Method of co-culturing mammalian muscle cells and motoneurons

Abstract

The invention provides a method of co-culturing mammalian muscle cells and mammalian motoneurons. The method comprises preparing one or more carriers coated with a covalently bonded monolayer of trimethoxysilylpropyl diethylenetriamine (DETA); suspending isolated fetal mammalian skeletal muscle cells in serum-free medium according to medium composition 1; suspending isolated fetal mammalian spinal motoneurons in serum-free medium according to medium composition 1; plating the suspended muscle cells onto the one or more carriers at a predetermined density and allowing the muscle cells to attach; plating the suspended motoneurons at a predetermined density onto the one or more carriers and allowing the motoneurons to attach; covering the one or more carriers with a mixture of medium composition 1 and medium composition 2; and incubating the carriers covered in the media mixture.

Claims

1. A co-culture for forming at least one synthetic mammalian neuromuscular junction, comprising: fetal mammalian skeletal muscle cells adhered to an artificial surface and overlayered with fetal mammalian spinal motoneurons in a serum-free medium, wherein the serum-free medium comprises a mixture of the components of Table 3 at the concentrations listed in Table 3, the components of Table 4 at the concentrations listed in Table 4, the components of Table 5 at the concentrations listed in Table 5, creatine, estrogen, and cholesterol.

2. The co-culture of claim 1, wherein one or more of the fetal mammalian spinal motoneurons is functionally linked to one or more of the fetal mammalian skeletal muscle cells.

3. The co-culture of claim 1, wherein the artificial surface comprises a silicon based monolayer substrate deposited thereon.

4. The co-culture of claim 3, wherein the silicon based monolayer substrate is deposited in a predetermined pattern.

5. The co-culture of claim 3, wherein the silicon based monolayer substrate comprises trimethoxysilylpropyl diethylenetriamine (DETA).

6. The co-culture of claim 1, wherein the fetal mammalian skeletal muscle cells have a density of about from 700 to about 1000 cells/mm.sup.2.

7. The co-culture of claim 1, wherein the fetal mammalian spinal motoneurons have a density of about 100 cells/mm.sup.2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Some of the features, advantages, and benefits of the present invention having been stated, others will become apparent as the description proceeds when taken in conjunction with the accompanying drawings, presented for solely for exemplary purposes and not with intent to limit the invention thereto, and in which:

(2) FIG. 1 is a protocol for long-term NMJ formation in a motoneuron and skeletal muscle co-culture, according to an embodiment of the present invention;

(3) FIG. 2 shows phase contrast micrographs of the motoneuron and skeletal muscle co-culture between days 12-15. (A-D); red arrows indicate the distinct morphology of the motoneuron and its processes; green arrows indicate the myotubes; scale bar=25 m;

(4) FIG. 3 provides phase contrast pictures of the co-cultures between days 25-30; (A, B) the myotubes exhibited characteristic striations; (C, D) myotubes with striations and myotubes without striations; red arrows indicate the motoneuron cell body and the processes; green arrows indicate the myotubes; scale bar for A, B=40 m; scale bar for C, D=25 m;

(5) FIG. 4 shows the immunocytochemistry of co-cultures at day 25; (A-B) NF-150 (red) indicates the large motoneurons and their processes (white arrows); the striated myotubes (green) stained for nMHC (N3.36); scale bar=50 m;

(6) FIG. 5 depicts neuromuscular junction (NMJ) formation between day 30-40; (A) phase picture of the myotube indicating the alpha-bungarotoxin staining in green; (B) triple stain, showing the close proximity of alpha-bungarotoxin (green) and synaptophysin (blue) indicating synapse formation at a specific confocal plane and myotube striations are indicated in red (nMHC); (C-D) NMJ observed at two different planes using confocal microscopy; a much more dense clustering of synaptophysin and alpha-bungarotoxin was observed in these planes;

(7) FIG. 6 shows striated myotube development in the absence of NMJ formation; (A, B) no NMJs were observed on this striated myotube; (A) a phase picture of the myotube; (B) immunostained picture of the same myotube with alpha-bungarotoxin, N3.36 and synaptophysin; scale bar=50 m; and

(8) FIG. 7 depicts NMJ formation on an N3.36 () myotube; (A) phase picture showing the different morphologies of myotubes in the co-culture; (B-D) show that NMJ formation was observed on a myotube that was negative for N3.36; culture stained with alpha-bungarotoxin, N3.36 and synaptophysin; possibly the myotube on which NMJ was formed was immature and had not yet expressed the neonatal myosin heavy chain marker (N3.36).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(9) 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 have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although 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. Any publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. 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 and 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.

(10) Materials and Methods

(11) Surface Modification and Characterization

(12) Glass coverslips (Thomas Scientific 6661F52, 2222 mm No. 1) were cleaned using an O2 plasma cleaner (Harrick PDC-32G) for 20 min at 100 mTorr. The DETA (United Chemical Technologies Inc. T2910KG) films were formed by the reaction of the cleaned glass surface with a 0.1% (v/v) mixture of the organosilane in freshly distilled toluene (Fisher T2904). The DETA coated coverslips were then heated to approximately 100 C., rinsed with toluene, reheated to approximately 100 C., and then oven dried (Das et al., 2006). Surfaces were characterized by contact angle measurements using an optical contact angle goniometer (KSV Instruments, Cam 200) and by X-ray photoelectron spectroscopy (XPS) (Kratos Axis 165). XPS survey scans, as well as high-resolution N1s and C1s scans utilizing monochromatic AI Ka excitation were obtained (Das et al., 2006).

(13) Skeletal Muscle Culture in Serum-Free Medium

(14) Skeletal muscle was dissected from the thighs of the hind limbs of fetal rat (17e18 days old). Briefly, rats were anaesthetized and killed by inhalation of an excess of CO.sub.2. This procedure was in agreement with the Animal Research Council of University of Central Florida, which adheres to IACUC policies. The tissue was collected in a sterile 15 mL centrifuge tube containing 1 mL phosphate-buffered saline (calcium- and magnesium-free) (Gibco 14200075). The tissue was enzymatically dissociated using 2 mL of 0.05% of trypsin-EDTA (Gibco 25300054) solution for 30 min in a 37 C. water bath at 50 rpm. After 30 min the trypsin solution was removed and 4 mL Hibernate E/10% fetal bovine serum (Gibco 16000044) was added to terminate the trypsin reaction. The tissue was then mechanically triturated with the supernatant being transferred to a 15 mL centrifuge tube. The same process was repeated two times by adding 2 mL of L15/10% FBS each time. The 6 mL cell suspension obtained after mechanical trituration was suspended on a 2 mL, 4% BSA (Sigma A3059) (prepared in L15 medium) cushion and centrifuged at 300 g for 10 min at 4 C. The pellet obtained was washed 5 times with L15 medium then resuspended in 10 mL of L15 and plated in 100 mm uncoated dishes for 30 min. The non-attached cells were removed and then centrifuged on a 4% BSA cushion (Das et al., 2006).

(15) The pellet was resuspended in serum-free medium according to the protocol illustrated in FIG. 1 and plated on the coverslips at a density of 700-1000 cells/mm.sup.2. The serum-free medium containing different growth factors and hormones was added to the culture dish after 1 h. The final medium was prepared by mixing medium 1 (Table 1) and medium 2 (Table 2) in a 1:1 v/v ratio. FIG. 1 indicates a flowchart of the culture protocol. Tables 1 and 2 list the growth factor and hormone supplement compositions of medium one and medium two. The cells were maintained in a 5% CO2 incubator (relative humidity 85%). The entire medium was replaced after four days with NbActiv4 medium according to the protocol in FIG. 1 (Brewer et al., 2008). As described in (Brewer et al., 2008), NbActiv4 (available from BrainBits LLC) comprises all of the ingredients in Neurobasal (Table 3), B27 (Table 4), and Glutamax (Table 5). NbActiv4 may also comprise creatine, estrogen, and cholesterol. Thereafter three-fourths of the medium was changed every three days with NbActiv4.

(16) TABLE-US-00001 TABLE 1 Composition of Medium 1 No. Component Amount Catalogue # Source References 1 Neurobasal A 500 mL 10888 Gibco/ Brewer et al., 1993 Invitrogen 2 Antibiotic- 5 mL 15240-062 Gibco/ Antimycotic Invitrogen 3 Glutamax 5 mL 35050-061 Gibco/ Invitrogen 4 B27 Supplement 10 mL 17504-044 Gibco/ Das et al., 2004; Invitrogen Brewer et al., 1993 5 G5 Supplement 5 mL 17503-012 Gibco/ Alterio et al., 1990; (100X) Invitrogen Clegg et al., 1987; Bottenstein 1981, 1988; Bottenstein et al., 1988; Morrow et al., 1990; Gonzalez et al., 1990; Moore et al., 1991; Anderson et al., 1991; Olwin et al., 1992 6 VEGF.sub.165 r Human 10 g P2654 Gibco/ Arsic et al., 2004; Invitrogen Germani et al., 2003; Lee et al., 2003; Lescaudron et al., 1999 7 Acidic FGF 12.5 g 13241-013 Gibco/ Alterio et al., 1990; Invitrogen Moore et al., 1991; Olwin et al., 1992; Motamed et al., 2003; Dusterhoft et al., 1999; Fu et al., 1995; Smith et al., 1994; Oliver et al., 1992; Dell'Era et al., 2003 8 Heparin 50 g D9809 Sigma Alterio et al., 1990; Sulphate Moore et al., 1991; Olwin et al., 1992; Motamed et al., 2003; Dusterhoft et al., 1999; Fu et al., 1995; Smith et al., 1994; Oliver et al., 1992; Dell'Era et al., 2003 9 LIF 10 g L5158 Sigma Husmann et al., 1996; Kurek et al., 1996; Megeney et al., 1996; Vakakis et al., 1995; Martinou et al., 1992; Sun et al., 2007; Malm et al., 2004; Zorzano et al., 2003; Sakuma et al., 2000 10 Vitronectin 50 g V0132 Sigma Biesecker 1990; (Rat Plasma) Gullberg et al., 1995 11 CNTF 20 g CRC 401B Cell Sciences Wang et al., 2008; Chen et al., 2003, 2005; Cannon 1998; Marques et al., 1997 12 NT 3 10 g CRN 500B Cell Sciences Oakley et al., 1997 13 NT 4 10 g CRN 501B Cell Sciences Carrasco et al., 2003; Simon et al., 2003 14 GDNF 10 g CRG 400B Cell Sciences Choi-Lundberg et al., 1995; Lin et al., 1993; Yang et al., 2004; Golden et al., 1999; Henderson et al., 1994 15 BDNF 10 g CRB 600B Cell Sciences Simon et al., 2003; Heinrich 2003; Mousavi et al., 2004 16 CT-1 10 g CRC 700B Cell Sciences Chen et al., 2004; Bordet et al., 2001; Dolcet et al., 2001; Lesbordes et al., 2002; Nishikawa et al., 2005; Mitsumoto et al., 2001; Oppenheim et al., 2001; Peroulakis et al., 2000; Sheng et al., 1996

(17) TABLE-US-00002 TABLE 2 Composition of Medium 2 No. Component(s) Amount Catalogue Source References 1 Neurobasal A 500 mL 10888 Invitrogen/ Brewer et al., 1993 Gibco 2 Glutamax 5 mL 35050-061 Invitrogen/ Gibco 3 Antibiotic- 5 mL 15240-062 Invitrogen/ Antimycotic Gibco 4 B27 Supplement 10 mL 17504-044 Invitrogen/ Das et al., 2004; Gibco Brewer et al., 1993 5 Cholesterol 5 mL 12531 Invitrogen/ Jaworska-Wilczynska (250X) Gibco et al., 2002 6 TNF-alpha, 10 g T6674 Sigma-Aldrich Caratsch et al., 1994; human Al-Shanti et al., 2008; Miller et al., 1988 7 PDGF BB 50 g P4056 Sigma-Aldrich Husmann et al., 1996; Jin et al., 1991; Kudla et al., 1995; Quinn et al., 1990; Yablonka-Reuveni et al., 1995 8 Vasoactive intestinal 250 g V6130 Sigma-Aldrich Gold 1982 peptide (VIP) 9 Insulin-like growth 25 g 12656 Sigma-Aldrich Malm et al., 2004; factor 1 Zorzano et al., 2003; Al-Shanti et al., 2008 10 NAP 1 mg 61170 AnaSpec. Inc. Gozes et al., 2004; Aracil et al., 2004 11 Recombinant 50 g P2002 Panvera Robertson et al., 2000 Apolipoprotein E2 12 Laminin, mouse 2 mg 08-125 Millipore Langen et al., 2003; purified Foster et al., 1987; Hantai et al., 1991; Kuhl et al., 1986; Lyles et al., 1992; Song et al., 1992; Swasdison et al., 1992 13 Beta amyloid 1 mg AG966 Millipore Wang et al., 2005; (1-40) Yang et al., 2007; Akaaboune et al., 2000 14 Human Tenascin-C 100 g CC065 Millipore Hall et al., 2000 protein 15 rr-Sonic hedgehog, 50 g 1314-SH R&D Systems Fan et al., 1994; Shh N-terminal Munsterberg et al., 1995; Nelson et al., 1996; Cossu et al., 1996; Currie et al., 1996; Norris et al., 2000; Brand-Saberi et al., 2005; Elia et al., 2007; Pagan et al., 1996; Bren-Mattison et al., 2002; Maves et al., 2007; Koleva et al., 2005 16 rr (Agrin C 50 g 550-AG-100 R&D Systems Bandi et al., 2008; terminal) Sanes 1997

(18) TABLE-US-00003 TABLE 3 Composition of Neurobasal medium Molecular Concentration Concentration No. Components Weight (mg/L) (mM) 1 Glycine 75 30 4.00E01 2 L-Alanine 89 2 2.25E02 3 L-Arginine 211 84 3.98E01 hydrochloride 4 L-Asparagine-H.sub.2O 150 0.83 5.53E03 5 L-Cysteine 121 31.5 2.60E01 6 L-Histidine 210 42 2.00E01 hydrochloride-H.sub.2O 7 L-Isoleucine 131 105 8.02E01 8 L-Leucine 131 105 8.02E01 9 L-Lysine 183 146 7.98E01 hydrochloride 10 L-Methionine 149 30 2.01E01 11 L-Phenylalanine 165 66 4.00E01 12 L-Proline 115 7.76 6.75E02 13 L-Serine 105 42 4.00E01 14 L-Threonine 119 95 7.98E01 15 L-Tryptophan 204 16 7.84E02 16 L-Tyrosine 181 72 3.98E01 17 L-Valine 117 94 8.03E01 18 Choline chloride 140 4 2.86E02 19 D-Calcium 477 4 8.39E03 pantothenate 20 Folic Acid 441 4 9.07E03 21 Niacinamide 122 4 3.28E02 22 Pyridoxal 204 4 1.96E02 hydrochloride 23 Riboflavin 376 0.4 1.06E03 24 Thiamine 337 4 1.19E02 hydrochloride 25 Vitamin B12 1355 0.0068 5.02E+06 26 i-Inositol 180 7.2 4.00E02 27 Calcium Chloride 111 200 1.80E+00 (CaCl2) (anhyd.) 28 Ferric Nitrate 404 0.1 2.48E+04 (Fe(NO3)39H2O) 29 Magnesium Chloride 95 77.3 8.14E01 (anhydrous) 30 Potassium Chloride 75 400 5.33E+00 (KCl) 31 Sodium Bicarbonate 84 2200 2.62E+01 (NaHCO3) 32 Sodium Chloride 58 3000 5.17E+01 (NaCl) 33 Sodium Phosphate 138 125 9.06E01 monobasic (NaH2PO4H2O) 34 Zinc sulfate 288 0.194 6.74E+04 (ZnSO47H2O) 35 D-Glucose (Dextrose) 180 4500 2.50E+01 36 HEPES 238 2600 1.09E+01 37 Phenol Red 376.4 8.1 2.15E02 38 Sodium Pyruvate 110 25 2.27E01

(19) TABLE-US-00004 TABLE 4 Composition of B27 Concentration No. Component (mg/L) 1 L-Alanine 2.00E+00 2 L-Glutamate 3.70E+00 3 L-Glutamine 4.41E+02 4 L-Proline 7.76E+00 5 Biotin 1.00E01 6 Vitamin B12 3.40E01 7 Corticosterone 2.00E02 8 Progesterone 6.30E03 9 Retinol, all trans (Vit. A) 1.00E01 10 Retinol, acetate 1.00E01 11 Insulin 4.00E+00 12 T3 (triodo-L-thyronine) 2.00E03 13 Na pyruvate 2.50E+01 14 Lipoic acid (thioctic acid) 4.70E02 15 D,L--Tocopherol (vit. E) 1.00E+00 16 D,L--Tocopherol acetate 1.00E+00 17 Catalase 2.50E+00 18 Glutathione (reduced) 1.00E+00 19 Superoxide dismutase 2.50E+00 20 L-Carnitine 2.00E+00 21 Ethanolamine 1.00E+00 22 D(+)-Galactose 1.50E+01 23 HEPES 2.60E+03 24 Putrescine 1.61E+01 25 Penicillin 50 IU/mL 26 Streptomycin 5.00E01 27 Selenium 1.60E02 28 Zinc sulfate 1.94E01 29 Linoleic acid 1.00E+00 30 Linolenic acid 1.00E+00 31 Albumin, bovine 2.50E+03 32 Transferrin 5.00E+00

(20) TABLE-US-00005 TABLE 5 Composition of GlutaMax No. Component Concentration 1 L-alanyl-L-glutamine dipeptide 200 mM 2 NaCl 0.85%
Rat Embryonic Motoneuron Isolation and Co-Culture

(21) Rat spinal motoneurons were purified from ventral cords of embryonic day 14 (E14) embryos. Briefly, rats were anaesthetized and killed by inhalation of an excess of CO.sub.2. This procedure was in agreement with the Animal Research Council of University of Central Florida, which adheres to IACUC policies. Ventral spinal cord cells from the embryo were collected in cold Hibernate E/GlutaMAX/antibiotic-antimycotic/B27. The cells were dissociated with 0.05% trypsin-EDTA (Invitrogen) treatment for 15 min. The dissociated cells were layered over a 4 mL step gradient Optiprep diluted 0.505:0.495 (v/v) with Hibernate E/GlutaMAX/antibiotic-antimycotic/B27 and then made to 15%, 20%, 25% and 35% (v/v) in Hibernate E/GlutaMAX/antibiotic-anti-mycotic/B27 followed by centrifugation for 15 min, using 200 g at 4 C. After centrifugation, four bands of cells were obtained. The motoneurons with large somas constituted the uppermost band. These cells present in the uppermost band were collected in fresh Hibernate E/GlutaMAX/antibiotic-anti-mycotic/B27 and centrifuged for 5 min at 200 g and 4 C. The pelleted motoneurons were re-suspended in plating medium then plated on top of muscle cells at a density of 100 cells/mm.sup.2. Motoneuron plating was performed 30 min after plating of the muscle cells.

(22) Immunocytochemistry

(23) Neonatal Myosin Heavy Chain (Neonatal MHC)

(24) Coverslips were rinsed with PBS, fixed in 20 C. methanol for 5-7 min, washed in PBS, incubated in PBS supplemented with 1% BSA and 0.05% saponin (permeabilization solution), and blocked for 30 min in a permeabilization solution+10% goat serum (blocking solution). Cells were incubated overnight with primary antibody against neonatal MHC (N3.36, IgG, Developmental Studies Hybridoma Bank) diluted (1:5) in the blocking solution. Cells were washed with PBS and incubated with AlexaFluor secondary antibody (Invitrogen) (diluted in PBS) for 2 h. The secondary antibody solution was removed and the cells were rinsed using PBS. The coverslips were dried and mounted on glass slides using VectaShield+DAPI mounting medium (Vector Laboratories H-1200) and viewed on a confocal microscope (UltraVIEW LCI, PerkinElmer).

(25) Double Staining with Neurofilament 150 and Neonatal Myosin Heavy Chain

(26) Co-cultures were processed for immunocytochemistry as described above. Next, cells were incubated overnight at 4 C. with rabbit anti-neurofilament M polyclonal antibody, 150 kD, (Chemicon, AB1981, diluted 1:2000) and neonatal MHC (N3.36, IgG, Developmental Studies Hybridoma Bank diluted 1:5). After overnight incubation, the coverslips were rinsed three times with PBS and then incubated with the AlexaFluor secondary antibodies (Invitrogen) for 2 h. After rinsing three times in PBS, the coverslips were mounted with Vectashield+DAPI mounting medium onto glass slides. The coverslips were visualized and images collected using a confocal microscope (UltraVIEW LCI, PerkinElmer). Controls without primary antibody were negative.

(27) AChR+Synaptophysin Co-Staining

(28) AChRs were labeled as described previously by incubating cultures with 510.sup.8 M of -bungarotoxin, Alexa Fluor 488 conjugate (Molecular Probes, B-13422) for 1.5 h at 37 C. before observation (Das et al., 2007 (Neuroscience)). Labeled cultures were fixed with glacial acetic acid and ethanol, washed with PBS, dried, mounted and examined by confocal microscopy. The coverslips which were used for double staining with AChR+synaptophysin for locating the NMJs were processed further. After 1.5 h of -bungarotoxin labeling of the AChR receptors, the coverslips were fixed, blocked, permeabilized and incubated overnight with synaptophysin antibody (MAB368, diluted 1:1000; Millipore/Chemicon), the pre-synaptic marker present in motoneuron axonal terminals.

(29) Data Analysis

(30) Statistics were calculated using the following procedure. One coverslip was randomly selected from each experiment (typically, there are six coverslips per experiment). 25 non-overlapping fields of view were used to characterize each coverslip. At the magnification used, 25 fields covers over 40% of the surface area of the coverslip.

(31) Results

(32) DETA Surface Modification and Characterization

(33) Static contact angle and XPS analysis were used for the validation of the surface modifications and for monitoring the quality of the surfaces. Stable contact angles (40.642.9/meanSD) throughout the study indicated high reproducibility and quality of the DETA surfaces and these characteristics were similar to previously published results (Das et al., 2004; Das et al., 2007 (Nat. Protocols); Das et al., 2007 (Neuroscience); Das et al., 2006; Das et al., 2003). Based on the ratio of the N is (401 and 399 eV) and the Si 2p.sub.3/2 peaks, XPS measurements indicated that a reaction-site limited monolayer of DETA was formed on the coverslips (Stenger et al., 1992).

(34) Temporal Growth Factor Application

(35) The formation of the maximal number of neuromuscular junctions was observed using the temporal growth factor application technique described in FIG. 1. Upon plating of the motoneurons and skeletal muscle, the cells were treated with medium containing factors that promoted both growth and survival as well as enhancement of NMJ formation (Table 1, Table 2). After 3 days in culture, the entire medium was removed and switched to a minimal formulation, NbActiv4, which facilitated both long-term survival and further development of the NMJs (FIG. 1). Further, three-fourths of the NbActiv4 medium per well was removed and replaced with an equal volume of fresh NbActiv4 medium. When compared to the continuous application of growth factors, the timed application resulted in cultures that lasted for up to 7 weeks as opposed to 10-12 days.

(36) Culture Morphology of Motoneuron and Skeletal Muscle Myotube Interactions

(37) Phase contrast microscopy was used to visualize motoneuron axons appearing to interact with skeletal muscle myotubes between days 12-15 (FIG. 2, A-D). Some of the axonal processes appear to branch and terminate on the myotubes. Furthermore, many of the myotubes exhibited characteristic striation patterns observed after sarcomere formation when the fibers reached approximately 25-30 days in culture (FIG. 3, A-D). Quantification of the appearance of striations after this time indicated that the co-cultures contained about twice the number of myotubes showing striations.

(38) Immunocytochemical Characterization of Motoneuron and Skeletal Muscle Co-Culture

(39) The characteristic protein expression patterns of the motoneurons and myotubes in co-culture were evaluated at day 25. Immunocytochemistry was used to visualize the neurofilament protein expression in the motoneurons and neonatal myosin heavy chain (MHC) expression for the myotubes (FIG. 4, A-B). Motoneuron processes were clearly indicated interacting with the skeletal muscle myotubes. A band/I band formation was more visible in the myotubes after staining with the neonatal myosin heavy chain antibody. The immunocytochemical analysis supported the morphological analysis, which had indicated the presence of striations in double the number of myotubes as observed with the muscle only controls.

(40) Neuromuscular Junction Formation

(41) In order to determine neuromuscular junction formation using this novel medium formulation, the clustering of AChRs using alpha-bungarotoxin and their colocalization with synaptophysin vesicles was analyzed immunocytochemically. The colocalization of these two synaptic markers indicated the proximity of pre-synaptic and post-synaptic structures and was a positive indication of NMJ formation. This technique was used to identify the colocalization of synaptophysin vesicles with the AChR clusters (FIG. 5, A-D). The axon+myotube interactions that did not result in the colocalization of pre-synaptic and post-synaptic structures were also identified (FIG. 6, A-B). The observation of the negative result defines the difference between colocalization and non-colocalization and emphasizes the positive result observed in this system. FIG. 7 illustrates NMJ formation between a myotube in culture that did not stain for neonatal myosin heavy chain and a motoneuron.

(42) Discussion

(43) This work documents the substantial improvement of an in vitro model system for NMJ formation. Specifically, we observed enhanced survivability of the culture resulting in our ability to conduct long-term studies on the motoneuron-skeletal muscle cocultures. This increased survivability resulted in maturation of the skeletal muscle myotubes and a significant improvement in the number of NMJs formed in culture.

(44) Previously, we developed the first defined culture model to coculture embryonic motoneuron and fetal skeletal muscle, however this model was not suitable for long-term tissue engineering studies and the myotubes in the culture only expressed an early muscle marker, i.e. fetal myosin heavy chain and none of the myotubes exhibited characteristic striations. In this study, significant improvement over our previous motoneuron-skeletal muscle co-culture model system was documented. This new culture model supported long-term co-culture of both motoneuron and muscle, resulted in a more adult-like morphology of the muscle and a higher density of neuromuscular junctions (NMJ). Our findings were supported by morphological and immunocytochemical data.

(45) We developed this serum-free medium, supplemented with growth factors that supported the survival, proliferation and fusion of fetal rat myoblasts into contractile myotubes, in a semi-empirical fashion. The rationale for selecting the growth factors was based on the distribution of their cognate receptors in the developing myotubes in rat fetus (Arnold et al., 1998; Brand-Saberi et al., 2005; Olson et al., 1992). Tables 1 and 2 reference the literature where these individual growth factors, hormones and neurotransmitters were observed to support muscle and neuromuscular junction development. The composition in Table 1 is the formulation used for a previously published medium utilized for motoneuron-muscle co-culture and adult spinal cord neuron culture (Das et al., 2007 (Neuroscience); Das et al., 2008 (Exp. Neurology); Das et al., 2005; Das et al., 2007 (Biomaterials)). Table 2 lists the twelve additional factors identified in muscle development and neuromuscular junction formation that enabled the increased survivability of the system. Further addition of the factors in Table 2 promoted formation of characteristic striation in the muscle in the culture. The use of NbActiv4 for the maintenance of the cells significantly improved the survival of the skeletal muscle derived myotubes despite the fact that the original purpose of the development of NbActiv4 was for the long-term maintenance and synaptic connectivity of fetal hippocampal neurons in vitro (Brewer et al., 2008).

(46) In our previous co-culture model, we did not observe the expression of neonatal MHC proteins in the myotubes. Interestingly, when this same medium and protocol was used to culture pure skeletal muscle we observed certain striking differences. The pure muscle culture survived longer, exhibited characteristic striations, but only a very small percentage of myotubes expressed N3.36 (Das et al., 2009 (Biomaterials)). To the best of our understanding, the N3.36 expression in skeletal muscle in culture is influenced by the motoneurons either physically or by certain trophic factors secreted in the presence of this modified medium and NbActiv4. This observation needs further studies in order to dissect the molecular pathways regulating N3.36 expression in pure skeletal muscle culture and in skeletal muscle-motoneuron co-culture. Also, the potential regulation of MHC class switching independent of neuronal innervation/denervation represents an interesting topic for further study. This system would have applications in developing therapies for muscle-nerve diseases such as ALS, spinal muscular atrophy, spinal cord injury and myasthenia gravis.

(47) Conclusions

(48) The development of robust NMJ formation, long-term survival of motoneuron skeletal muscle co-cultures and selective MHC class switching is documented in this research. This improved system supports the goal of creating a physiologically relevant tissue engineered motoneuron skeletal muscle construct and puts within reach the goal of developing functional bio-hybrid devices to analyze NMJ activity. This defined model can also be used to map the developmental pathways regulating NMJ formation and MHC class switching. Furthermore, we believe this serum free culture system will be a powerful tool in developing advanced strategies for regenerative medicine in ALS, stretch reflex arc development and integrating motoneuron+skeletal muscle with bio-hybrid prosthetic devices. Due to the use of a serum-free defined culture system this also has applications for new high-throughput screening systems for use in drug discovery research and toxicology investigations.

(49) 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.

REFERENCES CITED

(50) [1] Akaaboune M, et al. (2000) Developmental regulation of amyloid precursor protein at the neuromuscular junction in mouse skeletal muscle. Mol Cell Neurosci. 15(4):355-367. [2] Al-Shanti N, et al. (2008) Beneficial synergistic interactions of TNF.alpha and IL.6 in C2 skeletal myoblastspotential cross-talk with IGF system. Growth Factors. 26(2):61-73. [3] Alterio J, et al. (1990) Acidic and basic fibroblast growth factor mRNAs are expressed by skeletal muscle satellite cells. Biochem Biophys Res Commun. 166(3):1205-1212. [4] Anderson J E, et al. (1991) Distinctive patterns of basic fibroblast growth factor (bFGF) distribution in degenerating and regenerating areas of dystrophic (mdx) striated muscles. Dev Biol. 147(1):96-109. [5] Aracil A, et al. (2004) Proceedings of Neuropeptides 2004, the XIV European Neuropeptides Club meeting. Neuropeptides. 38 (6): 369-371. [6] Arnold H H, et al. (1998) more complexity to the network of myogenic regulators. Curr Opin Genet Dev. 8(5):539-44. [7] Arsic N, et al. (2004) Vascular endothelial growth factor stimulates skeletal muscle regeneration in vivo. Mol Ther. 10(5):844-854. [8] Bandi E, et al. (2008) Neural agrin controls maturation of the excitation-contraction coupling mechanism in human myotubes developing in vitro. Am J Physiol Cell Physiol. 294(1):C66-73. [9] Biesecker G, et al. (1990) The complement SC5b.9 complex mediates cell adhesion through a vitronectin receptor. J Immunol. 145(1):209-214. [10] Bordet T., et al. (2001) Protective effects of cardiotrophin-1 adenoviral gene transfer on neuromuscular degeneration in transgenic ALS mice. Hum Mol Genet. 10(18):1925-1933. [11] Bottenstein J E, et al. (1988) CNS neuronal cell line-derived factors regulate gliogenesis in neonatal rat brain cultures. J Neurosci Res. 20(3):291-303. [12] Bottenstein J E, et al. (1998) Advances in vertebrate cell culture methods. Science. 239(4841 Pt 2):G42, G48. [13] Bottenstein J E, et al. (1981) Proliferation of glioma cells in serum-free defined medium. Cancer Treat Rep. 65 Suppl 2:67-70. [14] Brand-Saberi B, et al. (2005) Genetic and epigenetic control of skeletal muscle development. Ann Anat. 187(3):199-207. [15] Bren-Mattison Y, et al. (2002) Sonic hedgehog inhibits the terminal differentiation of limb myoblasts committed to the slow muscle lineage. Dev Biol. 242(2):130-148. [16] Brewer G J, et al. (2008) NbActiv4 medium improvement to Neurobasal/B27 increases neuron synapse densities and network spike rates on multielectrode arrays. J Neurosci Methods. 170(2):181-187. [17] Brewer G J, et al. (1993) Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res. 35(5):567-576. [18] Cannon J G, et al. (1998) Intrinsic and extrinsic factors in muscle aging Ann NY Acad Sci. 854:72-77. [19] Caratsch C G, et al. (1994) Interferon-alpha, beta and tumor necrosis factor-alpha enhance the frequency of miniature end-plate potentials at rat neuromuscular junction. Neurosci Lett. 166(1):97-100. [20] Carrasco Dl, et al. (2003) Neurotrophin 4/5 is required for the normal development of the slow muscle fiber phenotype in the rat soleus. J Exp Bio. 206(Pt 13):2191-2200. [21] Chen J, et al. (2004) Role of exogenous and endogenous trophic factors in the regulation of extraocular muscle strength during development. Invest Ophthalmol Vis Sci. 45(10):3538-3545. [22] Chen X, et al. (2005) Dedifferentiation of adult human myoblasts induced by ciliary neurotrophic factor in vitro. Mol Bioi Cell. 16(7):3140-3151. [23] Chen X P, et al. (2003) [Exogenous rhCNTF inhibits myoblast differentiation of skeletal muscle of adult human in vitro]. Sheng Li Xue Bao. 55(4):464-468. [24] Choi-Lundberg D L, et al. (1995) Ontogeny and distribution of glial cell line-derived neurotrophic factor (GDNF) mRNA in rat. Brain Res Dev Brain Res. 85(1):80-88. [25] Chow I, et al. (1985) Release of acetylcholine from embryonic neurons upon contact with muscle cell. J Neurosci. 5(4):1076-82. [26] Ciegg C H, et al. (1987) Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor. J Cell Biol. 105(2):949-956. [27] Colomar A, et al. (2004) Glial modulation of synaptic transmission at the neuromuscular junction. Glia. 47(3):284-9. [28] Cossu G, et al. (1996) How is myogenesis initiated in the embryo? Trends Genet. 12(6):218-223. [29] Currie P D, et al. (1996) Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish. Nature. 382(6590):452-455. [30] Daniels M P, et al. (2000) Rodent nerve-muscle cell culture system for studies of neuromuscular junction development: refinements and applications. Microsc Res Tech. 49(1):26-37. [31] Daniels M P, (1997) Intercellular communication that mediates formation of the neuromuscular junction. Mol Neurobiol. 14(3):143-170. [32] Das M, et al. (2008) Temporal neurotransmitter conditioning restores the functional activity of adult spinal cord neurons in long-term culture. Exp Neurol. 209(1): 171-180. [33] Das M, et al. (2005) Adult rat spinal cord culture on an organosilane surface in a novel serum-free medium. In Vitro Cell Dev Bioi Anim. 41(10):343-348. [34] Das M, et al. (2003) Electrophysiological and morphological characterization of rat embryonic motoneurons in a defined system. Biotechnol Prog. 19(6):1756-1761. [35] Das M, et al. (2004) Long-term culture of embryonic rat cardiomyocytes on an organosilane surface in a serum-free medium. Biomaterials. 25(25):5643-5647. [36] Das M, et al. (2007) Auto-catalytic ceria nanoparticles offer neuroprotection to adult rat spinal cord neurons. Biomaterials. 28(10): 1918-1925. [37] Das M, et al. (2009) Skeletal muscle tissue engineering: an improved model promoting long term survival of myotubes, structural development of e-c coupling apparatus and neonatal myosin heavy chain (MHC) expression. Biomaterials. 30:5392-402. [38] Das M, et al. (2007) Embryonic motoneuron-skeletal muscle co-culture in a defined system. Neuroscience. 146(2):481-488. [39] Das M, et al. (2007) Differentiation of skeletal muscle and integration of myotubes with silicon microstructures using serum-free medium and a synthetic silane substrate. Nat Protoc. 2(7): 1795-1801. [40] Das M, et al. (2006) A defined system to allow skeletal muscle differentiation and subsequent integration with silicon microstructures. Biomaterials. 27(24):437 4-4380. [41] DellEra P, et al. (2003) Fibroblast growth factor receptor 1 is essential for in vitro cardiomyocyte development. Circ Res. 93(5):414-420. [42] Dolcet X, et al. (2001) Cytokines promote motoneuron survival through the Janus kinase-dependent activation of the phosphatidylinositol 3-kinase pathway. Mol Cell Neurosci. 18(6):619-631. [43] Dusterhoft S, et al. (1999) Evidence that acidic fibroblast growth factor promotes maturation of rat satellite-cell-derived myotubes in vitro. Differentiation. 65(3): 161-169. [44] Eisenberg T, et al. (2009) Induction of autophagy by spermidine promotes longevity. Nat Cell Biol. 11(11):1305-14. [45] Elia D, et al. (2007) Sonic hedgehog promotes proliferation and differentiation of adult muscle cells: Involvement of MAPK/ERK and P13K/Akt pathways. Biochim Biophys Acta. 1773(9):1438-1446. [46] English A W., et al. (2003) Cytokines, growth factors and sprouting at the neuromuscular junction. J Neurocytol. 32(5):943-60. [47] Fan C M, et al. (1994) Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog. Cell. 79(7):1175-1186. [48] Foster R F, et al. (1987) A laminin substrate promotes myogenesis in rat skeletal muscle cultures: analysis of replication and development using antidesmin and anti-BrdUrd monoclonal antibodies. Dev Bio. 122(1):11-20. [49] Fu X, et al. (1995) Acidic fibroblast growth factor reduces rat skeletal muscle damage caused by ischemia and reperfusion. Chin Med J (Engl). 108(3):209-214. [50] Germani A, et al. (2003) Vascular endothelial growth factor modulates skeletal myoblast function. Am J Pathol. 163(4):1417-1428. [51] Gold M R, et al. (1982) The effects of vasoactive intestinal peptide on neuromuscular transmission in the frog. J Physiol. 327:325-335. [52] Golden J P, et al. (1999) Expression of neurturin, GDNF, and GDNF family-receptor mRNA in the developing and mature mouse. Exp Neural. 158(2):504-528. [53] Gonzalez A M, et al. (1990) Distribution of basic fibroblast growth factor in the 18-day rat fetus: localization in the basement membranes of diverse tissues. J Cell Biol. 110(3):753-765. [54] Gozes I, et al. (2004) NAP mechanisms of neuroprotection. J Mol Neurosci. 24(1):67-72. [55] Gullberg D, et al. (1995) Analysis of fibronectin and vitronectin receptors on human fetal skeletal muscle cells upon differentiation. Exp Cell Res. 220(1):112-123. [56] Hall B K, et al. (2000) All for one and one for all: condensations and the initiation of skeletal development. Bioessays. 22(2): 138-147. [57] Hantai D, et al. (1991) Developmental appearance of thrombospondin in neonatal mouse skeletal muscle. Eur J Cell Biol. 55(2):286-294. [58] Heinrich G, et al. (2003) A novel BDNF gene promoter directs expression to skeletal muscle. BMC Neurosci. 4:11. [59] Henderson C E, et al. (1994) GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science. 266(5187):1062-1064. [60] Husmann I, et al. (1996) Growth factors in skeletal muscle regeneration. Cytokine Growth Factor Rev. 7(3):249-258. [61] Jaworska-Wilczynska M, et al. (2002) Three lipoprotein receptors and cholesterol in inclusion-body myositis muscle. Neurology. 58(3):438-445. [62] Jin P, et al. (1991) Recombinant platelet-derived growth factor-88 stimulates growth and inhibits differentiation of rat L6 myoblasts. J Biol Chem. 266(2):1245-1249. [63] Koleva M, et al. (2005) Pleiotropic effects of sonic hedgehog on muscle satellite cells. Cell Mol Life Sci. 62(16): 1863-1870. [64] Kudla A J, et al. (1995) A requirement for fibroblast growth factor in regulation of skeletal muscle growth and differentiation cannot be replaced by activation of platelet-derived growth factor signaling pathways. Mol Cell Biol. 15(6):3238-3246. [65] Kuhl U, et al. (1986) Role of laminin and fibronectin in selecting myogenic versus fibrogenic cells from skeletal muscle cells in vitro. Dev Biol. 117(2):628-635. [66] Kurek J B, et al. (1996) Leukemia inhibitory factor and interleukin-6 are produced by diseased and regenerating skeletal muscle. Muscle Nerve. 19(10):1291-1301. [67] Langen R C, et al. (2003) Enhanced myogenic differentiation by extracellular matrix is regulated at the early stages of myogenesis. In Vitro Cell Dev Bioi Anim. 39(3-4): 163-169. [68] Lee E W., et al. (2003) Neuropeptide Y induces ischemic angiogenesis and restores function of ischemic skeletal muscles. J Clin Invest. 111(12): 1853-1862. [69] Lesbordes J C, et al. (2002) In vivo electrotransfer of the cardiotrophin-1 gene into skeletal muscle slows down progression of motor neuron degeneration in pmn mice. Hum Mol Genet. 11(14):1615-1625. [70] Lescaudron L, et al. (1999) Blood borne macrophages are essential for the triggering of muscle regeneration following muscle transplant. Neuromuscul Disord. 9(2):72-80. [71] Li M X, et al. (2001) Opposing actions of protein kinase A and C mediate Hebbian synaptic plasticity. Nat Neurosci. 4(9):871-872. [72] Lin L F, et al. (1993) GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science. 260(5111):1130-1132. [73] Lyles J M, et al. (1992) Matrigel enhances myotube development in a serum-free defined medium. Int J Dev Neurosci. 10(1):59-73. [74] Malm C, et al. (2004) Leukocytes, cytokines, growth factors and hormones in human skeletal muscle and blood after uphill or downhill running J Physiol. 556(Pt 3):983-1000. [75] Marques M J, et al. (1997) Ciliary neurotrophic factor stimulates in vivo myotube formation in mice. Neurosci Lett. 234(1):43-46. [76] Martinou J C, et al. (1992) Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro. Neuron. 8(4):737-744. [77] Mayes L, et al. (2007) Pbx homeodomain proteins direct Myod activity to promote fast-muscle differentiation. Development. 134 (18):3371-3382. [78] Megeney L A, et al. (1996) bFGF and LIF signaling activates STAT3 in proliferating myoblasts. Dev Genet. 19(2):139-145. [79] Miller S C, et al. (1998) Tumor necrosis factor inhibits human myogenesis in vitro. Mol Cell Biol. 8(6):2295-2301. [80] Mitsumoto H, et al. (2001) Effects of cardiotrophin.1 (CT-1) in a mouse motor neuron disease. Muscle Nerve. 24(6):769-777. [81] Moore J W, et al. (1991) The mRNAs encoding acidic FGF, basic FGF and FGF receptor are coordinately downregulated during myogenic differentiation. Development. 111(3):741-748. [82] Morrow N G, et al. (1990) Increased expression of fibroblast growth factors in a rabbit skeletal muscle model of exercise conditioning. J Clin Invest. 85(6):1816-1820. [83] Motamed K, et al. (2003) Fibroblast growth factor receptor-1 mediates the inhibition of endothelial cell proliferation and the promotion of skeletal myoblast differentiation by SPARC: a role for protein kinase A. J Cell Biochem. 90(2):408-423. [84] Mousavi K, et al. (2004) BDNF rescues myosin heavy chain 118 muscle fibers after neonatal nerve injury. Am J Physiol Cell Physicl. 287(1):C22-29. [85] Munsterberg A E, et al. (1995) Combinatorial signaling by Sonic hedgehog and Wnt family members induces myogenic bHLH gene expression in the somite. Genes Dev. 9(23):2911-2922. [86] Nelson C E, et al. (1996) Analysis of Hox gene expression in the chick limb bud. Development. 122(5):1449-1466. [87] Nelson P G (1975) Nerve and muscle cells in culture. Physiol Rev. 55(1):1-61. [88] Nishikawa J, et al. (2005) Increase of Cardiotrophin-1 immunoreactivity in regenerating and overloaded but not denervated muscles of rats. Neuropathology. 25(1): 54-65. [89] Norris W, et al. (2000) Slow muscle induction by Hedgehog signalling in vitro. J Cell Sci. 113 (Pt 15):2695-2703. [90] Oliver L, et al. (1992) Acidic fibroblast growth factor (aFGF) in developing normal and dystrophic (mdx) mouse muscles. Distribution in degenerating and regenerating mdx myofibres. Growth Factors. 7(2):97-106. [91] Oakley R A, et al. (1997) Neurotrophin-3 promotes the differentiation of muscle spindle afferents in the absence of peripheral targets. J Neurosci. 17(11):4262-4274. [92] Olson E N., et al. (1992) Interplay between proliferation and differentiation within the myogenic lineage. Dev Biol. 154(2):261-72. [93] Olwin B B, et al. (1992) Repression of myogenic differentiation by aFGF, bFGF, and K.FGF is dependent on cellular heparan sulfate. J Cell Biol. 118(3):631-639. [94] Oppenheim R W, et al. (2001) Cardiotrophin-1, a muscle-derived cytokine, is required for the survival of subpopulations of developing motoneurons. J Neurosci. 21(4):1283-1291. [95] Pagan S M, et al. (1996) Surgical removal of limb bud Sonic hedgehog results in posterior skeletal defects. Dev Biol. 180(1):35-40. [96] Peroulakis M E, et al. (2000) Forger NG: Ciliary neurotrophic factor increases muscle fiber number in the developing levator ani muscle of female rats. Neurosci Lett. 296(2-3):73-76. [97] Quinn L S, et al. (1990) Paracrine control of myoblast proliferation and differentiation by fibroblasts. Dev Biol. 140(1):8-19. [98] Ravenscroft M S, et al. (1998) Developmental Neurobiology Implications from Fabrication and Analysis of Hippocampal Neuronal Networks on Patterned Silane-Modified Surfaces. J Am Chem Soc. 120(47):12169-12177. [99] Robertson T A, et al. (2000) Comparison of astrocytic and myocytic metabolic dysregulation in apolipoprotein E deficient and human apolipoprotein E transgenic mice. Neuroscience. 98(2):353-359. [100] Sakuma K, et al. (2000) Differential adaptation of growth and differentiation factor 8/myostatin, fibroblast growth factor 6 and leukemia inhibitory factor in overloaded, regenerating and denervated rat muscles. Biochim Biophys Acta. 1497(1):77-88. [101] Sandi E, et al. (2008) Neural agrin controls maturation of the excitation-contraction coupling mechanism in human myotubes developing in vitro. Am J Physiol, Cell Physiol. 294(1):C66-73. [102] Sanes J R, et al. (1997) Genetic analysis of postsynaptic differentiation at the vertebrate neuromuscular junction. Curr Opin Neurobiol. 7(1):93-100. [103] Sheng Z, et al. (1996) Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival. Development. 122(2):419-428. [104] Simon M, et al. (2003) Effect of NT-4 and BDNF delivery to damaged sciatic nerves on phenotypic recovery of fast and slow muscles fibres. Eur J Neurosci. 18(9):2460-2466. [105] Smith J, et al. (1994) The effects of fibroblast growth factors in long-term primary culture of dystrophic (mdx) mouse muscle myoblasts. Exp Cell Res. 210(1):86-93. [106] Song W K, et al. (1992) H36-alpha 7 is a novel integrin alpha chain that is developmentally regulated during skeletal myogenesis. J Cell Biol. 117(3):643-657. [107] Stenger D A, et al. (1992) Coplanar molecular assemblies of aminoalkylsilane and perfluorinated alkylsilanecharacterization and geometric definition of mammalian-cell adhesion and growth. J Am Chem Soc. 114(22):8435-42. [108] Sun L, et al. (2007) JAK1-STAT1.-STAT3, a key pathway promoting proliferation and preventing premature differentiation of myoblasts. J Cell Biol. 179(1): 129-138. [109] Swasdison S, et al. (1992) Formation of highly organized skeletal muscle fibers in vitro. Comparison with muscle development in vivo. J Cell Sci. 102 (Pt 3):643-652. [110] Torgan C E, et al. (2006) Calcineurin localization in skeletal muscle offers insights into potential new targets. J Histochem Cytochem. 54(1): 119-128. [111] Torgan C E, et al. (2001) Regulation of myosin heavy chain expression during rat skeletal muscle development in vitro. Mol Bioi Cell. 12(5): 1499-1508. [112] Vakakis N, et al. (1995) In vitro myoblast to myotube transformations in the presence of leukemia inhibitory factor. Neurochem Int. 27(4-5):329-335. [113] Vogel Z, et al. (1976) Ultrastructure of acetylcholine receptor clusters on cultured muscle fibers. J Cell Biol. 69(2):501-507. [114] Wang P, et al. (2005) Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2. J Neurosci. 25(5): 1219-1225. [115] Wang X, et al. (2008) Effects of interleukin-6, leukemia inhibitory factor, and ciliary neurotrophic factor on the proliferation and differentiation of adult human myoblasts. Cell Mol Neurobiol. 28(1): 113-124. [116] Witzemann V., (2006) Development of the neuromuscular junction. Cell Tissue Res. 326(2):263-71. [117] Yablonka-Reuveni Z, et al. (1995) Development and postnatal regulation of adult myoblasts. Microsc Res Tech. 30(5):366-380. [118] Yang L, et al. (2007) Increased asynchronous release and aberrant calcium channel activation in amyloid precursor protein deficient neuromuscular synapses. Neuroscience. 149(4):768-778. [119] Yang L X, et al. (2004) Glia cell line-derived neurotrophic factor regulates the distribution of acetylcholine receptors in mouse primary skeletal muscle cells. Neuroscience. 128(3):497-509. [120] Zorzano A, et al. (2003) Intracellular signals involved in the effects of insulin-like growth factors and neuregulins on myofibre formation. Cell Signal. 15(2):141-149.