD45, CD34, and CD117 suggest that the cell preparation consisted of MSC and was not contaminated with hemopoietic stem cells (Fig. 1). Immunocytochemical analysis revealed intensive expression of GJ Cx43 in both C6 glioma (Fig. 2, a) and MSC (Fig. 2, b). In both cell preparations, Cx43 was visualized in the cytoplasm and cell membrane in the form of typical placoids. The detected expression of Cx43 does not definitely indicate the formation of functional hemichannels and true GJ between the cells in Cx43+ cultures. To confirm cell–cell communication via GJ, experi-
Fig. 1. Immunophenotyping of MSC. a) FITC-labeled unspecific antibodies (negative control); b) FITC-labeled antibodies to CD90; c) PElabeled unspecific antibodies ; d, e) PE-labeled antibodies to CD34 and CD117, respectively; f) FITC-labeled antibodies to CD45.
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Fig. 2. Cx43 expression in fixed C6 glioma and MSC. Immunocytochemical analysis with primary monoclonal antibodies to extracellular loop 2 of Cx43 (MabCx43) and second Alexa Fluor 488-labeled anti-mouse immunoglobulin antibodies. Nuclei were poststained with DAPI.
ments with fluorescent dye transfer were carried out. Donor cells (C6 or MSC) were loaded with Calcein AM (green fluorescence) and stained with DiI (red fluorescence) that binds to the cell membrane. Dye transfer between glioma C6 cells served as the control (Fig. 3, a). The formation of GJ was followed by Calcein AM transfer between labeled and unlabeled cells and cells containing Calcein AM and not containing DiI appeared around labeled cells, because this membrane dye cannot be transported from one cell to another. For semiquantitative comparison of the dye transfer intensity, a histogram of fluorescence intensity profile was constructed (Fig. 3, b, c). Calcein AM fluorescence (curve 1) considerably increased in the zone of the recipient cell in comparison with fluorescence in point 0 (baseline), while DiI fluorescence (curve 2) remained unchanged. This confirms that unlabeled recipient cells received the dye from the donor cell. Intensive fluorescence from two labels, Calcein AM and DiI, was recorded in sites where scanning vector passed through the location of the donor cells (curves 1 and 2). In experiments with human glioma cells, dye transfer was recorded between MSC and human U87 and U373 glioma cells; however, dye transfer between
SVG p12 astrocytes and tumor cells served as the control in these experiments [36]. The objective was to prove that MSC and rat C6 glioma cells can form functional heterologous GJ characterized by the same activity as homologous GJ between tumor cells. In this case, Calcein AM is transported through heterologous GJ between these two cell types similar as in the control (Fig. 4, a). To compare the intensity of dye transfer between the two types of GJ, the same fluorescence intensity profile histogram for heterologous GJ was constructed (Fig. 4, b, c) The mean fluorescence intensity in the recipient cells (3500±150 arb. units) determined by dye transfer through heterologous GJ corresponded to the mean fluorescence intensity resultant from dye transfer through homologous GJ (4000±180 arb. units). This confirms functional activity of heterologous GJ between MSC and glioma C6 cells. In turn, glioma C6 cells can receive intracellular messengers Ca2+, cAMP, and cGMP from MSC, which can modulate proliferative activity of these cells and their invasive potential. As was already noted, MSC due to their high tropism to glioma cells are promising vectors for targeted drug delivery to tumor focus. Cell migration is usually evaluated using wound healing assay or Transwell migration assay [17,28,30,33]. In the first case, it is very
A. N. Gabashvili, V. P. Baklaushev, et al.
177
Fig. 3. Analysis of the function of homologous GJ in culture of C6 glioma cells. a) Visualization of Calcein AM transfer through GJ. Left: image superposition; center: Calcein AM green fluorescence; right: DiI red fluorescence. 1) Donor cell; 2) Recipient cell. b) Combined microphotograph of fluorescence intensity measurement vector (arrow). Red mark shows the spot where fluorescence intensity is taken as zero (baseline). Then, the vector passes through the location of recipient cell. c) Histogram of Calcein AM (1) and DiI (2) fluorescence intensity in the donor cell.
difficult to create a gradient of chemotactic factors modulating migration, and in the second, cell migration cannot be measured in real time. In our study, we tracked MSC migration towards C6 glioma cells under conditions of indirect co-culture providing a gradient of chemotactic factors using xCELLigence RTCA DP cell analyzer, which allowed us to evaluate the number of migrating MSC in real-time mode at 20-min intervals. We found that the process of MSC migration towards C6 glioma cells started as soon as in 5 h of indirect co-culturing (Fig. 5, curves 1 and 2) and active migration lasted for 25-30 h. In control
wells, no cell migration was recorded throughout the observation period. It has been experimentally proven that IL-8, SDF1α (stromal factor), HGH (human growth hormone), SCF (stem cell factor), uPa (urokinase plasminogen activator), and VEGF are involved in MSC tropism to glioma cells [13,16,21]. However, the mechanisms mediating MSC migration to glioma focus require further investigations. The authors are grateful to Alamed Company (Moscow) for presented xCELLigence RTCA DP demo version.
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Fig. 4. Analysis of the function of heterologous GJ between MSC and C6 glioma cells. a) Superposition of images of green and red fluorescent channels and differential interference contrast microscopy. b) Calcein AM fluorescence. 1) Donor cell; 2) recipient cell. c) DiI fluorescence (shows only primarily labeled cells). d) Combined microphotograph with fluorescence intensity measurement vector (arrow). Asterisk shows recipient cell. Red mark: point on the vector corresponding to donor cell. e) Histogram of Calcein AM (1) and DiI (2) fluorescence intensity in the donor cell. Asterisk shows the zone corresponding to recipient cell fluorescence.
Fig. 5. Analysis of migration activity of MSC towards glioma cells. Curves 1 and 2: MSC migration towards wells with C6 glioma cells. (1 – 20,000, 2 – 2000 MSC per well. Curves 3 and 4: migration of MSC towards wells with culture medium and HEK293 cells, respectively (control, no migration).
A. N. Gabashvili, V. P. Baklaushev, et al.
The study was supported by the Russian Foundation for Basic Research (grant No. 3-04-40202 NKOMPhI).
REFERENCES 1. G. M. Yusubalieva, V. P. Baklaushev, O. I. Gurina, et al., Bull. Exp. Biol. Med., 157, No. 4, 510-515 (2014). 2. K. S. Aboody, A. Brown, N. G. Rainov, et al., Proc. Natl Acad. Sci. USA, 97, No. 23, 12,846-12,851 (2000). 3. V. P. Baklaushev, N. F. Grinenko, E. A. Savchenko, et al., Bull. Exp. Biol. Med., 152, No. 4, 497-503 (2012). 4. V. P. Baklaushev, O. I. Gurina, G. M. Yusubalieva, et al., Bull. Exp. Biol. Med., 148, No. 4, 725-730 (2009). 5. V. Barresi, N. Belluardo, S. Sipione, et al., Cancer Gene Ther., 10, No. 5, 396-402 (2003). 6. R. Caltabiano, A. Torrisi, D. Condorelli, et al., Acta Histochem., 112, No. 6, 529-535 (2010). 7. S. R. Chirasani, A. Sternjak, P. Wend, et al., Brain, 133, Pt. 7, 1961-1972 (2010). 8. P. B. Dirks, Mol. Oncol., 4, No. 5, 420-430 (2010). 9. D. Friedmann-Morvinski, E. Bushong, E. Ke, et al., Science, 338, 1080-1084 (2012). 10. R. Glass, M. Synowitz, G. Kronenberg, et al., J. Neurosci., 25, No. 10, 2637-2646 (2005). 11. M. Gnecchi and L. G. Melo, Methods Mol. Biol., 482, 281-294 (2009). 12. G. S. Goldberg, J. F. Bechberger, and C. C. Naus, Biotechniques, 18, No. 3, 490-497 (1995). 13. M. Gutova, J. Najbauer, R. T. Frank, et al., Stem Cells, 26, No. 6, 1406-1413 (2008). 14. J. C. Hervé, N. Bourmeyster, D. Sarrouilhe, and H. S. Duffy, Prog. Biophys. Mol. Biol., 94, No. 1, 29-65 (2007). 15. B. E. Huber, E. A. Austin, C. A. Richards, et al., Proc. Natl Acad. Sci. USA, 91, No. 17, 8302-8306 (1994). 16. S. E. Kendall, J. Najbauer, H. F. Johnston, et al., Stem Cells, 26, No. 6, 1575-1586 (2008). 17. P. Kovaříková, E. Michalova, L. Knopfová, and P. Bouchal, Klin. Onkol., 27, Suppl. 1, S22-S27 (2014).
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