- Email: [email protected]
Embryonic stem cells inhibit expression of erythropoietin in the injured spinal cord
Neuroscience Letters 488 (2011) 55–59
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Embryonic stem cells inhibit expression of erythropoietin in the injured spinal cord Margarita Glazova ∗ , Sarah Hollis, Elena S. Pak, Alexander K. Murashov Departments of Physiology, The Brody School of Medicine, East Carolina University School of Medicine, Brody Building, 600 Moye Boulevard, Greenville, NC 27834, USA
a r t i c l e
i n f o
Article history: Received 10 August 2010 Received in revised form 8 October 2010 Accepted 1 November 2010 Key words: Embryonic stem cells Erythropoietin Regeneration Spinal cord injury
a b s t r a c t Recent observations have demonstrated neuroprotective role of erythropoietin (Epo) and Epo receptor in the central nervous system. Here we examined Epo function in the murine spinal cord after transplantation of pluripotent mouse embryonic stem (ES) cells pre-differentiated towards neuronal type following spinal cord injury. Expression of Epo was measured at both mRNA and protein levels in the ES cells as well as in the spinal cords after 1 and 7 days. Our data demonstrated that expression of Epo mRNA, as well as its protein content, in ES cells was significantly decreased after differentiation procedure. In the spinal cords, analysis showed that Epo mRNA level was significantly decreased after 1 day of ES cell injections in comparison to media-injected control. Epo protein level detected by Western blot was diminished as well. Examination of Epo production in the injured spinal cords after media or ES cells injections by indirect immunofluorescence showed increased Epo-immunopositive staining after media injections 1 day after injection. In contrast, ES cell transplantation did not induce Epo expression. Seven days after ES cell injections, Epo-immunopositive cells’ distribution in the ipsilateral side was not changed, while the intensity of immunostaining on the contralateral side was increased, approaching levels in control media-injected tissues. Our data let us to presume that previously described immediate positive effects of ES cells injected into the injured zone of spinal cord are not based on Epo, but on other factors or hormones, which should be elucidated further. © 2010 Elsevier Ireland Ltd. All rights reserved.
Recent animal studies have indicated that ES cells transplanted into the injured zone of the spinal cord significantly increase recovery rate [3,10,12,14,21]. Therefore, stem cells have an ability to successfully restore and maintain spinal cord function after injury. ES cells can secrete many factors like BDNF and IL-6 [7], which are involved in the recovery process. However, the precise mechanism of ES cells action after grafting into the injured host tissue still remains unknown and needs to be elucidated. Neuroprotective role of cytokines and growth factors let us to hypothesize that ES cells transplanted into the lesion can support the host tissue by secreting these active agents to promote recovery and neuronal survival [7]. Upregulation of Epo receptor expression after injury [1,9,24], and beneficial role of systemic administration of Epo directly into the lesion zone of the spinal cord [4] suggests neuroprotective function of Epo. In the central nervous system expression of Epo and Epo receptor has been demonstrated [1,11], and a positive neuroprotective role of Epo has been documented [8]. We hypothesized that transplanted ES cells could supply a host tissue with Epo or
∗ Corresponding author. Present address: Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 Thorez pr., 194223 Sankt-Petersburg, Russia. Tel.: +7 812 552 32 27; fax: +7 812 552 3012. E-mail address: [email protected] (M. Glazova). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.11.002
induce Epo expression in injured spinal cord. In the current study we asked the question of whether Epo system may play a role in mediating effects of pre-differentiated ES cells in the spinal cord after excitotoxic injury caused by Quisqualic acid [27]. Embryonic stem cell culture: The pGFP-transfected ES cells were used for injections. Briefly, undifferentiated, pluripotent mouse ES cells (D3 cell line, American Type Culture Collection Manassas, VA) ES cells were transfected with the pEGFP-N3 construct (Clontech, Palo Alto, CA) in the presence of FuGENE 6 (Roche, Indianapolis, IN) as described [13]. Transfected colonies were then selected in the presence of 200 mg/ml of G418 (Sigma) and FACS sorted on BD FACSVantage SE flow cytometry system (Becton Dickinson) to achieve 90% of GFP-labeled cells. Transgenic D3 cells were further used for induction of neural cells. pGFP-transfected ES cells were differentiated according to our published protocol [19,20]. Briefly, cells were cultured in Knock-out DMEM supplemented with 15% fetal bovine serum (FBS)–ES grade and 1400 U/ml murine leukemia inhibitory factor (Stem Cell Technologies, Vancouver, Canada), and 55 M -mercaptoethanol (Sigma Chemical Co., St. Louis, MO). To induce differentiation the medium was exchanged for differentiation medium (Iscove’s Modified Dulbecco’s Medium, 15% FBS, 2 mM l-glutamine, 0.1 mM non-essential amino acids) when the cells began to form embryoid bodies after 1–2 days. At day 5, alltrans retinoic acid (Sigma, St. Louis, MO) (1 × 10−6 M) was added
56
M. Glazova et al. / Neuroscience Letters 488 (2011) 55–59
to the culture. On day 9, embryoid bodies were collected and seeded on poly-l-orhithine/fibronectin coated flask in differentiation/Neurobasal plus B27 supplement 1:1 medium. The next day, the medium was replaced with Neurobasal medium plus B27 supplement. The cells were cultured for another 2–3 days and then harvested for the injections or analysis of Epo expression. Our early study demonstrated that this differentiating protocol gave rise for about 60% of neurons and 40% of glial cells [19,20]. Animal surgery: the animal surgical procedure was performed as reported previously [12,27]. Briefly, adult male mice (CD-1, Harlan, Indianapolis, IN) were anesthetized with isoflurane (2–3% inhalation) and unilateral microinjections were made in a single segment at spinal levels ranging from T12 to L2. Each animal received 0.6 l of 125 mM Quisqualic acid (QUIS) (Sigma, St. Louis, MO) (three tracks of 0.2 l separated by 0.3 mm, parallel to the long axis of the cord). Following a one-week period of recovery, pre-differentiated mouse ES cells (100,000 cells/l) were injected into the injury (three tracks of 0.2 l separated by 0.3 mm, parallel to the long axis of the cord). Control animals received similar microinjections of cell neurobasal media in equal volume and locations. At 1, or 7 days post-ES cell or media microinjection, the animals were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and tissues were collected: (1) in liquid nitrogen for biochemical analysis, (2) in RNA-later for RNA isolation and subsequent real time PCR study and (3) for histological studies, animal were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and then intracardially perfused first with cold PBS and then cold 4% paraformaldehyde in PBS (pH 7.4). Each thoracic and lumbar spinal cord was removed and postfixed in 4% paraformaldehyde for 2 h. Fixed tissue was stored in 30% sucrose solution at 4 ◦ C until sectioning. For cryosectioning, tissue was embedded in tissue freezing medium (Triangle Biomedical Science, Durham, NC), and sectioned into 14 m coronal slices
Fig. 1. Expression of Epo is decreased in ES cells after differentiation. (A) Quantitative real-time PCR results of Epo mRNA expression presented as a fold induction, Epo mRNA expression level in non-differentiated (n/d) ES cells was taken as 1. (B) The proteins from non-differentiated and differentiated cells were extracted and immunoblotted with affinity purified antibodies against Epo, and optical density was counted and the data are presented in Arbitrary Units. All data were normalized against GAPDH expression. Data are shown as mean ± SEM *P < 0.05.
using a cryostat microtome. All procedures and tissue collection methods were approved by the East Carolina University Animal Care and Use Committee. RNA preparation, reverse-transcription, and real-time PCR: total RNA was extracted from collected tissue and ES cells using TRIZOL (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Total RNA was exposed to RNAase-free DNAase I (Ambion, Austin, TX) and reverse transcribed to cDNA using Random Hexames and Cloned AMV firs-Strand cDNA Synthesis Kit (Invitrogen) according manufacturer’s protocol. Real-time PCR was performed using a BioRad iQ iCycler Detection System (BioRad Laboratories, Ltd., Hercules, CA) with SYBR green fluorophore. Reactions were performed in a total volume of 25 l, including 12.5 l 2× SYBR Green PCR Master Mix (BioRad), 10 M of each primer (Epo forward: 5 -GGA AAA GAA TGG AGG TGG AAG A3 , Epo reversed: 5 -TAC CCG AAG CAG TGA AGT GAG-3 ; GAPDH forward: 5 -CTTCACCACCATGGAGAAGGC-3 , GAPDH reversed: 5 GGCATGGACTGTGGTCATGAG-3 ) and 1 l of the cDNA template. The PCR conditions were 3 min at 95 ◦ C, followed by 40 cycles of 10 s at 95 ◦ C and 30 s at 56 ◦ C. Fluorescence data were specified for collecting during the 56 ◦ C step. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was taken as a normalization control. Western blot analysis: then the estimation of Epo protein expression was done by Western blotting analysis. Undifferentiated and differentiated ES cells were homogenized in SDS-stop buffer containing 3% beta-mercaptoethanol and denaturated at 95 ◦ C for 5 min, and when separated on a 12% Acrilamide/BisAcrilamide gel. The proteins on the gels were transferred to a PVDF
Fig. 2. Epo expression in neuronal tissue after spinal cord injury and ES cell injections. (A) Quantitative real-time PCR results of Epo mRNA expression presented as a fold induction, Epo mRNA expression level after 1 day of media treatment was taken as 1. (B) The protein extracts of spinal cords from different groups were immunoblotted with affinity purified antibodies against Epo. Optical density presented in Arbitrary Units where the Epo level after 1 day of media injection was taken as 1. All data was normalized against GAPDH expression. Data are shown as mean ± SEM *P < 0.05.
M. Glazova et al. / Neuroscience Letters 488 (2011) 55–59
membranes, which were then incubated overnight with rabbit antiEpo polyclonal antibody 1:500 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or with mouse anti-GAPDH monoclonal antibody 1:500 (Chemicon International, Inc., Temecula, CA) followed by chemiluminiscent detection by ECL-plus (Amersham, Piscataway, NJ).
57
Epo immunostaining: Epo production in the spinals cords after media or ES cells injections was examined by indirect immunofluorescence. Briefly, cryostat sections were first washed for 30 min in phosphate buffered saline with 0.1% Tween 20 (PBST), followed by 1-h incubation with blocking solution, 5% BSA in PBST. Then the sections were incubated with rabbit anti-Epo primary antibodies
Fig. 3. Epo-immunopositive cell distribution in the injected side of spinal cord. Strong immunoreactivity was observed after control media injections: (A) after 1 day; (C) after 7 days. (B) Transplanted pGFP-transfected ES cells (green) demonstrated weak staining for Epo (red) after 1 day, whereas in the host tissue Epo staining was not observed. (D) 7 days after ES cell treatment. Original magnification, 40×. Scale bar is 100 m.
58
M. Glazova et al. / Neuroscience Letters 488 (2011) 55–59
(Santa Cruz) 1:100 overnight at 4 ◦ C. Next day they were subsequently washed three times for 15 min with PBST and incubated with goat Taxes Red-conjugated AffiniPure goat anti-rabbit antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) 1:500 for 2 h. The sections were washed again three times in PBS for 15 min and coversliped with Gel/Mount aqueous mounting media with anti-fading agents (Biomeda Corp., Foster City, CA). Control samples were processed without primary antibody. To obtain an adequate yield of total RNA and protein for molecular and biochemical assays, at least three animals were pooled for one sample, and at least three independent samples were analyzed per time point. For histological analyses, at least three animals were used for tissue collection at every time point in every group. Statistical analysis was performed with Student’s t-test using Prism (GraphPad Prism version 3.00 for Windows; GraphPad Software, San Diego, CA). All values are represented as mean ± SEM, and p values of <0.05 were used for statistical significance. Analysis of RT-PCR data was done using Relative Expression Software Tool – 384 = Rest − 384 – version 1 (calculation software for the relative expression in real-time PCR using Pair Wise Fixed Reallocation Randomization Test [22]). We first examined the ability of ES cells to express the Epo before and after differentiation procedure. Evidence has indicated that neuronal tissue expresses Epo at very low level [11], however in developing nervous system the expression is markedly upregulated [6,15]. Thus, according to the published data, the differentiated ES cells should demonstrate decreased level of Epo expression in comparison to undifferentiated cells. Epo production was analyzed by real-time RT-PCR and Western blotting. Indeed, our data demonstrated that expression of Epo mRNA in ES cells was significantly
decreased after differentiation procedure (Fig. 1A), as well as the protein content (Fig. 1B). This finding tightly correlates with published observations demonstrating high expression of Epo and its receptor in the developing spinal cord [15] in comparison to adult spinal cord where expression of Epo is very low [11]. Also it has been found that Epo stimulates stem cells differentiation into neuronal progenitors [23] and glial cells [17,25]. To determine effects of ES cells on Epo production after spinal cord injury we analyzed Epo mRNA expression. The data analyses showed that, after 1-day post-injections of ES cell, Epo mRNA level were significantly decreased compared to media-injected control (Fig. 2A). However, after 7 days Epo mRNA level was the same in control and in ES cell-treated groups (Fig. 2A). Also, the analysis of Epo protein expression was done by Western blotting. Our data demonstrated significantly decreased level of Epo in the spinal cord in ES cell-treated group in comparison to media-injected control 1 day after injections (Fig. 2B). The histological analysis showed increased expression of Epo in the damaged tissue after media injections (Fig. 3A and C) that evenly overspread to the contralateral side (Fig. 4A and C). Interestingly, after 1 day of ES cell treatment Epo staining in the host tissue was not observed (Fig. 3B), and in the transplanted cell it was not extensive (Fig. 3B). However, 7 days after media injections (Fig. 3C) immunopositive cell distribution was not notably changed compared to 1-day time-point (Fig. 3A), while after ES cells transplantation the intensity of immunostaining on the contralateral side was increased (Fig. 4D). Published observations demonstrated some beneficial role of Epo in therapy of neurological diseases [5,26]. Although, Epo mainly participates in erythropoiesis, Epo and its receptor have
Fig. 4. (A and C) Epo-immunopositive cell distribution in the contralateral side of spinal cord after 1 and 7 days of media injections correspondently. (B) Weak immunoreactivity was observed after 1 day of ES cell injections in the contralateral side in comparison to contralateral side of media injected group (A), but (D) after 7 days post-ES cell treatment Epo immunopositive cells appeared in the same manner as it was observed after media injections (C). Original magnification, 40×. Scale bar is 100 m.
M. Glazova et al. / Neuroscience Letters 488 (2011) 55–59
been fond in the nervous system [11] suggesting other role then erythropoiesis. In the adult brain, constitutive expression of Epo and its receptor is low [11], but neuronal injury like hypoxia markedly induces expression of both Epo [1,9,16,18] and Epo receptor [1,9,24]. Positive neuroprotective effect of Epo has also been observed after Epo administration following spinal cord injury [2,24]. Moreover, recently published data demonstrated that Epo can promote neurogenesis [23]. On the other hand, it has been shown that ES cells have significant beneficial effect when injected directly into the injured spinal cord [3,7,10,12,14,21] and such positive effects have been detected at very early time-points [12]. It this study, examination of spinal cord demonstrated that after injections of pre-differentiated ES cells expression of Epo in the host tissue was not induce at both mRNA and protein levels at the early time-point. In conclusion, our data let us to suggest that early neuroprotective effects of ES cells injected into the injured zone [3,10,12,14,21] do not base on Epo system, but on other factors like BDNF, IL-6 [7] and others, which should be elucidated further. Acknowledgments This research was supported in part by the North Carolina Biotechnology Center (grant 2004-MRG-1104 to A.K.M.). References [1] M. Bernaudin, H.H. Marti, S. Roussel, D. Divoux, A. Nouvelot, E.T. MacKenzie, E. Petit, A potential role for erythropoietin in focal permanent cerebral ischemia in mice, J. Cereb. Blood Flow Metab. 19 (1999) 643–651. [2] M. Brines, G. Grasso, F. Fiordaliso, A. Sfacteria, P. Ghezzi, M. Fratelli, R. Latini, Q.W. Xie, J. Smart, C.J. Su-Rick, E. Pobre, D. Diaz, D. Gomez, C. Hand, T. Coleman, A. Cerami, Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 14907–14912. [3] Q.L. Cao, Y.P. Zhang, R.M. Howard, W.M. Walters, P. Tsoulfas, S.R. Whittemore, Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage, Exp. Neurol. 167 (2001) 48–58. [4] A. Cetin, K. Nas, H. Buyukbayram, A. Ceviz, G. Olmez, The effects of systemically administered methylprednisolone and recombinant human erythropoietin after acute spinal cord compressive injury in rats, Eur. Spine J. (2006). [5] H. Ehrenreich, M. Hasselblatt, C. Dembowski, L. Cepek, P. Lewczuk, M. Stiefel, H.H. Rustenbeck, N. Breiter, S. Jacob, F. Knerlich, M. Bohn, W. Poser, E. Ruther, M. Kochen, O. Gefeller, C. Gleiter, T.C. Wessel, M. De Ryck, L. Itri, H. Prange, A. Cerami, M. Brines, A.L. Siren, Erythropoietin therapy for acute stroke is both safe and beneficial, Mol. Med. Cambridge Mass 8 (2002) 495–505. [6] S. Erbayraktar, G. Grasso, A. Sfacteria, Q.W. Xie, T. Coleman, M. Kreilgaard, L. Torup, T. Sager, Z. Erbayraktar, N. Gokmen, O. Yilmaz, P. Ghezzi, P. Villa, M. Fratelli, S. Casagrande, M. Leist, L. Helboe, J. Gerwein, S. Christensen, M.A. Geist, L.O. Pedersen, C. Cerami-Hand, J.P. Wuerth, A. Cerami, M. Brines, Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 6741–6746. [7] M. Glazova, E.S. Pak, J. Moretto, S. Hollis, K.L. Brewer, A.K. Murashov, Pre-differentiated embryonic stem cells promote neuronal regeneration by cross-coupling of BDNF and IL-6 signaling pathways in the host tissue, J. Neurotrauma 26 (2009) 1029–1042. [8] G. Grasso, A. Sfacteria, A. Cerami, M. Brines, Erythropoietin as a tissueprotective cytokine in brain injury: what do we know and where do we go? Neuroscientist 10 (2004) 93–98.
59
[9] G. Grasso, A. Sfacteria, M. Passalacqua, A. Morabito, M. Buemi, B. Macri, M.L. Brines, F. Tomasello, Erythropoietin and erythropoietin receptor expression after experimental spinal cord injury encourages therapy by exogenous erythropoietin, Neurosurgery 56 (2005) 821–827, discussion 821–827. [10] S.S. Han, D.Y. Kang, T. Mujtaba, M.S. Rao, I. Fischer, Grafted lineage-restricted precursors differentiate exclusively into neurons in the adult spinal cord, Exp. Neurol. 177 (2002) 360–375. [11] M. Hasselblatt, H. Ehrenreich, A.L. Siren, The brain erythropoietin system and its potential for therapeutic exploitation in brain disease, J. Neurosurg. Anesthesiol. 18 (2006) 132–138. [12] W.A. Hendricks, E.S. Pak, J.P. Owensby, K.J. Menta, M. Glazova, J. Moretto, S. Hollis, K.L. Brewer, A.K. Murashov, Predifferentiated embryonic stem cells prevent chronic pain behaviors and restore sensory function following spinal cord injury in mice, Mol. Med. (2006). [13] M.R. Hoane, G.D. Becerra, J.E. Shank, L. Tatko, E.S. Pak, M. Smith, A.K. Murashov, Transplantation of neuronal and glial precursors dramatically improves sensorimotor function but not cognitive function in the traumatically injured brain, J. Neurotrauma 21 (2004) 163–174. [14] D.A. Kerr, J. Llado, M.J. Shamblott, N.J. Maragakis, D.N. Irani, T.O. Crawford, C. Krishnan, S. Dike, J.D. Gearhart, J.D. Rothstein, Human embryonic germ cell derivatives facilitate motor recovery of rats with diffuse motor neuron injury, J. Neurosci. 23 (2003) 5131–5140. [15] W. Knabe, A.L. Siren, H. Ehrenreich, H.J. Kuhn, Expression patterns of erythropoietin and its receptor in the developing spinal cord and dorsal root ganglia, Anat. Embryol. (Berl.) 210 (2005) 209–219. [16] M. Lee, E.S. Lee, Y.S. Kim, B.H. Choi, S.R. Park, H.S. Park, H.C. Park, S.W. Kim, Y. Ha, Ischemic injury-specific gene expression in the rat spinal cord injury model using hypoxia-inducible system, Spine 30 (2005) 2729–2734. [17] S.M. Lee, T.H. Nguyen, M.H. Park, K.S. Kim, K.J. Cho, D.C. Moon, H.Y. Kim, Y. Yoon do, J.T. Hong, EPO receptor-mediated ERK kinase and NF-kappaB activation in erythropoietin-promoted differentiation of astrocytes, Biochem. Biophys. Res. Commun. 320 (2004) 1087–1095. [18] H.H. Marti, R.H. Wenger, L.A. Rivas, U. Straumann, M. Digicaylioglu, V. Henn, Y. Yonekawa, C. Bauer, M. Gassmann, Erythropoietin gene expression in human, monkey and murine brain, Eur. J. Neurosci. 8 (1996) 666–676. [19] A.K. Murashov, E.S. Pak, W.A. Hendricks, J.P. Owensby, P.L. Sierpinski, L.M. Tatko, P.L. Fletcher, Directed differentiation of embryonic stem cells into dorsal interneurons, Faseb J. 19 (2005) 252–254. [20] A.K. Murashov, E.S. Pak, W.A. Hendricks, L.M. Tatko, 17beta-Estradiol enhances neuronal differentiation of mouse embryonic stem cells, FEBS Lett. 569 (2004) 165–168. [21] T.M. Myckatyn, S.E. Mackinnon, J.W. McDonald, Stem cell transplantation and other novel techniques for promoting recovery from spinal cord injury, Transpl. Immunol. 12 (2004) 343–358. [22] M.W. Pfaffl, G.W. Horgan, L. Dempfle, Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR, Nucleic Acids Res. 30 (2002) e36. [23] T. Shingo, S.T. Sorokan, T. Shimazaki, S. Weiss, Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells, J. Neurosci. 21 (2001) 9733–9743. [24] A.L. Siren, F. Knerlich, W. Poser, C.H. Gleiter, W. Bruck, H. Ehrenreich, Erythropoietin and erythropoietin receptor in human ischemic/hypoxic brain, Acta Neuropathol. (Berl.) 101 (2001) 271–276. [25] M. Sugawa, Y. Sakurai, Y. Ishikawa-Ieda, H. Suzuki, H. Asou, Effects of erythropoietin on glial cell development; oligodendrocyte maturation and astrocyte proliferation, Neurosci. Res. 44 (2002) 391–403. [26] M.Y. Tseng, P.J. Hutchinson, H.K. Richards, M. Czosnyka, J.D. Pickard, W.N. Erber, S. Brown, P.J. Kirkpatrick, Acute systemic erythropoietin therapy to reduce delayed ischemic deficits following aneurysmal subarachnoid hemorrhage: a Phase II randomized, double-blind, placebo-controlled trial. Clinical article, J. Neurosurg. 111 (2009) 171–180. [27] R.P. Yezierski, S. Liu, G.L. Ruenes, K.J. Kajander, K.L. Brewer, Excitotoxic spinal cord injury: behavioral and morphological characteristics of a central pain model, Pain 75 (1998) 141–155.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Embryonic stem cells inhibit expression of erythropoietin in the injured spinal cord Margarita Glazova ∗ , Sarah Hollis, Elena S. Pak, Alexander K. Murashov Departments of Physiology, The Brody School of Medicine, East Carolina University School of Medicine, Brody Building, 600 Moye Boulevard, Greenville, NC 27834, USA
a r t i c l e
i n f o
Article history: Received 10 August 2010 Received in revised form 8 October 2010 Accepted 1 November 2010 Key words: Embryonic stem cells Erythropoietin Regeneration Spinal cord injury
a b s t r a c t Recent observations have demonstrated neuroprotective role of erythropoietin (Epo) and Epo receptor in the central nervous system. Here we examined Epo function in the murine spinal cord after transplantation of pluripotent mouse embryonic stem (ES) cells pre-differentiated towards neuronal type following spinal cord injury. Expression of Epo was measured at both mRNA and protein levels in the ES cells as well as in the spinal cords after 1 and 7 days. Our data demonstrated that expression of Epo mRNA, as well as its protein content, in ES cells was significantly decreased after differentiation procedure. In the spinal cords, analysis showed that Epo mRNA level was significantly decreased after 1 day of ES cell injections in comparison to media-injected control. Epo protein level detected by Western blot was diminished as well. Examination of Epo production in the injured spinal cords after media or ES cells injections by indirect immunofluorescence showed increased Epo-immunopositive staining after media injections 1 day after injection. In contrast, ES cell transplantation did not induce Epo expression. Seven days after ES cell injections, Epo-immunopositive cells’ distribution in the ipsilateral side was not changed, while the intensity of immunostaining on the contralateral side was increased, approaching levels in control media-injected tissues. Our data let us to presume that previously described immediate positive effects of ES cells injected into the injured zone of spinal cord are not based on Epo, but on other factors or hormones, which should be elucidated further. © 2010 Elsevier Ireland Ltd. All rights reserved.
Recent animal studies have indicated that ES cells transplanted into the injured zone of the spinal cord significantly increase recovery rate [3,10,12,14,21]. Therefore, stem cells have an ability to successfully restore and maintain spinal cord function after injury. ES cells can secrete many factors like BDNF and IL-6 [7], which are involved in the recovery process. However, the precise mechanism of ES cells action after grafting into the injured host tissue still remains unknown and needs to be elucidated. Neuroprotective role of cytokines and growth factors let us to hypothesize that ES cells transplanted into the lesion can support the host tissue by secreting these active agents to promote recovery and neuronal survival [7]. Upregulation of Epo receptor expression after injury [1,9,24], and beneficial role of systemic administration of Epo directly into the lesion zone of the spinal cord [4] suggests neuroprotective function of Epo. In the central nervous system expression of Epo and Epo receptor has been demonstrated [1,11], and a positive neuroprotective role of Epo has been documented [8]. We hypothesized that transplanted ES cells could supply a host tissue with Epo or
∗ Corresponding author. Present address: Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 Thorez pr., 194223 Sankt-Petersburg, Russia. Tel.: +7 812 552 32 27; fax: +7 812 552 3012. E-mail address: [email protected] (M. Glazova). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.11.002
induce Epo expression in injured spinal cord. In the current study we asked the question of whether Epo system may play a role in mediating effects of pre-differentiated ES cells in the spinal cord after excitotoxic injury caused by Quisqualic acid [27]. Embryonic stem cell culture: The pGFP-transfected ES cells were used for injections. Briefly, undifferentiated, pluripotent mouse ES cells (D3 cell line, American Type Culture Collection Manassas, VA) ES cells were transfected with the pEGFP-N3 construct (Clontech, Palo Alto, CA) in the presence of FuGENE 6 (Roche, Indianapolis, IN) as described [13]. Transfected colonies were then selected in the presence of 200 mg/ml of G418 (Sigma) and FACS sorted on BD FACSVantage SE flow cytometry system (Becton Dickinson) to achieve 90% of GFP-labeled cells. Transgenic D3 cells were further used for induction of neural cells. pGFP-transfected ES cells were differentiated according to our published protocol [19,20]. Briefly, cells were cultured in Knock-out DMEM supplemented with 15% fetal bovine serum (FBS)–ES grade and 1400 U/ml murine leukemia inhibitory factor (Stem Cell Technologies, Vancouver, Canada), and 55 M -mercaptoethanol (Sigma Chemical Co., St. Louis, MO). To induce differentiation the medium was exchanged for differentiation medium (Iscove’s Modified Dulbecco’s Medium, 15% FBS, 2 mM l-glutamine, 0.1 mM non-essential amino acids) when the cells began to form embryoid bodies after 1–2 days. At day 5, alltrans retinoic acid (Sigma, St. Louis, MO) (1 × 10−6 M) was added
56
M. Glazova et al. / Neuroscience Letters 488 (2011) 55–59
to the culture. On day 9, embryoid bodies were collected and seeded on poly-l-orhithine/fibronectin coated flask in differentiation/Neurobasal plus B27 supplement 1:1 medium. The next day, the medium was replaced with Neurobasal medium plus B27 supplement. The cells were cultured for another 2–3 days and then harvested for the injections or analysis of Epo expression. Our early study demonstrated that this differentiating protocol gave rise for about 60% of neurons and 40% of glial cells [19,20]. Animal surgery: the animal surgical procedure was performed as reported previously [12,27]. Briefly, adult male mice (CD-1, Harlan, Indianapolis, IN) were anesthetized with isoflurane (2–3% inhalation) and unilateral microinjections were made in a single segment at spinal levels ranging from T12 to L2. Each animal received 0.6 l of 125 mM Quisqualic acid (QUIS) (Sigma, St. Louis, MO) (three tracks of 0.2 l separated by 0.3 mm, parallel to the long axis of the cord). Following a one-week period of recovery, pre-differentiated mouse ES cells (100,000 cells/l) were injected into the injury (three tracks of 0.2 l separated by 0.3 mm, parallel to the long axis of the cord). Control animals received similar microinjections of cell neurobasal media in equal volume and locations. At 1, or 7 days post-ES cell or media microinjection, the animals were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and tissues were collected: (1) in liquid nitrogen for biochemical analysis, (2) in RNA-later for RNA isolation and subsequent real time PCR study and (3) for histological studies, animal were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and then intracardially perfused first with cold PBS and then cold 4% paraformaldehyde in PBS (pH 7.4). Each thoracic and lumbar spinal cord was removed and postfixed in 4% paraformaldehyde for 2 h. Fixed tissue was stored in 30% sucrose solution at 4 ◦ C until sectioning. For cryosectioning, tissue was embedded in tissue freezing medium (Triangle Biomedical Science, Durham, NC), and sectioned into 14 m coronal slices
Fig. 1. Expression of Epo is decreased in ES cells after differentiation. (A) Quantitative real-time PCR results of Epo mRNA expression presented as a fold induction, Epo mRNA expression level in non-differentiated (n/d) ES cells was taken as 1. (B) The proteins from non-differentiated and differentiated cells were extracted and immunoblotted with affinity purified antibodies against Epo, and optical density was counted and the data are presented in Arbitrary Units. All data were normalized against GAPDH expression. Data are shown as mean ± SEM *P < 0.05.
using a cryostat microtome. All procedures and tissue collection methods were approved by the East Carolina University Animal Care and Use Committee. RNA preparation, reverse-transcription, and real-time PCR: total RNA was extracted from collected tissue and ES cells using TRIZOL (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Total RNA was exposed to RNAase-free DNAase I (Ambion, Austin, TX) and reverse transcribed to cDNA using Random Hexames and Cloned AMV firs-Strand cDNA Synthesis Kit (Invitrogen) according manufacturer’s protocol. Real-time PCR was performed using a BioRad iQ iCycler Detection System (BioRad Laboratories, Ltd., Hercules, CA) with SYBR green fluorophore. Reactions were performed in a total volume of 25 l, including 12.5 l 2× SYBR Green PCR Master Mix (BioRad), 10 M of each primer (Epo forward: 5 -GGA AAA GAA TGG AGG TGG AAG A3 , Epo reversed: 5 -TAC CCG AAG CAG TGA AGT GAG-3 ; GAPDH forward: 5 -CTTCACCACCATGGAGAAGGC-3 , GAPDH reversed: 5 GGCATGGACTGTGGTCATGAG-3 ) and 1 l of the cDNA template. The PCR conditions were 3 min at 95 ◦ C, followed by 40 cycles of 10 s at 95 ◦ C and 30 s at 56 ◦ C. Fluorescence data were specified for collecting during the 56 ◦ C step. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was taken as a normalization control. Western blot analysis: then the estimation of Epo protein expression was done by Western blotting analysis. Undifferentiated and differentiated ES cells were homogenized in SDS-stop buffer containing 3% beta-mercaptoethanol and denaturated at 95 ◦ C for 5 min, and when separated on a 12% Acrilamide/BisAcrilamide gel. The proteins on the gels were transferred to a PVDF
Fig. 2. Epo expression in neuronal tissue after spinal cord injury and ES cell injections. (A) Quantitative real-time PCR results of Epo mRNA expression presented as a fold induction, Epo mRNA expression level after 1 day of media treatment was taken as 1. (B) The protein extracts of spinal cords from different groups were immunoblotted with affinity purified antibodies against Epo. Optical density presented in Arbitrary Units where the Epo level after 1 day of media injection was taken as 1. All data was normalized against GAPDH expression. Data are shown as mean ± SEM *P < 0.05.
M. Glazova et al. / Neuroscience Letters 488 (2011) 55–59
membranes, which were then incubated overnight with rabbit antiEpo polyclonal antibody 1:500 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or with mouse anti-GAPDH monoclonal antibody 1:500 (Chemicon International, Inc., Temecula, CA) followed by chemiluminiscent detection by ECL-plus (Amersham, Piscataway, NJ).
57
Epo immunostaining: Epo production in the spinals cords after media or ES cells injections was examined by indirect immunofluorescence. Briefly, cryostat sections were first washed for 30 min in phosphate buffered saline with 0.1% Tween 20 (PBST), followed by 1-h incubation with blocking solution, 5% BSA in PBST. Then the sections were incubated with rabbit anti-Epo primary antibodies
Fig. 3. Epo-immunopositive cell distribution in the injected side of spinal cord. Strong immunoreactivity was observed after control media injections: (A) after 1 day; (C) after 7 days. (B) Transplanted pGFP-transfected ES cells (green) demonstrated weak staining for Epo (red) after 1 day, whereas in the host tissue Epo staining was not observed. (D) 7 days after ES cell treatment. Original magnification, 40×. Scale bar is 100 m.
58
M. Glazova et al. / Neuroscience Letters 488 (2011) 55–59
(Santa Cruz) 1:100 overnight at 4 ◦ C. Next day they were subsequently washed three times for 15 min with PBST and incubated with goat Taxes Red-conjugated AffiniPure goat anti-rabbit antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) 1:500 for 2 h. The sections were washed again three times in PBS for 15 min and coversliped with Gel/Mount aqueous mounting media with anti-fading agents (Biomeda Corp., Foster City, CA). Control samples were processed without primary antibody. To obtain an adequate yield of total RNA and protein for molecular and biochemical assays, at least three animals were pooled for one sample, and at least three independent samples were analyzed per time point. For histological analyses, at least three animals were used for tissue collection at every time point in every group. Statistical analysis was performed with Student’s t-test using Prism (GraphPad Prism version 3.00 for Windows; GraphPad Software, San Diego, CA). All values are represented as mean ± SEM, and p values of <0.05 were used for statistical significance. Analysis of RT-PCR data was done using Relative Expression Software Tool – 384 = Rest − 384 – version 1 (calculation software for the relative expression in real-time PCR using Pair Wise Fixed Reallocation Randomization Test [22]). We first examined the ability of ES cells to express the Epo before and after differentiation procedure. Evidence has indicated that neuronal tissue expresses Epo at very low level [11], however in developing nervous system the expression is markedly upregulated [6,15]. Thus, according to the published data, the differentiated ES cells should demonstrate decreased level of Epo expression in comparison to undifferentiated cells. Epo production was analyzed by real-time RT-PCR and Western blotting. Indeed, our data demonstrated that expression of Epo mRNA in ES cells was significantly
decreased after differentiation procedure (Fig. 1A), as well as the protein content (Fig. 1B). This finding tightly correlates with published observations demonstrating high expression of Epo and its receptor in the developing spinal cord [15] in comparison to adult spinal cord where expression of Epo is very low [11]. Also it has been found that Epo stimulates stem cells differentiation into neuronal progenitors [23] and glial cells [17,25]. To determine effects of ES cells on Epo production after spinal cord injury we analyzed Epo mRNA expression. The data analyses showed that, after 1-day post-injections of ES cell, Epo mRNA level were significantly decreased compared to media-injected control (Fig. 2A). However, after 7 days Epo mRNA level was the same in control and in ES cell-treated groups (Fig. 2A). Also, the analysis of Epo protein expression was done by Western blotting. Our data demonstrated significantly decreased level of Epo in the spinal cord in ES cell-treated group in comparison to media-injected control 1 day after injections (Fig. 2B). The histological analysis showed increased expression of Epo in the damaged tissue after media injections (Fig. 3A and C) that evenly overspread to the contralateral side (Fig. 4A and C). Interestingly, after 1 day of ES cell treatment Epo staining in the host tissue was not observed (Fig. 3B), and in the transplanted cell it was not extensive (Fig. 3B). However, 7 days after media injections (Fig. 3C) immunopositive cell distribution was not notably changed compared to 1-day time-point (Fig. 3A), while after ES cells transplantation the intensity of immunostaining on the contralateral side was increased (Fig. 4D). Published observations demonstrated some beneficial role of Epo in therapy of neurological diseases [5,26]. Although, Epo mainly participates in erythropoiesis, Epo and its receptor have
Fig. 4. (A and C) Epo-immunopositive cell distribution in the contralateral side of spinal cord after 1 and 7 days of media injections correspondently. (B) Weak immunoreactivity was observed after 1 day of ES cell injections in the contralateral side in comparison to contralateral side of media injected group (A), but (D) after 7 days post-ES cell treatment Epo immunopositive cells appeared in the same manner as it was observed after media injections (C). Original magnification, 40×. Scale bar is 100 m.
M. Glazova et al. / Neuroscience Letters 488 (2011) 55–59
been fond in the nervous system [11] suggesting other role then erythropoiesis. In the adult brain, constitutive expression of Epo and its receptor is low [11], but neuronal injury like hypoxia markedly induces expression of both Epo [1,9,16,18] and Epo receptor [1,9,24]. Positive neuroprotective effect of Epo has also been observed after Epo administration following spinal cord injury [2,24]. Moreover, recently published data demonstrated that Epo can promote neurogenesis [23]. On the other hand, it has been shown that ES cells have significant beneficial effect when injected directly into the injured spinal cord [3,7,10,12,14,21] and such positive effects have been detected at very early time-points [12]. It this study, examination of spinal cord demonstrated that after injections of pre-differentiated ES cells expression of Epo in the host tissue was not induce at both mRNA and protein levels at the early time-point. In conclusion, our data let us to suggest that early neuroprotective effects of ES cells injected into the injured zone [3,10,12,14,21] do not base on Epo system, but on other factors like BDNF, IL-6 [7] and others, which should be elucidated further. Acknowledgments This research was supported in part by the North Carolina Biotechnology Center (grant 2004-MRG-1104 to A.K.M.). References [1] M. Bernaudin, H.H. Marti, S. Roussel, D. Divoux, A. Nouvelot, E.T. MacKenzie, E. Petit, A potential role for erythropoietin in focal permanent cerebral ischemia in mice, J. Cereb. Blood Flow Metab. 19 (1999) 643–651. [2] M. Brines, G. Grasso, F. Fiordaliso, A. Sfacteria, P. Ghezzi, M. Fratelli, R. Latini, Q.W. Xie, J. Smart, C.J. Su-Rick, E. Pobre, D. Diaz, D. Gomez, C. Hand, T. Coleman, A. Cerami, Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 14907–14912. [3] Q.L. Cao, Y.P. Zhang, R.M. Howard, W.M. Walters, P. Tsoulfas, S.R. Whittemore, Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage, Exp. Neurol. 167 (2001) 48–58. [4] A. Cetin, K. Nas, H. Buyukbayram, A. Ceviz, G. Olmez, The effects of systemically administered methylprednisolone and recombinant human erythropoietin after acute spinal cord compressive injury in rats, Eur. Spine J. (2006). [5] H. Ehrenreich, M. Hasselblatt, C. Dembowski, L. Cepek, P. Lewczuk, M. Stiefel, H.H. Rustenbeck, N. Breiter, S. Jacob, F. Knerlich, M. Bohn, W. Poser, E. Ruther, M. Kochen, O. Gefeller, C. Gleiter, T.C. Wessel, M. De Ryck, L. Itri, H. Prange, A. Cerami, M. Brines, A.L. Siren, Erythropoietin therapy for acute stroke is both safe and beneficial, Mol. Med. Cambridge Mass 8 (2002) 495–505. [6] S. Erbayraktar, G. Grasso, A. Sfacteria, Q.W. Xie, T. Coleman, M. Kreilgaard, L. Torup, T. Sager, Z. Erbayraktar, N. Gokmen, O. Yilmaz, P. Ghezzi, P. Villa, M. Fratelli, S. Casagrande, M. Leist, L. Helboe, J. Gerwein, S. Christensen, M.A. Geist, L.O. Pedersen, C. Cerami-Hand, J.P. Wuerth, A. Cerami, M. Brines, Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 6741–6746. [7] M. Glazova, E.S. Pak, J. Moretto, S. Hollis, K.L. Brewer, A.K. Murashov, Pre-differentiated embryonic stem cells promote neuronal regeneration by cross-coupling of BDNF and IL-6 signaling pathways in the host tissue, J. Neurotrauma 26 (2009) 1029–1042. [8] G. Grasso, A. Sfacteria, A. Cerami, M. Brines, Erythropoietin as a tissueprotective cytokine in brain injury: what do we know and where do we go? Neuroscientist 10 (2004) 93–98.
59
[9] G. Grasso, A. Sfacteria, M. Passalacqua, A. Morabito, M. Buemi, B. Macri, M.L. Brines, F. Tomasello, Erythropoietin and erythropoietin receptor expression after experimental spinal cord injury encourages therapy by exogenous erythropoietin, Neurosurgery 56 (2005) 821–827, discussion 821–827. [10] S.S. Han, D.Y. Kang, T. Mujtaba, M.S. Rao, I. Fischer, Grafted lineage-restricted precursors differentiate exclusively into neurons in the adult spinal cord, Exp. Neurol. 177 (2002) 360–375. [11] M. Hasselblatt, H. Ehrenreich, A.L. Siren, The brain erythropoietin system and its potential for therapeutic exploitation in brain disease, J. Neurosurg. Anesthesiol. 18 (2006) 132–138. [12] W.A. Hendricks, E.S. Pak, J.P. Owensby, K.J. Menta, M. Glazova, J. Moretto, S. Hollis, K.L. Brewer, A.K. Murashov, Predifferentiated embryonic stem cells prevent chronic pain behaviors and restore sensory function following spinal cord injury in mice, Mol. Med. (2006). [13] M.R. Hoane, G.D. Becerra, J.E. Shank, L. Tatko, E.S. Pak, M. Smith, A.K. Murashov, Transplantation of neuronal and glial precursors dramatically improves sensorimotor function but not cognitive function in the traumatically injured brain, J. Neurotrauma 21 (2004) 163–174. [14] D.A. Kerr, J. Llado, M.J. Shamblott, N.J. Maragakis, D.N. Irani, T.O. Crawford, C. Krishnan, S. Dike, J.D. Gearhart, J.D. Rothstein, Human embryonic germ cell derivatives facilitate motor recovery of rats with diffuse motor neuron injury, J. Neurosci. 23 (2003) 5131–5140. [15] W. Knabe, A.L. Siren, H. Ehrenreich, H.J. Kuhn, Expression patterns of erythropoietin and its receptor in the developing spinal cord and dorsal root ganglia, Anat. Embryol. (Berl.) 210 (2005) 209–219. [16] M. Lee, E.S. Lee, Y.S. Kim, B.H. Choi, S.R. Park, H.S. Park, H.C. Park, S.W. Kim, Y. Ha, Ischemic injury-specific gene expression in the rat spinal cord injury model using hypoxia-inducible system, Spine 30 (2005) 2729–2734. [17] S.M. Lee, T.H. Nguyen, M.H. Park, K.S. Kim, K.J. Cho, D.C. Moon, H.Y. Kim, Y. Yoon do, J.T. Hong, EPO receptor-mediated ERK kinase and NF-kappaB activation in erythropoietin-promoted differentiation of astrocytes, Biochem. Biophys. Res. Commun. 320 (2004) 1087–1095. [18] H.H. Marti, R.H. Wenger, L.A. Rivas, U. Straumann, M. Digicaylioglu, V. Henn, Y. Yonekawa, C. Bauer, M. Gassmann, Erythropoietin gene expression in human, monkey and murine brain, Eur. J. Neurosci. 8 (1996) 666–676. [19] A.K. Murashov, E.S. Pak, W.A. Hendricks, J.P. Owensby, P.L. Sierpinski, L.M. Tatko, P.L. Fletcher, Directed differentiation of embryonic stem cells into dorsal interneurons, Faseb J. 19 (2005) 252–254. [20] A.K. Murashov, E.S. Pak, W.A. Hendricks, L.M. Tatko, 17beta-Estradiol enhances neuronal differentiation of mouse embryonic stem cells, FEBS Lett. 569 (2004) 165–168. [21] T.M. Myckatyn, S.E. Mackinnon, J.W. McDonald, Stem cell transplantation and other novel techniques for promoting recovery from spinal cord injury, Transpl. Immunol. 12 (2004) 343–358. [22] M.W. Pfaffl, G.W. Horgan, L. Dempfle, Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR, Nucleic Acids Res. 30 (2002) e36. [23] T. Shingo, S.T. Sorokan, T. Shimazaki, S. Weiss, Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells, J. Neurosci. 21 (2001) 9733–9743. [24] A.L. Siren, F. Knerlich, W. Poser, C.H. Gleiter, W. Bruck, H. Ehrenreich, Erythropoietin and erythropoietin receptor in human ischemic/hypoxic brain, Acta Neuropathol. (Berl.) 101 (2001) 271–276. [25] M. Sugawa, Y. Sakurai, Y. Ishikawa-Ieda, H. Suzuki, H. Asou, Effects of erythropoietin on glial cell development; oligodendrocyte maturation and astrocyte proliferation, Neurosci. Res. 44 (2002) 391–403. [26] M.Y. Tseng, P.J. Hutchinson, H.K. Richards, M. Czosnyka, J.D. Pickard, W.N. Erber, S. Brown, P.J. Kirkpatrick, Acute systemic erythropoietin therapy to reduce delayed ischemic deficits following aneurysmal subarachnoid hemorrhage: a Phase II randomized, double-blind, placebo-controlled trial. Clinical article, J. Neurosurg. 111 (2009) 171–180. [27] R.P. Yezierski, S. Liu, G.L. Ruenes, K.J. Kajander, K.L. Brewer, Excitotoxic spinal cord injury: behavioral and morphological characteristics of a central pain model, Pain 75 (1998) 141–155.