Myelination of Motor Neurons Derived from Mouse Embryonic Stem Cells by Oligodendrocytes Derived from Mouse Embryonic Stem Cells

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Research Article Open Access Myelination of Motor Neurons Derived from Mouse Embryonic Stem Cells by Oligodendrocytes Derived from Mouse Embryonic Stem Cells in a Microfluidic Compartmentalized Platform Su Liu2#, Ping Xiang1, Aysel Cetinkaya Fisgin3, Visar Belegu2, Nitish V Thakor1,4, John W McDonald2* and In Hong Yang1,4*# 1Singapore Institute for Neurotechnology, National University of Singapore, Singapore 2The International Centre for Spinal Cord Injury, Hugo Moser Research Institute at Kennedy Krieger Institute, USA 3Institute of Biomedical Engineering, Bogazici University, Istanbul, Turkey 4Department of Biomedical Engineering, Johns Hopkins University, School of Medicine, Baltimore, USA #indicating co-first author *Equal Contribution

Abstract Neuronal cell death and demyelination are devastating aspects of neurological diseases, such as multiple sclerosis, and spinal cord injury. Stem cell derived neurons and oligodendrocytes have shown potential as therapeutics for replacement of damaged neurons and remyelination of demyelinated axons in the central nervous system (CNS). However, in some cases, the neurons and axons are damaged so severely that they should be replaced. In this paper, we examined the hypothesis that stem cell derived oligodendrocytes can myelinate axons of stem cell derived motor neurons in a microfluidic platform, which mimics the isolated in vivo environment. The polydimethylsiloxane (PDMS) microfluidic platform achieves compartmentalization of mouse embryonic stem cells (mESCs) derived motor neurons and mESCs derived oligodendrocytes, while allowing the axons of the motor neurons to pass through microchannels and reach the oligodendrocytes. As results show, axons of mESCs derived motor neurons were subjected to myelination by mESCs derived oligodendrocytes shown by myelin basic protein immunostaining and electron microscopy. These functioning neuron and oligodendrocyte units may be a very useful tool to study stem cell replacement therapies for nerve injuries where nerve reconstruction would be beneficial.

Keywords: Myelination; Embryonic stem cells (ESCs); Mouse ESCs Motor neurons are very sensitive to injury. Neurons may die or derived motor neurons; Mouse ESCs derived oligodendrocytes their axons may be damaged upon injury, thus leading to the loss of neurological function. In the damaged spinal cord, the innate ability to Abbreviations: ALS: Amyotrophic Lateral Sclerosis; BDNF: Brain- replace lost cells, repair damaged myelin, and regenerate axons is very Derived Neurotrophic Factor; CNS: Central Nervous System; EBs: limited. The formation of glial scar is the main physical and chemical Embryonic Bodies; ECM: Extracellular Matrix; ESCs: Embryonic Stem barrier to the regeneration of axons. Reactive astrocytes, the major Cells; FGF2: Basic Fibroblast Growth Factor; GDNF: Glial Cell Line- component of glial scar [5], secrete extracellular matrix (ECM) and Derived Neurotrophic Factor; mESCs: mouse Embryonic Stem Cells; inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs), MNs: Motor Neurons; MS: Multiple Sclerosis; NF: Neurofilament; which supress axonal growth [6,7]. Recent experimental interventions OPCs: Oligodendrocyte Progenitor Cells; PDGF: Platelet-Derived have been tried to overcome the inhibitory environment and to make Growth Factor; PDL: Poly-D-Lysine; PDMS: Polydimethylsiloxane; Pur: it more conducive to neuronal growth, such as neutralization of myelin Purmorphamine; RA: Retinoic Acid; SCI: Spinal Cord Injury; SMA: inhibitors [8] and degradation of ECM [9]. However, this solution is spinal Muscular Atrophy; TEM: Transmission Electron Microscopy far from clinical applications because only a small portion of neuron fibers regenerate and the length of regenerated axons is still limited Introduction [10]. Nevertheless, transplantation of stem cell derived neurons and oligodendrocytes is a potentially effective approach for cell replacement Severe Spinal cord injury (SCI) results in a serious central nervous in SCI. system damage that interrupts ascending and descending axonal pathways and results in the loss of neurological function below the site of injury. Neuronal cell death occurs in the primary injury, first by mechanical damage and is then followed by a secondary degenerative *Corresponding author: John W Mcdonald, The International Centre for Spinal Cord Injury, Hugo Moser Research Institute at Kennedy Krieger Institute, USA, stage due to the ensuing inflammation and demyelination processes E-mail: [email protected] [1]. Demyelination is the loss of myelin sheath surrounding the axons, In Hong Yang, Department of Biomedical Engineering, Johns Hopkins University, which renders CNS axons incapable of passing signals from the brain School of Medicine, Baltimore, USA, Tel: 4432875407; Fax: 410 9551498; E-mail: to other neurons. This demyelination occurs mainly due to the death [email protected] of endogenous oligodendrocytes, the myelin forming cells in the CNS Received September 03, 2015; Accepted September 14, 2015; Published [2,3]. Oligodendrocyte progenitor cells (OPCs) are potential targets September 16, 2015 to replace endogenous oligodendrocytes, as the OPCs are widely Citation: Liu S, Xiang P, Fisgin AC, Belegu V, Thakor NV, et al. (2015) Myelination considered precursor cells that differentiate into oligodendrocytes of Motor Neurons Derived from Mouse Embryonic Stem Cells by Oligodendrocytes during the development and adult stages [4]. However, the mechanism Derived from Mouse Embryonic Stem Cells in a Microfluidic Compartmentalized Platform. J Stem Cell Res Ther 5: 304. doi:10.4172/2157-7633.1000304 involved in the regulation of OPCs differentiation into oligodendrocytes is not fully understood. Moreover, whether the newly formed Copyright: © 2015 Liu S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted oligodendrocytes can survive in the demyelinating microenvironment use, distribution, and reproduction in any medium, provided the original author and and myelinate axons is still an open question. source are credited.

J Stem Cell Res Ther ISSN: 2157-7633 JSCRT, an open access journal Volume 5 • Issue 9 • 1000304 Citation: Liu S, Xiang P, Fisgin AC, Belegu V, Thakor NV, et al. (2015) Myelination of Motor Neurons Derived from Mouse Embryonic Stem Cells by Oligodendrocytes Derived from Mouse Embryonic Stem Cells in a Microfluidic Compartmentalized Platform. J Stem Cell Res Ther 5: 304. doi:10.4172/2157-7633.1000304

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Embryonic stem cells (ESCs) are derived from the inner cell mass This in vitro co-culture system for MNs and oligodendrocytes not only of the late blastocyst-stage embryos and are capable of self–renewal and aids differentiation of mESCs into MNs and oligodendrocytes, but also differentiation into all cell types in the body [11]. The availability of provides a good system to study the mechanisms of myelination that ESCs shows great prospect for clinical therapies in neurodegenerative can provide new knowledge to improve ESC-based myelination therapy. diseases because of their potential to differentiate into the specialized cell types. Stem cell therapy strategies include: replacement of the Material and Methods damaged neurons and glial cells; re-establishment of neuronal network; Cell culture production of neurotrophic factors which are conducive to the survival of host cells and axonal regeneration [12]. Several studies have shown The general process of mESCs differentiation into either successful transplantation of ESCs into an animal SCI model and oligodendrocytes or MNs is shown in Figure 1. Briefly, mESCs were reported motor function recovery [13,14]. Although stem cell derived cultured in the ESC growth medium that consisted of DMEM, 10% OPCs and MNs have shown neuroprotective effects in neurological FBS, 10% new-born calf serum, 1x Nucleosides, 2 mM L-glutamine diseases, the ability of stem cell derived OPCs to myelinate stem cell (Gibco), 0.1 mM 2-mercaptoethanol (Sigma) and human recombinant 6 derived motor neuron axons in order to regenerate new neurons with leukemia inhibitory factor (LIF 10 units Chemicon). Aggregates were myelination has not been examined. then induced. For motor neuron differentiation, embryoid bodies (EBs) were firstly induced from aggregates in DFK-10 medium consisted In this study, a novel myelination system in a two-compartmentalized DMEM/F12 medium, 10% knockout serum replacement, 1% N2 polydimethylsiloxane (PDMS) microfluidic platform has been supplement, glucose (4500mg/L), 2 mM L-glutamine (Gibco), Heparin established, in which the two compartments are connected through (1U/µl), 0.1 mM 2-mercaptoethanol (Sigma) for 2 days and then by microchannels. Two cell types, oligodendrocytes and MNs, were adding retinoic acid (RA) and purmorphamine (Pur) for another 6 days successfully induced from mESCs in the microfluidic device, and (2-/6+ RA, Pur). At day 9, glial cell derived neurotrophic factor (GDNF) myelination of MNs was observed through MBP expression as well as and Brain-derived neurotrophic factor (BDNF) (10 ng/ml; Peprotech) myelin observation with an transmission electron microscope (TEM). were added to the medium for 5-7 days to induce differentiation of EBs

Figure 1: Diagram showing differentiation process of mESCs into either motor neurons or oligodendrocytes and schematic of two-compartmentalized microfluidic platform. (A) Diagram showing motor neuron (MNs) and oligodendrocytes induction from mESCs. (B) For MN differentiation, EBs were induced from mESCs by adding 2-/6+RA and Pur for 8 days and followed treatment with GDNF and BDNF for 5 to 7 days. OPCs were derived from EBs in the presence of FGF2 and PDGF for 12 to 14 days. (C) Schematic overview of MNs and oligodendrocytes co-culture system in the two compartmentalized PDMS platform. Soma and axons of MNs are separated in this microfluidic platform. Only the axons pass through the microchannels and reach the OPCs compartment.

Page 3 of 6 into motor neurons. For oligodendrocyte lineage, EBs were induced (Pur), mESCs were induced to form embryonic bodies (EBs) at day in the ESC induction medium (ES cell growth medium absence of 8. From EBs, MNs were differentiated by adding growth factors as leukemia inhibitory factor) for 4 days and then treated with RA for GDNF+BDNF for continuously 5 to 7 days. Similarly, EBs induced from another 4 days (4-/4+ RA). At day 9, EBs were trypsinized and re- mESCs by treatment with RA only differentiated into oligodendrocyte suspended in the modified OPC differentiation medium (DMEM with lineage after around 14 days culture in the presence of FGF2 and BSA, Pyruvate, progesterone, putrescine, thyroxine, triiodothryonine, PDGF. MNs proceeded to mature in the culture and pass through the insulin, transferring, sodium selenite with basic fibroblast growth microchannel to reach the other compartment where oligodendrocytes factor (FGF2) and Platelet-derived growth factor (PDGF) treatment were fully differentiated from OPCs and ready to co-culture with (10 ng/ml, Peprotech, USA) for 12-14 days. To achieve further MNs. According to this schedule, differentiation of MNs and OPCs oligodendrocyte maturation, the growth factors were removed, and the can be started at the same time from mESCs and then co-cultured in cells were cultured in OPC medium with 0.5% FBS and 0.5% HS (Sato this microfluidic system. This co-culture system provides an easy and 0.5+0.5) for 1-2 weeks. flexible way to study the myelination of MNs. Diagram in Figure 1C shows how co-culture of MNs and Oligodendrocytes is achieved in Microfluidic Platform Preparation the microfluidic platform. Cell soma and axons are separated in two Two-compartmentalized microfluidic platforms were prepared compartments which are connected through an array of microchannels. according to Yang et al., [15,16]. Briefly, the master mold was fabricated Only the axons of MNs pass through the microchannels and reach the by a two-step photolithographic process. The platform contains 100 oligodendrocytes, while the cell soma are blocked. This system mimics parallel microchannels, each with 10 µm-width, 500 µm-length and 2.5 the in vivo isolated environment, where axons are myelinated by µm-height. Complementary PDMS replicas were formed by pouring oligodendrocytes far away from cell bodies, thus showing advantages PDMS prepolymer (mixed in a 10:1 ration with a cross linking catalyst, in myelination studies compared with the traditional in vitro model. Dow Corning, USA) over the silicon master, degasing for 1h and then Differentiation of mESCs into motor neurons and curing at 70°C in an oven for 1h. Wells were cut at both sides of the channels through a puncher. The PDMS cast was then plasma bonded oligodendrocytes to the cover glass (22×40 mm, #1 thickness, Menzel-Glaser, Germany) Motor neurons and oligodendrocyes are differentiated separately and left in the oven overnight to improve the bonding. in the presence of different growth factors. With GDNF and BDNF The wells were sterilized by autoclaving and further cleaned with treatment for around 5-7 days, mESCs derived EBs differentiated into motor neurons, which was verified through immunofluorescence ethanol and washed with dd water for several times before use. For + mESCs culture, the wells were coated with 100 µg/ml Poly-D-Lysine staining with various motor neuron markers. Extensive Tuj1 neuronal (PDL) overnight at RT and matrigel for 1h at 37°C. processes grew out from mESCs derived EBs (Figure 2A). Nuclear expression of motor neuron marker HB9 was also observed, suggesting Immunocytochemistry formation of MNs (Figure 2B). EBs were further dissociated and allowed to mature for 7 days. Formation of MNs was confirmed by Differentiated mESCs cultures were washed with 1x PBS for immunostaining with anti-Tuj1 (Figure 2C) and anti-Islet1, a specific 1 time and then fixed with 4% paraformaldehyde for 20 mins. After motor neuron marker (Figure 2D). washing 3 times with 1x PBS , the samples were blocked with 10% goat serum (Sigma, USA) and incubated with primary antibodies as Induction of oligodendrocytes from mESCs needs a long time anti-Tuj1 (Covance,USA), anti-HB9, anti-Islet1 (DSHB,USA), anti- compared with MNs. It takes around two weeks to induce OPCs from GFAP (ImmunoStar, USA), anti-A2B5, anti-O1, anti-O4 (gift, USA), mESCs derived EBs at the beginning and then around 7 days to induce anti-MBP, anti-CNPase and anti-neurofilament (Chemicon, USA) OPCs differentiation into mature oligodendrocytes by removal of overnight at 4°C. Next day, the cultures were incubated with secondary growth factors. After 14 days in the presence of growth factors FGF2 antibodies as Alexa Fluor 488 (Invitrogen, USA) and Cy3-conjugated and PDGF to induce oligodendrogenesis, cells were characterized Affinipure (Jackson ImmunoResearch, USA) after three washes with 1× by various makers of oligodendrocytes lineage. Most of the cells PBS. The nuclei were counterstained with Hoechst 33342 (Molecular are positively stained by anti-O1 antibody, a marker for immature Probes, USA). Images were then taken through a confocal microscope oligodendrocytes (Figure 3A and 3C) and few cells are immunostained (Olympus FluoviewTM FV-1000). with GFAP (Figure 3B and 3C), suggesting mESCs are differentiating into oligodendrocyte lineage. Different stages of oligodendrogenesis Electron Microscopy from mESC derived EBs were verified by various markers: bipolar Three weeks after co-culture of mESCs derived motor neurons and shaped A2B5 positive (Figure 3D) and multipolar shaped NG2 positive oligodendrocytes, cultures were fixed with 2% paraformaldehyde/2% cells (Figure 3E) for the immature oligodendrocyte progenitor cells, glutaraldehyde and then prepared for transmission electron microscopy O4 positive cells for the pre-oligodendrocytes (Figure 3F), O1 positive as previous described [15]. Images were taken on a Hitachi, H-7600 cells for the pre-myelinating oligodendrocytes (Fgiure 3G) and MBP/ TEM at the Electron Microscope unit. CNPase positive cells for the terminally mature oligodendrocytes (Figure 3H and 3I). Results Myelination of axons of motor neurons by oligodendrocytes Co-culture system of motor neurons and OPCs derived from Myelination of axons of motor neurons was confirmed in the mESCs in a two-compartmentalized platform microfluidic platform through two methods: immunostaining with In this study, a novel co-culture system of MNs and OPCs has been anti-MBP and myelin sheath observed by TEM. Many axons of MNs established in the microfluidic platform. Schematic diagram shows stretching out from EBs at 7 days after inducing the differentiation in the differentiation process of MNs and OPCs from mESCSc in Figure the MN compartment (Figure 4A). These axons successfully passed 1A and 1B. In the presence of retinoic acid (RA) and purmorphamine through the microchannels into the oligodendrocyte compartment,

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Figure 2: Characterization of mESCs derived motor neurons. Immunofluorescence staining for various motor neurons markers: anti-Tuj1 (green, A) and anti-HB9 (red, B) in EBs derived from mESCs; anti-Tuj1 (green, C) and anti- Islet-1 (red, D) in dissociated MN cultures by higher magnification image. Hoechst (blue) was used to identify the nuclei. Scale bar: 50 μm and 100 μm.

Figure 3: Immunofluorescence staining of different markers of oligodendrocyte lineage. After 12-14 days of GDNF and BDNF treatment, the cells were stained with anti-O1 (green, A) and anti-GFAP (red, B). Most of the cells are O1+, indicating mESCs are successfully induced to differentiate into oligodendrocytes. Oligodendrogenesis is further confirmed by immunostaining with oligodendrocyte lineage markers like anti-A2B5 (D), anti-NG2 (E), anti-O4 (F), anti-O1 (G), anti-MBP (H) and anti-CNPase (I). All cells are nuclear counterstained with Hoechst (blue). Scale bar: 100 µm.

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Figure 4: Myelination of MN axons by oligodendrocytes. (A) Axons from mESCs derived MNs are guided by the microchannels and reach to the oligodendrocyte compartment. (B) Myelin sheaths (yellow colour) are defined as completely overlap between MBP+ oligodendrocyte processes (green) and NF+ axons (red) (Ba and Bc) and higher magnification image (Bb and Bd, enlarged image from Ba and Bc in dotted lines). (C) The electron microscope image of a myelinated axon fiber. Multi-layer of myelin around the axon fiber is observed. Scale bar: 20 μm and 100 μm. while cell bodies were blocked. This compartmentalized platform without limitation in cell number. Likewise, the differentiation protocol separates the axons from cell bodies, which is advantageous for the study for mESCs into oligodendrocytes has been well established, so it is also of myelination with axons only. After successful establishment of the convenient to obtain oligodendrocytes derived from mESCs without above MN culture, mESC derived OPCs were placed into the other well. limitation in cell number [19,20]. Myelination of MN axons was studied through immunocytochemistry Moreover, an easy but efficientin vitro system to study the with anti-MBP (Figure 4B). Myelin sheaths were defined as structures of myelination of stem cell derived motor neurons by oligodendrocytes completely overlapped oligodendrocyte processes expressing MBP on has also been described. In the microfluidic platform, two Neurofilament (NF)-expressing axon fibers. As showing in the Figure compartments, a soma compartment and an axon/oligodendrocyte 4B, processes of MBP+ oligodendrocytes are partially co-localized with the NF+ axons of MNs (yellow colour) indicating that oligodendrocytes compartment are connected by arrays of microchannels separating are myelinating the axons to form myelin segments. Furthermore, TEM axons from neuronal soma and dendrites, allowing only the axons to imaging also confirms that these segments are myelin sheaths, showing interact with oligodendrocytes. Therefore, this compartmentalized by the thickness of axons (Figure 4C). system closely mimics the in vivo environment, where axons are myelinated by oligodendrocytes far away from cell bodies. This cannot Discussion be accomplished in conventional mixed culture experiments. With this novel culture platform, study of the mechanisms of myelination, such In this study, a highly reproducible in vitro model for myelination as the effects of growth factors and axon-glia signalling networks, can of MNs by oligodendrocytes, both derived from mESCs has been be easily achieved. established. This method has some advantages over other techniques. Firstly, mESCs provides a reliable source and large quantities of This study may provide some new knowledge useful in ESC both dissociated MNs and oligodendrocytes. It is well known that therapies. Studies have reported successful transplantation of ESCs into motor neurons are difficult to maintain for extended periods of time animals and subsequent improvement of motor function [13]. However, in dissociated tissues from mouse/rat spinal cord. As the protocols it is still not clear how to control the proliferation and differentiation of inducing the differentiation of mESCs into motor neurons are widely ESCs into the specific cell types and prevent tumor formation. Moreover, established [17,18], it provides a reproducible method to culture MNs the microenvironment around the injury site might not be conducive

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J Stem Cell Res Ther ISSN: 2157-7633 JSCRT, an open access journal Volume 5 • Issue 9 • 1000304