Received date September 03, 2015; Accepted date September 14, 2015; Published date September 16, 2015
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
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 use, distribution, and reproduction in any medium, provided the original author and source are credited.
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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.
Myelination; Embryonic stem cells (ESCs); Mouse ESCs derived motor neurons; Mouse ESCs derived oligodendrocytes
ALS: Amyotrophic Lateral Sclerosis; BDNF: Brain- Derived Neurotrophic Factor; CNS: Central Nervous System; EBs: Embryonic Bodies; ECM: Extracellular Matrix; ESCs: Embryonic Stem Cells; FGF2: Basic Fibroblast Growth Factor; GDNF: Glial Cell Line- Derived Neurotrophic Factor; mESCs: mouse Embryonic Stem Cells; MNs: Motor Neurons; MS: Multiple Sclerosis; NF: Neurofilament; OPCs: Oligodendrocyte Progenitor Cells; PDGF: Platelet-Derived Growth Factor; PDL: Poly-D-Lysine; PDMS: Polydimethylsiloxane; Pur: Purmorphamine; RA: Retinoic Acid; SCI: Spinal Cord Injury; SMA: spinal Muscular Atrophy; TEM: Transmission Electron Microscopy
Severe Spinal cord injury (SCI) results in a serious central nervous 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 stage due to the ensuing inflammation and demyelination processes . Demyelination is the loss of myelin sheath surrounding the axons, which renders CNS axons incapable of passing signals from the brain to other neurons. This demyelination occurs mainly due to the death of endogenous oligodendrocytes, the myelin forming cells in the CNS [2,3]. Oligodendrocyte progenitor cells (OPCs) are potential targets to replace endogenous oligodendrocytes, as the OPCs are widely considered precursor cells that differentiate into oligodendrocytes during the development and adult stages . However, the mechanism involved in the regulation of OPCs differentiation into oligodendrocytes is not fully understood. Moreover, whether the newly formed oligodendrocytes can survive in the demyelinating microenvironment and myelinate axons is still an open question.
Motor neurons are very sensitive to injury. Neurons may die or their axons may be damaged upon injury, thus leading to the loss of neurological function. In the damaged spinal cord, the innate ability to replace lost cells, repair damaged myelin, and regenerate axons is very limited. The formation of glial scar is the main physical and chemical barrier to the regeneration of axons. Reactive astrocytes, the major component of glial scar , secrete extracellular matrix (ECM) and inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs), which supress axonal growth [6,7]. Recent experimental interventions have been tried to overcome the inhibitory environment and to make it more conducive to neuronal growth, such as neutralization of myelin inhibitors  and degradation of ECM . However, this solution is far from clinical applications because only a small portion of neuron fibers regenerate and the length of regenerated axons is still limited . Nevertheless, transplantation of stem cell derived neurons and oligodendrocytes is a potentially effective approach for cell replacement in SCI.
Embryonic stem cells (ESCs) are derived from the inner cell mass of the late blastocyst-stage embryos and are capable of self–renewal and differentiation into all cell types in the body . The availability of ESCs shows great prospect for clinical therapies in neurodegenerative diseases because of their potential to differentiate into the specialized cell types. Stem cell therapy strategies include: replacement of the damaged neurons and glial cells; re-establishment of neuronal network; production of neurotrophic factors which are conducive to the survival of host cells and axonal regeneration . Several studies have shown successful transplantation of ESCs into an animal SCI model and reported motor function recovery [13,14]. Although stem cell derived OPCs and MNs have shown neuroprotective effects in neurological diseases, the ability of stem cell derived OPCs to myelinate stem cell derived motor neuron axons in order to regenerate new neurons with myelination has not been examined.
In this study, a novel myelination system in a two-compartmentalized polydimethylsiloxane (PDMS) microfluidic platform has been established, in which the two compartments are connected through microchannels. Two cell types, oligodendrocytes and MNs, were successfully induced from mESCs in the microfluidic device, and myelination of MNs was observed through MBP expression as well as myelin observation with an transmission electron microscope (TEM). This in vitro co-culture system for MNs and oligodendrocytes not only aids differentiation of mESCs into MNs and oligodendrocytes, but also provides a good system to study the mechanisms of myelination that can provide new knowledge to improve ESC-based myelination therapy.
The general process of mESCs differentiation into either oligodendrocytes or MNs is shown in Figure 1. Briefly, mESCs were cultured in the ESC growth medium that consisted of DMEM, 10% FBS, 10% new-born calf serum, 1x Nucleosides, 2 mM L-glutamine (Gibco), 0.1 mM 2-mercaptoethanol (Sigma) and human recombinant leukemia inhibitory factor (LIF 106 units Chemicon). Aggregates were then induced. For motor neuron differentiation, embryoid bodies (EBs) were firstly induced from aggregates in DFK-10 medium consisted DMEM/F12 medium, 10% knockout serum replacement, 1% N2 supplement, glucose (4500mg/L), 2 mM L-glutamine (Gibco), Heparin (1U/μl), 0.1 mM 2-mercaptoethanol (Sigma) for 2 days and then by adding retinoic acid (RA) and purmorphamine (Pur) for another 6 days (2-/6+ RA, Pur). At day 9, glial cell derived neurotrophic factor (GDNF) and Brain-derived neurotrophic factor(BDNF) (10 ng/ml; Peprotech) were added to the medium for 5-7 days to induce differentiation of EBs into motor neurons. For oligodendrocyte lineage, EBs were induced in the ESC induction medium (ES cell growth medium absence of leukemia inhibitory factor) for 4 days and then treated with RA for another 4 days (4-/4+ RA). At day 9, EBs were trypsinized and resuspended in the modified OPC differentiation medium (DMEM with BSA, Pyruvate, progesterone, putrescine, thyroxine, triiodothryonine, insulin, transferring, sodium selenite with basic fibroblast growth factor (FGF2) and Platelet-derived growth factor (PDGF) treatment (10 ng/ml, Peprotech, USA) for 12-14 days. To achieve further oligodendrocyte maturation, the growth factors were removed, and the cells were cultured in OPC medium with 0.5% FBS and 0.5% HS (Sato 0.5+0.5) for 1-2 weeks.
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.
Microfluidic Platform Preparation
Two-compartmentalized microfluidic platforms were prepared according to Yang et al., [15,16]. Briefly, the master mold was fabricated by a two-step photolithographic process. The platform contains 100 parallel microchannels, each with 10 μm-width, 500 μm-length and 2.5 μm-height. Complementary PDMS replicas were formed by pouring PDMS prepolymer (mixed in a 10:1 ration with a cross linking catalyst, Dow Corning, USA) over the silicon master, degasing for 1h and then 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 to the cover glass (22×40 mm, #1 thickness, Menzel-Glaser, Germany) and left in the oven overnight to improve the bonding.
The wells were sterilized by autoclaving and further cleaned with 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 (PDL) overnight at RT and matrigel for 1h at 37°C.
Differentiated mESCs cultures were washed with 1x PBS for 1 time and then fixed with 4% paraformaldehyde for 20 mins. After washing 3 times with 1x PBS , the samples were blocked with 10% goat serum (Sigma, USA) and incubated with primary antibodies as anti-Tuj1 (Covance,USA), anti-HB9, anti-Islet1 (DSHB,USA), anti- GFAP (ImmunoStar, USA), anti-A2B5, anti-O1, anti-O4 (gift, USA), anti-MBP, anti-CNPase and anti-neurofilament (Chemicon, USA) overnight at 4°C. Next day, the cultures were incubated with secondary antibodies as Alexa Fluor 488 (Invitrogen, USA) and Cy3-conjugated Affinipure (Jackson ImmunoResearch, USA) after three washes with 1× PBS. The nuclei were counterstained with Hoechst 33342 (Molecular Probes, USA). Images were then taken through a confocal microscope (Olympus Fluoview™ FV-1000).
Three weeks after co-culture of mESCs derived motor neurons and oligodendrocytes, cultures were fixed with 2% paraformaldehyde/2% glutaraldehyde and then prepared for transmission electron microscopy as previous described . Images were taken on a Hitachi, H-7600 TEM at the Electron Microscope unit.
Co-culture system of motor neurons and OPCs derived from mESCs in a two-compartmentalized platform
In this study, a novel co-culture system of MNs and OPCs has been established in the microfluidic platform. Schematic diagram shows the differentiation process of MNs and OPCs from mESCSc in Figure 1A and 1B. In the presence of retinoic acid (RA) and purmorphamine (Pur), mESCs were induced to form embryonic bodies (EBs) at day 8. From EBs, MNs were differentiated by adding growth factors as GDNF+BDNF for continuously 5 to 7 days. Similarly, EBs induced from mESCs by treatment with RA only differentiated into oligodendrocyte lineage after around 14 days culture in the presence of FGF2 and PDGF. MNs proceeded to mature in the culture and pass through the microchannel to reach the other compartment where oligodendrocytes were fully differentiated from OPCs and ready to co-culture with MNs. According to this schedule, differentiation of MNs and OPCs can be started at the same time from mESCs and then co-cultured in this microfluidic system. This co-culture system provides an easy and flexible way to study the myelination of MNs. Diagram in Figure 1C shows how co-culture of MNs and Oligodendrocytes is achieved in the microfluidic platform. Cell soma and axons are separated in two compartments which are connected through an array of microchannels. Only the axons of MNs pass through the microchannels and reach the oligodendrocytes, while the cell soma are blocked. This system mimics the in vivo isolated environment, where axons are myelinated by oligodendrocytes far away from cell bodies, thus showing advantages in myelination studies compared with the traditional in vitro model.
Differentiation of mESCs into motor neurons and oligodendrocytes
Motor neurons and oligodendrocyes are differentiated separately in the presence of different growth factors. With GDNF and BDNF treatment for around 5-7 days, mESCs derived EBs differentiated into motor neurons, which was verified through immunofluorescence staining with various motor neuron markers. Extensive Tuj1+ neuronal processes grew out from mESCs derived EBs (Figure 2A). Nuclear expression of motor neuron marker HB9 was also observed, suggesting formation of MNs (Figure 2B). EBs were further dissociated and allowed to mature for 7 days. Formation of MNs was confirmed by immunostaining with anti-Tuj1 (Figure 2C) and anti-Islet1, a specific motor neuron marker (Figure 2D).
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.
Induction of oligodendrocytes from mESCs needs a long time compared with MNs. It takes around two weeks to induce OPCs from mESCs derived EBs at the beginning and then around 7 days to induce OPCs differentiation into mature oligodendrocytes by removal of growth factors. After 14 days in the presence of growth factors FGF2 and PDGF to induce oligodendrogenesis, cells were characterized by various makers of oligodendrocytes lineage. Most of the cells are positively stained by anti-O1 antibody, a marker for immature oligodendrocytes (Figure 3A and 3C) and few cells are immunostained with GFAP (Figure 3B and 3C), suggesting mESCs are differentiating into oligodendrocyte lineage. Different stages of oligodendrogenesis from mESC derived EBs were verified by various markers: bipolar shaped A2B5 positive (Figure 3D) and multipolar shaped NG2 positive cells (Figure 3E) for the immature oligodendrocyte progenitor cells, O4 positive cells for the pre-oligodendrocytes (Figure 3F), O1 positive cells for the pre-myelinating oligodendrocytes (Fgiure 3G) and MBP/ CNPase positive cells for the terminally mature oligodendrocytes (Figure 3H and 3I).
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.
Myelination of axons of motor neurons by oligodendrocytes
Myelination of axons of motor neurons was confirmed in the microfluidic platform through two methods: immunostaining with anti-MBP and myelin sheath observed by TEM. Many axons of MNs stretching out from EBs at 7 days after inducing the differentiation in the MN compartment (Figure 4A). These axons successfully passed through the microchannels into the oligodendrocyte compartment, while cell bodies were blocked. This compartmentalized platform separates the axons from cell bodies, which is advantageous for the study of myelination with axons only. After successful establishment of the above MN culture, mESC derived OPCs were placed into the other well. Myelination of MN axons was studied through immunocytochemistry with anti-MBP (Figure 4B). Myelin sheaths were defined as structures of completely overlapped oligodendrocyte processes expressing MBP on Neurofilament (NF)-expressing axon fibers. As showing in the Figure 4B, processes of MBP+ oligodendrocytes are partially co-localized with the NF+ axons of MNs (yellow colour) indicating that oligodendrocytes are myelinating the axons to form myelin segments. Furthermore, TEM imaging also confirms that these segments are myelin sheaths, showing by the thickness of axons (Figure 4C).
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.
In this study, a highly reproducible in vitro model for myelination of MNs by oligodendrocytes, both derived from mESCs has been established. This method has some advantages over other techniques. Firstly, mESCs provides a reliable source and large quantities of both dissociated MNs and oligodendrocytes. It is well known that motor neurons are difficult to maintain for extended periods of time in dissociated tissues from mouse/rat spinal cord. As the protocols inducing the differentiation of mESCs into motor neurons are widely established [17,18], it provides a reproducible method to culture MNs without limitation in cell number. Likewise, the differentiation protocol for mESCs into oligodendrocytes has been well established, so it is also convenient to obtain oligodendrocytes derived from mESCs without limitation in cell number [19,20].
Moreover, an easy but efficient in vitro system to study the myelination of stem cell derived motor neurons by oligodendrocytes has also been described. In the microfluidic platform, two compartments, a soma compartment and an axon/oligodendrocyte compartment are connected by arrays of microchannels separating axons from neuronal soma and dendrites, allowing only the axons to interact with oligodendrocytes. Therefore, this compartmentalized system closely mimics the in vivo environment, where axons are myelinated by oligodendrocytes far away from cell bodies. This cannot be accomplished in conventional mixed culture experiments. With this novel culture platform, study of the mechanisms of myelination, such as the effects of growth factors and axon-glia signalling networks, can be easily achieved.
This study may provide some new knowledge useful in ESC therapies. Studies have reported successful transplantation of ESCs into animals and subsequent improvement of motor function . However, it is still not clear how to control the proliferation and differentiation of ESCs into the specific cell types and prevent tumor formation. Moreover, the microenvironment around the injury site might not be conducive to the neuronal differentiation of ESCs due to the acute inflammatory condition after injury. The expression of inflammatory cytokines such as TNF-α, IL-1 and IL-6, which are neurotoxic, increase sharply after injury, as reported . It seems to be difficult or impossible to control the environmental signals at the site of implantation. In order to avoid these potential problems, some researchers directly transplanted OPCs into the injury site instead of ESCs and observed enhanced remyelination and recovery of motor function . Since studies have reported that astroglial scar is correlated with the failure of remyelination [23,24], it suggested transplantation of OPCs at the early post-injury period after SCI. However, at the acute phase of injury, macrophages infiltrating from the blood and constitutive microglia are activated and may be toxic to the OPCs. Studies have shown that activated microglia are harmful to OPCs and reduce their survival [25,26].
Since the final goal of ESC therapy is to form myelinated axon fibers and restore neuronal circuitry in the injury site, one may consider cotransplantation of ESCs derived motor neurons and OPCs. Transplanted motor neurons may replace the lost MNs. Studies have shown that transplanted ESCs derived MNs supported the endogenous neurons survival through secretion of beneficial growth factors in the SCI model , spinal muscular atrophy (SMA) and Amyotrophic lateral sclerosis (ALS) model . Transplanted OPCs may differentiate into mature oligodendrocytes to myelinate both ESCs derived MNs and constitutive MNs. As shown in this study, ESCs derived oligodendrocytes could myelinate MNs to form myelin around axons. Moreover, ECSs derived OPCs may contribute to functional recovery in SCI through secretion of growth factors [29,30]. Therefore, it may beneficial to re-connect the neuron network in the injury centre through co-transplantation of MNs and OPCs.
In conclusion, the current results have shown that mESCs derived motor neurons can be myelinated by mESCs derived oligodendrocytes in a microfluidic platform. This in vitro co-culture system may be helpful for further study of the mechanisms of demyelination and remyelination that can be used in new ESC based therapies for SCI.