Polarity Induced in Human Stem Cell Derived Motoneurons on

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Polarity Induced in Human Stem Cell Derived Motoneurons on Patterned Self-Assembled Monolayers Mercedes Gonzalez, Xiufang Guo, Min Lin, Maria Stancescu, Peter Molnar, Severo Spradling, and James J. Hickman ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00682 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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ACS Chemical Neuroscience

Polarity Induced in Human Stem Cell Derived Motoneurons on Patterned Self-Assembled Monolayers Authors: Mercedes Gonzalez1,†, Xiufang Guo1,†, Min Lin1, Maria Stancescu1,2, Peter Molnar1, Severo Spradling3 and James J. Hickman 1,2,3, *

Affiliations: 1 Hybrid

Systems Lab, NanoScience Technology Center, University of Central Florida, 12424

Research Parkway, Suite 400, Orlando, FL 32826, USA 2 Department

of Chemistry, 4000 Central Florida Blvd., Physical Sciences Building (PS) Room

255, University of Central Florida, Orlando, FL 32816-2366, USA 3 Biomolecular

Science Center, Burnett School of Biomedical Sciences, University of Central

Florida, 12722 Research Parkway, Orlando, FL 32826, USA † These authors contribute equally to this work.

* Corresponding author University of Central Florida, NanoScience Technology Center 12424 Research Parkway, Suite 400, Orlando, FL 32826 USA Phone: 407-823-1925 Fax: 407-882-2819 E-mail: [email protected]

1 ACS Paragon Plus Environment

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Abstract: The control of polarized human neurite/axon development at the single neuron level is critical in geographically directing signal propagation in engineered neural networks, both for in vitro and in vivo applications. While there is an increasing need to exert control over axonal growth for the successful development and establishment of integrative and functional in vitro systems, controlled, polarized distribution of either human-derived neurons or motoneurons in vitro has yet to be reported. In this study, we established the polarized distribution of stem-cell derived human motoneurons, using a patterned surface, and maintained the cells in a serum-free system. A surface pattern with defined polarity was developed using self-assembled monolayers (SAM). A cell permissive SAM, DETA (trimethoxysilyl propyldiethylenetri-amine), combined with photolithography and a non-permissive fluorinated silane, 13F (tridecafluoro-1,1,2,2tetrahydroctyl-1-dimethylchloro-silane), generated a surface where neurons only adhered to the designed attachment sites and did so with preferred orientation. In addition, 75% of the cells attached to the patterns were motoneurons compared to their percentage in the standard unpatterned surface which was used as a control condition (20%), demonstrating the preference of these human motoneurons in adhering to the patterns. The ability to dictate the distribution and polarity of human motoneurons will be essential to the engineering of human-based functional in vitro systems in which the control of signal propagation is necessary but more importantly for cell implantation studies. Such systems will greatly benefit the study of motor function as well as aid the development of high-throughput systems for drug screening and test beds for use in preclinical studies related to conditions such as spinal cord injury, ALS and muscular dystrophy.


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Keywords: motoneurons; human; polarity; surface chemistry.

Introduction Functional in vitro neural networks are in high demand for both the study of systems biology as well as developing high information throughput systems for drug screening. Humancell-based functional systems are especially desired due to their elimination of the species gap, thus providing a shorter time for the translation of these studies to clinic. Understanding how to induce polarity for neurons in situ is not only essential for designing neural network in vitro, but also important for developing therapeutics for some neuronal disorders.1-2 The development of human cell-based in vitro neuronal systems was slow to develop, due to issues such as limited tissue quantity, variability of tissues from different sources and strict regulations. Fortunately, the stem cell field has opened a new avenue for the development of advanced in vitro systems and is playing an increasingly important role in etiology, drug discovery and for therapeutic treatments.3 Stem cells have the potential to reproduce themselves indefinitely and to be differentiated into cells with various functions in certain conditions, thus providing an excellent source for generating human neurons.4-7 Many types of neurons have been successfully induced from stem cells including motoneurons,8-11 peripheral neurons,12-14 cortical pyramidal neurons15-16 and dopaminergic neurons.17 These studies provide a foundation for using stem cells to build in vitro neural networks engineered to optimize the study of various related and unrelated conditions such as Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease, spinal cord injury and neuropathic pain. However, the lack of control that can currently be exerted over neuronal localization and orientation, as well as an insufficient influence on the formation of functional synapses with correct cell partners, and thus the



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direction of signal propagation, are two significant problems researchers face when attempting to engineer novel in vitro neural network models as well as for in vivo applications. Controlling polarized neurite/axon distribution at the single neuron level is a key step in engineering neural networks, since it allows directional control of signal propagation in the circuit. Motoneurons are highly polarized cells both structurally and functionally, with each motoneuron possessing a single axon and multiple dendrites. The axon is typically long, unbranched and specialized for neurotransmitter release, whereas dendrites are highly branched and express postsynaptic receptors for signal reception. After plating, most neurons extend undifferentiated neurites within 24 hours of adhesion to the substrate. One of the neurites eventually grows longer than the others becoming the axon, while the remaining neurites become dendrites.18 In culture, the absence of the fine scale molecular cues present in vitro, leads axons and dendrites to extend in random directions. They form en passant synapses with other neurons in culture whenever they physically reach appropriate physiological partners, thus making it virtually impossible to establish reproducible synaptic interactions and geographically controlled functional circuits in vitro or to reestablish functional connections in vivo. Controlling the distribution of dendritic/axonal orientation at a single neuron level on the culture surface is therefore critical for developing high resolution and reproducible neural networks in vitro. Multiple strategies have been investigated in order to achieve geometrically controlled neuronal distribution in vitro. One major strategy to control the directionality of neuronal network outgrowth is by generating chemical or biochemical surface patterns, by utilizing the technologies such as photolithography 19, microcontact printing 20, and microfluidic patterning 21,22

Another strategy is the utilization of 3D physical guidance23, such as topographical features

generated by laser fabrication 24, microprinting 25, microfluidics 26 and electrospinning 27. In


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work with mouse embryonic hippocampal neurons it was demonstrated that Axo-dendritic polarity on poly-L-Lysine micropatterns aligned above silicon nanowires , for which the micropatterns were generated using classical photolithography techniques 28. Our group, by combining silane-based self-assembled-monolayers (SAMs) with lithographic technique, has been able to create patterned surfaces that generated polarized distribution of adult 29 and embryonic rat hippocampal neurons 30,31, but such a culture system has yet to be reported for motoneurons or for human neurons derived from stem cells. Self-assembled monolayers (SAM) provides great flexibility for controlling surface properties and cell attachment.32 The utility of SAM surface modifications over other types of substrates is based on their ability to generate geometric patterns without having to etch off the surface,33-35 as well as the wide availability of functional groups.36 One type of SAM, trimethoxysilyl propyldiethylenetri-amine (DETA) was previously shown to support the growth of multiple cell types including neurons.34, 37-38 Morphological, immunocytochemical and electrophysiological evidence suggests that neurons grown on a DETA substrate are indistinguishable from those grown on traditional poly-D-lysine coated surfaces.32, 37 DETA combined with photolithography and a non-permissive, hydrophobic fluorinated silane surface, such as tridecafluoro-1,1,2,2-tetrahydroctyl-1-dimethylchloro-silane (13F), has been utilized successfully in developing high-resolution, in vitro patterned circuits of embryonic hippocampal neurons.31, 39-40 Compared to other surface patterning strategies, the SAM lithographic approach is relatively easier to perform, applicable to high throughput and less expensive. In addition, the generated surface patterns can be monitored for their quality with surface analysis methods, and they are stable in long-term culture. In this study, we established a high-resolution SAM surface which enabled pattern derived polarity of motoneurons derived from human fetal spinal cord



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stem cells, in a defined system. The system was characterized by surface analysis, immunocytochemistry and electrophysiological analysis. The percentage of motoneurons was enriched in the cell population attached to the patterns compared to those on unpatterned surface (75% vs. 20%). The development of such a system is a vital step in the movement towards fabrication of controllable, high-resolution neural networks for the study of axonal regeneration which is essential for the treatment of spinal cord injury, neuromuscular junction formation as well as application in investigations of the motor control system and its associated disorders.

Results and Discussion Surface Modification and Patterning The polarity pattern utilized in this study was visualized using the metallization procedure described in the Methods and is presented in Fig 1A. This pattern induced a polarized distribution of motoneuron neuritic outgrowth, by providing one long, unbroken line of DETA. The favored pathway for rapid neurite elongation was a solid line of DETA, since the less favored paths contained molecular ‘speed bumps’ of fluorinated silane interspersed in the remaining DETA. The dimension and the distribution of patterns on the surface are displayed as in Figure 1, A and B respectively. The masks and the dimension of the patterns were derived from previous work that demonstrated geometric control of neuronal polarity 19 and network formation 41 using different SAM combinations for embryonic and adult rat hippocampal neurons 30, 29,41 . A study concerning the

ability of neurons to extend projections and to form physical connections utilized rodent hippocampal neurons on poly-D-lysine with adhesion spots of 23 μm in diameter, and found out that the maximum gap the axons could span was ~40 μm 42. Since this is a proof of principle study for establishing controlled polarized distribution of differentiated human motoneurons on the surface with SAM technology, the dimensions described in Fig 1A were utilized. The somal 6 ACS Paragon Plus Environment

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adhesion site, illustrated in Fig 1A, had a diameter of 20 μm, while a neurite path width of 5 μm was used throughout the study, since it was found to be narrow enough to minimize the attachment of cell bodies, yet wide enough to allow for normal process outgrowth.34, 43

Fig 1. Surface characterization: A and B) Optical image of palladium-catalyzed metallization of patterned DETA/13F monolayers. The dimension of each pattern is indicated in A: soma, 20 μm in diameter; axonal line length 200 μm; dendritic line length, 60 μm in total, interspersed with gaps of 10 μm; line widths of 2 μm. B) Distribution of patterns on the surface. The distribution of the patterns on the surface is demonstrated in B. C and D) Typical water contact angle on DETA (C) and 13F (D) surfaces. E and F) Typical DETA (E) and 13F (F) XPS survey spectra.



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A contact angle of less than 5 degrees was obtained for clean substrates. An average of 49 ± 2o was measured for DETA substrates confirming their hydrophilicity (Fig 1B), while an average of 90 ± 2o was recorded for 13F control slides, confirming their hydrophobicity (Fig 1C). XPS measurements of the DETA and 13F coated coverslips indicated a complete monolayer formation after the self-assembly process. Representative DETA and 13F XPS survey spectra are shown in Fig 1D & E respectively. The quality of the surfaces was in agreement with previously reported results.39, 44 For DETA SAMs, the normalized area values of N1s peak (399 ± 0.3 eV) to the Si 2p3/2 peak (103.3 ± 0.3 eV) were stable throughout the study at 1300-2300 and were similar to previously published results for DETA.37, 45-48 Stable XPS readings and contact angles across coverslips throughout the study indicate uniformity and reproducibility of the SAM patterning protocol. Pattern Optimization Analysis Human motoneurons utilized in this study were first differentiated from human fetal stem cells, then harvested and replated on the DETA/ 13F patterned coverslips at a density of 50~75 cells/mm2 (See Methods) for the investigation of their distribution and regrowth on the polarity pattern. After plating, the cells attached to the provided DETA patterns and avoided the 13F backfilled areas. Since the fidelity of attachment depends closely on the dimensions of the pattern features, the somal diameter of the patterns was first optimized to determine the most favorable attachment sizes for the growth of human motoneurons. Patterns with four different soma sizes, 10, 15, 20 and 25 μm were tested. Correct attachment of cell somata on intended somal adhesion sites was assessed for the different soma size patterns and the number of correct cell attachments (single cell on a pattern) was found to be the highest on patterns with a soma size of 20 μm. Cell numbers on patterns with smaller soma sizes (10, 15 μm) was suboptimal, so


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was not quantified. Those patterns with the larger soma size (25 μm) tended to have more than one cell on a single patterned soma site. Overall, both 20 μm and 25 μm soma sizes generated a significant number of correct cell attachments. This is to be expected considering the diameter of human motoneurons is generally between 15 and 25 μm. To further quantify this observation, a correctness index was established, defined as the number of cells correctly adhered to each pattern as a percentage of the total number of cells on the pattern. The correctness index for cells on the 20 μm and 25 μm pattern was 47 ± 7% and 38 ± 6% (mean +/- SEM) respectively. While there was no statistically significant difference between these 2 values (t-test, p = 0.3; Fig. 2), a somal adhesion area of 20 µm was utilized for the rest of this study in an attempt to achieve the maximum number of sites containing only 1 cell.

Fig 2. Optimization of soma adhesion site by analyzing correctness index. The correctness index is defined by the total number of correctly positioned cells on each pattern as a percentage of the total number of cells on the pattern. A) Phase images displaying examples of the incorrect cell attachment on the patterns: non-neuronal attachment (yellow arrow), cell attachment on the dendrites (red arrow), and multiple cell attachment (green arrow). B) Based on results from Day 6 analysis from multiple patterned chips in one experiment, the correctness index for the cells on the 20 μm patterns was 0.47 and 0.38 for the cells on the 25 μm patterns. The number of patterns



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analyzed for each soma size are indicated at the top of each bar. Data represented as mean + SEM.

Population Characterization of Cell Attachment on the Patterns The cell attachment on patterns was monitored using phase contrast microscopy beginning right after plating. The cell number that attached to each pattern and the correctness index of the patterns were quantified from Day 1 to Day 9 (Figure 3). As in Fig 3A, cells already selectively attached to the patterns 30 min after plating, displaying ~45% index of correctness starting from Day 1, and increasing slightly over the subsequent days and reaching ~49% on Day 9. Cell attachment on the 13F area was obvious right after plating (30 min), but was significantly reduced at day 1 and any remaining cells gradually released over time and were absent by day 6. During this period, the percentage of patterns containing one or more cells, as well as those empty patterns were also quantified as in Fig 3C. About 20% of patterns didn’t have any cells attached (empty), while about 40% of the patterns had one cell, and the remaining patterns had two or more cells attached. For all the patterns that had cells attached, more than 80% of the patterns had one correctly adhered neuron (Fig 3D).


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Fig 3. Longitudinal characterization of the neuronal culture on patterns. A) Sample images of cell attachment on the patterned surfaces from Day 0-9. The green arrow points a pattern with a neuron fully grown, and the yellow arrow points to one where the neuron has started to detach. Error bar: 100 μm. B) Correctness Index of the patterns from Day 0-9. C and D) Characterization of cell attachment on patterns based on data pooled from Day 5 to Day9 of culture. Percentage of patterns that have different number of cell attachment (C) and the percentage of patterns containing correctly attached neurons out of the total patterns occupied with correspondent number of cells (D) were quantified based on results from 4 coverslips of 2 platings, in total of 274 patterns. Data represented as mean + SEM.



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Growth of Motoneurons on Polarity Patterns The growth of the attached neurons on the described patterns was monitored for 12 DIV using phase contrast microscopy in order to determine how long correctly attached neurons survived. Figure 4A depicts a neuron after 2 DIV beginning to follow the provided pattern for neurite growth. The neurite extended the entire length of the pattern following 4 DIV, and some detachment of neurons from the pattern was observed from day 8 onwards. A representative picture for an 8 DIV neuron is shown in Figure 4B. Between Day 2 and Day 8, motoneuron attachment on the patterns were similar but with a distribution of axonal lengths.

Fig 4. A and B) Phase contrast image of human stem cell derived motoneuron on DETA/13F polarity patterns at 2 and 8 DIV, respectively. C and D) Metallization images of the corresponding polarity patterns with 20 μm somal adhesion site corresponding to the image in A and B, respectively.


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Since previous work has demonstrated that the described protocol only induces motoneuron differentiation in roughly 20% of the cultured stem cells10, immunostaining with βIII-Tubulin and Islet 1 antibodies was conducted to determine the identity of the cells on the DETA/ 13F polarity patterns. β-III-Tubulin was used to identify neurons, as well as their processes, and Islet 1 was used to identify motoneurons specifically. Co-staining of these two on a single pattern was taken as evidence of motoneuron presence on the patterns. Sample pictures from the immunostaining of 1 DIV (Fig. 5A) and 2 DIV (Fig. 5B) cultures demonstrate the attachment of single motoneurons on the patterns, and their polarized dendritic/axonal distribution following the designed pathways. Quantification of the immunostaining indicated that 75% of the cells correctly adhered to the patterned somal adhesion site were motoneurons (512 motoneurons out of 685 pattern-attached cells from 2 batches of platings). Moreover, while not all cells present on the patterns were motoneurons, every cell-occupied pattern examined (n=194) was found to contain at least one motoneuron. The observation that the population of cells attached to these polarity patterns is enriched for motoneurons (75%) when compared to identical cells plated on un-patterned controls (~20%), indicates that human motoneuron’s preferred adherence for the patterns provided in this study is substantially greater than that of other cell types (glia, interneurons, neuronal precursors etc.) within the examined population. This assumption is reasonable since the dimensions of the polarity pattern is designed to mimic the physical features of motoneurons and therefore would select against alternative cellular morphologies.



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Fig 5. Polarized distribution of human motoneurons on designed surface patterns in 1 DIV (A) and 2 DIV (B) cultures respectively. The cells were co-stained with β III Tubulin for neuronal identity and HB9 for motoneuron identity. The nuclei were identified using the fluorescent marker Dapi.

Polarized distribution of motoneurons on SAM patterns To further determine whether or not the attached motoneuron neurites developed the desired polarity as directed by the pattern design, the cultures were immunostained with Neurofilament and Islet 1 antibodies. Neurofilament is a type IV intermediate filament found specifically in neurons and has been used as a marker for axons as opposed to dendrites.49-50 Islet 1 was utilized to ensure the motoneuron identity of the analyzed cells. The immunostaining demonstrated that human motoneurons grew on patterns followed the designed polarity with correct soma and axonal positioning (Fig 6), indicating that the polarity of motoneurons could be controlled at the single neuron level using geometric cues of differentially adhesive surfaces.


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Fig 6. The polarity of the motoneurons attached to designed patterns was demonstrated by the immunostaining with Neurofilament antibody, which specifically recognize axons. The motoneuron identity of the attached cell was confirmed by the co-staining with Islet 1 antibody. Nuclei were visualized with Dapi.

Electrophysiology The functional maturation of the human motoneurons on DETA/ 13F polarity patterns was compared with that of cells on un-patterned DETA control areas using patch-clamp electrophysiological recordings performed from 6 DIV. Inward and outward currents were evoked using 250-ms voltage steps ranging from −70 to +170 mV in 10 mV increments from a holding potential of −70 mV (Figure 7). The outward currents were composed of a rapidly ascending and inactivating transient component and a persistent component (Figure 7A and C). APs were evoked using a 3-ms depolarizing current injection with an intensity that generated a single spike. Figure 7B and D show example AP traces from a neuron located in an un-patterned DETA control area and one from a polarity patterned area respectively. Based on the recordings from more than 12 cells per group, generated from two independent platings, AP amplitude, peak



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inward and outward currents as well as other monitored electrophysiological parameters were not significantly different between the two groups (T-test, p > 0.05, Table 1), and both are comparable to the data reported in a previous characterization of stem cell-derived human motoneurons 51. This indicates that motoneurons polarized on patterns were functionally equivalent to those on regular surfaces based on the patch clamp analysis.

Fig 7. Whole-cell patch-clamp recordings. A and C) Samples of the superimposed current traces in response to voltage steps (10 mV, 250 ms, every 5 s) from a holding potential of −70 to +170 mV in a cell at non-patterned control area (A) and a cell attached to polarity patterns (C). B and D) Sample traces of Action potential (AP) evoked by a 3 ms depolarization current pulse of sufficient intensity to generate a single AP (mV) in a cell at non-patterned control area (B) and a cell attached to polarity patterns (D).


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Control Polarity p-value

Outward current (pA) 614 +/- 92 471 +/- 40 0.09

Inward Current (pA) -201 +/- 54 -181 +/- 28 0.37

AP amplitude (mV) 86 +/-6 86+/- 4 0.36

RMP (mV) -39+/- 2 -38 +/- 2 0.35

Rm (MΩ)

Cm (pF)

1105 +/- 193 1357+/- 188 0.18

12 +/-2 8 +/- 0.6 0.06

Table 1. Comparison of electrophysiological properties between the motoneurons on polarity patterns and those in control areas. Data were based on the recordings from neurons between 5 and 6 DIV. AP: Action Potential; RMP: Resting Membrane Potential; Cm: Membrane Capacitance; Rm: Membrane Resistance (n = 12). Data represented as mean +/- SEM.

This study demonstrated that the polarized (axonal/ dendritic) distribution of human stem cell-derived motoneurons can be controlled at the single neuron level using patterned SAM surfaces. The study also indicated that the patterns were selective for motoneurons over other cell types in the culture when a 20 µm somal diameter adhesion site was utilized. The surface patterns consisted of a permissive substrate, DETA, on a background of a non-permissive, hydrophobic fluorinated silane (13F). The design of the patterns induced polarity by supporting the growth of six neurites, of which one was encouraged to develop into the axon using geometrical and molecular cues. The survival and differentiation of the motoneurons were monitored and analyzed by phase contrast microscopy (Fig 4.), immunocytochemistry (Fig 5 and 6) and electrophysiology (Fig 7). Cells located on somal adhesion sites began to extend neurites within 24 hours and filled the complete length of the pattern after approximately 4 DIV. Between 4 and 8 DIV the cell’s electrical properties were equivalent to those growing on DETA controls. However, the cells began to detach from the patterns after approximately 1 week in culture. The isolation of the cells on these patterns has been determined as one of the causes for early onset of detachment, since the cells on un-patterned DETA control areas on the same coverslip survived longer (more



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than 20 days in general). Furthermore, it was also observed that the cells on patterns adjacent to the DETA control areas generally survived longer than those further away, although there was no physical connection between the patterned cells and the control areas in either case. The collected data suggest cells may need a certain level of trophic factor(s) support released from the “cellular neighborhood” for survival. Therefore, increasing interspersed control areas in the vicinity of patterns, or feeding the cultures with conditioned medium may promote longer cell survival on patterns in future studies. This study is significant since it reports the control of polarized distribution of human stem cell-derived motoneurons at the single cell level using SAM technology in a defined system. This human cell-based system would be advantageous over animal cell systems in translational studies. Moreover, compared to using primary human tissue as the source, the utilization of stem cells would bypass the limitations of cell quantity and tissue source variation, thereby offering a system with the potential for use in reproducible, high-throughput applications3. In addition, with the implantation of stem cell derived neurons for therapeutic applications, the ability to orient biomaterials to control neuronal polarity could be used for applications such as spinal cord repair, Parkinson’s and other neurodegenerative diseases. The utilization of SAMs as the substrate makes this system compatible to bio-MEMS (biological microelectro-mechanical systems) technology such as MEAs (microelectrode arrays)52 and cantilevers for automatic, non-invasive monitoring and analysis.53 The defined nature of the culture protocols used in this study is beneficial for the controlled regulation of the in vitro system, as well as the reproducibility of the culture.54 To establish the patterned distribution, human motoneurons were differentiated from stem cells and then replated for re-growth. This replating procedure mimics spinal cord injury in


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which the axons are broken or lost and have to be regenerated and axonal regeneration has been a major therapeutic target for this type of injury. Spinal neurons are capable of regenerating axons which has been demonstrated in peripheral nerve bridge experiments.55 However, the CNS environment poses multiple hostile factors hindering the axonal regeneration. To date, almost all the mechanistic studies and therapeutic investigations have been relying on animal models, especially mouse spinal cord injury models combined with nerve grafting, genetic modification or reagent treatment.56-58 It is hoped that these discoveries can be tested in human systems. In the system reported here, the controlled polarized distribution of human motoneurons on the predesigned pattern greatly facilitate the accurate and high throughput analysis of axonal regeneration. The defined nature of this system, from culture medium composition to surface distribution, enables a suitable platform for investigating the factors that hinder CNS axonal regeneration as well as for testing the effectiveness of potential therapeutic strategies. Controlled polarization of motoneurons in vitro, integrated with other cellular components, such as the upstream inputs or downstream targets, would enable the establishment of functional and integrative neural networks associated with motoneurons with controlled signal propagation. These cells could be co-cultured with dorsal root ganglion (DRG) neurons or higher level motor systems at fine scales to allow high resolution dissection of the input systems to motoneurons. They could also be combined with muscle cultures with regulated distribution to investigate neuromuscular junction (NMJ) properties and repair mechanisms. All these systems would provide valuable platforms for the study of motor control and ultimately the treatment of spinal cord injury or diseases such as ALS and muscular dystrophy. In general, this study will facilitate the integration of human motoneurons into the construction of human-based in vitro systems, with high resolution and controllable signal propagation, for both the study of systems



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biology and for the development of high information throughput systems for drug screening and test beds in preclinical studies.

Methods DETA Preparation Glass cover slips (VWR cat. nr. 48366067, 22×22 mm2 No. 1) were chemically cleaned using serial acid baths. First, the surfaces were soaked in a 50:50 solution of concentrated hydrochloric acid (VWR cat. nr. EM1.00314.2503) in methanol (VWR cat. nr. BJLP230-4) for 2 hours. Second, the surfaces were immersed in concentrated H2SO4 for at least 2 hours. After each acid soak, the surfaces were carefully washed 3 times with DI water. The final water rinse was followed by boiling the glass slides in DI water for 30 minutes. The cleaned glass slides were dried in an oven set at 110 °C overnight. The DETA (N-1 -[3(trimethoxysilyl) propyl] diethylenetriamine, United Chemical Technologies Inc., Bristol, PA, T2910) monolayer was formed by the reaction of the cleaned and dried cover slips with a 0.1% (v/v) solution of the organosilane in dry toluene (VWR cat. nr. BDH1151). The cover slips were heated to 100 °C for 30 min in DETA-toluene solution, cooled down to 38°C, rinsed with toluene, reheated to 100 °C in toluene for another 30 min, and then oven dried. DETA/ 13F SAM Pattern Preparation To generate DETA/13F patterns, photolithographic masks were designed for fabricating molecular patterns of DETA on a background of non-adhesive fluorinated silane using deep UV lithography.35 The DETA/13F SAM patterns were made by first applying the DETA monolayer, using the method described above, followed by a second treatment to derivatize laser ablated areas of the coverslip with 13F. The DETA SAMs were patterned using a


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deep UV (193 nm) excimer laser (Lambda Physik) at a pulse power of about 200 mJ and a frequency of 10 Hz for 45 seconds through a quartz photo-mask (Bandwidth Foundry, Eveleigh, Australia). The energy dosage (8-15 J/cm2) was sufficient to ablate the regions of the SAM exposed to the UV light, and yield reactive hydroxyl groups. The irradiated DETA SAMs were subsequently derivatized with 13F (tridecafluoro-1,1,2,2-tetrahydroctyl-1-dimethylchlorosilane, Gelest, SIT8174.0). The derivatization was carried out in an inert atmosphere, by the reaction of the irradiated surfaces with a 0.1% (v/v) solution of 13F in dry chloroform (Sigma Aldrich, cat# 439142) for 5 min, followed by 3 rinses in chloroform. Surface Characterization Surfaces were characterized by contact angle goniometry using a Ramé Hart (Netcong, NJ) contact angle goniometer, and by X-ray Photoelectron Spectroscopy (XPS). The surfaces that were examined using these techniques were: un-modified clean glass cover slips, DETA, 13F, and DETA backfilled with 13F. Contact angle goniometry analysis In all cases, the contact angle of a static sessile drop (5 µl) of water placed on the sample was measured three times and averaged. X-Ray Photoelectron Spectroscopy In order to monitor monolayer formation on clean glass coverslips, both DETA and 13F control surfaces were characterized by X-ray Photoelectron Spectroscopy (XPS) using a VG ESCALAB 220i-XL spectrometer equipped with an aluminum anode and a quartz monochromator. The spectrometer was calibrated against the reference binding energies of clean Cu, Ag and Au samples. XPS survey scans were recorded in order to determine the relevant elements (pass energy of 50 eV, step size of 1 eV). Si 2p, C 1s, N 1s, and O 1s high resolution



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spectra were recorded in order to determine the quality of the surfaces (pass energy of 20 eV, step size of 0.1 eV). The fitting of the peaks was performed with Avantage version 3.25 software provided by Thermo Electron Corporation. Palladium-catalyzed metallization of patterned silane monolayers Patterned samples were visualized using a palladium-catalyzed copper reduction reaction, modified from Kind et al.59 In this reaction, copper is deposited in regions containing the amine terminated silane, DETA. Cell Culture The NSI-566RSC neural stem cell line was isolated from the tissue of an 8-week old human fetal spinal cord (Neuralstem Inc).60-62 The use of this tissue was approved by the Institutional Review Board (IRB) and is in full compliance with all of the guidelines and regulations as set forth by the National Institute of Health (NIH) and the Food and Drug Administration (FDA). For each differentiation round, one aliquot of cryopreserved stem cells (~0.5x106 cells) was seeded into a T25 flask pre-coated with 100 ug/mL Poly-D-Lysine (PDL) in serum-free N2 medium in the presence of fibronectin. The N2 medium was changed every other day, and 10 ng/mL basic Fibroblast Growth Factor (bFGF) was added daily. The cells were allowed to proliferate to 60-80% confluence (6-8 days) and then harvested with Trypsin-EDTA and seeded onto pre-coated PDL and Fibronectin 60 mm permanox cell culture dishes (Nunc, Cat. 174888) at a density of 400~500 cells/mm2. Differentiation of the seeded stem cells into motoneurons was performed as described in Guo et al 51. Briefly, the stem cells were cultured in priming medium for four days and then in differentiation medium for six to eight days in a 37 oC, 5% CO2 incubator. Half the medium was changed every other day during this time. After 10 days differentiation, cells were harvested from dishes with 0.05% Trypsin-EDTA and re-plated on


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DETA or patterned coverslips at 50~75 cells/mm2. Studies have shown that the SAM, DETA, supports the growth of embryonic and adult motoneurons.37, 44, 63 Motoneurons were maintained on coverslips for 5, 7, 10 and 12 days and then characterized using electrophysiological analysis and immunocytochemistry. Immunocytochemistry Cells on coverslips were fixed in freshly prepared 4% paraformaldehyde for 15 min. The cells were washed twice in 1X Phosphate Buffered Saline (PBS) (pH 7.2, without Mg2+, Ca2+) for 10 minutes each at room temperature (RT), and permeabilized with 0.05% Triton X-100/PBS (T-PBS) for 15 minutes. The non-specific binding sites were blocked with Blocking Buffer (2.5% Donkey serum, 2.5% Goat serum and 0.5% Bovine Serum Albumin in 1X PBS) for one hour at room temperature. The cells were then incubated with the primary antibodies overnight at 4oC. After being washed with 1X PBS three times for 10 minutes, the cells were incubated with secondary antibodies for three hours in the dark at RT. Afterwards, the cells were washed in 1X PBS, three times for 10 minutes each. The cells were then mounted with Vectashield mounting medium containing the minor groove DNA binging probe DAPI for nuclei visualization. The primary antibodies used in this study were: Mouse-anti-Islet 1 (1:20; Hybridoma Bank, 39.4D5), an early marker for motoneuron differentiation, Rabbit-anti-β-III-Tubulin (1:1500; Millipore 041049), a neuron-specific marker, and Mouse anti-Neurofilament (1:1500; Millipore MAB1621), an axonal marker in neurons. Secondary antibodies were: Donkey-anti-Mouse-Alexa 488 (Invitrogen, 1:300), Donkey-anti-Mouse-Alexa 594 (Invitrogen, 1:300), Donkey-anti-RabbitAlexa 488 (Invitrogen, 1:300). All antibodies were diluted in blocking buffer solution. The immunostained cells were imaged using a Carl Zeiss Confocal Microscope (Zeiss Axioskop 2mot plus, Hal 100) at the magnification of 40X.



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Electrophysiology The electrophysiological properties of human motoneurons on DETA control surfaces, as well as polarity patterned surfaces, were examined using whole-cell patch-clamp recording techniques. Cells were transferred to a recording chamber filled with a medium, Neurobasal A (Invitrogen), maintained at room temperature (22-25oC). Patch electrodes used for rupture patch recordings were fabricated from borosilicate glass (1.5 mm outer diameter; Sutter, Novato, CA) with a Flaming Brown horizontal puller (P-97, Sutter Instruments, Novato, CA). Electrodes were pulled to a final tip resistance of 5-6 M and filled with pipette solution containing: 140 mM Kgluconate, 2 mM MgCl2, 1 mM EGTA, 10 mM HEPES, 2 mM Na2ATP. The pH of the pipette solution was adjusted to 7.3 with KOH and the osmolarity was measured at 276 mOsm/ kg. To generate a single action potential (AP), a 3 ms depolarizing current pulse of sufficient intensity (50 - 200 pA) to trigger a single AP was applied. In certain cases, neurons were hyperpolarized to a holding potential of -80 mV by DC application through the recording electrode. Inward and outward currents were evoked by a series of 250 ms depolarizing steps from 70 to +170 mV with +10 mV increments. The peak amplitude of the transient inward and outward currents was measured as the values for comparison in control and drug applications. The series resistance was in the range of 5 – 10 M (typically about 5 M) and was compensated roughly 60% on-line. Membrane potential measurement was not corrected for the liquid junction potential (about 15 mV). The holding potentials were not corrected for liquid junction potentials. Leak currents were subtracted using a standard P/4 protocol.64 Before seals (5 G) were made on cells, offset potentials were nulled. Capacitance subtraction was used in all recordings.


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Statistics Values are given as mean ± standard error of the mean. A Student’s T-test was performed to compare neuronal growth on patterns with 20 and 25 μm somal adhesion sites. Results with a p-value of p < 0.05 were selected as significant.

Author Contributions M.G. performed the human motoneuron differentiation, plating and culture on the patterns, certain aspects experimental design and most of the data analysis for the polarity of motoneurons on patterns, and drafted the manuscript. X.G. participated in the experimental design, the analysis of all the data and manuscript revisions. M.L. performed all electrophysiology recordings. M.S. prepared the patterned surfaces. P.M. participated in the experimental design. S.S. participated in the data analysis. J.J.H. is the P.I who initiated the project, led the experimental design, and directed the work as well as edited the manuscript until it was in its final form.

Acknowledgements: This research was funded by NIH grant R01-NS050452. We wish to thank NeuralStem Inc for providing the human fetal spinal cord stem cells. We also wish to thank Dr. Steven Lambert for his thoughtful insights during this research. The authors confirm that no competing financial interests exist and there has been no financial support for this research that could have influenced its outcome.

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Fig 1. Surface characterization: A and B) Optical image of palladium-catalyzed metallization of patterned DETA/13F monolayers. The dimension of each pattern is indicated in A: soma, 20 μm in diameter; axonal line length 200 μm; dendritic line length, 60 μm in total, interspersed with gaps of 10 μm; line widths of 2 μm. B) Distribution of patterns on the surface. The distribution of the patterns on the surface is demonstrated in B. C and D) Typical water contact angle on DETA (C) and 13F (D) surfaces. E and F) Typical DETA (E) and 13F (F) XPS survey spectra. 167x191mm (150 x 150 DPI)

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Fig 2. Optimization of soma adhesion site by analyzing correctness index. The correctness index is defined by the total number of correctly positioned cells on each pattern as a percentage of the total number of cells on the pattern. A) Phase images displaying examples of the incorrect cell attachment on the patterns: nonneuronal attachment (yellow arrow), cell attachment on the dendrites (red arrow), and multiple cell attachment (green arrow). B) Based on results from Day 6 analysis from multiple patterned chips in one experiment, the correctness index for the cells on the 20 μm patterns was 0.47 and 0.38 for the cells on the 25 μm patterns. The number of patterns analyzed for each soma size are indicated at the top of each bar. Data represented as mean + SEM. 220x82mm (150 x 150 DPI)


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Fig 3. Longitudinal characterization of the neuronal culture on patterns. A) Sample images of cell attachment on the patterned surfaces from Day 0-9. The green arrow points a pattern with a neuron fully grown, and the yellow arrow points to one where the neuron has started to detach. Error bar: 100 μm. B) Correctness Index of the patterns from Day 0-9. C and D) Characterization of cell attachment on patterns based on data pooled from Day 5 to Day9 of culture. Percentage of patterns that have different number of cell attachment (C) and the percentage of patterns containing correctly attached neurons out of the total patterns occupied with correspondent number of cells (D) were quantified based on results from 4 coverslips of 2 platings, in total of 274 patterns. Data represented as mean + SEM. 211x184mm (150 x 150 DPI)



Fig 4. A and B) Phase contrast image of human stem cell derived motoneuron on DETA/13F polarity patterns at 2 and 8 DIV, respectively. C and D) Metallization images of the corresponding polarity patterns with 20 μm somal adhesion site corresponding to the image in A and B, respectively. 231x170mm (150 x 150 DPI)


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Fig 5. Polarized distribution of human motoneurons on designed surface patterns in 1 DIV (A) and 2 DIV (B) cultures respectively. The cells were co-stained with β III Tubulin for neuronal identity and HB9 for motoneuron identity. The nuclei were identified using the fluorescent marker Dapi. 239x129mm (150 x 150 DPI)



Fig 6. The polarity of the motoneurons attached to designed patterns was demonstrated by the immunostaining with Neurofilament antibody, which specifically recognize axons. The motoneuron identity of the attached cell was confirmed by the co-staining with Islet 1 antibody. Nuclei were visualized with Dapi. 184x104mm (150 x 150 DPI)


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Fig 7. Whole-cell patch-clamp recordings. A and C) Samples of the superimposed current traces in response to voltage steps (10 mV, 250 ms, every 5 s) from a holding potential of −70 to +170 mV in a cell at nonpatterned control area (A) and a cell attached to polarity patterns (C). B and D) Sample traces of Action potential (AP) evoked by a 3 ms depolarization current pulse of sufficient intensity to generate a single AP (mV) in a cell at non-patterned control area (B) and a cell attached to polarity patterns (D). 208x142mm (150 x 150 DPI)



TOC graphic 225x117mm (150 x 150 DPI)


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Polarity Induced in Human Stem Cell Derived Motoneurons on

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