Method for culturing skeletal muscle for tissue engineering

Abstract

The invention provides a nutrient medium composition and associated methods for lengthening the useful life of a culture of muscle cells. Disclosed is a method of culturing mammalian muscle cells, including preparing one or more carriers coated with a covalently bonded monolayer of trimethoxy-silylpropyl-diethylenetriamine (DETA); verifying DETA monolayer formation by one or more associated optical parameters; suspending isolated fetal rat skeletal muscle cells in serum-free medium according to medium composition 1; plating the suspended cells onto the prepared carriers at a predetermined density; leaving the carriers undisturbed for cells to adhere to the DETA monolayer; covering the carriers with a mixture of medium 1 and medium 2; and incubating. A cell nutrient medium composition includes Neurobasal, an antibiotic-antimycotic composition, cholesterol, human TNF-alpha, PDGF BB, vasoactive intestinal peptides, insulin-like growth factor 1, NAP, r-Apolipoprotein E2, purified mouse Laminin, beta amyloid, human tenascin-C protein, rr-Sonic hedgehog Shh N-terminal, and rr-Agrin C terminal.

Claims

1. A method of maintaining a muscle cell culture, the method comprising: maintaining the muscle cell culture in a serum-free maintenance medium comprising the components at the concentrations listed in Table 3, the components at the concentrations listed in Table 4, the components at the concentrations listed in Table 5, creatine, estrogen, and cholesterol wherein the cells in the muscle cell culture consist of muscle cells.

2. The method of claim 1, wherein the muscle cell culture is maintained in the serum-free maintenance medium for at least 30 days.

3. The method of claim 2, wherein the muscle cell culture is maintained in the serum-free maintenance medium for at least 50 days.

4. The method of claim 1, further comprising plating the muscle cell culture onto a non-biological growth substrate prior to maintaining the muscle cell culture in the serum-free maintenance medium.

5. The method of claim 4, wherein the non-biological growth substrate comprises a silane molecule.

6. The method of claim 5, wherein the non-biological growth substrate is trimethoxy-silylpropyl-diethylenetriamine (DETA).

7. The method of claim 1, further comprising plating the muscle cell culture at a density of 700 to 1000 muscle cells per square millimeter prior to maintaining the muscle cell culture in the serum-free maintenance medium.

8. The method of claim 1, further comprising replenishing the serum-free maintenance medium every 1 to 5 days.

9. The method of claim 8, further comprising replenishing the serum-free maintenance medium every 3 days.

10. The method of claim 1, further comprising incubating the muscle cell culture in a first serum-free medium prior to maintaining the muscle cell culture in the serum-free maintenance medium.

11. The method of claim 10, wherein the first serum-free medium comprises a factor selected from the group consisting of vascular endothelial growth factor (VEGF), acidic fibroblast growth factor (FGF), heparin sulfate, leukemia inhibitory factor (LIF), vitronectin, ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), cardiotrophin-1 (CT-1), cholesterol, tumor necrosis factor alpha (TNF-alpha), platelet derived growth factor (PDGF), vasoactive intestinal peptide, insulin-like growth factor 1, neutrophil activating protein (NAP), r-Apolipoprotein, laminin, beta amyloid, tenascin-C protein, rr-sonic hedgehog, and rr-agrin.

12. The method of claim 11, wherein the first serum-free medium comprises vitronectin.

13. The method of claim 12, wherein the first serum-free medium is a mixture of approximately equal volumes of the medium composition of Table 1 and the medium composition of Table 2.

14. The method of claim 10, wherein the muscle cell culture is maintained in the first serum-free medium from 2 to 6 days.

15. The method of claim 14, wherein the muscle cell culture is maintained in the first serum-free medium for 4 days.

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 schematic diagram of a culture protocol according to an embodiment of the present invention;

(3) FIG. 2 A, B, C and D provide phase pictures of 50 day old myotubes in culture; red arrows showing characteristic striations in most of the myotubes; scale bar equals 75 m;

(4) FIG. 3 shows myotubes stained with antibodies against embryonic myosin heavy chain (F 1.652) proteins at day 50; scale bar is 75 m; A) panel showing phase+fluorescence picture of the myotubes; B) another view of panel A but observed only under fluorescence; white arrows showing the striations; C) panel showing image of myotubes under phase+fluorescence illumination; D) shows panel C observed only under fluorescence illumination; E) panel showing phase+fluorescence picture of the myotubes (white arrow indicating the striations); F) panel E observed only in fluorescence light (white arrow indicating the striations); G) panel showing phase+fluorescence picture of the myotubes (white arrow indicating the striations); H) panel G observed only under fluorescence light (white arrow indicating the striations);

(5) FIG. 4 shows myotubes immunostained with neonatal myosin heavy chain (N3.36) and alpha-bungarotoxin at day 50; scale bar is 75 m; A) is a phase picture of 2 myotubes indicated by white arrows; B) both the myotubes shown in phase (panel A) have acetylcholine receptor clustering indicated by alpha-bungarotoxin staining; C) only one myotube out of the two seen in panel A stained for N3.36; D) double stained image of panel A with alpha-bungarotoxin and N3.36; E) phase image of 6 myotubes indicated by white arrows; F) all the myotubes shown in phase (panel E) have acetylcholine receptor clustering shown by alpha-bungarotoxin staining; G) none of the myotubes in panel E stained for N3.36; H) I) and J) show differential staining of the myotubes with N3.36; K) L) and M) showing differential staining of the myotubes with N3.36;

(6) FIG. 5 illustrates ryanodine receptor and DHPR receptor clustering in 30 days old skeletal muscle culture (scale bar 75 m); A) phase and fluorescent-labeled picture of the myotubes; B) merged fluorescence picture of the ryanodine receptor (green) and DHPR receptor (red) clustering on the myotubes shown in panel A; C) ryanodine receptor (green) on the myotubes shown in panel A; D) DHPR receptors on the myotubes shown in panel A; E) phase and fluorescent-labeled picture of the myotubes; F) merged fluorescent picture of the ryanodine receptor (green) and DHPR receptor (red) clustering on the myotubes (panel E); G) ryanodine receptor (green) on the myotubes (panel E); H) DHPR receptors on the myotubes (panel E); I) phase and fluorescent-labeled picture of the myotubes; J) K) and L) show merged fluorescence pictures of the ryanodine receptor (green) and DHPR receptor (red) clustering on the myotubes (panel I) at three different planes (white arrows indicate the striations and the receptor clustering);

(7) FIG. 6 shows ryanodine receptor and DHPR receptor clustering in 100 days old skeletal muscle culture (scale bar: 75 m); A) shows phase and fluorescent-labeled picture of the myotubes; B) is a merged fluorescence picture of the ryanodine receptor (green) and DHPR receptor (Red) clustering on the myotubes (panel A); C) shows ryanodine receptor (green) on the myotubes (panel A); D) shows DHPR receptors on the myotubes (panel A); E and F show views of the same panels at different planes showing the merged fluorescent picture of the ryanodine receptor (green) and DHPR receptor (red) clustering on the myotubes;

(8) FIG. 7 depicts patch clamp electrophysiology of the myotubes, wherein A shows representative voltage clamp trace obtained after patching a 48 days old myotube in culture; B shows representative current clamp trace of the same myotube for which voltage clamp trace had been obtained (inset showing the picture of patched myotubes).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(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, or 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, however, 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 O.sub.2 plasma cleaner (Harrick PDC-32G) for 20 minutes 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 QQ 100 C., rinsed with toluene, reheated to approximately 100 C., and then oven dried [28]. 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 Al K excitation were obtained [28].

(13) Skeletal Muscle Culture and Serum Free Medium

(14) The skeletal muscle was dissected from the thighs of the hind limbs of fetal rats (17-18 days old). The tissue was collected in a sterile 15 mL centrifuge tube containing 1 mL of 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 minutes in a 37 C. water bath at 50 rpm. After 30 minutes the trypsin solution was removed and 4 mL of 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 300g for 10 minutes at 4 C. The pellet obtained was washed 5 times with L15 medium then resuspended in 10 mL of L15 and plated in 100mm uncoated dishes for 30 minutes. The non-attached cells were removed and then centrifuged on a 4% BSA cushion [28]. 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 one hour. The final medium was prepared by mixing medium one (Table 1) and medium two (Table 2) in a 1:1 v/v ratio. FIG. 1 indicates the flowchart of the culture protocol. Tables 1 and 2 indicated the growth factor and hormone supplement compositions of medium one and medium two. The cells were maintained in a 5% CO.sub.2 incubator (relative humidity 85%). The full medium was replaced after four days with NBactiv4 medium according to the protocol in FIG. 1 [34]. Thereafter three-fourth of the medium was changed every three days with NBactiv4.

(15) NbActiv4 (available from BrainBits LLC) comprises all of the ingredients in Neurobasal, B-27, and GlutaMAX. NbActiv4 also comprises creatine, estrogen, and cholesterol.

(16) Immunocytochemistry of Skeletal Muscle Myotubes

(17) Coverslips were prepared for immunocytochemical analysis as previously described. Briefly, 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) for 10 minutes, and blocked for 2 h with 10% goat serum and 1% BSA. Cells were incubated overnight with primary antibodies against embryonic myosin heavy chain (F1.652) (dilution>1:5), neonatal myosin heavy chain (N3.36) (1:5) (Developmental Studies Hybridoma Bank), ryanodine receptor (AB9078, Millipore) (1:500) and dihydropyridine binding complex (1-Subunit) (MAB 4270, Millipore) (1:500) diluted in the blocking solution. Cells were washed with PBS and incubated with the appropriate secondary antibodies for two hours in PBS. After two hours the coverslips were rinsed with PBS and mounted on glass slides and evaluated using confocal microscopy [25, 28, 31].

(18) AChR Labeling of Myotubes

(19) AChRs were labeled as described previously by incubating cultures with 510-8 M of -bungarotoxin, Alexa Fluor 488 conjugate (B-13422; Invitrogen) for 1.5 h at 37 C. [12, 31]. Following incubation in -bungarotoxin, the cultures were fixed as above for subsequent staining with embryonic myosin heavy chain (F1.652) antibodies.

(20) Patch Clamp Electrophysiology of the Myotubes

(21) Whole-cell patch clamp recordings were performed in a recording chamber located on the stage of a Zeiss Axioscope 2FS Plus upright microscope as described previously [25, 33]. The chamber was continuously perfused (2 ml/min) with the extracellular solution (Leibovitz medium, 35 C.). Patch pipettes were prepared from borosilicate glass (BF150-86-10; Sutter, Novato, Calif.) with a Sutter P97 pipette puller and filled with intracellular solution (K-gluconate 140 mM, EGTA 1 mM, MgCl.sub.2 2 mM, Na.sub.2ATP 2 mM, phosphocreatine 5 mM, phosphocreatine kinase 2.4 mM, Hepes 10 mM; pH=7.2). The resistance of the electrodes was 6-8M. Voltage clamp and current clamp experiments were performed with a Multiclamp 700A amplifier (Axon Laboratories, Union City, Calif.). Signals were filtered at 2 kHz and digitized at 20 kHz with an Axon Digidata 1322A interface. Data recording and analysis were done with pClamp 8 software (Axon Laboratories). Membrane potentials were corrected by subtraction of a 15 mV tip potential, which was calculated using Axon's pClamp 8 program. Sodium and potassium currents were measured in voltage clamp mode using voltage steps from a 85 mV holding potential. Action potentials were evoked with 1 second depolarizing current injections from a -85 mV holding potential [25, 28].

(22) Results

(23) DETA Surface Modification and Characterization

(24) Static contact angle and XPS analysis was 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 were similar to previously published results [24, 25, 28, 29, 31]. Based on the ratio of the N (401 and 399 eV) and the Si 2p3/2 peaks, XPS measurements indicated that a reaction-site limited monolayer of DETA was formed on the coverslips [35].

(25) Development of the Serum Free Medium Formulation and Culture Timeline for Long-Term Survival and Maturation of Myotubes

(26) The serum free medium composition was developed empirically. The final medium is derived from two different medium compositions described in Tables 1 and 2. Table 1 constitutes the same medium composition used previously for a motoneuron-muscle co-culture and adult spinal cord neurons culture [26, 27, 30, 31]. Table 2 is composed of twelve additional factors that had been shown to promote skeletal muscle maturation and neuromuscular junction formation separately. The final medium was prepared by mixing these two media in a 1:1 v/v ratio. After first 4 days of culture the whole medium was replaced with NBactiv4 medium [34]. hereafter, every three days three-fourth medium was changed with NBactiv4. The culture technique has been illustrated in the flowchart (FIG. 1).

(27) Using this new medium formulation and timeline, myotubes were successfully cultured for more than 50 days. FIG. 2 indicates 50 days old myotubes in culture. As the myotubes aged and grew they began to form the characteristic anisotropic (A band) and isotropic (I band) banding pattern observed with in vivo muscle fibers [22, 23]. This banding pattern is caused by differential light diffraction due to the organization of myofibril proteins forming sarcomeres within the myotubes [22, 23]. The arrowheads in the images (FIG. 2A-D) indicate myotubes where sarcomeric organization has occurred and is visualized by the appearance of A and I bands.

(28) Myotube Expression of Fetal Myosin Heavy Chain

(29) The myotubes formed were evaluated for the expression of fetal MHC to establish a baseline as comparison to our previous results [28]. In FIG. 3, the myotubes phenotypes formed at approximately day 50 in vitro are shown. The myotubes ranged from having clustered nuclei (FIG. 3A-D) to having diffuse nuclear organization (FIG. 3E-H). The arrowheads in the images indicate the characteristic striations.

(30) Differential Expression of Neonatal MHC Protein in the Myotubes

(31) In order to determine if the myotubes were maturing in a physiologically relevant way as they aged in vitro, the expression of neonatal MHC protein was evaluated. After approximately 50 days in vitro 25% of the myotubes expressed neonatal MHC (FIG. 4A-M). Additionally, the myotubes were stained for clustering of acetylcholine receptors (AChR) using alpha bungarotoxin (FIG. 5B,F). This clustering of the AChR receptors, induced by the motoneuron protein agrin in vivo, are locations on the myotube where neuromuscular junction formation occurs.

(32) Formation of the ExcitationContraction Coupling Apparatus

(33) The presence of ryanodine (RyR) receptor and dihydropyridine (DHPR) receptor clusters, as well as their colocalization in vivo, represents the development of excitation-contraction coupling apparatus in skeletal muscle myotubes [19, 21-23]. The clustering of both RyR and DHPR receptors was observed on the myotubes after 30 days in culture (FIG. 5A-D). The clustering and colocalization of the RyR+DHPR clusters was observed with different myotube morphologies (FIG. 5E-L). This functional adaptation illustrated that the medium formulation facilitated not only the structural maturation but also the functional maturation of myotubes in this in vitro system. The clustering of the RyR+DHPR receptors was also observed in the 70 day old myotubes, indicating that the older myotubes maintained their functional integrity (FIG. 6A-F).

(34) Myotube Electrophysiology

(35) The myotubes contracted spontaneously in the culture and the contractions began generally by day four and continued throughout the life of the culture. Most of the myotubes expressed functional voltage gated sodium, potassium and calcium ion channels as reported previously [28]. The voltage clamp electrophysiology of the myotubes indicated the inward and outward currents that demonstrate functional sodium and potassium channels (FIG. 7A). The current clamp study indicated the single action potential fired by the myotubes (FIG. 7B).

(36) Discussion

(37) Herein we have documented the development of a system for long-term in vitro functional, skeletal muscle culture. This system was developed in response to a need for more physiologically relevant skeletal muscle myotubes for functional in vitro systems. For our specific research, they were needed for a realistic model of the stretch reflex arc development and to be integrated with bio-MEMS cantilevers for screening appications. The results indicate we achieved three significant structural modifications within the myotubes, causing both the developmental profile and functionality of the fibers to better mimic in vivo physiology. It is believed that this skeletal muscle maturation resulted from modifications to the cell culture technique, a new medium formulation and the use of NBactiv4 as the maintenance medium.

(38) The presently described serum-free medium supplemented with growth factors was developed to support the survival, proliferation and fusion of fetal rat myoblasts into contractile myotubes. The rationale for selecting the growth factors was based on the distribution of their cognate receptors in the developing myotubes in rat fetus [1-11]. 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 used for motoneuron-muscle co-culture and adult spinal cord neuron culture [26, 27, 30, 31]. Table 2 lists the twelve additional factors we have identified in muscle development and neuromuscular junction formation. The use of NBactiv4 for the maintenance of the cells provided unexpected results in that it significantly improved the survival of the skeletal muscle derived myotubes despite the original development of NBactiv4 for the long-term maintenance and synaptic connectivity of fetal hippocampal neurons in vitro [34].

(39) We observed a ratio of 25% neonatal to 75% embryonic MHC expression of the myotubes, which contrasts with the previous study in which MHC expression was strictly embryonic. We believe that the myotubes matured in this culture system because the long-term survival provided adequate time for the myotubes to respond to the additional growth factors, which activated the necessary signaling pathways to achieve MHC class switching [20]. This suggests that a different growth factor profile could be utilized to activate alternative signaling pathways and drive myotube differentiation down other pathways. For example, the effects of adding steroid hormones like testosterone to the system could be critically examined.

(40) The colocalization of RyR and DHPR clusters in the myotubes indicated the formation of excitation-contraction coupling apparatus and was another indicator of functional maturation in the fibers. Excitation-contraction coupling is the signaling process in muscle by which membrane depolarization causes a rapid elevation of the cytosolic Ca.sup.2+ generating contractile force [36]. The close proximity of the DHPR and RyR complexes occurs at specialized junctions established between the transverse tubule and sarcoplasmic reticulum (SR) membranes in skeletal muscle myotubes [37]. At these junctions, T-tubule depolarization is coupled to Ca.sup.2+ release from the SR resulting in muscle contraction [38-40]. This structural adaptation represents a significant functional change due to the fact that excitation-contraction coupling is required for successful extrafusal muscle fiber development as well as neuromuscular junction formation [19, 21-23]. This improved model provides the potential to study excitation-contraction coupling in a defined system as well as myotonic and myasthenic diseases.

(41) Conclusion

(42) The development of sarcomeric structures, the excitation-contraction coupling apparatus and MHC class switching in the skeletal muscle myotubes is a result of the improvements to the model system documented in this research. This improved system along with the new findings support the goal of creating physiologically relevant tissue engineered muscle constructs and puts within reach the goal of functional skeletal muscle grafts. Furthermore, we believe this serum-free culture system will be a powerful tool in developing advanced strategies for regenerative medicine in muscular dystrophies, stretch reflex arc development and integrating skeletal muscle with bio-hybrid prosthetic devices.

(43) 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.

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(45) TABLE-US-00001 TABLE 1 Medium Composition 1 S. Cata- Refer- No Component Amount logue # Source ences 1. Neurobasal 500 ml 10888 Gibco/ [41] Invitrogen 2. Antibiotic- 5 ml 15240-062 Gibco/ Antimycotic Invitrogen 3. G5 Supplement 5 ml 17503-012 Gibco/ [42-51] (100X) Invitrogen 4. VEGF.sub.165 r Human 10 g P2654 Gibco/ [52-55] Invitrogen 5. Acidic FGF 12.5 g 13241-013 Gibco/ [42, 49, Invitrogen 51, 56-61] 6. Heparin Sulfate 50 g D9809 Sigma [42, 49, 51, 56-61] 7. LIF 10 g L5158 Sigma [62-70] 8. Vitronectin 50 g V0132 Sigma [71, 72] (Rat Plasma) 9. CNTF 20 g CRC 401B Cell [73-77] Sciences 10. NT-3 10 g CRN 500B Cell [15] Sciences 11. NT-4 10 g CRN 501B Cell [78, 79] Sciences 12. GDNF 10 g CRG 400B Cell [80-84] Sciences 13. BDNF 10 g CRB 600B Cell [79, 85, Sciences 86] 14. CT-1 10 g CRC 700B Cell [87-95] Sciences

(46) TABLE-US-00002 TABLE 2 Medium Composition 2 Refer- No Component(s) Amount Catalog Source ences 1 Neurobasal 500 ml 10888 Invitrogen/ [41] Gibco 2 Antibiotic- 5 ml 15240- Invitrogen/ antimycotic 062 Gibco 3 Cholesterol 5 ml 12531 Invitrogen/ [96] (250X) Gibco 4 TNF-alpha, 10 g T6674 Sigma- [97-99] human Aldrich 5 PDGF BB 50 g P4056 Sigma- [62, 100- Aldrich 103] 6 Vasoactive 250 g V6130 Sigma- [104] intestinal Aldrich peptide (VIP) 7 Insulin-like 25 g I2656 Sigma- [68, 69, 98] growth Aldrich factor 1 8 NAP 1 mg 61170 AnaSpec, [105, 106] Inc. 9 r- 50 g P2002 Panvera, [107] Apolipoprotein Madison, WI E2 10 Laminin, 2 mg 08-125 Millipore [108-114] mouse purified 11 Beta amyloid 1 mg AG966 Millipore [115-117] (1-40) 12 Human 100 g CC065 Millipore [118] Tenascin- C protein 13 rr-Sonic 50 g 1314-SH R&D [7, 119-129] hedgehog, Systems Shh N-terminal 14 rr-Agrin 50 g 550-AG- R&D [130, 131] (C terminal) 100 Systems

(47) TABLE-US-00003 TABLE 3 B-27 Serum-Free Supplement Media ingredients Concentration Components (mg/L) Vitamins Biotin 0.10 DL Alpha Tocopherol Acetate 1.0 DL Alpha-Tocopherol 1.0 Vitamin A 0.1-0.2 Proteins BSA, fatty acid free Fraction V 2500.0 Catalase 2.5 Human Recombinant Insulin 4.0 Human Transferrin 5.0 Superoxide Dismutase 2.5 Other Components Corticosterone 0.02 D-Galactose 15.0 Ethanolamine HCl 1.0 Glutathione (reduced) 1.0 L-Carnitine HCl 2.0 Linoleic Acid 1.0 Linolenic Acid 1.0 Progesterone 0.0063 Putrescine 2HCl 16.1 Sodium Selenite 0.035 T3 (triodo-I-thyronine) 0.002

(48) TABLE-US-00004 TABLE 4 Neurobasal media formulation Concentration Components (mg/L) Amino Acids Glycine 30.0 L-Alanine 2.0 L-Arginine hydrochloride 84.0 L-Asparagine-H2O 0.83 L-Cysteine 31.5 L-Histidine hydrochloride-H2O 42.0 L-Isoleucine 105.0 L-Leucine 105.0 L-Lysine hydrochloride 146.0 L-Methionine 30.0 L-Phenylalanine 66.0 L-Proline 7.76 L-Serine 42.0 L-Threonine 95.0 L-Tryptophan 16.0 L-Tyrosine 72.0 L-Valine 94.0 Vitamins Choline chloride 4.0 D-Calcium pantothenate 4.0 Folic Acid 4.0 Niacinamide 4.0 Pyridoxal hydrochloride 4.0 Riboflavin 0.4 Thiamine hydrochloride 4.0 Vitamin B12 0.0068 i-Inositol 7.2 Inorganic Salts Calcium. Chloride (CaCl2) (anhyd.) 200.0 Ferric Nitrate (Fe(NO3)39H2O) 0.1 Magnesium Chloride (anhydrous) 77.3 Potassium Chloride (KCl) 400.0 Sodium Bicarbonate (NaHCO3) 2200.0 Sodium Chloride (NaCl) 3000.0 Sodium Phosphate monobasic 125.0 (NaH2PO4H2O) Zinc sulfate (ZnSO47H2O) 0.194 Other D-Glucose (Dextrose) 4500.0 Components HEPES 2600.0 Phenol Red 8.1 Sodium Pyruvate 25.0

(49) TABLE-US-00005 TABLE 5 Glutamax media formulation Components Concentration (mM) Peptides L-alanyl-L-glutamine 200 Inorganic Salts Sodium Chloride (NaCl) 145