ll Re Ce sea m rc te h & S f Liu et al., J Stem Cell Res Ther 2015, 5:9 T o h l e a r n a Stem Cell r p http://dx.doi.org/10.4172/2157-7633.1000304 u y o J ISSN: 2157-7633 Research & Therapy 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 Page 2 of 6 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