An integrated biomanufacturing platform for the large-scale expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells

Accepted Manuscript Full length article An integrated biomanufacturing platform for the large-scale expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells Gayathri Srinivasan, Daylin Morgan, Divya Varun, Nicholas Brookhouser, David A. Brafman PII: DOI: Reference:

S1742-7061(18)30277-0 https://doi.org/10.1016/j.actbio.2018.05.008 ACTBIO 5461

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

26 January 2018 3 May 2018 7 May 2018

Please cite this article as: Srinivasan, G., Morgan, D., Varun, D., Brookhouser, N., Brafman, D.A., An integrated biomanufacturing platform for the large-scale expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells, Acta Biomaterialia (2018), doi: https://doi.org/10.1016/j.actbio.2018.05.008

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TITLE An integrated biomanufacturing platform for the large-scale expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells AUTHORS Gayathri Srinivasan1, Daylin Morgan1, Divya Varun1, Nicholas Brookhouser1, and David A. Brafman1 1

School of Biological and Health Systems Engineering, Arizona State University

*CORRESPONDING AUTHOR David Brafman, (480) 727-2859, [email protected] 501 E. Tyler Mall ECG 334A Tempe, AZ 85287 RUNNING TITLE Human neural progenitor biomanufacturing platform FUNDING SUPPORT NIH-NIBIB 5R21EB020767-02

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ABSTRACT Human pluripotent stem cell derived neural progenitor cells (hNPCs) have the unique properties of long-term in vitro expansion as well as differentiation into the various neurons and supporting cell types of the central nervous system (CNS). Because of these characteristics, hNPCs have tremendous potential in the modeling and treatment of various CNS diseases and disorders. However, expansion and neuronal differentiation of hNPCs in quantities necessary for these applications is not possible with current two dimensional (2-D) approaches. Here, we used a fully defined peptide substrate as the basis for a microcarrier (MC)-based suspension culture system. Several independently derived hNPC lines were cultured on MCs for multiple passages as well as efficiently differentiated to neurons. Finally, this MC-based system was used in conjunction with a low shear rotating wall vessel (RWV) bioreactor for the integrated, large-scale expansion and neuronal differentiation of hNPCs. Overall, this fully defined and scalable biomanufacturing system will facilitate the generation of hNPCs and their neuronal derivatives in quantities necessary for basic and translational applications.

KEYWORDS Pluripotent stem cells; human neural progenitor cells; neuronal differentiation; bioreactor; largescale; chemically defined peptide substrate

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STATEMENT OF SIGNIFICANCE In this work, we developed a microcarrier (MC)-based culture system that allows for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells (hNPCs) under defined conditions. In turn, this MC approach was implemented in a rotating wall vessel (RWV) bioreactor for the large-scale expansion and neuronal differentiation of hNPCs. This work is of significance as it overcomes current limitations of conventional two dimensional (2-D) culture systems to enable the generation of hNPCs and their neuronal derivatives in quantities required for downstream applications in disease modeling, drug screening, and regenerative medicine.

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1. INTRODUCTION Human pluripotent stem cell (hPSC) derived neural progenitor cells (hNPCs) are capable of extensive in vitro expansion as well as directed differentiation into the various neuronal and neuroglial cell types of the central nervous system (CNS). As such, hNPCs and their derivatives have broad applications including understanding the mechanisms of neurodevelopmental fate choices [1-3], engineering accessible models of complex neurodegenerative diseases [4, 5], and developing cell therapies to replace dysfunctional or damaged neural cells [6-8]. However, future progress in the application of hNPCs in these areas will require the development of platforms for their reproducible large-scale expansion and differentiation. Moving forward, such biomanufacturing platforms should have several important design criteria [9]: (i) Fully-defined, xeno-free culture conditions. Current culture systems often employ undefined substrates, such as MatrigelTM, or animal-derived proteins, such as laminin (LN) (which is a common substrate for hNPC growth and differentiation [10-12]) that are difficult to isolate and heterogeneous in composition. In turn, this can lead to variability in cell expansion and differentiation as well as the exposure of cells to xenogeneic components which may limit subsequent clinical applications [13]; (ii) Scalability. Several downstream applications of hNPCs such as high-throughput drug screening and cell-based therapies will require large quantities of cells. For example, previous pharmacology and toxicology screens that have implemented hNPCs or their neuronal derivatives have required 10,000-15,000 cells per well of a 384-well plate [14-17]. Similarly, it has been estimated that hPSC-based cell therapies will require 1-10 x 109 cells per treatment [9, 18-20]. Yet, the limited surface-area-to-volume ratio offered by conventional two dimensional (2-D) culture systems limits cell expansion and differentiation to these quantities. (iii) Adaptable integration of hNPC expansion and differentiation. It remains uncertain whether hNPCs [21-26] or their differentiated neuronal progeny [6, 27-29] represent the best therapeutic target for transplantation strategies as both cell populations have shown efficacy in pre-clinical models of various neurodegenerative diseases. Along similar lines, both hNPCs [30-35] and hNPC-derived neuronal populations [4, 5, 36-38] have been employed in CNS-disease models and drug screening. To that end, an adaptable biomanufacturing platform that allows for the integrated hNPC expansion and subsequent neuronal differentiation is highly desirable. Previously, we developed a fully defined peptide-based substrate, referred to as vitronectinderived peptide (VDP), that allowed for the long-term expansion and directed neuronal differentiation of multiple hNPC lines in completely defined medium conditions [39]. In this work, we use this defined substrate as a basis for a microcarrier (MC)-based suspension system that 4

enables the long-term growth and highly efficient neuronal differentiation of several hNPC lines. In turn, we were able to use this MC-based approach in a low-shear rotating wall vessel (RWV) bioreactor [40, 41] to enable the large-scale expansion and differentiation of hNPCs. Overall, this highly adaptable and fully integrated biomanufacturing platform allows for the growth and neuronal differentiation of hNPCs and in sufficient quantities necessary for disease modeling, drug screening, and regenerative medicine applications.

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2. MATERIALS AND METHODS 2.1 Human neural progenitor cell (hNPC) generation, expansion, and differentiation. HES3- [42], RiPSC- [43], AD (ASUi003; [44]), and NDC (ASUi004, [44]) hPSCs were differentiated to hNPCs as previously described with some minor modifications [3]. Briefly, to initiate neural differentiation hPSCs were cultured in feeder-free conditions (MatrigelTM [BD Biosciences]; Essential 8™ Medium (E8); ThermoFisher) for a minimum of 2 passages. Cells were then detached with Accutase (ThermoFisher) and resuspended in E8 media supplemented with 5 µM Rho kinase inhibitor (ROCKi; Y-27632 [BioGems]). Next, 1-2 x 106 cells were pipetted to each well of a 6-well ultra-low attachment plate (Corning). The plates were then placed on an orbital shaker set at 95 rpm in a 37OC/5% CO2 tissue culture incubator. The next day, the cells formed spherical cultures (embryoid bodies [EBs]) and the media was changed to neural induction media (NIM) [1X DMEM-F12 (ThermoFisher), 0.5% (v/v) N2 supplement (ThermoFisher), 1% (v/v) B27 supplement (ThermoFisher), 1% (v/v) GlutaMAX supplement (ThermoFisher), 1% (v/v) Penicillin Streptomycin, 50 ng/ml recombinant human Noggin (R&D Systems), 0.5 µM Dorsomorphin (Tocris Bioscience)]. Half of the media was subsequently changed every day. After 6 days in suspension culture, the EBs were then transferred to a 10 cm dish (1-2 6 wells per 10 cm dish) coated with Matrigel™. The plated EBs were cultured in NIM for an additional 5-7 days. Cells were then plated on surfaces that had to be coated first with poly-L-ornithine (PLO; Sigma) and then with mouse laminin (LN; 4µg/mL; ThermoFisher) as described as follows: Tissue culture plates were coated with 4 µg/mL PLO at 37°C for 4 hours. After 4 hours of incubation, the PLO solution was aspirated and the plates were washed 2 times with PBS. The plates were then coated with 4 µg/mL LN at 37°C overnight and washed 2 times with PBS prior to use. For simplicity, in the manuscript and figures these plates are simply referred to as ‘LN-coated’. Plated cells were cultured in LN-coated dishes in neural induction media supplemented with 30 ng/ml recombinant human FGF2 (Peprotech/STEMCELL Technologies) and 30 ng/ml recombinant human EGF (ThermoFisher). For routine maintenance, hNPCs were passaged onto LN-coated plates at a density of 1-5 x 104 cells/cm2 in neural expansion media (NEM; [1X DMEM-F12, 0.5% (v/v) N2 supplement, 1% (v/v) B27 supplement, 1% (v/v) GlutaMAX supplement, 1% (v/v) Penicillin Streptomycin, 30 ng/ml FGF2, and 30 ng/ml EGF]). For two dimensional (2-D) neuronal differentiation, hNPCs were grown to confluence and the media was changed to neuronal differentiation media (NDM; [1X DMEMF12, 0.5% (v/v) N2 supplement, 1% (v/v) B27 supplement, 1% (v/v) GlutaMAX, 1% (v/v) Penicillin Streptomycin, 20 ng/ml BDNF (R&D Systems/ STEMCELL Technologies), 20 ng/ml

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GDNF (R&D Systems/STEMCELL Technologies), 1 µM DAPT (Tocris Bioscience), and 0.1 mM dibutyrl-cAMP (cAMP) (Sigma Aldrich)]). 2.2 Coating of microcarriers (MCs) with vitronectin derived peptide (VDP) and laminin (LN). Vitronectin derived peptides (VDP; CGKKQRFRHRNRKG) were custom synthesized by AnaSpec. The linear peptide sequences were synthesized on a resin using standard Fmoc chemistries. Analysis of the peptides by analytical HPLC and MALDI-TOF confirmed that the peptides had the correct expected masses. The peptides were then subjected to HPLC using C18 columns to remove any impurities. Analytical HPLC and ESI-MS were used to confirm the purity and mass, respectively. The solvents used to dissolve the peptide were Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in acetonitrile). Over a run time of 8.5 min, the step-wise gradient increased the percentage of Buffer B from 1% to 30%. The analytical HPLC was monitored at 220 nm. To coat microcarriers (MCs; Corning Enhanced Attachment Microcarriers) with VDP, microcarriers were incubated in 0.5mM solution of VDP for 48 hours in 37˚C. To coat MCs with LN, MCs were suspended in 4µg/mL PLO solution and incubated overnight in 37˚C, after which the MCs were washed twice with PBS. The MCs were then coated with 4µg/mL LN solution at 37˚C overnight. For simplicity, in the manuscript and figures these MCs are simply referred to as ‘LN-coated’. Coated MCs were washed once with PBS and once with culture media prior to use. 2.3 Small-scale serial expansion of hNPCs on MCs. HNPCs were seeded on LN- or VDP-coated MCs in NEM containing 5µM Rho kinase inhibitor (ROCKi Y-27632) at a density of 1.5 x 106 cells per well and a MC density of 1 mg/mL in 6-well ultra-low attachment plates (Corning). The plates were placed under static conditions with 2 mL of NEM in each well to allow for cell attachment on MCs for 12 hours after which an additional 2 mL of NEM was added to the wells. The plates were placed on an orbital shaker (Dura-Shaker, VWR) at 95 RPM. Three-fourths of the media (~3 mL) was changed after 24 hours of culture to remove the ROCKi and half of the media (~2mL) was changed every day thereafter. Upon confluence, hNPCs were detached from the MCs by incubating in Accutase for 10 minutes (5 minutes with no agitation and 5 minutes on the orbital shaker) and then triturated after which the cell- suspension was passed through a 40µm cell strainer to remove the MCs and obtain a single cell suspension. The cells were counted using a hemocytometer and seeded on freshly coated MCs.

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2.4 Small-scale differentiation of hNPCs on MCs. For neural differentiation of hNPCs on MCs, hNPCs were expanded on LN- and VDP-coated MCs for 4-5 days until 80-90% confluent after which the media was switched to NDM. After 18 days, the MCs were maintained in NDM without BDNF and GDNF for the rest of the differentiation period with a minimum of half media change each day. Cells were differentiated for a minimum of 30 days prior to analysis or replating. 2.5 Large-scale expansion of hNPCs on MCs in RWV bioreactor. RCCS-1 or RCCS4-HD systems (Synthecon) with 55mL rotating wall vessels (RWV) (Synthecon) were used for the large-scale expansion and differentiation of hNPCs. The vessels were prepared according to the manufacturer’s instructions. HNPCs expanded on LN- or VDPcoated MCs in small scale cultures (6 well ultra-low attachment plates) were dissociated and seeded in the RWV bioreactor at a density of 3.6 x 105 cells/mL with 10 mg/mL LN- or VDPcoated MCs. The initial rotation was set to 10 RPM for cultures on LN-coated MCs and 8 RPM for cultures on VDP-coated MCs. Rotation speed was adjusted as necessary throughout the culture period to the keep the MCs suspended in the RWV vessel. HNPCs were expanded in NEM. For the first 24 hours of culture, 5µM ROCKi was added to aid with cell viability. Threefourths (~42 mL) of the media was changed every day thereafter except for day 2 of culture when half the media was changed. Samples were collected each day and cells were dissociated from the MCs by incubating in Accutase for 10 min without agitation. Cell viability was assessed by propidium iodide (PI; 0.01mg/mL, ThermoFisher) and cell counts were obtained. 2.6 Large-scaled differentiation of hNPCs on MCs in RWV bioreactor. For the large-scale differentiation of hNPCs, hNPCs were expanded in the RWV bioreactor (as described above). After 4 days, the media was changed to NDM. The MCs were maintained in NDM for 18 days and NDM without BDNF and GDNF for the rest of the differentiation period. For the first 18 days of differentiation, three-fourth media changes (~42 mL) were performed daily after which half media changes (~27 mL) were performed for the remainder of the differentiation period. Cells were differentiated for a minimum of 30 days prior to analysis or replating. 2.7 Replating of neuronal cultures on astrocyte feeders. HPSC-derived astrocytes were generated as previously described [45]. Briefly, hNPCs (> passage 6) were cultured on LN-coated plates in astrocyte differentiation medium composed of 8

astrocyte medium (ScienCell) supplemented with human recombinant BMP4 (10ng/ml), Heregulin-β (10ng/ml), and CNTF (10ng/ml) (STEMCELLTechnologies). Cells were passaged throughout the differentiation process when they reached confluence. Cells were differentiated for a minimum of 45 days prior to being used as astrocyte feeders. Prior to seeding with MCderived neurons, astrocyte feeder layers were plated on MatrigelTM-coated plates (11.5 x 103 cells/cm2). Astrocytes were allowed to adhere and grow for a minimum of 1 day prior to seeding with MC-derived neurons. For seeding onto astrocyte feeder layers, neurons cultured on MCs were incubated in Accutase at 37˚C for 5 minutes. The MCs were gently pipetted up and down (using a P1000 pipette). The dissociated cells were plated on astrocytes in astrocyte media (ScienCell) for a minimum of 5 days prior to analysis. 2.8 Quantitative PCR (qPCR). RNA was isolated from cells using the NucleoSpin RNA Kit (Clontech). For neurons cultured in the bioreactor, cells were lysed using ice-cold TRIzol reagent (ThermoFisher) and incubated at 65˚C for 5 minutes. 200µL chloroform per mL of TRIzol reagent was added to the samples and mixed well. The samples were incubated on ice for 5 minutes and centrifuged at 1000g for 5 minutes. The aqueous phase was collected and one volume of 70% ethanol was added, after which the NucleoSpin RNA Kit columns were used to isolate the RNA. Reverse transcription was performed with iScript RT Supermix (Bio-Rad). Quantitative PCR was carried out using SYBR green dye on a CFX384 Touch™ Real-Time PCR Detection System. QPCR experiments run with SYBR green dye were carried out using iTaq Universal SYBR Green Supermix (Bio-Rad). For qPCR experiments run with SYBR green dye, a 2 min gradient to 95 °C followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s was used. The list of primer sequences used is provided in Supplementary Table 1. Gene expression was normalized to 18S rRNA levels. Delta Ct values were calculated as Cttarget − Ct18s. Relative fold changes in gene expression were calculated using the 2− ∆∆Ct method [46]. 2.9 RNA-seq analysis. All sequencing was performed at BGI Americas Corporation. Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit. The workflow consists of mRNA enrichment, cDNA generation, and end repair to generate blunt ends, A-tailing, adaptor ligation and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on BGISEQ-500 for a single end 50 bp run. Reads were filtered were to remove reads which contain adapter sequence, high content of unknown bases and low quality reads. Bowtie2 [47] was used to map reads to the latest UCSC reference human genome and HISAT [48] was used for read alignment. A minimum of 20 M mapped reads were 9

collected for each sample. RSEM [49] was used for transcript quantification. FPKM [50] were calculated for each gene and used as an estimate of expression levels. Differentially expressed genes (DEGs) were identified using Possion distribution based algorithm [51]. Genes showing altered expression with FDR < 0.05 and more than 2 fold changes were considered differentially expressed. All DEGs were mapped to gene ontology (GO) terms (http://www.geneontology.org/) and gene numbers were calculated for each test. The following hypergeometric test to find significantly enriched GO terms in the input list of DEGs, based on 'GO::TermFinder':

where N is the number of all genes with GO annotation; n is the number of DEGs in N; M is the number of all genes that are annotated to certain GO terms; m is the number of DEGs in M. The calculated p-value were adjusted through Bonferroni Correction, taking corrected p-value ≤ 0.05 as a statistically significant threshold. GO terms fulfilling this condition are defined as significantly enriched GO terms in DEGs. 2.10 Immunofluorescence. Cultures were gently washed twice with PBS prior to fixation. Cultures were then fixed for 15 min at room temperature (RT) with BD Cytofix Fixation Buffer (BD Biosciences). HNPC-MCs were fixed for 30 minutes at RT with BD Cytofix Fixation Buffer (BD Biosciences). The cultures were then washed twice with PBS and permeabilized with BD Phosflow Perm Buffer III (BD Biosciences) for 30 min at 4OC. Cultures were then washed twice with PBS. Primary antibodies were incubated overnight at 4OC and then washed twice with PBS at RT. Secondary antibodies were incubated at RT for 1 hr. Antibodies used are listed in Supplementary Table 2. Nucleic acids were stained for DNA with Hoechst 33342 (2 µg/mL; ThermoFisher) for 10 min at RT and then washed twice with PBS. For imaging hNPC-MCs, the MCs were transferred to double cavity slides and mounted in Vectashield (Vector Labs) mounting medium. Imaging was performed using an automated confocal microscope (Leica TCS SP5) or EVOS microscope (ThermoFisher). Z-stack images were acquired and maximum projection images were obtained from these z-stacks using the Leica Application Suite. 2.11 Flow cytometry. Cells were dissociated with Accutase for 10 min at 37OC, triturated, and passed through a 40 µm cell strainer. Cells were then washed twice with stain buffer (BD Biosciences) and resuspended at a maximum concentration of 5 x 106 cells per 100 µL. For staining of intracellular proteins, cells were fixed for 30 min at RT with BD Cytofix Fixation Buffer (BD Biosciences). The cells were then washed twice with stain buffer and permeabilized with 10

BD Phosflow Perm Buffer III (BD Biosciences) for 30 min on ice. Cells were then washed twice with stain buffer and one test volume of antibody was added for each 100 µL of cell suspension. Cells were stained with primary antibodies overnight on ice, washed, and resuspended in stain buffer. Cells were stained with secondary antibodies for 1 hour at RT, washed and resuspended in stain buffer. For propidium iodide staining, cells were resuspended in PBS and stained with 0.01mg/mL propidium iodide (ThermoFisher) and incubated in the dark for 1-2 minutes. Cells were analyzed on a ACCURI C6 (BD Biosciences). Antibodies and isotype negative controls are listed in Supplementary Table 2. 2.12 Population doubling time. Population doubling time of hNPCs was calculated using the following equation: PDT (h) = (T2 − T1) / (3.32 * [log(N2) − log(N1)]). 2.13 Statistical analysis. Data were analyzed using Student’s t-test and ANOVA statistical methods. Where appropriate, a Bonferroni post hoc correction was employed. A p-value < 0.05 was considered significant. Unless otherwise noted, all data are displayed as mean ± standard error of the mean (S.E.M.).

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3. RESULTS 3.1 Long-term passaging of hNPCs in small-scale microcarrier (MC)-based cultures To systematically optimize the conditions required for culture of hNPCs on microcarriers (MC), hNPCs derived from a human induced pluripotent stem cell (hiPSC) line (RiPSCs [43]; herein referred to as RiPSC-hNPCs) or a human embryonic stem cell (hESC) line (HES3 [42]; herein referred to as HES3-hNPCs) were enzymatically dissociated from 2-D cultures onto vitronectin-derived peptide (VDP) or laminin (LN)-coated MCs in 6-well ultra-low attachment plates

(RiPSC-hNPCs:

Figure

1A

and

Supplementary

Figure

1A;

HES3-hNPCs:

Supplementary Figure 2A). HNPCs did not adhere when cultured on uncoated MCs (Supplementary Figure 3A and 3B). In addition, a concentration gradient analysis revealed that 500 µM was the minimum concentration of VDP necessary to promote cell adhesion similar to that of hNPCs cultured on LN MCs (Supplementary Figures 3A and 3B). Although hNPCs cultured on MCs and 2-D cultures displayed a similar doubling time (RiPSC-hNPCs: Figure 1C and Supplementary Figure 1B; HES3-hNPCs: Supplementary Figure 2B), the higher surface-area to volume ratio of MCs allowed for a high level of cell expansion in culture wells of the same size. In fact, by varying the initial cell seeding and MC density we were able to achieve a greater than 8-fold RiPSC-hNPC expansion (16.47 ± 2.27 x 106 cells) using MCbased cultures than by using conventional 2-D culture. RiPSC-hNPCs and HES3-hNPCs were expanded for greater than 20 and 10 passages, respectively, on both VDP- and LN-coated MCs. Notably, the hNPC growth on the VDP- and LN-coated MCs was consistent at low (i.e. no adaptation to the MCs was required) and high (i.e. no growth senescence) passages. Finally, immunofluorescence (RiPSC-hNPCs: Figure 1D and Supplementary Figure 1C) and flow cytometry (RiPSC-hNPCs: Figure 1E and Supplementary Figure 1D; HES3-hNPCs: Supplementary Figure 2C) demonstrated that hNPCs cultured on VDP and LN MCs for multiple passages (RiPSC-hNPCs > 10 passages, HES3-hNPCs > 10 passages) continued to express high levels (> 95% positive) of the hNPC markers (SOX1, SOX2, NESTIN) in a manner similar to hNPCs cultured on VDP- and LN-coated 2-D surfaces (Supplementary Figures 4A and 4B). Together, these results demonstrate that VDP- and LN-coated MCs can support the long-term culture of hNPCs as well as facilitate their expansion at significantly higher levels than traditional 2-D culture methods. 3.2 Neuronal differentiation of hNPCs in small-scale microcarrier (MC)-based cultures Next, we determined if hNPCs could be differentiated to neurons in MC-based cultures. To achieve neuronal differentiation of MCs, RiPSC-hNPCs were seeded onto VDP- or LN-coated 12

MCs and expanded in the presence of fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) (see Materials and Methods). After 5 days of expansion, neuronal differentiation was induced through the removal of FGF2 and EGF and the addition of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), and DAPT. From day 23 onward, cells were cultured in the absence of these neuronal induced factors. Gene expression analysis by RNA-sequencing (RNA-seq) (Figure 2A and Supplementary Tables 3 and 4) and quantitative PCR (qPCR) demonstrated that the expression of markers associated with hNPC state such as SOX1, SOX2, NESTIN, and PAX6 were significantly downregulated in MC-based neuronal cultures (Figure 2A and 2B and Supplementary Figure 5A). On the other hand, genes associated with mature neurons (e.g. MAP2, MAPT, RBFOX3, TUBB3) and the formation of functional synapses (e.g. DLG4, SNAP25, SYN1) were highly expressed in neurons generated on MCs (Figure 2A and 2B and Supplementary Figure 5A). In addition, quantification of MC-based neuronal cultures by flow cytometry revealed that >75% expressed the neuronal marker TUBB3 with a high percentage of these cells (>50%) expressed NEUN, a marker for mature post-mitotic neurons [52] (Figure 2C and Supplementary Figure 5B). We also demonstrated that neurons could be enzymatically dissociated from the MCs and replated onto 2-D surfaces with high levels of cell viability (as measured by propidium iodide (PI); right panel Figure 2C and Supplementary Figure 5B). Counts of viable cells dissociated from MCs revealed that over 3 x 106 live TUBB3-positive neurons could be obtained from MCs cultured in a single well of a low-binding 6 well plate (VDP: 3.01 ± 0.06 x 106 neurons; LN: 2.59 ± 0.52 x 106 neurons) which is almost a 10-fold increase of the number of TUBB3-positive neurons obtained using standard 2-D approaches (VDP: 0.33 ± 0.03 x 106 neurons; LN: 0.34 ± 0.03 x 106 neurons). Finally, immunofluorescence analysis of replated neurons demonstrated that they displayed a neuronal-like morphology and maintained expression of pan-neuronal markers MAP2, NEUN, and TUBB3 as well as the forebrain marker FOXG1 (Figure 2D and Supplementary Figure 5C). It is well-established that in vivo cells reside within a complex three dimensional (3-D) microenvironment that plays a significant role in regulating cell behavior [53]. Signaling and other cellular functions, such as gene expression and differentiation potential, differ in 3-D cultures compared with 2-D substrates [54-58]. Therefore, we hypothesized that MC-based cultures would provide a 3-D like environment that may result in neuronal cultures that were more mature than those generated with conventional 2-D methods. To that end, we performed RNA-seq on neuronal cultures that had been generated on VDP-coated MC and 2-D surfaces (Figure 3A and Supplementary Tables 3 and 5). This analysis revealed that genes related to 13

post-mitotic neurons (Figure 3A, center panel and Supplementary Figure 6A) and synapse formation (Figure 3A, right panel and Supplementary Figure 6A), as well as the synthesis, release, and transmission of various neurotransmitters such as acetylcholine (ACH), γaminobutyric acid (GABA), and glutamate (GLU) (Supplementary Figure 6B), and numerous potassium (K+), calcium (Ca2+), sodium (Na+), and chloride (Cl-) ion channels involved in action potential generation (Supplementary Figure 6C) were significantly upregulated in the neuronal cultures generated on MCs compared to those on conventional 2-D surfaces (Supplementary Table 6). In addition, gene ontology (GO) analysis revealed that the top ten statistical significant cellular component (Figure 3B) and molecular function/biological process (Figure 3C) GO terms that were elevated in MC-based neuronal cultures were associated with mature neuron formation and function (see also Supplementary Table 7). Finally, qPCR analysis was also used to confirm that genes associated with mature, post-mitotic neurons (e.g. NEFM, NCAM, RBFOX3) were elevated in neuronal cultures generated on both VDP- and LN-coated MCs (Supplementary Figure 6D). We wanted to extend our MC-based neuronal differentiation strategies to hNPCs generated from hiPSCs derived from patient samples. Using our established protocols we generated hNPCs from hiPSCs derived from an Alzheimer’s disease patient (herein referred to as ADhNPCs) and hiPSC derived from a healthy non-demented control patient (herein referred to as NDC-hNPCs) [44]. Similar to our other established hNPC cell lines, both AD- and NDC-hNPCs expressed high levels of the hNPC markers SOX1, SOX2, and NESTIN (Supplementary Figure 7A and 7B). NDC- and AD-hNPCs were cultured on VDP- and LN-coated MCs and then subsequently differentiated to neuronal cultures in a manner similar to as described above. MCbased neuronal cultures generated from both NDC- and AD-hNPCs expressed high levels of genes associated with mature neurons (e.g. MAP2, MAPT, RBFOX3, TUBB3) (Figure 4A). Quantification of MC-based neuronal cultures by flow cytometry demonstrated that high percentage of the neurons were TUBB3 (83.2%-95.7%) and NEUN (39.3%-55.4%) positive (Figure 4B). Finally, neurons were dissociated from MCs with high levels of cell viability (Figure 4B) and replated on 2-D surfaces while maintaining their neuronal phenotype as measured by expression of FOXG1, MAP2, and TUBB3 (Figure 4C). Overall, these data demonstrate that MC-based systems not only facilitate the highly efficient generation of post-mitotic neuronal cultures from hNPCs derived from several hiPSC lines (i.e. RiPSC, NDC-hiPSCs, AD-hiPSCs) but also result in neuronal cultures with a higher degree of maturity than those generated using traditional 2-D approaches.

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3.3 Integrated large-scale expansion and differentiation of hNPCs in rotating wall vessel (RWV) bioreactors After establishing small-scale methods for hNPC expansion, we next sought to develop methods for large-scale MC-based expansion using rotating wall vessel (RWV) bioreactors, which produce a low fluid shear stress environment and limit the mechanical stress on cells in MC suspension culture [40, 41]. RiPSC-hNPCs expanded using small-scale MC-based cultures were enzymatically dissociated and seeded onto VDP- or LN-coated MCs in 55 mL RWV bioreactors (Figure 5A and 5B and Supplementary Figure 8A). After a minimum of 6 days in culture, RWV-based culture on VDP- and LN-coated MCs allowed for the expansion of hNPCs to over 160 million viable cells (VDP: 249.61 ± 23.74 x 106 cells; LN: 162.21 ± 28.18 x 106 cells; n=4 independent bioreactor runs; Figure 5C and Supplementary Figure 8B). In addition, immunofluorescence (Figure 5D and Supplementary Figure 8C) and flow cytometry analysis (Figure 5E and Supplementary Figure 8D) revealed that hNPCs expanded in RWV continued to express high levels (~95% positive) of the canonical hNPC markers SOX1, SOX2, NESTIN. We next assessed if RWV bioreactors could facilitate the neuronal differentiation of expanded hNPCs. RiPSC-hNPCs were expanded on VDP-coated MCs in the RWV bioreactor and then subsequently differentiated to neuronal cells in similar manner as described for the small-scale MC-based differentiation experiments (see Materials and Methods). Differentiation of RiPSC-hNPCs in the RWV bioreactors allowed for the generation of over 125 million viable neurons (135.24 ± 20.56 x 106 neurons; n=4 independent bioreactor runs; Figure 6A). Analysis of RWV-generated cultures by RNA-seq (Figure 6B and Supplementary Tables 8 and 9) and qPCR (Figure 6C) revealed that similar to neurons generated in small-scale MC- based cultures, neurons generated with RWV cultures expressed high levels of genes associated with mature neurons (e.g. MAP2, MAPT, RBFOX3, TUBB3) as well as the formation of functional synapses (e.g. SNAP25, SYN1, SYP). Along similar lines, quantification by flow cytometry of neuronal cultures generated in RWV bioreactors demonstrated that >80% expressed the neuronal marker TUBB3 with a high percentage (>50%) also expressing NEUN (Figure 6D). In addition, neurons could be enzymatically dissociated from MC cultures generated in RWV bioreactors with low levels of cell death (<10% PI positive; right panel Figure 6D). Finally, neurons dissociated from MCs and replated onto 2-D surfaces maintained high levels of expression of pan-neuronal markers including MAP2, NEUN, and TUBB3 as well as the forebrain marker FOXG1 (Figure 6E). Overall, the use of MCs in RWV-based bioreactors allowed for the seamless large-scale and subsequent neuronal differentiation of hNPCs under defined conditions. 15

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4. DISCUSSION In this study, we developed a microcarrier (MC)-based approach for the long-term expansion and neuronal differentiation of hNPCs. This MC-based approach is compatible with laminin (LN)-based coatings, which are commonly used as substrates for the growth and differentiation of hNPCs [10-12], as well as our previously described defined substrate, vitronectin derived peptide (VDP) [39]. In addition, similar to our previous work with 2-D surfaces, in this study we show that compared to hNPCs cultured on LN-coated MCs, hNPCs grown on VDP-coated MCs have similar growth rates, expression of hNPC multipotency markers, and ability to differentiate to mature neurons. Moreover, in our previous work [39], we demonstrated that the ability of VDP to bind to both integrin ανβ5 as well as cell surface proteoglycans highly expressed by hNPCs allowed for VDP to support the long-term expansion of hNPCs on 2-D surfaces. We speculate that the mechanism by which VDP supports hNPC growth on MCs is similar to these mechanisms for growth on 2-D surfaces. Building upon this previous work, we demonstrated that hNPCs cultured on MCs expressed similar levels of multipotent hNPC markers as cells cultured using 2-D approaches. As such, the use of small scale MC cultures enabled almost a 10-fold increase in cell expansion (per passage) when compared with 2-D methods. In turn, hNPCs could be enzymatically dissociated, cryopreserved, and later thawed directly back onto VDP- or LN-coated MCs. In addition, expanded hNPCs, including those from patient-specific hiPSCs, could be differentiated with high efficiency to neuronal cultures. In fact, neurons generated on MCs expressed higher levels of genes associated with maturity, synapse formation, neurotransmitter synthesis, release, and transmission, and ion channels associated with action potential generation. Neurons generated on MCs could also be dissociated and replated onto 2-D surfaces with minimal cell death, which will be important for downstream highcontent phenotypic drug screening assays where the culture of neurons in 2-D will be required. Significantly, the small-scale MC-based approaches developed as part of this study have the benefits of off-the-shelf availability and ease-of-use that will enable the facile application by researchers that do not have extensive expertise with suspension culture methods. In turn, we used these small-scale MC-based approaches as a test-bed as means to adapt to large-scale rotating wall vessel (RWV) bioreactors. Importantly, our analysis of hNPC and neuronal cultures using RNA-seq, qPCR, flow cytometery and immunofluorescence did not reveal any significant phenotypic differences between cells generated in small-scale MC-based cultures with those generated in RWV bioreactors. Specifically, using a 55 mL bioreactor vessel, we were able to reproducibly generate over 250 million hNPCs and 125 million hNPC-derived neurons. By comparison, based upon our previous experience using traditional 2-D approaches, we estimate 17

that the generation of cells in these numbers would require over 40 10-cm plates. In the future, the use of multiple vessels in parallel (the RWV bioreactor used in this study can simultaneously accommodate 4 vessels) or vessels of larger volume (the RWV bioreactor used in this study can be used with vessels up to 500 mL), could lead to the facile generation over 1 billion hNPCs or hNPC-derived neurons, a target cell number needed for most downstream applications in disease modeling, drug screening, or regenerative medicine applications. Because of the small surface-area-to-volume ratio, conventional 2-D culture systems are not adequate in generating the large number of hPSC-derived cell types needed for downstream preclinical and clinical applications. Scale-up methods such as stacked plates, hollow fibers and packed bed reactors often result in heterogeneous cell expansion due to the difficultly in maintaining homogenous environmental conditions [59]. Alternatively, microcarrier (MC)-based culture in suspension bioreactors has emerged as an attractive approach for the large-scale expansion of a variety of cell populations [60-63]. However, current approaches often rely on the use of MCs that have been coated with animal-derived or undefined matrix proteins. Additionally, these MCs are often used in stirred suspension bioreactors which may expose cells to high levels of shear stress which may negatively influence cell growth and differentiation [63-65]. By comparison, the rotating-wall vessel (RWV) bioreactors that were used in this study produce a low shear stress environment [40] and limit the mechanical stresses on cells in MCbased suspension culture [41]. In addition, previous studies with hPSCs have shown that RWV bioreactors have similar levels of scalability to these other high shear producing bioreactor systems [66]. As described in this study, the use of VDP-coated MCs in these RWVs provided for a robust, defined, and scalable biomanufacturing process for expansion and directed neuronal differentiation of hNPCs. Previously, several groups have reported the development of bioreactor-based approaches for the large-scale expansion of neural stem cells (NSCs) derived from primary and fetal neural tissue [67-69]. However, it is important to note that while these NSCs share some similarities to hNPCs, NSCs are biologically and developmentally distinct from hNPCs [70, 71]. Specifically, the differentiation potential and, thereby, the research and clinical application of NSCs is much more limited than hNPCs, which can differentiate to all of the cell types of the CNS [70]. Nonetheless, the development of approaches for large-scale growth and differentiation of hPSC-neural derivatives have been limited [72, 73]. For example, Bardy and colleagues described the use of undefined MatrigelTM-coated MCs in high shear stirred spinner flasks for 18

the differentiation of hPSCs into hNPCs [73]. However, the serial passaging of the hNPCs on the MCs as well as their subsequent differentiation to mature neurons in the bioreactors were not described in this study. In addition, the direct generation of hNPCs from hPSCs, without enrichment or purification, may limit subsequent clinical application as these cultures may contain residual pluripotent cells with tumorigenic potential. The biomanufacturing system described in this study improves on these previous approaches through the use of defined substrates along with low shear stress RWV bioreactors. In addition, this system provides for both the expansion and neuronal differentiation of hNPCs, an important feature given that both hNPCs and their neuronal derivatives have applications in disease modeling [4, 5, 30-38] and regenerative medicine [6, 21-29] applications. The term ‘neuron’ is often used in a general sense but, in fact, the CNS is composed of many different neuronal subtypes, each with defined anatomical locations (e.g. cortex, midbrain, spinal cord) and specialized functions (e.g. sensory, motor, communication, computation). It has been well-established that many neurodegenerative disorders are characterized by selective neuronal death—neurons of one subtype are completely unaffected while neurons of another subtype become diseased [74]. In fact, recent studies have demonstrated that the ability to use hiPSC-derived neurons to model neurodegenerative diseases is enhanced by the ability to generate neurons of the affected subtype as disease-related phenotypes are absent or significantly diminished in unaffected neuronal subtypes [75, 76]. In addition, many diseaserelated neuronal phenotypes are mediated by their interactions and signals from non-neuronal cell types such as astrocytes [77-79]. Through this study, we provide an adaptable and modular framework that can be used in conjunction with established differentiation protocols [4, 80] for the large-scale differentiation of hNPCs into astrocytes or neuronal cell populations of specific regional identities. In the future, such methods can also be applied with hNPCs generated from hiPSCs derived from patients suffering from neurodegenerative diseases, such as Alzheimer’s disease, to allow for the dissection of disease-related phenotypes as well investigation of the mechanisms that regulate disease onset and progression. It has been well-established that current 2-D differentiation strategies do not reflect the 3-D complexity and architecture of in vivo neural tissue. An interesting observation of this study was that MC-based approaches provided a 3-D like environment that resulted in neuronal cell populations of greater maturity than those generated using conventional 2-D approaches. Specifically, genome-wide expression analysis by RNA-seq showed that compared to neurons 19

generated on 2-D surfaces, MC-generated neurons had significantly higher levels of genes related to post-mitotic maturation, synapse formation, neurotransmitter synthesis, release, and transmission, and ion channels involved in action potential generation. This observation is consistent with other approaches that have emerged to model complex 3-D neural tissue physiology in vitro [81-84]. In particular, ‘organoid’-based methods have emerged as a robust and adaptable platform to model human brain development and complexity [83, 85-88]. However, these organoid approaches rely exclusively on cell aggregation in which necrosis may occur in center of larger spheroids (> 300 µm diameter) due to oxygen and nutrient diffusion restrictions [64]. Although the MC-based cultures generated in this study form small aggregates, these aggregates do not contain a cellular core that could be prone to necrosis (i.e. the MC is at the center of these ‘aggregates’). In fact, our analysis revealed that MC-generated hNPCs and neurons displayed no significant necrosis (as measured by propidium iodide [89]). As such, we speculate that the MC approach described in this study provides the benefits of a 3-D environment without mass transfer limitations of organoid-based strategies. Nonetheless, sideby-side studies will be necessary to compare and contrast the various advantages and disadvantages of organoid- and MC-based approaches for generation of neuronal cell populations. In addition, future studies that compare the neurons generated using the bioreactor-based approaches described in this study with primary human neurons are needed to assess the extent to which these in vitro derived neurons represent functionally mature cell types.

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5. CONCLUSION In conclusion, in this study we developed a MC-based system that employs a completely defined substrate for the long-term growth and directed neuronal differentiation of several independent hNPC lines. When combined with low shear RWV bioreactors, we were able to use this MC approach to facilitate large-scale hNPC expansion and neuronal differentiation. In the future, this fully defined and integrated biomanufacturing system will provide a platform for the generation of hNPCs and neuronal derivatives under GMP/GLP standards in numbers (>109) necessary for many downstream drug screening and regenerative medicine applications.

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ACKNOWLEDGEMENTS Funding for this work was provided by the NIH-NIBIB (5R21EB020767) and the Arizona Biomedical Research Commission. N.B. was supported by a fellowship from the International Foundation for Ethical Research. We would like to thank Jing Zhao and Guojun Bu for assistance with the astrocyte differentiation protocols.

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FIGURE LEGENDS Figure 1. Long-term culture and expansion of RiPSC-hNPCs on VDP-coated microcarriers (MCs). (A) Representative phase contrast and Hoechst images of RiPSC-hNPCs cultured on VDP-coated MCs (scale bar = 100 µm). (B) Cell counts of RiPSC-hNPCs cultured on VDP- and LN-coated MCs at various cell seeding and MC densities (mean ± S.D). For 2-D culture, 0.2 x 106 cells were seeded into a single well of a 6-well plate. (C) Doubling time of RiPSC-hNPCs cultured on VDP-coated 2-D and MC surfaces. Data is presented as the mean ± S.D of the doubling time over the course of 20 passages. There was no statistical difference in the doubling time of hNPCs grown on MC and 2-D surfaces (Student’s t-test, p = 0.626). (D) SOX1, SOX2, and NESTIN immunofluorescence of RiPSC-hNPCs cultured on VDP-coated MCs (scale bar = 100 µm; passage 13 shown). (E) Flow cytometry analysis for SOX1, SOX2, and NESTIN expression in RiPSC-hNPCs cultured on VDP-coated MCs (passage 11 shown). Gates were determined using isotype or secondary antibody only controls listed in Supplementary Table 2. Figure 2. Neuronal differentiation of RiPSC-hNPCs on VDP-coated microcarriers (MCs). (A) RNA sequencing (RNA-seq) analysis comparing gene expression profiles of hNPC and differentiated neuronal cultures. Genes with FPKM values greater than 1 were plotted. Genes showing altered expression with FDR < 0.001 and more than 2 fold changes were considered significantly upregulated (red) or downregulated (green). Genes related to hNPC identity (upper right panel), post-mitotic neurons (lower left panel), and synapse formation (lower right panel) are highlighted. Arrows are used to indicate gene names associated with particular data points. The complete RNA-seq data set is provided in Supplementary Table 3. The set of genes with statistically different levels of expression levels is provided in Supplementary Table 4. RNAseq data sets were obtained by sequencing equimolar amounts of mRNA from 3 biologically independent samples. (B) Quantitative PCR (qPCR) analysis for expression of hNPC multipotency and pan-neuronal markers in differentiated cultures. Gene expression fold changes were calculated relative to expression levels in undifferentiated hNPCs (n=3). (C) Flow cytometry analysis for TUBB3, NEUN, and propidium iodide (PI) in differentiated cultures. Gates were determined using isotype or secondary antibody only controls listed in Supplementary Table 2. Y-axis of flow cytometry plots captures a control non-fluorescent channel (NFC). (D) Immunofluorescence analysis for expression of MAP2, TUBB3, FOXG1, NEUN in replated differentiated cultures (scale bar = 200 µm).

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Figure 3. Comparison of differentiation of RiPSC-hNPCs on VDP-coated 2-D and microcarrier (MC) surfaces. (A) RNA sequencing (RNA-seq) analysis comparing gene expression profiles of hNPCs differentiated on 2-D and MC surfaces. Genes with FPKM values greater than 1 were plotted. Genes showing altered expression with FDR < 0.05 and more than 2 fold changes were considered significantly upregulated (red) or downregulated (green). The set of genes with statistically different levels of expression levels is provided in Supplementary Table 5. Arrows are used to indicate gene names associated with particular data points. Gene ontology (GO) analysis identified (B) cellular components and (C) biological processes related to neuron maturation, axon generation, and synapse formation were significantly enriched in differentiated cultures generated on MCs. The complete GO data set is provided in Supplementary Table 6. Figure 4. Neuronal differentiation of NDC- and AD-hNPCs on VDP- and LN-coated microcarriers (MCs). (A) Quantitative PCR (qPCR) analysis for expression of pan-neuronal markers in differentiated cultures. Gene expression fold changes were calculated relative to expression levels in undifferentiated hNPCs. (B) Flow cytometry analysis for TUBB3, NEUN, and propidium iodide (PI) in differentiated cultures. Gates were determined using isotype or secondary antibody only controls listed in Supplementary Table 2. Y-axis of flow cytometry plots captures a control non-fluorescent channel (NFC). (C) Immunofluorescence analysis for expression of MAP2, TUBB3, FOXG1 in replated differentiated cultures (scale bar = 200 µm). Figure 5. Large-scale culture and expansion of RiPSC-hNPCs on VDP-coated microcarriers (MCs) in rotating wall vessel (RWV) bioreactor. (A) Rotating wall vessel (RWV) bioreactor with 4 x 55mL culture vessels. (B) Representative phase contrast and Hoechst images of RiPSC-hNPCs cultured on VDP-coated MCs in 55mL RWV bioreactor (scale bar = 100 µm). (C) In the 55 mL RWV bioreactor, 3.6 x 105 cells/mL were seeded with 10 mg/mL of VDP-coated MCs. Cells were allowed to expand for a minimum of 6 days prior to analysis. Live cell count of RiPSC-hNPCs expanded on VDP-coated MCs in RWV bioreactor (n=4 independent bioreactor experiments). (D) Representative SOX1, SOX2, and NESTIN immunofluorescence of RiPSC-hNPCs cultured on VDP-coated MCs in RWV bioreactor (scale bar = 100 µm). (E) Representative flow cytometry analysis for SOX1, SOX2, NESTIN, propidium iodide (PI) in RiPSC-hNPCs cultured on VDP-coated MCs in RWV bioreactor. Gates were determined using isotype or secondary antibody only controls listed in Supplementary Table 2. Y-axis of flow cytometry plots captures a control non-fluorescent channel (NFC). 24

Figure 6. Large-scale neuronal differentiation of RiPSC-hNPCs on VDP-coated microcarriers (MCs). (A) Live cell count of RiPSC-hNPC derived neurons generated on VDPcoated MCs in RWV bioreactor (n=4 independent bioreactor experiments). (B) RNA sequencing (RNA-seq) analysis comparing gene expression profiles of hNPC and differentiated neuronal cultures (n=4). The average values of FPKM values from four independent neuronal samples are shown. Genes showing altered expression with FDR < 0.001 and more than 2 fold changes were considered significantly upregulated (red) or downregulated (green). Genes related to hNPC identity, post-mitotic neurons, and synapse formation are highlighted (right panel). Arrows are used to indicate gene names associated with particular data points. The complete RNA-seq data set is provided in Supplementary Table 8. The set of genes with statistically different levels of expression levels is provided in Supplementary Table 9. (C) Quantitative PCR (qPCR) analysis for expression of pan-neuronal markers in differentiated neuronal cultures. Gene expression fold changes were calculated relative to expression levels in undifferentiated hNPCs (n=3). (D) Flow cytometry analysis for TUBB3, NEUN, and propidium iodide (PI) in differentiated cultures. Gates were determined using isotype or secondary antibody only controls listed in Supplementary Table 2. Y-axis of flow cytometry plots captures a control nonfluorescent channel (NFC). (E) Immunofluorescence analysis for expression of MAP2, TUBB3, FOXG1, NEUN in replated differentiated cultures (scale bar = 200 µm).

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SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1. Long-term culture and expansion of RiPSC-hNPCs on LNcoated microcarriers (MCs). (A) Representative phase contrast and Hoechst images of RiPSC-hNPCs cultured on LN-coated MCs (scale bar = 100 µm). (B) Doubling time of RiPSChNPCs cultured on LN-coated 2-D and MC surfaces. Data is presented as the mean ± S.D of the doubling time. There was no statistical difference in the doubling time of hNPCs grown on MC and 2-D surfaces (Student’s t-test, p = 0.358). (C) SOX1, SOX2, and NESTIN immunofluorescence of RiPSC-hNPCs cultured on LN-coated MCs (scale bar = 100 µm; passage 13 shown). (D) Flow cytometry analysis for SOX1, SOX2, and NESTIN expression in RiPSC-hNPCs cultured on LN-coated MCs (passage 11 shown). Gates were determined using isotype or secondary antibody only controls. Isotype and secondary antibody controls used are listed in Supplementary Table 2. Y-axis of flow cytometry plots captures a control nonfluorescent channel (NFC). Supplementary Figure 2. Long-term culture and expansion of HES3-hNPCs on VDP- and LN-coated microcarriers (MCs). (A) Representative phase contrast images of HES3-hNPCs cultured on VDP- (left panel) and LN-coated (right panel) for 5 passages. (B) Doubling time of HES3-hNPCs cultured on 2-D and MC surfaces coated with VDP (left panel) and LN (right panel). Data is presented as the mean ± S.D of the doubling time. There was no statistical difference in the doubling time of hNPCs grown on MC and 2-D surfaces coated with VDP (Student’s t-test, p = 0.941) and LN (Student’s t-test, p = 0.542). (C) Flow cytometry analysis for SOX1, SOX2, and NESTIN expression in HES3-hNPCs cultured on VDP- (top panels) and LNcoated MCs (passage 11 shown). Gates were determined using isotype or secondary antibody only controls. Isotype and secondary antibody controls used are listed in Supplementary Table 2. Y-axis of flow cytometry plots captures a control non-fluorescent channel (NFC). Supplementary Figure 3. Effect of VDP concentration on growth and adhesion of hNPCs on MCs. (A) Representative phase contrast images of RiPSC-hNPCs grown on surfaces coated with various concentrations of VDP (scale = 250 µm). (B) Percentage of MCs after 24 hours of culture with cell attachment on un-, LN-, and VDP-coated MCs. Data is presented as the mean ± S.D. of four independent fields of view. Supplementary Figure 4. Culture of RiPSC-hNPCs on VDP- and LN-coated 2-D surfaces. (A) Immunofluorescence and (B) flow cytometry analysis of RiPSC-hNPCs cultured on VDP26

and LN-coated 2-D surfaces for hNPC markers SOX1, SOX2, and NESTIN. Gates were determined using isotype or secondary antibody only controls. Isotype and secondary antibody controls used are listed in Supplementary Table 2. Y-axis of flow cytometry plots captures a control non-fluorescent channel (NFC). Supplementary Figure 5. Neuronal differentiation of RiPSC-hNPCs on LN-coated microcarriers (MCs). (A) Quantitative PCR (qPCR) analysis for expression of hNPC multipotency and pan-neuronal

markers in differentiated cultures. Gene expression fold

changes were calculated relative to expression levels in undifferentiated hNPCs (n=3). (B) Flow cytometry analysis for TUBB3, NEUN, and propidium iodide (PI) in differentiated cultures. Gates were determined using isotype or secondary antibody only controls listed in Supplementary Table 2. Y-axis of flow cytometry plots captures a control non-fluorescent channel (NFC). (C) Immunofluorescence analysis for expression of MAP2, TUBB3, FOXG1, NEUN in replated differentiated cultures (scale bar = 200 µm). Supplementary Figure 6. Gene expression analysis of differentiation of RiPSC-hNPCs on VDP- and LN-coated 2-D and microcarrier (MC) surfaces. Heatmap displaying subset of genes from RNA-seq analysis that were significantly upregulated in differentiated cultures generated on VDP-coated MC surfaces compared to 2-D surfaces including those related to (A) post-mitotic neurons and synapse formation, (B) acetylcholine (ACH), γ-aminobutyric acid (GABA), and glutamate (GLU) neurotransmitter synthesis, release, and transmission, (C) potassium (K+), calcium (Ca2+), sodium (Na+), and chloride (Cl-) ion channels involved in action potential generation. The data associated with this subset of genes is provided in Supplementary Table 6. (D) Quantitative PCR (qPCR) comparing gene expression of hNPCs differentiated on 2-D and MC surfaces (mean ± S.E.M). Expression of these genes was statistically significantly higher in the differentiated cultures generated on MCs compared to 2-D surfaces (Student’s t-test, *p<0.05, **p<0.01, ***p<0.001). Supplementary

Figure

Immunofluorescence

7.

Characterization

of

NDC-

and

AD-hNPCs.

(A)

and (B) flow cytometry analysis of NDC- and AD-hNPCs for hNPC

markers SOX1, SOX2, and NESTIN. Gates were determined using isotype or secondary antibody only controls. Isotype and secondary antibody controls used are listed in Supplementary Table 2. Y-axis of flow cytometry plots captures a control non-fluorescent channel (NFC). 27

Supplementary Figure 8. Large-scale culture and expansion of RiPSC-hNPCs on LNcoated microcarriers (MCs) in rotating wall vessel (RWV) bioreactor. (A) Representative phase contrast and Hoechst images of RiPSC-hNPCs cultured on LN-coated MCs in 55mL RWV bioreactor (scale bar = 100 µm). (B) In the 55 mL RWV bioreactor, 3.6 x 105 cells/mL were seeded with 10 mg/mL of LN-coated MCs. Cells were allowed to expand for a minimum of 6 days prior to analysis. Live cell count of RiPSC-hNPCs expanded on LN-coated MCs in RWV bioreactor (n=4 independent bioreactor experiments). (C) Representative SOX1, SOX2, and NESTIN immunofluorescence of RiPSC-hNPCs cultured on LN-coated MCs in RWV bioreactor (scale bar = 100 µm). (D) Representative flow cytometry analysis for SOX1, SOX2, NESTIN, propidium iodide (PI) in RiPSC-hNPCs cultured on LN-coated MCs in RWV bioreactor. Gates were determined using isotype or secondary antibody only controls listed in Supplementary Table 2. Y-axis of flow cytometry plots captures a control non-fluorescent channel (NFC).

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SUPPLEMENTARY TABLES Supplementary Table 1. List of qPCR primers used in this study. Supplementary Table 2. List of antibodies used in this study. Supplementary Table 3. RNA-seq data set for hNPC and neurons generated on VDPcoated 2-D surfaces and small scale microcarriers (MCs). Supplementary Table 4. List of genes that are expressed at statistically (FDR <0.05, Fold change > 2) different levels between hNPCs and neurons in small scale VDP-coated microcarrier (MC) cultures. Supplementary Table 5. List of genes that are expressed at statistically (FDR <0.05, Fold change > 2) different levels between neurons generated on VDP-coated 2-D and microcarrier (MC) surfaces. Supplementary Table 6. List of subset of genes related to post-mitotic neurons, and synapse formation, acetylcholine (ACH), γ-aminobutyric acid (GABA), and glutamate (GLU) neurotransmitter synthesis, release, and transmission, potassium (K+), calcium (Ca2+), sodium (Na+), and chloride (Cl-) ion channels involved in action potential generation that are expressed at statistically (FDR <0.05, Fold change > 2) different levels between neurons generated on VDP-coated 2-D and microcarrier (MC) surfaces. Supplementary Table 7. Enrichment for Gene Ontology (GO) terms that are enriched in neurons generated on VDP-coated MCs compared to neurons generated on VDP-coated 2-D surfaces. Supplementary Table 8. RNA-seq data set for hNPC and neurons generated on VDP-coated microcarriers (MCs) in RWV bioreactors. Supplementary Table 9. List of genes that are expressed at statistically (FDR <0.05, Fold change > 2) different levels between hNPCs and neurons generated on VDP-coated microcarriers (MCs) in RWV bioreactors.

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