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Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds
Biologicals xxx (2017) 1e6
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Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds Simin Jamali a, Hossein Mostafavi a, Ghasem Barati b, Mehdi Eskandari a, *, Samad Nadri b, c, ** a b c
Department of Physiology and Pharmacology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran Department of Medical Biotechnology and Nanotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran Cancer and Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 May 2017 Received in revised form 6 September 2017 Accepted 11 September 2017 Available online xxx
Parkinson's disease (PD) is a neurodegenerative disorder of the brain which is produced by the damage to dopaminergic neurons. Stem cell transplantation with a nanofibrous scaffold is one of the encouraging strategies for Parkinson's disease therapy. In this study, human mesenchymal stem cells (MSCs) from eye trabecular meshwork (TM) were differentiated into dopaminergic neurons on nanofibrous scaffold. After Trabecular meshwork biopsy, MSCs were isolated, cultivated on Poly-L-Lactide Acid (PLLA) nanofibrous scaffold (fabricated by electrospinning methods) and treated with medium containing DMEM supplemented with RA, IBMX and forskolin for 7 days. Scanning electron microscopy imaging, qPCR and immunostaining were used to analyze differentiated TM-MSCs on scaffold and their expression of dopaminergic-specific markers such as TH and Nurr-1. qPCR analysis revealed the expression of dopaminergic neuron genes such as TH, Nurr-1 on fibrous scaffold as well as TCPS. Immunostaining revealed that the differentiated TM-MSCs on TCPS and Scaffold not only express TH and Nurr-1 genes, but also express TH protein. In conclusion, the results indicate that TM-MSCs might be a suitable source for cell transplantation therapy. In addition the nanofibrous scaffold reported herein could be used as a potential cell carrier for the central system diseases such as PD. © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Trabecular meshwork mesenchymal stem cells Dopaminergic neurons Nanofibrous scaffold
1. Introduction Parkinson's disease (PD) is a neurodegenerative disorder of the brain which is produced by damage to dopaminergic neurons and a reduction of dopamine in the substantia nigra and in the striatum, respectively [1e3]. Although many treatment strategies such as administration of drugs, have been designed for this disease, they have shown an imperfect efficacy [4]. In recent years, stem cell therapy has emerged as a promising alternative treatment for Parkinson's disease (PD) [5].
* Corresponding author. Physiology and Pharmacology Department, P.O.Box: 14115-111, Zanjan, Iran. ** Corresponding author. Medical Biotechnology and Nanotechnology Department, P.O.Box: 14115-111, Zanjan, Iran. E-mail addresses: [email protected] (M. Eskandari), [email protected] (S. Nadri).
Despite the fact that human embryonic stem cells (ESCs) and neural stem cells (NSCs) are considered well-accepted cell sources with the capacity to differentiate into dopaminergic neurons [6], The clinical use of ESCs and NSCs has presented problems including teratoma formation as well as eliciting immune rejection after transplantation, low efficiency of differentiation and lineage polarization [7]. Based on these facts, additional studies are necessary to identify other stem cell sources with the ability to produce dopaminergic neurons with high efficiency. Mesenchymal stem cells (MSCs) have been shown to be a reliable cell source for PD treatment [8]. MSCs are multipotent stem cells that have been isolated from a wide variety of tissues and can be differentiated into neurons [9]. MSCs are currently in clinical trials for a number of disorders such as multiple sclerosis [10,11] and PD [11]. Human brain microenvironments are highly complex, with threedimensional (3D) structures involving the organized interaction
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Please cite this article in press as: Jamali S, et al., Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.09.002
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of multiple neural cell types with other cells (microglia and endothelial cells) [12]. One option for improving the physiological relevance of human neuronal culture models is to use these cells in combination with 3D scaffolds. Several studies have indicated that cell transplantation with a biocompatible polymer scaffold increases survival [13] and differentiation [14] of the cells. Scaffolds are the key constituents of tissue engineering for stem cells delivery. To date, several studies have been conducted on the effects of 3D electrospun scaffolds of a variety of biomaterials to support neural differentiation of stem cells [15]. While 3D scaffolds have been used to engineer neurocyte celltype specific constructs, there have been few reports on the successful application of a single 3D scaffold to support cell differentiation into specific types of neurocyte cells [16]. In the present study, Trabecular meshwork Mesenchymal stem cells (TM-MSCs) were differentiated into dopaminergic-like neuron on a nanofibrous scaffold in vitro. Taken together, a combination of TM-MSCs (as a new source of cells) and a nanofibrous scaffold could provide a strategy for Parkinson disease therapy. 2. Materials and methods
20 min and treated under UV irradiation for 10 min. Nanofibrous PLLA scaffolds were placed in 70 mm2 culture dishes and 3 105 of the TM-MSCs were cultivated on them. For differentiation, the cells were incubated in an induction medium that consisted of DMEM (low glucose), supplemented with 10 mM retinoic acid (RA, sigma), 0.5 mM IBMX and 10 mM Forskolin for 7 days. Cultivated cells on a tissue culture polystyrene (TCPS) plate were maintained in induction medium and culture medium as a two dimensional culture group (as a control group). At the end of this period, the cells were used for qPCR and Immunofluorescence analysis. The expression levels of the genes and proteins were compared with untreated cells. 2.4. Immunofluorescence analysis The cells were fixed (with 4% paraformaldehyde) and permeabilized with 0.5% Triton X-100 for 10 min. Then the cells were reacted with primary antibodies for TH (SANTA CRUZ BIOTECHNOLOGY,INC) at 4 C for 24 h, and subsequently reacted with the FITC-conjugated IgG as the secondary antibody (SANTA CRUZ BIOTECHNOLOGY,INC) at room temperature for 1 h. In addition, the cells were incubated with diaminobenzidine (DAB) solution (Sigma Chemical Co.) for 30 s for nuclear staining.
2.1. Isolation and differentiation into mesenchymal lineage cells 2.5. Gene expression This research was performed in accordance with the Helsinki Declaration and with approval of the ethics board of the Zanjan University of medical Sciences. Eye globes were obtained from the Central Eye Bank of Iran. TM-MSCs were isolated according to a protocol modified by Nadri et al. [17]. TM biopsy was incubated in PBS (GIBCO-BRL, Grand Island, NY) supplemented with 40 mg/mL BSA and 4 mg/mL collagenase for 1 h. The isolated stromal tissue segment was cultured in low glucose DMEM (GIBCO-BRL, Grand Island, NY) supplemented with 20% serum (GIBCO-BRL, Grand Island, NY), 200 ng/mL basic-FGF (Peprotech, Rocky Hill, NJ), and was incubated at 37 C with 5% CO2 in a humidified chamber. After 2 weeks, the cells were trypsinized (0.25% Trypsin e GIBCO-BRL, Grand Island, NY) and expanded by two passages. To demonstrate the multipotent nature of the isolated cells, the cells were differentiated down three lineages over a period of 21 days as follows: osteogenic (DMEM including 50 mg/mL ascorbic acid 2-phosphate (Sigma Chemical Co. St Louis, MO), 10 nM dexamethasone (Sigma Chemical Co.), 10 mM b-glycerol phosphate (Sigma Chemical Co.), adipogenic (DMEM supplemented with 50 mg/mL indomethacine (Sigma Chemical Co) and 100 nM dexamethasone (Sigma Chemical Co), and chondrogenic (DMEM supplemented with 10 ng/mL transforming growth factor- b3 (TGF-b3; Sigma Chemical Co), bone morphogenetic protein- 6 (BMP-6), 107 M dexamethasone (Sigma Chemical Co.), 50 mg/mL ascorbate2-phosphate (Sigma Chemical Co.), and 50 mg/mL insulintransferrin- selenium (ITS; GIBCO- BRL) medium.
The total RNA was extracted by RNXPLUS (Sinaclon, Iran). Quantification and purity of RNA was determined using a spectrophotometer (nanodrop 2000, Wilmington, USA). cDNA was synthesized using PrimeScript 1st strand cDNA synthesis Kit (Takara. Japan) according to the manufacturer's procedure. Real-time PCR was performed with an Applied BiosystemsTM Real-Time PCR System (Life Technologies Corporation, USA). All genes were subjected to 40 cycles, which consisted of a 15 min for initial denaturation at 95 C, followed by forty cycles of 15 s at 95 C, 30s annealing at 57 C, and a final 30s extension at 72 C. Related specific primers are presented in Table 1. The rest software from the rotor-gene Q, based on Pfaffl mathematical methods, was used for statistical analysis of real time data and relative gene expression [18]. 3. Results Cell culture and In vitro differentiation of trabecular meshwork mesenchymal stem cells (TM-MSCs) into mesenchymal lineages. After 2 weeks, fibroblast-like cells with spindle-shaped morphology appeared on TCPS dishes. These cells were utilized for future experiments. To confirm their mesenchymal nature, the isolated cells were treated with appropriate osteo-, chondro- and adipoinduction media, and their differentiation was confirmed via appropriate staining including, alizarin red (for osteogenic differentiation), alcian blue (for chondrogenic differentiation) and oil red (for adipogenic differentiation) staining (Fig. 1 AeD).
2.2. Fabrication of electrospun nanofibrous PLLA scaffolds 3.1. Dopaminergic differentiation of TM-MSCs Nanofibrous PLLA scaffolds were fabricated using an electrospinning technique [18]. PLLA was dissolved in chloroform (9% w/ w) and added to the chloroform/N,N-dimethylformamide (DMF) (Sigma, Steinheim, Germany) solution (10:1). Aligned nanofibrous scaffolds were obtained using high (3000 rpm) speed rotating disk. 2.3. In vitro differentiation of TM-MSCs to dopaminergic neuron on PLLA scaffold For sterilization, the scaffolds were placed in 70 C alcohol for
Next, we studied the potential of TM-MSCs to differentiate into dopaminergic neural cells. To elucidate the dopaminergic neural differentiation potential of CJMSCs, these cells were cultured in neuroinductive medium including DMEM supplemented With RA, IBMX and Forskolin. During differentiation more morphological changes in cells were observed (Fig. 2). Following induction, bi and multipolar cells with multiple dendrites and cytoplasmic branches were observed 2, 5 and 7 days after induction in TM-MSCs (see Fig. 2). The SEM results indicated that the TM-MSCs were well
Please cite this article in press as: Jamali S, et al., Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.09.002
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Table 1 Primer sequences employed in this project. Genes
Primer sequences
Size (bp)
Annealing melt ( C)
TH
F: 50 - CAGTTCTCGCAGGACATTG-30 R: 50 - CCCGTAGGAGGACAGCAG-30 F: 50 - TGGGATGGTCAAAGAAGTG-30 R: 50 - GCCTGGAACCTGGAATAG-30 F: 50 -CCTGGCGTCGTGATTAGTG - 30 R: 50 - TCAGTCCTGTCCATAATTAGTCC-30
156
51
189
50
124
56
Nurr1 HPRT1
Fig. 1. Morphological characteristics and in vitro differentiation of trabecular meshwork cells into mesenchymal lineages. (A) TM cells have been differentiated into (B) mineralizing cells stained with alizarin red (C) Adipocytes stained with Oil red O (D) Chondrocytic lineage stained with alcian blue.
Fig. 2. Morphological changes in TM-MSCs after induction. The morphological changes were observed at 2 (AeB), 5(C) and 7 (D) days after induction.
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attached on electrospun PLLA scaffolds and produce neurite outgrowth along the direction of fiber orientation (under differentiation media) (Fig. 3). After 7 days, we examined the expression of dopaminergic protein and genes by immunofluorescence and qPCR. Immunofluorescence was used to analyze the intracellular Tyrosine hydroxylase (TH) protein. This protein was identified in the TM-MSCs cultivated on PLLA scaffold and TCPS after treatment in neuroinductive medium for 7 days (Fig. 4). 3.2. Gene expression The results related to the expression of two genes on PLLA scaffold and TCPS are shown in Fig. 5. As shown in Fig. 5, gene expression of TH and Nurr-1 was significantly higher in treated TM-MSCs in comparison with untreated cells. As shown in Fig. 5, qPCR analysis demonstrated that TH (1.343 fold; p 0.3) and Nurr-1 (1.564 fold; p 0.16) gene expression was higher on PLLA scaffolds compared with TCPS. 4. Discussion Parkinson's disease (PD) is the neurodegenerative disorder for
which a variety of different options such as levodopa drug administration [19,20] are currently used to reverse the symptoms of PD. However, the efficacy of such treatments gradually diminishes over time. New methods have been explored applying stem cells to repair the damaged dopaminergic system [21]. To date, different stem cells such as embryonic stem cells (ESCs) [22e24], and mesenchymal stem cells (MSCs) have been investigated as potential therapies for the treatment of PD diseases. However, the difficulties encountered using human ESCs have led to the search for other types of cells for treatment of PD disease [5]. Dopaminergic differentiation of MSCs has also been described [25]. The clinical use of MSCs from well-accepted sources such as bone marrow has presented problems including painful aspiration, contamination with non-MSCs, and the low yield of cells [26]. This has caused many scientists to consider alternative sources of MSCs for treatment of PD disease. This work aimed to differentiate TMMSCs into dopamine secreting cells. Eye Trabecular meshwork tissue has been identified as a source of multipotent stromal MSCs, which can be obtained by a less invasive method without any contamination with non-MSCs. In this work, we demonstrated that TM-MSCs expressed dopaminergic neuron genes after treatment with DMEM containing 10% FBS, IBMX, forskolin, and 10 mM retinoic acid for 1 week. Previous investigators have differentiated MSCs over 2 weeks, using a
Fig. 3. SEM micrographs of PLLA nanofibrous scaffolds. The PLLA scaffold contains aligned (A) fibers and TM-MSCs differentiated on it 7 days after induction(B).
Fig. 4. Immunostaining of cultivated cells on tissue culture polystyrene (TCPS) and PLLA fibrous scaffold. The cultivated cells on scaffold and TCPS were maintained in induction medium for 7 days and analyzed for expression of TH proteins. The cells on PLLA and TCPS were co-stained with 4,6-diamidino-2-phenylindole to visualize nuclei (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Jamali S, et al., Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.09.002
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Fig. 5. Investigation of dopaminergic-specific gene expression. Expression of dopaminergic genes in control (day 0) and differentiation culture (day 7) of cultivated cells on TCPS and PLLA. HPRT-1 is shown as a control for RNA sample quality. Rest software was used for gene expression analysis using real-time PCR data from the rotor-gene Q.
multistep protocol in a cocktail of dopaminergic medium consisting of DMEM supplemented with 10% FBS, sonic hedgehog (SHH), fibroblast growth factor, epidermal growth factor, N2 supplement, butylated hydroxyanisole, IBMX, RA and dbcAMP [25,27]. MSC-derived adipose tissue were differentiated into dopaminergic neuron-like cells using neural induction media containing FGF2, EGF, BMP-9, retinoic acid and heparin [28]. According to the results presented in this paper it seems that TM-MSCs were differentiated into dopaminergic neurons by a straightforward method compared with BMMSCs, human umbilical mesenchymal stem cells and MSCs-derived adipose tissue. In the present study, qPCR analysis demonstrated that dopaminergic genes including TH and Nurr-1 were expressed in TMMSCs. Furthermore, immunofluorescence revealed that TH protein was expressed in TM-MSCs. Several studies of dopaminergic induction reported TH expression in MSCs after induced differentiation [29e31]. A TH protein is the initial enzyme in the catecholamine synthesis pathway, required for the generation of dopamine and is considered the principal regulator of dopamine biosynthesis in the central neuron system and as a marker for dopaminergic neurons [32,33]. Nurr-1 plays a main role in the maintenance of the dopaminergic system of the brain [34]. Nurr1 induces tyrosine hydroxylase (TH) expression, which leads to differentiation into dopaminergic neurons [35]. The results of this work showed that TM-MSCs were acquired through a simple approach and expressed TH and Nurr-1 genes and protein. The detection of TH and some other dopamine-associated genes such as Nurr1 necessary for both the survival and differentiation of the dopaminergic precursor's neurons [36,37] demonstrated that TM-MSCs can be differentiated into dopaminergic neurons and are good candidates for neurological cell therapy and for treating Parkinson's disease. For tissue engineering applications, it is essential to recognize the scaffold properties that on this construct cell
material interactions may influence the lineage fate decision of the stem cells [38,39]. Investigation of stem cell behaviour and the functional response to fibrous scaffolds has proved to be significantly challenging [15]. In the present study, we studied the influence of PLLA nanofibrous scaffold on differentiation of TM-MSCs to dopaminergic cells. To date several studies have been conducted on the effects of 3D electrospun scaffolds (as scaffold surfaces) of a variety of biomaterials including polyamide [40], poly (l-lactic acid) (PLLA) [41], poly (l-lactide-co-glycolide) (PLGA) [42] and polyurethane [43] to support neural differentiation of stem cells. In the present study, PLLA scaffolds were fabricated using electrospinning methods. In this work, after exposure to induction media, qPCR analysis and immunofluorescence indicated that specific dopaminergic genes including TH and Nurr1 were expressed in the TM-MSCs cultivated on the scaffold. Both the non-cultivation and cultivation of TM-MSCs on scaffold promotes the expression of dopaminergic markers such as TH and Nurr-1. It is important to note that two dimensional culture on TCPS, which is considered standard to show cellular behavior, is not a perfect condition. Scaffolds have 3D characteristics which best recapitulate the native environment of cells and mimic some aspects of the natural Extra Cellular Matrix (ECM) [44]. In this work, the TM-MSCs cultivated on the scaffolds can alter the expression of the analyzed genes in comparison to TCPS. Although these differences are not significant, it is possible that the interaction between the TM-MSCs and the scaffolds can increase the expression of the specific neuronal genes as a result of physical and chemical signals current in 3D culture [45]. This result has called into question the clinical applicability of TM-MSCs cultured on PLLA-scaffold for dopaminergic cell engineering. This study is a first to report dopaminergic differentiation of TM-MSCs cultivated in a PLLA nanofibrous scaffold.
Please cite this article in press as: Jamali S, et al., Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.09.002
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Together, the results in this study demonstrate that nanofiber scaffolds support differentiation of TM-MSCs to dopaminergic-like cells. Acknowledgment This work was supported by Zanjan University of Medical Science, Deputy of Research and Technology (Grant No: A-10-129-6, Ethical No: ZUMS.REC.1394.144). The results presented in this article were extracted from MSc thesis of Mrs Simin Jamali. References [1] Dauer W, Przedborski S. Parkinson's disease: mechanisms and models. Neuron 2003;39:889e909. [2] Bjarkam CR, Sorensen JC, Sunde NA, Geneser FA, Ostergaard K. New strategies for the treatment of Parkinson's disease hold considerable promise for the future management of neurodegenerative disorders. Biogerontology 2001;2: 193e207. [3] Kleiner-Fisman G, Fisman DN, Sime E, Saint-Cyr JA, Lozano AM, Lang AE. Longterm follow up of bilateral deep brain stimulation of the subthalamic nucleus in patients with advanced Parkinson disease. J Neurosurg 2003;99:489e95. [4] Mandel S, Grunblatt E, Riederer P, Gerlach M, Levites Y, Youdim MB. Neuroprotective strategies in Parkinson's disease : an update on progress. CNS Drugs 2003;17:729e62. [5] Nadri S, Soleimani M, Mobarra Z, Amini S. Expression of dopamine-associated genes on conjunctiva stromal-derived human mesenchymal stem cells. Biochem Biophys. Res Commun 2008;377:423e8. [6] Lie DC, Dziewczapolski G, Willhoite AR, Kaspar BK, Shults CW, Gage FH. The adult substantia nigra contains progenitor cells with neurogenic potential. J Neurosci 2002;22:6639e49. [7] Snyder BJ, Olanow CW. Stem cell treatment for Parkinson's disease: an update for 2005. Curr Opin Neurol 2005;18:376e85. [8] Wang Y, Chen S, Yang D, Le WD. Stem cell transplantation: a promising therapy for Parkinson's disease. J Neuroimmune Pharmacol 2007;2:243e50. [9] Nadri S, Soleimani M, Kiani J, Atashi A, Izadpanah R. Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells. Differentiation 2008;76:223e31. [10] Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001;19:180e92. [11] Schwarz EJ, Alexander GM, Prockop DJ, Azizi SA. Multipotential marrow stromal cells transduced to produce L-DOPA: engraftment in a rat model of Parkinson disease. Hum Gene Ther 1999;10:2539e49. [12] Kunze A, Giugliano M, Valero A, Renaud P. Micropatterning neural cell cultures in 3D with a multi-layered scaffold. Biomaterials 2011;32:2088e98. [13] Tezel TH, Del Priore LV. Reattachment to a substrate prevents apoptosis of human retinal pigment epithelium. Graefes Arch Clin Exp Ophthalmol 1997;235:41e7. [14] Neeley WL, Redenti S, Klassen H, Tao S, Desai T, Young MJ, et al. A microfabricated scaffold for retinal progenitor cell grafting. Biomaterials 2008;29:418e26. [15] Soman P, Tobe BT, Lee JW, Winquist AA, Singec I, Vecchio KS, et al. Threedimensional scaffolding to investigate neuronal derivatives of human embryonic stem cells. Biomed Microdevices 2012;14:829e38. [16] Soleimani M, Nadri S, Shabani I. Neurogenic differentiation of human conjunctiva mesenchymal stem cells on a nanofibrous scaffold. Int J Dev Biol 2010;54:1295e300. [17] Nadri S, Yazdani S, Arefian E, Gohari Z, Eslaminejad MB, Kazemi B, et al. Mesenchymal stem cells from trabecular meshwork become photoreceptorlike cells on amniotic membrane. Neurosci Lett 2013;541:43e8. [18] Nadri S, Kazemi B, Eslaminejad MB, Yazdani S, Soleimani M. High yield of cells committed to the photoreceptor-like cells from conjunctiva mesenchymal stem cells on nanofibrous scaffolds. Mol Biol Rep 2013;40:3883e90. [19] Cotzias GC, Van Woert MH, Schiffer LM. Aromatic amino acids and modification of parkinsonism. N Engl J Med 1967;276:374e9. [20] Olanow CW, Tatton WG. Etiology and pathogenesis of Parkinson's disease. Annu Rev Neurosci 1999;22:123e44. [21] Bjorklund A, Lindvall O. Cell replacement therapies for central nervous system
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Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds Simin Jamali a, Hossein Mostafavi a, Ghasem Barati b, Mehdi Eskandari a, *, Samad Nadri b, c, ** a b c
Department of Physiology and Pharmacology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran Department of Medical Biotechnology and Nanotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran Cancer and Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 May 2017 Received in revised form 6 September 2017 Accepted 11 September 2017 Available online xxx
Parkinson's disease (PD) is a neurodegenerative disorder of the brain which is produced by the damage to dopaminergic neurons. Stem cell transplantation with a nanofibrous scaffold is one of the encouraging strategies for Parkinson's disease therapy. In this study, human mesenchymal stem cells (MSCs) from eye trabecular meshwork (TM) were differentiated into dopaminergic neurons on nanofibrous scaffold. After Trabecular meshwork biopsy, MSCs were isolated, cultivated on Poly-L-Lactide Acid (PLLA) nanofibrous scaffold (fabricated by electrospinning methods) and treated with medium containing DMEM supplemented with RA, IBMX and forskolin for 7 days. Scanning electron microscopy imaging, qPCR and immunostaining were used to analyze differentiated TM-MSCs on scaffold and their expression of dopaminergic-specific markers such as TH and Nurr-1. qPCR analysis revealed the expression of dopaminergic neuron genes such as TH, Nurr-1 on fibrous scaffold as well as TCPS. Immunostaining revealed that the differentiated TM-MSCs on TCPS and Scaffold not only express TH and Nurr-1 genes, but also express TH protein. In conclusion, the results indicate that TM-MSCs might be a suitable source for cell transplantation therapy. In addition the nanofibrous scaffold reported herein could be used as a potential cell carrier for the central system diseases such as PD. © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Trabecular meshwork mesenchymal stem cells Dopaminergic neurons Nanofibrous scaffold
1. Introduction Parkinson's disease (PD) is a neurodegenerative disorder of the brain which is produced by damage to dopaminergic neurons and a reduction of dopamine in the substantia nigra and in the striatum, respectively [1e3]. Although many treatment strategies such as administration of drugs, have been designed for this disease, they have shown an imperfect efficacy [4]. In recent years, stem cell therapy has emerged as a promising alternative treatment for Parkinson's disease (PD) [5].
* Corresponding author. Physiology and Pharmacology Department, P.O.Box: 14115-111, Zanjan, Iran. ** Corresponding author. Medical Biotechnology and Nanotechnology Department, P.O.Box: 14115-111, Zanjan, Iran. E-mail addresses: [email protected] (M. Eskandari), [email protected] (S. Nadri).
Despite the fact that human embryonic stem cells (ESCs) and neural stem cells (NSCs) are considered well-accepted cell sources with the capacity to differentiate into dopaminergic neurons [6], The clinical use of ESCs and NSCs has presented problems including teratoma formation as well as eliciting immune rejection after transplantation, low efficiency of differentiation and lineage polarization [7]. Based on these facts, additional studies are necessary to identify other stem cell sources with the ability to produce dopaminergic neurons with high efficiency. Mesenchymal stem cells (MSCs) have been shown to be a reliable cell source for PD treatment [8]. MSCs are multipotent stem cells that have been isolated from a wide variety of tissues and can be differentiated into neurons [9]. MSCs are currently in clinical trials for a number of disorders such as multiple sclerosis [10,11] and PD [11]. Human brain microenvironments are highly complex, with threedimensional (3D) structures involving the organized interaction
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Please cite this article in press as: Jamali S, et al., Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.09.002
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of multiple neural cell types with other cells (microglia and endothelial cells) [12]. One option for improving the physiological relevance of human neuronal culture models is to use these cells in combination with 3D scaffolds. Several studies have indicated that cell transplantation with a biocompatible polymer scaffold increases survival [13] and differentiation [14] of the cells. Scaffolds are the key constituents of tissue engineering for stem cells delivery. To date, several studies have been conducted on the effects of 3D electrospun scaffolds of a variety of biomaterials to support neural differentiation of stem cells [15]. While 3D scaffolds have been used to engineer neurocyte celltype specific constructs, there have been few reports on the successful application of a single 3D scaffold to support cell differentiation into specific types of neurocyte cells [16]. In the present study, Trabecular meshwork Mesenchymal stem cells (TM-MSCs) were differentiated into dopaminergic-like neuron on a nanofibrous scaffold in vitro. Taken together, a combination of TM-MSCs (as a new source of cells) and a nanofibrous scaffold could provide a strategy for Parkinson disease therapy. 2. Materials and methods
20 min and treated under UV irradiation for 10 min. Nanofibrous PLLA scaffolds were placed in 70 mm2 culture dishes and 3 105 of the TM-MSCs were cultivated on them. For differentiation, the cells were incubated in an induction medium that consisted of DMEM (low glucose), supplemented with 10 mM retinoic acid (RA, sigma), 0.5 mM IBMX and 10 mM Forskolin for 7 days. Cultivated cells on a tissue culture polystyrene (TCPS) plate were maintained in induction medium and culture medium as a two dimensional culture group (as a control group). At the end of this period, the cells were used for qPCR and Immunofluorescence analysis. The expression levels of the genes and proteins were compared with untreated cells. 2.4. Immunofluorescence analysis The cells were fixed (with 4% paraformaldehyde) and permeabilized with 0.5% Triton X-100 for 10 min. Then the cells were reacted with primary antibodies for TH (SANTA CRUZ BIOTECHNOLOGY,INC) at 4 C for 24 h, and subsequently reacted with the FITC-conjugated IgG as the secondary antibody (SANTA CRUZ BIOTECHNOLOGY,INC) at room temperature for 1 h. In addition, the cells were incubated with diaminobenzidine (DAB) solution (Sigma Chemical Co.) for 30 s for nuclear staining.
2.1. Isolation and differentiation into mesenchymal lineage cells 2.5. Gene expression This research was performed in accordance with the Helsinki Declaration and with approval of the ethics board of the Zanjan University of medical Sciences. Eye globes were obtained from the Central Eye Bank of Iran. TM-MSCs were isolated according to a protocol modified by Nadri et al. [17]. TM biopsy was incubated in PBS (GIBCO-BRL, Grand Island, NY) supplemented with 40 mg/mL BSA and 4 mg/mL collagenase for 1 h. The isolated stromal tissue segment was cultured in low glucose DMEM (GIBCO-BRL, Grand Island, NY) supplemented with 20% serum (GIBCO-BRL, Grand Island, NY), 200 ng/mL basic-FGF (Peprotech, Rocky Hill, NJ), and was incubated at 37 C with 5% CO2 in a humidified chamber. After 2 weeks, the cells were trypsinized (0.25% Trypsin e GIBCO-BRL, Grand Island, NY) and expanded by two passages. To demonstrate the multipotent nature of the isolated cells, the cells were differentiated down three lineages over a period of 21 days as follows: osteogenic (DMEM including 50 mg/mL ascorbic acid 2-phosphate (Sigma Chemical Co. St Louis, MO), 10 nM dexamethasone (Sigma Chemical Co.), 10 mM b-glycerol phosphate (Sigma Chemical Co.), adipogenic (DMEM supplemented with 50 mg/mL indomethacine (Sigma Chemical Co) and 100 nM dexamethasone (Sigma Chemical Co), and chondrogenic (DMEM supplemented with 10 ng/mL transforming growth factor- b3 (TGF-b3; Sigma Chemical Co), bone morphogenetic protein- 6 (BMP-6), 107 M dexamethasone (Sigma Chemical Co.), 50 mg/mL ascorbate2-phosphate (Sigma Chemical Co.), and 50 mg/mL insulintransferrin- selenium (ITS; GIBCO- BRL) medium.
The total RNA was extracted by RNXPLUS (Sinaclon, Iran). Quantification and purity of RNA was determined using a spectrophotometer (nanodrop 2000, Wilmington, USA). cDNA was synthesized using PrimeScript 1st strand cDNA synthesis Kit (Takara. Japan) according to the manufacturer's procedure. Real-time PCR was performed with an Applied BiosystemsTM Real-Time PCR System (Life Technologies Corporation, USA). All genes were subjected to 40 cycles, which consisted of a 15 min for initial denaturation at 95 C, followed by forty cycles of 15 s at 95 C, 30s annealing at 57 C, and a final 30s extension at 72 C. Related specific primers are presented in Table 1. The rest software from the rotor-gene Q, based on Pfaffl mathematical methods, was used for statistical analysis of real time data and relative gene expression [18]. 3. Results Cell culture and In vitro differentiation of trabecular meshwork mesenchymal stem cells (TM-MSCs) into mesenchymal lineages. After 2 weeks, fibroblast-like cells with spindle-shaped morphology appeared on TCPS dishes. These cells were utilized for future experiments. To confirm their mesenchymal nature, the isolated cells were treated with appropriate osteo-, chondro- and adipoinduction media, and their differentiation was confirmed via appropriate staining including, alizarin red (for osteogenic differentiation), alcian blue (for chondrogenic differentiation) and oil red (for adipogenic differentiation) staining (Fig. 1 AeD).
2.2. Fabrication of electrospun nanofibrous PLLA scaffolds 3.1. Dopaminergic differentiation of TM-MSCs Nanofibrous PLLA scaffolds were fabricated using an electrospinning technique [18]. PLLA was dissolved in chloroform (9% w/ w) and added to the chloroform/N,N-dimethylformamide (DMF) (Sigma, Steinheim, Germany) solution (10:1). Aligned nanofibrous scaffolds were obtained using high (3000 rpm) speed rotating disk. 2.3. In vitro differentiation of TM-MSCs to dopaminergic neuron on PLLA scaffold For sterilization, the scaffolds were placed in 70 C alcohol for
Next, we studied the potential of TM-MSCs to differentiate into dopaminergic neural cells. To elucidate the dopaminergic neural differentiation potential of CJMSCs, these cells were cultured in neuroinductive medium including DMEM supplemented With RA, IBMX and Forskolin. During differentiation more morphological changes in cells were observed (Fig. 2). Following induction, bi and multipolar cells with multiple dendrites and cytoplasmic branches were observed 2, 5 and 7 days after induction in TM-MSCs (see Fig. 2). The SEM results indicated that the TM-MSCs were well
Please cite this article in press as: Jamali S, et al., Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.09.002
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Table 1 Primer sequences employed in this project. Genes
Primer sequences
Size (bp)
Annealing melt ( C)
TH
F: 50 - CAGTTCTCGCAGGACATTG-30 R: 50 - CCCGTAGGAGGACAGCAG-30 F: 50 - TGGGATGGTCAAAGAAGTG-30 R: 50 - GCCTGGAACCTGGAATAG-30 F: 50 -CCTGGCGTCGTGATTAGTG - 30 R: 50 - TCAGTCCTGTCCATAATTAGTCC-30
156
51
189
50
124
56
Nurr1 HPRT1
Fig. 1. Morphological characteristics and in vitro differentiation of trabecular meshwork cells into mesenchymal lineages. (A) TM cells have been differentiated into (B) mineralizing cells stained with alizarin red (C) Adipocytes stained with Oil red O (D) Chondrocytic lineage stained with alcian blue.
Fig. 2. Morphological changes in TM-MSCs after induction. The morphological changes were observed at 2 (AeB), 5(C) and 7 (D) days after induction.
Please cite this article in press as: Jamali S, et al., Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.09.002
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attached on electrospun PLLA scaffolds and produce neurite outgrowth along the direction of fiber orientation (under differentiation media) (Fig. 3). After 7 days, we examined the expression of dopaminergic protein and genes by immunofluorescence and qPCR. Immunofluorescence was used to analyze the intracellular Tyrosine hydroxylase (TH) protein. This protein was identified in the TM-MSCs cultivated on PLLA scaffold and TCPS after treatment in neuroinductive medium for 7 days (Fig. 4). 3.2. Gene expression The results related to the expression of two genes on PLLA scaffold and TCPS are shown in Fig. 5. As shown in Fig. 5, gene expression of TH and Nurr-1 was significantly higher in treated TM-MSCs in comparison with untreated cells. As shown in Fig. 5, qPCR analysis demonstrated that TH (1.343 fold; p 0.3) and Nurr-1 (1.564 fold; p 0.16) gene expression was higher on PLLA scaffolds compared with TCPS. 4. Discussion Parkinson's disease (PD) is the neurodegenerative disorder for
which a variety of different options such as levodopa drug administration [19,20] are currently used to reverse the symptoms of PD. However, the efficacy of such treatments gradually diminishes over time. New methods have been explored applying stem cells to repair the damaged dopaminergic system [21]. To date, different stem cells such as embryonic stem cells (ESCs) [22e24], and mesenchymal stem cells (MSCs) have been investigated as potential therapies for the treatment of PD diseases. However, the difficulties encountered using human ESCs have led to the search for other types of cells for treatment of PD disease [5]. Dopaminergic differentiation of MSCs has also been described [25]. The clinical use of MSCs from well-accepted sources such as bone marrow has presented problems including painful aspiration, contamination with non-MSCs, and the low yield of cells [26]. This has caused many scientists to consider alternative sources of MSCs for treatment of PD disease. This work aimed to differentiate TMMSCs into dopamine secreting cells. Eye Trabecular meshwork tissue has been identified as a source of multipotent stromal MSCs, which can be obtained by a less invasive method without any contamination with non-MSCs. In this work, we demonstrated that TM-MSCs expressed dopaminergic neuron genes after treatment with DMEM containing 10% FBS, IBMX, forskolin, and 10 mM retinoic acid for 1 week. Previous investigators have differentiated MSCs over 2 weeks, using a
Fig. 3. SEM micrographs of PLLA nanofibrous scaffolds. The PLLA scaffold contains aligned (A) fibers and TM-MSCs differentiated on it 7 days after induction(B).
Fig. 4. Immunostaining of cultivated cells on tissue culture polystyrene (TCPS) and PLLA fibrous scaffold. The cultivated cells on scaffold and TCPS were maintained in induction medium for 7 days and analyzed for expression of TH proteins. The cells on PLLA and TCPS were co-stained with 4,6-diamidino-2-phenylindole to visualize nuclei (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Jamali S, et al., Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.09.002
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Fig. 5. Investigation of dopaminergic-specific gene expression. Expression of dopaminergic genes in control (day 0) and differentiation culture (day 7) of cultivated cells on TCPS and PLLA. HPRT-1 is shown as a control for RNA sample quality. Rest software was used for gene expression analysis using real-time PCR data from the rotor-gene Q.
multistep protocol in a cocktail of dopaminergic medium consisting of DMEM supplemented with 10% FBS, sonic hedgehog (SHH), fibroblast growth factor, epidermal growth factor, N2 supplement, butylated hydroxyanisole, IBMX, RA and dbcAMP [25,27]. MSC-derived adipose tissue were differentiated into dopaminergic neuron-like cells using neural induction media containing FGF2, EGF, BMP-9, retinoic acid and heparin [28]. According to the results presented in this paper it seems that TM-MSCs were differentiated into dopaminergic neurons by a straightforward method compared with BMMSCs, human umbilical mesenchymal stem cells and MSCs-derived adipose tissue. In the present study, qPCR analysis demonstrated that dopaminergic genes including TH and Nurr-1 were expressed in TMMSCs. Furthermore, immunofluorescence revealed that TH protein was expressed in TM-MSCs. Several studies of dopaminergic induction reported TH expression in MSCs after induced differentiation [29e31]. A TH protein is the initial enzyme in the catecholamine synthesis pathway, required for the generation of dopamine and is considered the principal regulator of dopamine biosynthesis in the central neuron system and as a marker for dopaminergic neurons [32,33]. Nurr-1 plays a main role in the maintenance of the dopaminergic system of the brain [34]. Nurr1 induces tyrosine hydroxylase (TH) expression, which leads to differentiation into dopaminergic neurons [35]. The results of this work showed that TM-MSCs were acquired through a simple approach and expressed TH and Nurr-1 genes and protein. The detection of TH and some other dopamine-associated genes such as Nurr1 necessary for both the survival and differentiation of the dopaminergic precursor's neurons [36,37] demonstrated that TM-MSCs can be differentiated into dopaminergic neurons and are good candidates for neurological cell therapy and for treating Parkinson's disease. For tissue engineering applications, it is essential to recognize the scaffold properties that on this construct cell
material interactions may influence the lineage fate decision of the stem cells [38,39]. Investigation of stem cell behaviour and the functional response to fibrous scaffolds has proved to be significantly challenging [15]. In the present study, we studied the influence of PLLA nanofibrous scaffold on differentiation of TM-MSCs to dopaminergic cells. To date several studies have been conducted on the effects of 3D electrospun scaffolds (as scaffold surfaces) of a variety of biomaterials including polyamide [40], poly (l-lactic acid) (PLLA) [41], poly (l-lactide-co-glycolide) (PLGA) [42] and polyurethane [43] to support neural differentiation of stem cells. In the present study, PLLA scaffolds were fabricated using electrospinning methods. In this work, after exposure to induction media, qPCR analysis and immunofluorescence indicated that specific dopaminergic genes including TH and Nurr1 were expressed in the TM-MSCs cultivated on the scaffold. Both the non-cultivation and cultivation of TM-MSCs on scaffold promotes the expression of dopaminergic markers such as TH and Nurr-1. It is important to note that two dimensional culture on TCPS, which is considered standard to show cellular behavior, is not a perfect condition. Scaffolds have 3D characteristics which best recapitulate the native environment of cells and mimic some aspects of the natural Extra Cellular Matrix (ECM) [44]. In this work, the TM-MSCs cultivated on the scaffolds can alter the expression of the analyzed genes in comparison to TCPS. Although these differences are not significant, it is possible that the interaction between the TM-MSCs and the scaffolds can increase the expression of the specific neuronal genes as a result of physical and chemical signals current in 3D culture [45]. This result has called into question the clinical applicability of TM-MSCs cultured on PLLA-scaffold for dopaminergic cell engineering. This study is a first to report dopaminergic differentiation of TM-MSCs cultivated in a PLLA nanofibrous scaffold.
Please cite this article in press as: Jamali S, et al., Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.09.002
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Together, the results in this study demonstrate that nanofiber scaffolds support differentiation of TM-MSCs to dopaminergic-like cells. Acknowledgment This work was supported by Zanjan University of Medical Science, Deputy of Research and Technology (Grant No: A-10-129-6, Ethical No: ZUMS.REC.1394.144). The results presented in this article were extracted from MSc thesis of Mrs Simin Jamali. References [1] Dauer W, Przedborski S. Parkinson's disease: mechanisms and models. Neuron 2003;39:889e909. [2] Bjarkam CR, Sorensen JC, Sunde NA, Geneser FA, Ostergaard K. New strategies for the treatment of Parkinson's disease hold considerable promise for the future management of neurodegenerative disorders. Biogerontology 2001;2: 193e207. [3] Kleiner-Fisman G, Fisman DN, Sime E, Saint-Cyr JA, Lozano AM, Lang AE. Longterm follow up of bilateral deep brain stimulation of the subthalamic nucleus in patients with advanced Parkinson disease. J Neurosurg 2003;99:489e95. [4] Mandel S, Grunblatt E, Riederer P, Gerlach M, Levites Y, Youdim MB. Neuroprotective strategies in Parkinson's disease : an update on progress. CNS Drugs 2003;17:729e62. [5] Nadri S, Soleimani M, Mobarra Z, Amini S. Expression of dopamine-associated genes on conjunctiva stromal-derived human mesenchymal stem cells. Biochem Biophys. Res Commun 2008;377:423e8. [6] Lie DC, Dziewczapolski G, Willhoite AR, Kaspar BK, Shults CW, Gage FH. The adult substantia nigra contains progenitor cells with neurogenic potential. J Neurosci 2002;22:6639e49. [7] Snyder BJ, Olanow CW. Stem cell treatment for Parkinson's disease: an update for 2005. Curr Opin Neurol 2005;18:376e85. [8] Wang Y, Chen S, Yang D, Le WD. Stem cell transplantation: a promising therapy for Parkinson's disease. J Neuroimmune Pharmacol 2007;2:243e50. [9] Nadri S, Soleimani M, Kiani J, Atashi A, Izadpanah R. Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells. Differentiation 2008;76:223e31. [10] Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001;19:180e92. [11] Schwarz EJ, Alexander GM, Prockop DJ, Azizi SA. Multipotential marrow stromal cells transduced to produce L-DOPA: engraftment in a rat model of Parkinson disease. Hum Gene Ther 1999;10:2539e49. [12] Kunze A, Giugliano M, Valero A, Renaud P. Micropatterning neural cell cultures in 3D with a multi-layered scaffold. Biomaterials 2011;32:2088e98. [13] Tezel TH, Del Priore LV. Reattachment to a substrate prevents apoptosis of human retinal pigment epithelium. Graefes Arch Clin Exp Ophthalmol 1997;235:41e7. [14] Neeley WL, Redenti S, Klassen H, Tao S, Desai T, Young MJ, et al. A microfabricated scaffold for retinal progenitor cell grafting. Biomaterials 2008;29:418e26. [15] Soman P, Tobe BT, Lee JW, Winquist AA, Singec I, Vecchio KS, et al. Threedimensional scaffolding to investigate neuronal derivatives of human embryonic stem cells. Biomed Microdevices 2012;14:829e38. [16] Soleimani M, Nadri S, Shabani I. Neurogenic differentiation of human conjunctiva mesenchymal stem cells on a nanofibrous scaffold. Int J Dev Biol 2010;54:1295e300. [17] Nadri S, Yazdani S, Arefian E, Gohari Z, Eslaminejad MB, Kazemi B, et al. Mesenchymal stem cells from trabecular meshwork become photoreceptorlike cells on amniotic membrane. Neurosci Lett 2013;541:43e8. [18] Nadri S, Kazemi B, Eslaminejad MB, Yazdani S, Soleimani M. High yield of cells committed to the photoreceptor-like cells from conjunctiva mesenchymal stem cells on nanofibrous scaffolds. Mol Biol Rep 2013;40:3883e90. [19] Cotzias GC, Van Woert MH, Schiffer LM. Aromatic amino acids and modification of parkinsonism. N Engl J Med 1967;276:374e9. [20] Olanow CW, Tatton WG. Etiology and pathogenesis of Parkinson's disease. Annu Rev Neurosci 1999;22:123e44. [21] Bjorklund A, Lindvall O. Cell replacement therapies for central nervous system
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Please cite this article in press as: Jamali S, et al., Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds, Biologicals (2017), http://dx.doi.org/10.1016/j.biologicals.2017.09.002