Mol Biotechnol DOI 10.1007/s12033-013-9722-0
RESEARCH
Oligoprogenitor Cells Derived from Spermatogonia Stem Cells Improve Remyelination in Demyelination Model M. Nazm Bojnordi • M. Movahedin • T. Tiraihi M. Javan • H. Ghasemi Hamidabadi
•
Ó Springer Science+Business Media New York 2013
Abstract Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells
M. Nazm Bojnordi (&) M. Movahedin (&) T. Tiraihi Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Jalale-Ale-Ahmad Highway, P.O. Box 14115-175, Tehran, Iran e-mail: [email protected] M. Movahedin e-mail: [email protected] M. Nazm Bojnordi H. Ghasemi Hamidabadi Department of Anatomy & Cell Biology, Faculty of Medicine, Mazandaran University of Medical Sciences, P.O. Box 48471-91971, Sari, Iran M. Javan Department of Physiology, Faculty of Medical Sciences, Tarbiat Modares University, Jalale-Ale-Ahmad Highway, P.O. Box 14115-175, Tehran, Iran
was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells. Keywords Spermatogonia cells ES like cells Pluripotency Oligoprogenitor cells Demyelination model Transplantation
Introduction Demyelination, a feature of neurodegenerative diseases such as multiple sclerosis (MS) is characterized by damage of myelin sheath of axons and causes sever neurological symptoms [1]. New treatments focused on transplantation of exogenous cells that can promote myelin synthesis to restore action potential conduction [2, 3]. To this aim, different sources of cell therapy such as bone marrow, hematopoietic, or embryonic stem cells (ESCs) are used to repair myelin [4, 5]. But until now, an efficient and inexhaustible cell source has not been introduced. New evidences based on extensive proliferation activity and pluripotency of spermatogonial stem cells (SSCs) have suggested these cells as a new, natural, and unlimited source for the replacement of neurons and glia [6, 7]. Recent researches have proved that the functional glia cells-derived SSCs have therapeutic use in the reparation of demyelination models [8, 9]. There are several animal models used to study MS, but among these models, local models of demyelinations are especially useful for monitoring the mechanisms of
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demyelination and remyelination processes [10]. Lysophosphatidylcholine (LPC) is a toxin which cusses the loss of oligodendrocyte but not axons. Injection of gliotoxins into the white matter of central nervous system, cusses local demyelination which is followed by a remyelination process [11, 12]. Some criteria such as large mass of myelinated axons and oligodendrocyte suggest that the rat corpus callosum provide an excellent model for investigating myelinogenesis [13]. Moreover, MS frequently affects by corpus callosum so we designed a local demyelination model by the injection of LPC into the rat corpus callosum. To date, there are only two reports showed that glia cells derived SSCs improves remyelination activity in organotypic slice cultures of demyelinated models [8, 9]. But there is no report that evaluates the myelogenesis activity of transplanted glia cells derived SSCs in a demyelination animal model. The aim of his study was the generation of oligoprogenitor cells (OPCs) from SSCs in vitro; and investigation of capacity of transplanted cells to form myelin and improve demyelinated corpus callosum in a rat model, which induced by LPC.
Materials and Methods In vitro Differentiation of ES Like Cells into OP Like Cells ES like cells were generated from neonatal mouse testis as reported previously [14]. ES like cells differentiated to OP cells using neural differentiation methods of ESCs [4]. Cells were transferred to gelatin-coated dishes (0.1 %; Sigma) to eliminate feeder cell. ES like cells were differentiated into neuroprogenitor like cells using a technique called 4-/4? RA. After that cells were cultured in neural stem cells (NSCs) medium that consists of neurobasal medium added N2 and B27 supplements, 20 ng/ml bFGF and 10 ng/ml epidermal growth factor (EGF) for 8–12 days. At the end of each stage of induction phase, cells were harvested for evaluating the specific markers: Nestin, NF68, Olig2, and NG2 using immunocytochemistry.
This treatment induces demyelination after one week. However, spontaneous remyelination will occur over longer durations because of endogenous stem cells [12]. Therefore, 1 week post lesion, animals were divided into three groups: first group that received 200,000 OP cells in 2 ll medium (n = 5); the vehicle-transplanted group (n = 5) which received only 2 ll medium; and control group (n = 5) received no vehicle and no cells. For tracing of transplanted cells they were labeled with the plasma membrane marker, CM-DiI (Invitrogen, C7001). Then cells were transplanted in situ to the corpus callosum using a Hamilton syringe, connected to a 30 gauge needle. Tissue Preparation and Histologic Assessment The procedure was done as reported previously [15]. After 1 week post-lesion, animals were anesthetized and perfused transcardiacally with 0.1 M phosphate-buffered saline (PBS) followed by 4 % paraformaldehyde in 0.1 M PBS. Coronal serial sections of brains were made (5-lm thickness) using a microtome (Leica, Vienna, Austria). Myelin specific staining of sections were made using 0.1 % Luxol Fast Blue (British Drug House, UK) solution at 60 °C for 3 h. After the preparation of adequate contrast by immersion of slides in 0.05 % lithium carbonate and several changes of alcohol, the sections were counterstained with 0.1 % Safranin (Merck, Germany) for 1 min. Finally, sections were screened for demyelination and subsequent quantitative analyses. The extent of demyelination and remyelination intensity were evaluated similar to the previous studies of demyelination. Images of serial sections of 21 points corpus callosum were prepared and analyzed using light microscopy. The extent of demyelination and remyelination intensity were measured by Image Jsoftware and quantified, and Ratio of demyelinated area extention and remyelination intensity to the total area of the corpus callosum in each picture was measured. Average of 21 points obtained from each animal was calculated and (n = 5) animals for each experiment was reported [16]. Immunohistochemistry for Evaluation of Myelin Intensity
Demyelination Induction Female Sprague–Dawley rats were used in this study. The animals were maintained in rooms with a 12/12 h light/ dark cycle with no limitation for food and water. This research was performed after obtaining animal ethical clearance from Tarbiat Modares University, Ethical Committee for Animal Research. In order to induce demyelination model, animals received stereotaxical injection of 2 ll of 1 % LPC (Sigma, USA) into the corpus callosum.
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Expression of PLP as an important protein in mature oligodendrocytes and a marker of myelinating oligodendrocytes as well as myelination level was evaluated by immunohistochemical analysis. For immunostaining, sections were deparaffinized and rehydrated. Slides were then blocked with 10 % goat serum for 1 h and incubated overnight with rat anti-PLP 1:200 at 4 °C. At the following day, sections were incubated with secondary antibodies (goat antirat IgG; Abcam,Cambridge, UK) for 1 h at room temperature. Slides were washed with PBS. The intensity of PLP staining was
Mol Biotechnol
Fig. 1 Generation of oligoprogenitor like cells from ES like cells. For glial commitment, undifferentiated ES like colonies (a) were exposed to RA and differentiated into neuroeprogenitor cells expressed neural morphology (b1, c1) and immunopositive reactivity to specific neural markers; Nestin (b2) and NF68 (c2). Further
propagation in defined media supplemented with bFGF and EGF support the generation of oligoprogenitor like cells (d1, e1) which were immunopositive to Olig2 (d2) and NG2 (e2). ES like cells embryonic stem like cells; Scale bars 100 lm
considered as a remyelination intensity evaluated similar to the previous studies of demyelination [16].
with RA, ES like cells differentiated into neuroprogenitor like cells. Differentiation into neuroprogenitor like cells was confirmed by both appearance of the neural morphology with elongated soma and various processes (Fig. 1b1–c1) and immunoreactivity to the neuroprogenitor cells markers, Nestin and NF68 (Fig. 1b2–c2). The differentiation of ES like cells into OP like cells was confirmed by the expression of Olig2 and NG2 markers using immunocytochemistry at the end of the induction stage (Fig. 1d1–e2).
Statistical Analysis The results were expressed as mean ± SEM. Data were analyzed and compared using one-way analysis of variance (ANOVA) followed by Tukey post hoc test. (p \ 0.05 was considered significant).
Results
The Extent of Demyelination
Generation of OP Like Cells from ES Like Cells
Myelin staining using luxol fast blue showed demyelination following local injection of LPC into corpus callosum. Histologic evaluation of coronal and serial sections from corpus callosum showed myelin atrophy after gliotoxin
ES like colonies with sharp edge resembled ES cell was appeared after 3 weeks in culture (Fig. 1a). After induction
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Fig. 2 Effect of transplanted OPs on the extent of demyelination induced in corpus callosum by injection of LPC. a Quantitative data for the extent of demyelination in cell transplanted and vehicle groups. b Representative pictures for different data points.
(a) Positive control, (b) 1 week post lesion, (c, d) vehicle groups 2 and 4 weeks post Tx. (e, f) cell transplanted groups 2 and 4 weeks post Tx. OPs oligoprogenitor cells, LPC lysophosphatidylcholine, Tx transplantation, SC spermatogonia cells, W week. Scale bars 200 lm
injection. Demyelination was considered as areas of reduced Luxol fast blue-staining within corpus callosum. The amounts of demyelination extension and remyelination intensity were histologically evaluated using Luxol fast blue myelin-specific staining in mentioned groups. Maximum demyelination was observed 1 week post lesion, while less demyelination extension and obvious remyelination intensity were seen in cell transplanted groups when compared with vehicle groups (Fig. 2).
As shown in Fig. 4, a demyelination was seen 1 week post LPC injection. Monitoring remyelination process showed that the higher amount of remyelination was observed in cell transplanted group when compared with vehicle group (Fig. 4).
PLP Immunostaining Two weeks after transplantation, tracing of DiI-labeled cells showed that the transplanted cells survived and integrated within corpus callosum (Fig. 3). Double labeling for DiI and PLP, showed that the majority of the grafted cells were PLP ? which demonstrated differentiation of ES like cells-derived OPs into mature oligodendrocytes in vivo and confirmed remyelination by the transplanted cells (Fig. 3). PLP immunostaining was used to monitor changes in histological myelin staining. PLP immunoreactivity was used to estimate the level of myelination ,the corpus callosum, and to evaluate remyelination in vehicle and cell transplanted groups.
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Discussion New research has proved the generation of pluripotent stem cells from neonatal and adult mouse testis that can differentiate to all cell types [17, 18]. These researches showed that ES like cells were generated without any genetic manipulation, but under culture condition [19]. The perspective of the results obtained in mice has clinical application in other species, particularly in humans [19]. Following pervious researches, in this study, we isolated ES like cells from mouse testis but under simple co-culture condition with Sertoli cells which is a novelty of our research. The importance of our method will be clear when we compare it with other complex and time consuming methods. Generation of ES like cells from testis has clinical use in the production of cell line from each patient, hence has the effective role in regenerative therapy [1]. New researches
Mol Biotechnol
Fig. 3 Immunostaining of cell transplanted groups. a Oligoprogenitor like cells labeled with DiI, b DAPI of cells, c DiI-labeled cells showed PLP immunoreactivity. Scale bars 20 lm
Fig. 4 Effect of transplanted Ops on PLP immunoreactivity following LPC-induced demyelination in rat corpus callosum. a Quantitative data for the intensity of PLP-immunostaining in cell transplanted and vehicle groups. b Representative pictures for different data points.
(a) positive control, (b) 1 week post-lesion, (c, d) vehicle groups 2 and 4 weeks post-Tx. (e, f) cell transplanted groups 2 and 4 weeks post-Tx. OPs oligoprogenitor cells, LPC lysophosphatidylcholine, Tx transplantation, W week. Scale bars 200 lm
propose the therapeutic use of SSCs in regenerative medicine especially as a new and unlimited source for neurodegenerative disease treatment and may represent a possible alternative to the replacement of neurons and glia [2]. One of the most prevalent diseases is MS, in which the myelin sheaths surrounding the axons are lost, leading to demyelination and disability, especially in young adults [20]. Some factors such as complicated etiology and unpredictable course of this disease make its treatment a challenging subject in the medicine up to now [10, 21]. Cell therapy has been considered as an
effective solution usable for treatment of MS [22, 23]. The capacity of transplanted OPCs or oligodendrocyte precursors cells in remyelination has yielded new insights of cell therapy for demyelinating diseases [8, 20]. However, to date, an efficient and reliable cell source has not been introduced. The ES like cells derived from testis offer an inexhaustible source of transplantable neural and glial progenitors for cell therapy [8, 9]. Hence, in this study, we used spermatogonial cells for generation of OPCs and described an in vitro growth factor
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mediated procedure for the generation population of OP cells from mouse ESCs. At first induction stage using RA, ES like cells differentiated to neuroprogenitor cells, which were immunoreactive to Nestin and NF68. RA used in our research is a derivative of vitamin A and is essential factor in the normal cellular growth and development [12]. In addition, its receptors are found in different types of tissues, in particular, in the nervous system, where they increase neuronal differentiation [24]. Our result confirmed the previous studies which showed that RA induced neural differentiation in both ESC and ES like cells [7, 24]. Our results proved high level expression of Nestin and NF68 in neuroprogenitor like cells. In fact, alteration in these proteins expression pattern after induction, confirms the neuronal differentiation ability of ES like cells. Our results also showed that these cells differentiated to OP cells following additional differentiation in vitro. The differentiation of ES like cells into OP like cells was confirmed by Olig2 and NG2 expression using immunocytochemistry technique at the end of the induction stage. The results of in vivo part show that transplanted OPC derived SSCs have a significant effect in myelin synthesis as well as remyelination process of demyelinated plaques in a local model of MS. Significant remyelination on MS plaques following transplantation of OPC has been demonstrated in two ways in our research, first by demonstration of myelin repair in demyelinated plaques using specific myelin staining, Luxol Fast Blue, and second by PLP immunostaining in celltreated animals. In fact, any treatment for MS should target oligodendrocytes protection and also leads to generation of new oligodendrocytes pools [25]. Reduced demyelination following cell transplantation in demyelinated corpous callosum suggests that exogenous OPs can promote myelination in the context of demyelinating diseases like MS. These results support previous researches reported the neuroprotective effects of exogenous OPs [26, 27]. Several researches showed the positive effects of cell therapy in treatment of CNS disorders so makes it usable in the prevention and reparation of brain damages [ 22, 26]. Recognition of stem cell source that can proliferate and migrate toward the lesions and replace damaged myelin sheets has a new therapeutic perspective for neurodegenerative diseases [28]. Transplantation of stem or progenitor cells is an effective way to conquer these problems that lead to repair of MS diseases. In our research, we transplanted the OPCs derived SSCs into the demyelinated lesion in corpous callosum that effects functional remyelination of lesions as mentioned by specific myelin staining and PLP immunostaining. It was previously demonstrated that transplanted OPCs promotes
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in vitro myelogenesis in organo culture [8, 9]. In total, we have demonstrated that OP cells can be derived from SSCs that these SC-derived cells can now be tested for their ability to restore the function of damaged brains in animal models which opens a new therapeutic window in organ regeneration without the ethical and immunological problems accompanied with other stem cells-based therapy. Acknowledgments This research is derived from PhD thesis of Anatomical science with funding support from Medical Faculty of Tarbiat Modares University and Iranian council of stem cell technology.
References 1. Noseworthy, J. H., Lucchinetti, C., Rodriguez, M., & Weinshenker, B. G. (2000). Multiple sclerosis. The New England Journal of Medicine, 343, 938–952. 2. Manganas, L. N., & Maletic Savatic, M. (2005). Stem cell therapy for central nervous system demyelinating disease. Current Neurology and Neuroscience Reports, 5(3), 225–231. 3. Siddharthan, C., David, H., Alexis, J., Chao, Z., Alastair, C., & Robin, J. M. F. (2008). Myelin repair: the role of stem and precursor cells in multiple sclerosis. Philosophical Transactions of the Royal Society B, 363, 171–183. 4. Brustle, O., Jones, K. N., Learish, R. D., Karram, K., Choudhary, K., Wiestler, O. D., et al. (1999). Embryonic stem cell-derived glial precursors: a source of myelinating transplants. Science, 285, 754–756. 5. Martino, G., Franklin, R. J., Van Evercooren, A. B., & Kerr, D. A. (2010). Stem cell transplantation in multiple sclerosis: current status and future prospects. Nature Reviews Neurology, 6, 247–255. 6. Mizrak, S. C., Chikhovskaya, J. V., Sadri-Ardekani, H., van Daalen, S., Korver, C. M., Hovingh, S. E., et al. (2010). Embryonic stem cell-like cells derived from adult human testis. Human Reproduction, 25(1), 158–167. 7. Nayernia, K. (2007). Stem cells derived from testis show promise for treating a wide variety of medical conditions. Cell Research, 17, 895–897. 8. Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., Miki, H., Ogura, A., Toyokuni, S., et al. (2003). Long-termproliferation in culture and germline transmission of mouse male germline stem cells. Biology of Reproduction, 69, 612–616. 9. Streckfuss-Bo¨meke, K., Vlasov, A., Hu¨lsmann, S., Yin, D., Nayernia, K., Engel, E., et al. (2009). Generation of functional neurons and glia from multipotent adult mouse germ-line stem cells. Stem Cell Research, 2, 139–154. 10. Aditya, G. S., & Chakrabarty, A. (2007). Multiple sclerosis and demyelination. Current Diagnostic Pathology, 13, 193–202. 11. Kotter, M. R., Setzu, A., Sim, F. J., Van Rooijen, N., & Franklin, R. J. (2001). Macrophage depletion impairs oligodendrocyte remyelination following lysolecithin-induced demyelination. Glia, 35(3), 204–212. 12. Woodruff, R., & Franklin, R. (1999). Demyelination and remyelination of the caudal cerebellar peduncle of adult rats following stereotaxic injections of lysolecithin, ethidium bromide, and complement/anti-galactocerebroside: a comparative study. Glia, 25, 216–228. 13. Yaldizli, O., Atefy, R., Gass, A., Sturm, D., Glassl, S., Tettenborn, B., et al. (2010). Corpus callosum index and long-term disability in multiple sclerosis patients. Journal of Neurology, 257, 1256–1264.
Mol Biotechnol 14. Nazm Bojnordi, M., Movahedin, M., Tiraihi, T., & Javan, M. (2013). Alteration in genes expression patterns during in vitro differentiation of mouse spermatogonial cells into neuroepithelial like cells. Cytotechnology, 65(1), 97–104. 15. Sherafat, M. A., Heibatollahi, M., Mongabadi, S., Moradi, F., Javan, M., & Ahmadiani, (2012). A electromagnetic field stimulation potentiates endogenous myelin repair by recruiting subventricular neural stem cells in an experimental model of white matter demyelination. Journal of Molecular Neuroscience, 48, 144–153. 16. Mozafari, S., Sherafat, M. A., Javan, M., Mirnajafi-Zadeh, J., & Tiraihi, T. (2010). Visual evoked potentials and MBP gene expression implyendogenous myelin repair in adult rat optic nerve and chiasma following local lysolecithin induced demyelination. Brain Research, 1351, 50–56. 17. Guan, K., Nayernia, K., Maier, L. S., Wagner, S., Dressel, R., Lee, J. H., et al. (2006). Pluripotency of spermatogonial stem cells from adult mouse testis. Nature, 440(7088), 1199–1203. 18. Hasenfuss, G. (2007). Generation of functional cardiomyocytes from adult mouse spermatogonial stem cells. Circulation Research, 100, 1615–1625. 19. De Rooij, D. J., & Mizrak, C. (2008). Deriving multipotent stem cells from mouse spermatogonial stem cells: A new tool for developmental and clinical research. Development, 135(430), 2207–2213. 20. Annapurna, A., Krishna, K. V., Murali Mohan Rao, P., Someswara Rao, K., & Rajasekhar, J. (2002). Multiple sclerosis: The disease and its treatment. Indian Journal of Pharmacology, 34, 3–15. 21. Caon, C., Saunders, C., Smrtka, J., Baxter, N., & Shoemaker, J. (2010). Injectable disease-modifying therapy for relapsing-
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remitting multiple sclerosis: A review of adherence data. Journal of Neuroscience Nursing, 42(5), S5–S9. Chandran, S., Hunt, D., Joannides, A., Zhao, C., Compston, A., & Franklin, R. J. (2008). Myelin repair: The role of stem and precursor cells in multiple sclerosis. Philosophical transactions of the Royal Society of London Series B Biological Sciences, 363(1489), 171–183. Stangel, M., & Hartung, H. P. (2002). Remyelinating strategies for the treatment of multiple sclerosis. Progress in Neurobiology, 68(5), 361–376. Maden, M., Sonneveld, E., van der Saag, P. T., & Gale, E. (1998). The distribution of endogenous retinoic acid in the chick embryo: Implications for developmental mechanisms. Development, 125, 4133–4144. Tirotta, E., Carbajal, K. S., Schaumburg, C. S., Whitman, L., & Lane, T. E. (2010). Cell replacement therapies to promote remyelination in a viral model of demyelination. Journal of Neuroimmunology, 224(1–2), 101–107. Blakemore, W. F., Chari, D. M., Gilson, J. M., & Crang, A. J. (2002). Modelling large areas of demyelination in the rat reveals the potential and possible limitations of transplanted glial cells for remyelination in the CNS. Glia, 38(2), 155–168. Chari, D. M., & Blakemore, W. F. (2002). New insights into remyelination failure in multiple sclerosis: Implications for glial cell transplantation. Multiple Sclerosis, 8(4), 271–277. Mardanpour, P., Guan, K., Nolte, J., Lee, J., Hasenfuss, G., Engel, W., et al. (2008). Potency of germ cells and its relevance for regenerative medicine. Journal of Anatomy, 213(26–29), 457.
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RESEARCH
Oligoprogenitor Cells Derived from Spermatogonia Stem Cells Improve Remyelination in Demyelination Model M. Nazm Bojnordi • M. Movahedin • T. Tiraihi M. Javan • H. Ghasemi Hamidabadi
•
Ó Springer Science+Business Media New York 2013
Abstract Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells
M. Nazm Bojnordi (&) M. Movahedin (&) T. Tiraihi Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Jalale-Ale-Ahmad Highway, P.O. Box 14115-175, Tehran, Iran e-mail: [email protected] M. Movahedin e-mail: [email protected] M. Nazm Bojnordi H. Ghasemi Hamidabadi Department of Anatomy & Cell Biology, Faculty of Medicine, Mazandaran University of Medical Sciences, P.O. Box 48471-91971, Sari, Iran M. Javan Department of Physiology, Faculty of Medical Sciences, Tarbiat Modares University, Jalale-Ale-Ahmad Highway, P.O. Box 14115-175, Tehran, Iran
was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells. Keywords Spermatogonia cells ES like cells Pluripotency Oligoprogenitor cells Demyelination model Transplantation
Introduction Demyelination, a feature of neurodegenerative diseases such as multiple sclerosis (MS) is characterized by damage of myelin sheath of axons and causes sever neurological symptoms [1]. New treatments focused on transplantation of exogenous cells that can promote myelin synthesis to restore action potential conduction [2, 3]. To this aim, different sources of cell therapy such as bone marrow, hematopoietic, or embryonic stem cells (ESCs) are used to repair myelin [4, 5]. But until now, an efficient and inexhaustible cell source has not been introduced. New evidences based on extensive proliferation activity and pluripotency of spermatogonial stem cells (SSCs) have suggested these cells as a new, natural, and unlimited source for the replacement of neurons and glia [6, 7]. Recent researches have proved that the functional glia cells-derived SSCs have therapeutic use in the reparation of demyelination models [8, 9]. There are several animal models used to study MS, but among these models, local models of demyelinations are especially useful for monitoring the mechanisms of
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demyelination and remyelination processes [10]. Lysophosphatidylcholine (LPC) is a toxin which cusses the loss of oligodendrocyte but not axons. Injection of gliotoxins into the white matter of central nervous system, cusses local demyelination which is followed by a remyelination process [11, 12]. Some criteria such as large mass of myelinated axons and oligodendrocyte suggest that the rat corpus callosum provide an excellent model for investigating myelinogenesis [13]. Moreover, MS frequently affects by corpus callosum so we designed a local demyelination model by the injection of LPC into the rat corpus callosum. To date, there are only two reports showed that glia cells derived SSCs improves remyelination activity in organotypic slice cultures of demyelinated models [8, 9]. But there is no report that evaluates the myelogenesis activity of transplanted glia cells derived SSCs in a demyelination animal model. The aim of his study was the generation of oligoprogenitor cells (OPCs) from SSCs in vitro; and investigation of capacity of transplanted cells to form myelin and improve demyelinated corpus callosum in a rat model, which induced by LPC.
Materials and Methods In vitro Differentiation of ES Like Cells into OP Like Cells ES like cells were generated from neonatal mouse testis as reported previously [14]. ES like cells differentiated to OP cells using neural differentiation methods of ESCs [4]. Cells were transferred to gelatin-coated dishes (0.1 %; Sigma) to eliminate feeder cell. ES like cells were differentiated into neuroprogenitor like cells using a technique called 4-/4? RA. After that cells were cultured in neural stem cells (NSCs) medium that consists of neurobasal medium added N2 and B27 supplements, 20 ng/ml bFGF and 10 ng/ml epidermal growth factor (EGF) for 8–12 days. At the end of each stage of induction phase, cells were harvested for evaluating the specific markers: Nestin, NF68, Olig2, and NG2 using immunocytochemistry.
This treatment induces demyelination after one week. However, spontaneous remyelination will occur over longer durations because of endogenous stem cells [12]. Therefore, 1 week post lesion, animals were divided into three groups: first group that received 200,000 OP cells in 2 ll medium (n = 5); the vehicle-transplanted group (n = 5) which received only 2 ll medium; and control group (n = 5) received no vehicle and no cells. For tracing of transplanted cells they were labeled with the plasma membrane marker, CM-DiI (Invitrogen, C7001). Then cells were transplanted in situ to the corpus callosum using a Hamilton syringe, connected to a 30 gauge needle. Tissue Preparation and Histologic Assessment The procedure was done as reported previously [15]. After 1 week post-lesion, animals were anesthetized and perfused transcardiacally with 0.1 M phosphate-buffered saline (PBS) followed by 4 % paraformaldehyde in 0.1 M PBS. Coronal serial sections of brains were made (5-lm thickness) using a microtome (Leica, Vienna, Austria). Myelin specific staining of sections were made using 0.1 % Luxol Fast Blue (British Drug House, UK) solution at 60 °C for 3 h. After the preparation of adequate contrast by immersion of slides in 0.05 % lithium carbonate and several changes of alcohol, the sections were counterstained with 0.1 % Safranin (Merck, Germany) for 1 min. Finally, sections were screened for demyelination and subsequent quantitative analyses. The extent of demyelination and remyelination intensity were evaluated similar to the previous studies of demyelination. Images of serial sections of 21 points corpus callosum were prepared and analyzed using light microscopy. The extent of demyelination and remyelination intensity were measured by Image Jsoftware and quantified, and Ratio of demyelinated area extention and remyelination intensity to the total area of the corpus callosum in each picture was measured. Average of 21 points obtained from each animal was calculated and (n = 5) animals for each experiment was reported [16]. Immunohistochemistry for Evaluation of Myelin Intensity
Demyelination Induction Female Sprague–Dawley rats were used in this study. The animals were maintained in rooms with a 12/12 h light/ dark cycle with no limitation for food and water. This research was performed after obtaining animal ethical clearance from Tarbiat Modares University, Ethical Committee for Animal Research. In order to induce demyelination model, animals received stereotaxical injection of 2 ll of 1 % LPC (Sigma, USA) into the corpus callosum.
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Expression of PLP as an important protein in mature oligodendrocytes and a marker of myelinating oligodendrocytes as well as myelination level was evaluated by immunohistochemical analysis. For immunostaining, sections were deparaffinized and rehydrated. Slides were then blocked with 10 % goat serum for 1 h and incubated overnight with rat anti-PLP 1:200 at 4 °C. At the following day, sections were incubated with secondary antibodies (goat antirat IgG; Abcam,Cambridge, UK) for 1 h at room temperature. Slides were washed with PBS. The intensity of PLP staining was
Mol Biotechnol
Fig. 1 Generation of oligoprogenitor like cells from ES like cells. For glial commitment, undifferentiated ES like colonies (a) were exposed to RA and differentiated into neuroeprogenitor cells expressed neural morphology (b1, c1) and immunopositive reactivity to specific neural markers; Nestin (b2) and NF68 (c2). Further
propagation in defined media supplemented with bFGF and EGF support the generation of oligoprogenitor like cells (d1, e1) which were immunopositive to Olig2 (d2) and NG2 (e2). ES like cells embryonic stem like cells; Scale bars 100 lm
considered as a remyelination intensity evaluated similar to the previous studies of demyelination [16].
with RA, ES like cells differentiated into neuroprogenitor like cells. Differentiation into neuroprogenitor like cells was confirmed by both appearance of the neural morphology with elongated soma and various processes (Fig. 1b1–c1) and immunoreactivity to the neuroprogenitor cells markers, Nestin and NF68 (Fig. 1b2–c2). The differentiation of ES like cells into OP like cells was confirmed by the expression of Olig2 and NG2 markers using immunocytochemistry at the end of the induction stage (Fig. 1d1–e2).
Statistical Analysis The results were expressed as mean ± SEM. Data were analyzed and compared using one-way analysis of variance (ANOVA) followed by Tukey post hoc test. (p \ 0.05 was considered significant).
Results
The Extent of Demyelination
Generation of OP Like Cells from ES Like Cells
Myelin staining using luxol fast blue showed demyelination following local injection of LPC into corpus callosum. Histologic evaluation of coronal and serial sections from corpus callosum showed myelin atrophy after gliotoxin
ES like colonies with sharp edge resembled ES cell was appeared after 3 weeks in culture (Fig. 1a). After induction
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Fig. 2 Effect of transplanted OPs on the extent of demyelination induced in corpus callosum by injection of LPC. a Quantitative data for the extent of demyelination in cell transplanted and vehicle groups. b Representative pictures for different data points.
(a) Positive control, (b) 1 week post lesion, (c, d) vehicle groups 2 and 4 weeks post Tx. (e, f) cell transplanted groups 2 and 4 weeks post Tx. OPs oligoprogenitor cells, LPC lysophosphatidylcholine, Tx transplantation, SC spermatogonia cells, W week. Scale bars 200 lm
injection. Demyelination was considered as areas of reduced Luxol fast blue-staining within corpus callosum. The amounts of demyelination extension and remyelination intensity were histologically evaluated using Luxol fast blue myelin-specific staining in mentioned groups. Maximum demyelination was observed 1 week post lesion, while less demyelination extension and obvious remyelination intensity were seen in cell transplanted groups when compared with vehicle groups (Fig. 2).
As shown in Fig. 4, a demyelination was seen 1 week post LPC injection. Monitoring remyelination process showed that the higher amount of remyelination was observed in cell transplanted group when compared with vehicle group (Fig. 4).
PLP Immunostaining Two weeks after transplantation, tracing of DiI-labeled cells showed that the transplanted cells survived and integrated within corpus callosum (Fig. 3). Double labeling for DiI and PLP, showed that the majority of the grafted cells were PLP ? which demonstrated differentiation of ES like cells-derived OPs into mature oligodendrocytes in vivo and confirmed remyelination by the transplanted cells (Fig. 3). PLP immunostaining was used to monitor changes in histological myelin staining. PLP immunoreactivity was used to estimate the level of myelination ,the corpus callosum, and to evaluate remyelination in vehicle and cell transplanted groups.
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Discussion New research has proved the generation of pluripotent stem cells from neonatal and adult mouse testis that can differentiate to all cell types [17, 18]. These researches showed that ES like cells were generated without any genetic manipulation, but under culture condition [19]. The perspective of the results obtained in mice has clinical application in other species, particularly in humans [19]. Following pervious researches, in this study, we isolated ES like cells from mouse testis but under simple co-culture condition with Sertoli cells which is a novelty of our research. The importance of our method will be clear when we compare it with other complex and time consuming methods. Generation of ES like cells from testis has clinical use in the production of cell line from each patient, hence has the effective role in regenerative therapy [1]. New researches
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Fig. 3 Immunostaining of cell transplanted groups. a Oligoprogenitor like cells labeled with DiI, b DAPI of cells, c DiI-labeled cells showed PLP immunoreactivity. Scale bars 20 lm
Fig. 4 Effect of transplanted Ops on PLP immunoreactivity following LPC-induced demyelination in rat corpus callosum. a Quantitative data for the intensity of PLP-immunostaining in cell transplanted and vehicle groups. b Representative pictures for different data points.
(a) positive control, (b) 1 week post-lesion, (c, d) vehicle groups 2 and 4 weeks post-Tx. (e, f) cell transplanted groups 2 and 4 weeks post-Tx. OPs oligoprogenitor cells, LPC lysophosphatidylcholine, Tx transplantation, W week. Scale bars 200 lm
propose the therapeutic use of SSCs in regenerative medicine especially as a new and unlimited source for neurodegenerative disease treatment and may represent a possible alternative to the replacement of neurons and glia [2]. One of the most prevalent diseases is MS, in which the myelin sheaths surrounding the axons are lost, leading to demyelination and disability, especially in young adults [20]. Some factors such as complicated etiology and unpredictable course of this disease make its treatment a challenging subject in the medicine up to now [10, 21]. Cell therapy has been considered as an
effective solution usable for treatment of MS [22, 23]. The capacity of transplanted OPCs or oligodendrocyte precursors cells in remyelination has yielded new insights of cell therapy for demyelinating diseases [8, 20]. However, to date, an efficient and reliable cell source has not been introduced. The ES like cells derived from testis offer an inexhaustible source of transplantable neural and glial progenitors for cell therapy [8, 9]. Hence, in this study, we used spermatogonial cells for generation of OPCs and described an in vitro growth factor
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mediated procedure for the generation population of OP cells from mouse ESCs. At first induction stage using RA, ES like cells differentiated to neuroprogenitor cells, which were immunoreactive to Nestin and NF68. RA used in our research is a derivative of vitamin A and is essential factor in the normal cellular growth and development [12]. In addition, its receptors are found in different types of tissues, in particular, in the nervous system, where they increase neuronal differentiation [24]. Our result confirmed the previous studies which showed that RA induced neural differentiation in both ESC and ES like cells [7, 24]. Our results proved high level expression of Nestin and NF68 in neuroprogenitor like cells. In fact, alteration in these proteins expression pattern after induction, confirms the neuronal differentiation ability of ES like cells. Our results also showed that these cells differentiated to OP cells following additional differentiation in vitro. The differentiation of ES like cells into OP like cells was confirmed by Olig2 and NG2 expression using immunocytochemistry technique at the end of the induction stage. The results of in vivo part show that transplanted OPC derived SSCs have a significant effect in myelin synthesis as well as remyelination process of demyelinated plaques in a local model of MS. Significant remyelination on MS plaques following transplantation of OPC has been demonstrated in two ways in our research, first by demonstration of myelin repair in demyelinated plaques using specific myelin staining, Luxol Fast Blue, and second by PLP immunostaining in celltreated animals. In fact, any treatment for MS should target oligodendrocytes protection and also leads to generation of new oligodendrocytes pools [25]. Reduced demyelination following cell transplantation in demyelinated corpous callosum suggests that exogenous OPs can promote myelination in the context of demyelinating diseases like MS. These results support previous researches reported the neuroprotective effects of exogenous OPs [26, 27]. Several researches showed the positive effects of cell therapy in treatment of CNS disorders so makes it usable in the prevention and reparation of brain damages [ 22, 26]. Recognition of stem cell source that can proliferate and migrate toward the lesions and replace damaged myelin sheets has a new therapeutic perspective for neurodegenerative diseases [28]. Transplantation of stem or progenitor cells is an effective way to conquer these problems that lead to repair of MS diseases. In our research, we transplanted the OPCs derived SSCs into the demyelinated lesion in corpous callosum that effects functional remyelination of lesions as mentioned by specific myelin staining and PLP immunostaining. It was previously demonstrated that transplanted OPCs promotes
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in vitro myelogenesis in organo culture [8, 9]. In total, we have demonstrated that OP cells can be derived from SSCs that these SC-derived cells can now be tested for their ability to restore the function of damaged brains in animal models which opens a new therapeutic window in organ regeneration without the ethical and immunological problems accompanied with other stem cells-based therapy. Acknowledgments This research is derived from PhD thesis of Anatomical science with funding support from Medical Faculty of Tarbiat Modares University and Iranian council of stem cell technology.
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