Patent Application: US-78873210-A

Abstract:
the present invention provides a method of inducing myelination of isolated motoneurons by preparing a non - biological substrate having thereon a covalently attached monolayer of deta ; depositing isolated motoneurons on the substrate in a defined serum - free medium ; plating isolated schwann cells cultured in the defined serum - free medium onto the motoneurons , thereby initiating a co - culture ; and passaging the co - culture as necessary into fresh , defined serum - free medium supplemented with l - ascorbic acid at least until the motoneurons form nodes of ranvier indicative of myelination . the invention also includes a method of testing for new drugs effective in demyelinating diseases . additionally , cellular products provided by the invention include an isolated motoneurons myelinated or remyelinated in vitro according to the methods disclosed .

Description:
the present invention will now be described more fully hereinafter with reference to the accompanying drawings , in which preferred embodiments of the invention are shown . unless otherwise defined , all technical and scientific terms used herein are intended to have the same meaning as commonly understood in the art to which this invention pertains and at the time of its filing . although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention , suitable methods and materials are described below . however , the skilled should understand that the methods and materials used and described are examples and may not the only ones suitable for use in the invention . moreover , it should also be understood that as measurements are subject to inherent variability , any temperature , weight , volume , time interval , ph , salinity , molarity or molality , range , concentration and any other measurements , quantities or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary . hence , where appropriate to the invention and as understood by those of skill in the art , it is proper to describe the various aspects of the invention using approximate or relative terms and terms of degree commonly employed in patent applications , such as : so dimensioned , about , approximately , substantially , essentially , consisting essentially of , comprising , and effective amount . further , any publications , patent applications , patents , and other references mentioned herein are incorporated by reference in their entirety as if they were part of this specification . however , in case of conflict , the present specification , including any definitions , will control . in addition , the materials , methods and examples given are illustrative in nature only and not intended to be limiting . accordingly , this invention may be embodied in many different forms and should not be construed as limited to the illustrated embodiments set forth herein . rather , these illustrated embodiments are provided so that this disclosure will be thorough , complete , and will fully convey the scope of the invention to those skilled in the art . other features and advantages of the invention will be apparent from the following detailed description , and from the claims . glass coverslips ( vwr 48366067 , 22 × 22 mm 2 no . 1 ) were first cleaned using 1 : 1 hcl - methanol followed by a concentrated h2so4 soak for 2 h . the deta ( united chemical technologies inc . t2910 - kg ) film was formed by the reaction of the cleaned surfaces with a 0 . 1 % ( v / v ) mixture of the organosilane in freshly distilled toluene ( vwr bdh1151 ). the cleaned surfaces were heated to about 100 ° c . in the organosilane mixture , rinsed with toluene , reheated to about 100 ° c . in toluene , and then dried in the oven overnight ( 100 ° c .). surfaces were characterized by static water contact angle measurements using a rame - hart model 250 goniometer , and by x - ray photoelectron spectroscopy ( xps ) using an escalab 200i spectrometer ( vg scientific ) by monitoring the n1s peak [ 15 - 17 ]. the values are reported as the mean ± sem . dated pregnant sprague - dawley rats were housed in an animal facility at the university of central florida . all research was approved by the institutional animal care and use committee at the university of central florida and conformed to nih guidelines . pregnant rats were anesthetized and sacrificed at embryonic day 15 , embryos were removed by caesarean section and fetuses dissected under a stereo microscope ( carl zeiss , stemi , 2000 ). rat spinal cord motoneurons were purified from the ventral horn cords from embryonic day 15 ( e15 ) embryos as described by henderson et al . [ 18 ]. briefly , pregnant rats were anaesthetized and killed by inhalation of excess co 2 . spinal cords were removed from the e15 pups and the ventral horn tissue was dissected out and digested in 0 . 05 % trypsin - edta for 15 min in a 37 ° c . water bath ( gibco 25300 - 120 ). following incubation , the trypsin - edta was aspirated and the cells suspended in dissection media 10 % fbs and the tissue gently triturated . the dissociated cell suspension was then centrifuged at 500 g for 10 min at 4 ° c . to pellet the cells . next , the tissue was layered on a density gradient of opti - prep ( sigma d1556 ) solution and centrifuged at 500 g for 15 min at 4 ° c . after centrifugation , the resulting top two bands were collected in a 15 ml tube and the pellet discarded . the ventral horn cells were then applied to an immuopanning dish coated with goat affinity purified antibody to rat igg and the low affinity nerve growth factor receptor p75 ( chemicon mab365 ) in dissection medium for 45 min . this positive selection process provides attachment for the motoneurons while the other cells remain in suspension . after immuopanning the non - adherent cells were aspirated and the adherent motoneurons were removed from the dish in dissection medium to a 15 ml tube . lastly , the neurons were pelleted by centrifugation at 500 g for 10 min and then resuspended in culture medium and plated at 100 cells / mm 2 ( table 1 ). primary rat schwann cells ( sc ) were cultured from neonatal rat sciatic nerves as described originally by brockes et al . [ 19 ]. briefly , sciatic nerves from newly born sprague - dawley ( charles river : raleigh , n . c .) rat pups were dissected from the hind limb and then digested with 0 . 3 % collagenase in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem )+ 10 % fbs , forskolin and pituitary extract on poly - l - lysine coated 100 mm tissue culture dishes . after two days in culture , fibroblasts were eliminated using thy1 . 1 antibody / complement mediated lysis ( chemicon mab1406 ). purified sc cultures were passaged no more than three times before plating with the embryonic motoneurons for the myelination experiments . the co - cultures were fixed in fresh 4 % paraformaldehyde in pbs for 5 min and then rinsed twice with pbs . next , cells were permeabilized with a solution of 0 . 5 % triton - x 100 in pbs + 5 % bovine serum albumin ( bsa ) for 5 min . rinsed once with pbs and then blocked with permeabilization solution + 5 % donkey serum . the cells were then incubated with primary antibody solutions in blocking buffer overnight at 4 ° c . the following primary antibodies were obtained commercially from chemicon : anti - neurofilament heavy chain ( 1 : 12 , 000 ) ( ab5539 ), anti - voltage - gated sodium channel pan ( 1 : 200 ) ( ab5210 ), anti - voltage gated potassium channel ( 1 : 200 ) ( ab5483 ) and mbp ( 1 : 40 ) ( mab382 ). the anti - caspr antibody ( 1 : 500 ) ( sc - 14340 ) was obtained from santa cruz biotechnology , inc . the next day primary antibody solutions were aspirated and the cells rinsed three times with pbs . then , alexa - fluor 488 nm , 594 nm and 647 nm secondary antibodies diluted 1 : 200 in blocking solution were added to the cells and incubated for 2 h at room temperature in the dark . the secondary antibody solution was then aspirated and the coverslips rinsed three times in pbs and allowed to dry . finally , coverslips were mounted on glass slides using vectashield mounting medium with dapi ( vector labs , h - 1200 ) and fixed using clear nail polish . the aminosilane , trimethoxy - silylpropyl - diethylenetriamine ( deta ), functions efficiently as a non - biological substrate due to its self - assembling monolayer properties and the multiple amines contained in the terminal group . this group confers hydrophilic properties to the surface , and that combined with the partial positive change on the amines at physiological ph make it an ideal surface for neuronal cellular attachment and survival . the system is similar to poly - d - lysine , but has been found to be more robust and consistent [ 11 ]. xps measurements of the deta coated coverslips indicated a complete monolayer formed during the self - assembly process ( fig1 ). the normalized area values of n1s ( 401 and 399 ev ) to the si 2p 3 / 2 peaks were stable throughout the study at 1500 200 and were similar to previously published results ( fig1 a - c ) [ 11 , 14 , 15 , 17 , 20 ]. static contact angle measurements of 45 . 6 ± 2 validated the hydrophilicity of the deta surfaces ( fig1 d ). stable xps readings and contact angles across coverslips throughout the study indicate uniformity and reproducibility of the self - assembly of the deta monolayer . as previously reported , embryonic and adult motoneurons , grown in serum - free medium on deta recovered morphologically and electrically , firing repetitive action potentials under patch clamp conditions [ 12 ]. in this study , rat motoneurons and schwann cells were isolated and grown in serum - free medium on deta substrates . the defined medium formulation described in table 1 supported the growth and development of motoneurons and schwann cells as shown in fig2 . rat motoneurons and schwann cells were first individually isolated and grown separately as controls to ensure suitable morphology . in the individual cultures these motoneurons developed a singular axonal process and branching dendritic field ( fig2 a ). schwann cells exhibited a spindle - like morphology characteristic of this cell type ( fig2 b ). cultured together , motoneurons and schwann cells exhibited similar morphologies to the individual cultures ( fig2 c ). furthermore , with the temporal supplementation of ascorbic acid , schwann cells formed myelin sheaths and this also resulted in the subsequent clustering of the nodal proteins ( fig3 and 4 ). as compact myelin forms around neuronal axons , schwann cells express mbp as a component of the myelin sheath . using immunocytochemistry , mbp expression was evaluated as a standard for compact myelin formation in the culture system for day 25 to day 30 . the neuronal processes were imaged using anti - neurofilament - h ( nf - h ) antibodies and then the fluorescence co - localization was determined using the two antibodies . myelin segments were observed in motoneuron + schwann cell co - cultures ( fig3 ). after staining , myelin segments were quantified in order to determine the efficiency of schwann cell myelination in the co - culture system . as shown in table 2 , 63 . 11 ± 1 . 70 myelinated segments per coverslip were identified in the motoneuron + schwann cell co - culture . additionally , myelination resulted in the rearrangement and clustering of voltage - gated sodium channels ( vgsc &# 39 ; s ) and voltage - gated potassium channels ( vgpc &# 39 ; s ) in the axonal segment . this clustering resulted in the formation of physiologically correct nodes of ranvier as defined below . in order to visualize nodal development in this system , immunocytochemistry was used to stain for vgsc &# 39 ; s , vgpc &# 39 ; s and caspr localized at the nodes . as shown in fig4 , vgsc &# 39 ; s were found clustered between two myelinated segments of a motoneuron axon , verifying node of ranvier formation ( fig4 a , b ). additionally , clusters of caspr ( fig4 c ) and vgpc &# 39 ; s ( fig4 d ) were also seen in this culture system . the presence of these nodal proteins indicates maturation of the nodes into the physiologically correct morphologies . after staining , the number of nodes was quantified in order to determine the efficiency of schwann cell myelination and node formation in the co - culture system . as shown in table 2 , the formation of 20 . 67 ± 0 . 61 nodes of ranvier was identified per coverslip . the development of an in vitro system defining the minimum requirements for the survival , maturation and myelination of a motoneuron + schwann cell co - culture represents a significant scientific and technological breakthrough . these experiments indicate that this medium formulation is sufficient to not only recover cellular functionality , but also to provide an environment conducive to further cell - cell interactions and relevant physiological development that results in physiologically correct node of ranvier formation . using this basic serum - free medium formulation we have also shown the ability to grow dorsal root ganglia sensory neurons and both intrafusal and extrafusal muscle fibers [ 21 - 23 ]. the ability of the same basic serum - free medium formulation to sustain growth and facilitate myelination of a variety of interacting cell types facilitates future studies where all cells could be combined ( table 1 ). for example , studying motoneuron / sensory neuron electrical connectivity or recreating the stretch reflex arc in vitro will require all of these cell types to be in close proximity and will be more easily achieved using one basic medium formulation . this also is an essential requirement for drug discovery applications . furthermore , the reported importance of culturing motoneurons , sensory neurons and schwann cells together with muscle to form a significant number of neuromuscular junctions in vitro makes this basic medium even more critical [ 24 , 25 ]. schwann cell interaction with axons in the periphery is essential for efficient myelin sheath formation . here we have shown both myelin sheath formation and subsequent development of nodes of ranvier using this defined in vitro system ( fig3 and 4 ). the quantity of myelinated segments relative to nodes of ranvier indicate that not all myelinated segments formed in such a fashion as to result in the clustering of nodal proteins . while the processing of nodal proteins is influenced by the presence of myelinating schwann cells opposing the initial segment , it is not known what regulates the schwann cell “ decision ” to elongate an initial myelin segment or begin the process of forming a new segment . the likely candidate are interactions between the motoneuron and the extra - nodal proteins of the myelinating schwann cell [ 26 ]. due to the significant level of physiological development , the system also provides a model for further investigation into the potential molecular differences between schwann cell interaction with motoneurons and sensory neurons . for example , it could be useful in the evaluation of additional factors that could play a role in enhancing motoneuron myelination and node formation relative to sensory neurons . this is especially true for evaluating factors that are normally abundant in serum infused medium formulations typically used to facilitate schwann cell myelination of sensory neurons . deta &# 39 ; s utility from a bioengineering standpoint stems from its defined and reproducible nature . its role here , as a biomimetic , hydrophilic growth substrate , is especially useful because we believe it is not degraded by the cells plated on it and because it easily facilitates the study of deposited extracellular matrix molecules on the growth surface by the cells . deta can be coated onto any hydroxylated surface or material . all of these features make deta a useful substrate for bioengineering applications , a major goal in hybrid electronic systems , tissue engineering and cell - based biosensors . consequently , deta coated micro - electro - mechanical systems ( mems ) devices like multi - electrode arrays ( meas ) can provide a high throughput system for evaluating the electrical differences between myelinated and non - myelinated neurons . as previous studies have indicated , the deposition of a basal lamina and the subsequent modification of that layer are required for the formation of schwann cell myelin [ 6 , 7 , 27 ]. therefore , the use of deta as the growth substrate for these experiments suggests that the neurons and / or the schwann cells are secreting sufficient extracellular matrix ( ecm ) components necessary for the formation of the myelin sheath . this raises the questions of which cells generate the basal lamina , which cells secrete what ecm proteins , and how the ecm deposition influences cell - cell interaction between neurons and schwann cells . these questions are currently under investigation in our laboratory . we have used a completely defined in vitro system to demonstrate node of ranvier formation by schwann cells on motoneurons with concurrent k channel clustering and caspr formation . the development of this system , one where motoneurons are myelinated by schwann cells , is a critical breakthrough in understanding the interactions between these two cell types and represents significant progress towards culturing a stretch reflex arc in vitro [ 24 , 28 , 29 ]. additionally , it provides a novel system to evaluate the utility of a variety of factors not easily analyzed using an in vivo model . such a system could provide enhancement to or recovery of myelin segments for patients suffering from demyelinating neuropathies . this defined system provides a reproducible model for studying schwann cell interactions with motoneurons as well as the myelination process , and most importantly , remyelination . accordingly , in the drawings and specification there have been disclosed typical preferred embodiments of the invention and although specific terms may have been employed , the terms are used in a descriptive sense only and not for purposes of limitation . the invention has been described in considerable detail with specific reference to these illustrated embodiments . it will be apparent , however , that various modifications and changes can be made within the spirit and scope of the invention as described in the foregoing specification and as defined in the appended claims . hickman j j . bhatia s k , quong j n , shoen p , stenger d a , pike c j , et al . rational pattern design for in - 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