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A hydrogel-based bioprocess for scalable manufacturing of human pluripotent stem cells-derived neural stem cells Haishuang Lin, Qian Du, Qiang Li, Ou Wang, Zhanqi Wang, Kan Liu, Christian Elowsky, Chi Zhang, and Yuguo Lei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05780 • Publication Date (Web): 09 Aug 2018 Downloaded from http://pubs.acs.org on August 10, 2018

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ACS Applied Materials & Interfaces

A hydrogel-based bioprocess for scalable manufacturing of human pluripotent stem cells-derived neural stem cells

Haishuang Lin1,8, Qian Du2,8, Qiang Li1,3, Ou Wang1,3, Zhanqi Wang4, Kan Liu2, Christian Elowsky5, Chi Zhang2 and Yuguo Lei1,3,6,7,*

1. Department of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, Nebraska, USA 2. Department of Biological Systems Engineering, University of Nebraska-Lincoln, Nebraska, USA 3. Biomedical Engineering Program, University of Nebraska-Lincoln, Nebraska, USA 4. Department of Vascular Surgery, Beijing Anzhen Hospital of Capital Medical University, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing, China 5. Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Nebraska, USA 6. Mary and Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center, Omaha, Nebraska, USA 7. Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA 8. These authors contribute equally to this work. * Corresponding Author Yuguo Lei 820 N 16th St Lincoln, NE 68588 Email : [email protected] Phone: 402-472-5313 Fax: 402-472-6989

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ABSTRACT Neural stem cells derived from human pluripotent stem cells (hPSC-NSCs) are of great value for modeling diseases, developing drugs and treating neurological disorders. However, manufacturing high quantity and quality hPSC-NSCs, especially for clinical applications, remains a challenge. Here, we report a chemically-defined, high yield and scalable bioprocess for manufacturing hPSC-NSCs. hPSCs are expanded and differentiated into NSCs in microscale tubes made with alginate hydrogels. The tubes are used to isolate cells from the hydrodynamic stresses in the culture vessel and limit the radial diameter of the cell mass less than 400 μm to ensure efficient mass transport during the culture. The hydrogel tubes provide uniform, reproducible and cell-friendly microspaces and microenvironments for cells. With this new technology, we showed hPSC-NSCs could be produced in 12 days with high viability (~95%), high purity (>90%) and high yield (~5x108 cells/mL of microspace). The volumetric yield is about 250 times more than the current-state-of-the-art. Whole transcriptome analysis and qRT-PCR showed hPSC-NSCs made in this process had similar gene expression to hPSC-NSCs made in the conventional culture technology. The produced hPSC-NSCs could mature into both neurons and glial cells in vitro and in vivo. The process developed in this paper can be used to produce large numbers of hPSC-NSCs for various biomedical applications in the future. KEYWORDS: human pluripotent stem cells; neural stem cells; alginate hydrogel tube; cell differentiation; 3D microenvironment


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INTRODUCTION Millions of people are suffering from neurological disorders1. Neural stem cells (NSCs), the stem cells of the nervous system, have capability to proliferate and differentiate into neurons and glial cells in vitro and in vivo2. NSCs also secrete abundant neurotrophic factors that can protect or enhance the healing of the nervous system3–6. Further, NSCs can sense and migrate toward lesion sites7. These properties make NSCs highly promising for treating various neurological disorders either through replacing the lost neurons or through protecting the remaining neurons via the neurotrophic factors or using as vehicles to deliver drugs (e.g. drugs for killing brain tumors8). Clinical studies using NSCs to treat amyotrophic lateral sclerosis9,10, Parkinson’s11,12, multiple sclerosis13, spinal cord injury14,15, stroke16, cerebral palsy17,18 and brain tumors19 have generated highly encouraging results20,21.

To date, clinical studies mainly use NSCs isolated from fetal tissues and expanded in vitro20,21. Fetal tissue-derived NSCs have significant problems that limit their future success in clinics22. First, there are ethical and religious concerns on using fetal tissues in many countries20,21. Second, it is very challenging to produce them in large quantity required for clinical applications due to their slow growth rate and limited proliferation capability. Third, the cell products have large batch-to-batch variations22. Typically, each batch is derived from a different donor with unique genetics. It is well known that the NSC number, proliferation and differentiation capability all highly depend on the origin locations and the developmental stage of the donor23,24. Additionally, NSCs dynamically respond to the in vitro culture microenvironment and change their phenotypes significantly during



the in vitro culture25,26. Together, these lead to large product variations between batches. For instance, recent studies using fetal tissue-derived NSCs to treat spinal cord injury and Alzheimer’s disease found the research-grade NSCs had high efficacy in animal models, while the clinical-grade NSCs from the same vendor had no efficiency27,28. In short, fetal tissue-derived NSCs can hardly meet the need of various biomedical applications.

Human pluripotent stem cells (hPSCs) provides a potential solution to this challenge 29. hPSCs, including human embryonic stem cells (hESCs)30 and induced pluripotent stem cells (iPSCs)31,32, have unlimited proliferation capacity that can be expanded to generate large quantity of cells in vitro. hPSCs can be efficiently differentiated into high purity NSCs under chemically-defined condition33,34, and these hPSC-derived NSCs (hPSC-NSCs) have shown high safety and efficacy for treating various neurological disorders such as Parkinson’s disease (PD)34–36, Alzheimer’s disease (AD)37, stroke38, spinal cord injury (SCI)39–42 in animal models. In short, hPSC-NSCs can overcome the problems of the fetal tissue-derived NSCs. In the future, universal hPSC-NSCs can be produced in massive numbers (e.g. >1014 cells) in a single batch, purified and systematically characterized, and offered to many researchers for testing efficacy for treating various diseases in animal models and clinical studies11,13–15,43–47. If succeed, cells from the same batch can be used for treating patients. This strategy can avoid the batch-to-batch variation problems as encountered by the fetal tissue derived NSCs27,28.

While making small-scale hPSC-NSCs for laboratorial research can be readily done with current cell culture technologies33,34, generating large numbers of hPSC-NSCs for large


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animal studies, clinical trials and clinics is still very challenging. Current 2 dimensional (2D) cell culture methods (e.g. cell culturing flasks, in which cells are cultured as adherent cells on 2D surfaces) are labor-, time-, and cost-intensive, and are considered unsuitable for culturing cells in large scales48,49. 3D suspension culture methods (e.g. using stirredtank bioreactors) have been proposed as a solution for scaling up the cell production 48– 50.

However, recent researches find using 3D suspension culturing to produce large

numbers of cells is also very challenging51–54. hPSCs have strong cell-to-cell adhesions that drive them aggregate to form large cell agglomerates in 3D suspension cultures48. The agglomeration leads to impaired mass transport. For cells at the center of large agglomerates (e.g. > 400 µm diameter), the transport of nutrient, growth factor, and nutrients becomes limited, leading to slow cell growth, cell death and phenotype changes48. Agitation can be used to reduce the cell agglomeration. However, agitation generates critical shear force that induce significant cell death48,55,56. As a result, both the cell growth and volumetric yield are low in 3D suspension culturing50. For instance, hPSCs typically expand 4-fold in 4 days per passage to yield about 2.0x106 cells/mL in 3D suspension culturing51–54. Therefore, there is critical need for novel technologies that allow cost-effective and scalable manufacturing of high quality hPSCs and hPSC-NSCs.

To address the need, we previously developed a scalable, high-yield, and current Good Manufacturing Practice (cGMP) compliant cell culture method for expanding hPSCs57. With this method, cells are cultured in microscale tubes made with alginate hydrogels. The tubes can protect cells from shear stresses in the culture vessel and limit the cell mass smaller than the diffusion limit of human tissue. Together, the hydrogel tubes



provide highly uniform and cell-friendly microenvironments for cells to grow. This novel design leads to significant improvements in cell culture efficiency and consistency. Under optimized culture conditions, hPSCs could be cultured in these hydrogel tubes for longterm with high cell viability, high growth rate (1000-fold expansion/10 days/passage), high purity (>95% OCT4+) and high yield (~5x108 cells/mL of microspace). The yield is about 250 times of the yield of 3D suspension culturing. As discussed later, this high yield makes large-scale cell production technically and economically feasible. However, if hPSCs can be efficiently differentiated into NSCs in the alginate hydrogel tubes and if hPSC-NSCs made in alginate hydrogel tubes are similar to hPSC-NSCs made with conventional culture methods have not been studied. In this paper, we systematically studied the differentiation of hPSCs into NSCs in the hydrogel tubes and compared the gene expression of hPSC-NSCs made in hydrogel tubes and 2D cultures. We found hPSCNSCs could be made in the hydrogel tubes with extremely high yield and purity and these cells were similar to hPSC-NSCs made in 2D cultures. In short, we developed a scalable bioprocess for making clinical-grade hPSC-NSCs with high volumetric yield, high viability and highly purity.


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EXPERIMENTAL SECTION Culturing Human Pluripotent Stem Cells (hPSCs) in 2D. H9 hESCs were purchased from WiCell Research Institute. Fib-iPSCs reprogrammed from human fibroblasts are a gift from George Q. Daley laboratory (Children’s Hospital Boston, Boston)58. hPSCs (H9s and iPSCs) were maintained in 6-welll plate coated with Matrigel (BD Biosciences) in Essential 8TM medium (E8, Invitrogen)59. Cells were passaged every 3-4 days with 0.5 mM EDTA (Invitrogen). Medium was changed daily. Cells were routinely checked for the expression of pluripotency markers (OCT4 and NANOG), their capability to form teratomas in immunodeficient mice, their karyotypes, and bacterial or mycoplasma contamination. Processing Alginate Hydrogel Tube. Alginate hydrogel tubes are processed as described in our previous publication57. We made the hydrogel tubes with diameter of 120, 250 and 330 μm and found 250 μm was optimal for cell culture. We decided to use 250 μm hydrogel tubes with 40 μm shell thickness for all the cell culture studies except in Figure S2. To make these tubes, the flow rates for the cell suspension and alginate solution were 30 μL/min and 30 μL/min, respectively. Culturing hPSCs in Alginate Hydrogel Tube. The method for a typical cell culture using alginate hydrogel tubes has been described in our previous publication 57. hPSC Differentiation in 2D. Single hPSCs (iPSCs and H9s) were plated on Matrigelcoated 6 well plates (2.0x106 cells/well) and cultured in E8 medium with ROCK inhibitor overnight to reach >90% confluency. E8 medium was removed and replaced with neural induction medium consisting of Essential 6TM medium (E6, Invitrogen) without ROCK inhibitor supplied with 100 nM LDN193189 (Selleckchem, #S2618) and 10 µM SB431542 7 ACS Paragon Plus Environment


(Selleckchem, #S1067) for 7 days. Note that E6 medium is equal to E8 medium minus the bFGF and TGF-β proteins. Medium was changed daily33. hPSC Differentiation in Alginate Hydrogel Tube. Single hPSCs were encapsulated in alginate hydrogel tube (1.0x106 cells/mL) and cultured in E8 medium with ROCK inhibitor for 5 days. E8 medium was removed and replaced with neural induction medium without ROCK inhibitor for 7 days. Medium was changed daily. To differentiate NSCs into the cortical neurons, NSCs in alginate hydrogel tube were harvested on day 12 and plated on Matrigel-coated 6 well plates, and cultured in neural differentiation medium consisting of Neurobasal® Media (Life Technologies), B27 (50x, Life Technologies), BDNF (20 ng/mL, PeproTech), GDNF (10 ng/mL, PeproTech), L-ascorbic acid (200 µM, Sigma), DAPT (2.5 µM, Tocris), Dibutyryl-cAMP (0.5 mM, Santa Cruz Biotechnology) for another 19 days. Half medium was changed every two days. NSCs Production in Alginate Hydrogel Tube with Bioreactors. 2 mL of hPSCs suspension in alginate hydrogel tube were suspended in a bioreactor. hPSCs were first cultured in E8 medium for 5 days. Medium was stored in a bellow bottle. The bellow bottle was periodically pressed to flow the medium into the bioreactor and released to flow the medium out of the bioreactor. On day 5, E8 medium was removed and replaced with neural induction medium for 7 days. On day 12, hydrogel tubes were dissolved by adding 0.5 mM EDTA buffer. Cell masses were pelleted by centrifugation. Cell masses were treated with Accutase at 37 °C for 10 minutes and dissociated into single cells. Magnetic beads coated with anti-SSEA4 antibodies were added to pull down the undifferentiated SSEA4+ hPSCs with a magnetic cell separator. The supernatant was transferred into a new tube. Cells were pelleted by spinning at 300 g for 5 minutes and transported to the


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surgery room for injection. NSCs Cryopreservation. The freezing medium consists of KnockOutTM DMEM/F-12 (ThermoFisher,

#12660012)

with

StemPro

Neural

Supplement

(ThermoFisher,

#1050801), GlutaMAXTM-I (#1286001, ThermoFisher) and 10% DMSO. Single NSCs were suspended at 2.0x106 cells/mL and frozen in liquid nitrogen. Transplanting hPSC-Derived NSCs to Rat Brains. The Animal Care and Use Committee of the University of Nebraska, Lincoln approved the animal research protocol and all procedures followed the approved protocol. Sprague Dawley female rats were obtained from Charles River. Animals received intraperitoneal cyclosporine A (10 mg/kg, LC Laboratories) injection starting 1 day before transplantation. For transplantation, animals were anesthetized with 2-4% isoflurane. 2.0x105 cells suspended in 4 µL PBS were injected into the striatum (AP+0.5 mm; ML±3.0 mm; DV-6 mm) at 0.5 µl/minute using a 10 µL Hamilton syringe (Hamilton Company, USA) with a stereotaxic frame (RWD Life Science Inc). 30 days post transplantation, rats were anesthetized with ketamine/xylazine and perfused with PBS followed by 4% paraformaldehyde. After fixation, the brain was serially sectioned (40 µm in thickness) with a Leica cryosection machine, and free-floating sections were stained with antibodies. Six rats were used for the transplantation study. Immunostaining and Imaging. Details of immunostaining and imaging can be found in our previous publication60. For flow cytometry analysis, the harvested cells were dissociated into single cells with Accutase and fixed with 4% PFA solution for 10 minutes. Single cells were stained with primary antibodies (Table S1) at 4 °C overnight. After 3 times washing with 1% BSA in PBS, secondary antibodies (Table S1) were added and incubated at room temperature for 2 hours. Cells were washed with 1% BSA in PBS and 9 ACS Paragon Plus Environment


analyzed using Cytek flow cytometry. LIVE/DEAD® Cell Viability kit (Invitrogen) was used to stain the live and dead cells following the product manual. Quantitative Real Time PCR (qRT-PCR). Total RNA was extracted from the harvested cells using Trizol (Invitrogen, Carlsbad, CA, USA). Reverse transcription is done with the Maxima First Strand cDNA Synthesis Kit. Quantitative real-time PCR was carried out in an Eppendorf Master Cycler RealPlex4 (ThermoFisher Scientific) using the Power SYBR Green PCR Master Mix (ThermoFisher). The data were normalized to the endogenous GAPDH. Primer sequence were listed in Table S2. Embryoid Body (EB) Differentiation and Teratoma Formation in vivo. The Animal Care and Use Committee of the University of Nebraska, Lincoln approved the animal research protocol and all procedures followed the approved protocol. Three mice (two teratomas per mouse) were used for teratoma assay for each hPSC line. Details of EB differentiation and teratoma assays can be found in our previous publication57. RNA Sequencing and Data Analysis. Total RNA of day 8 NSCs cultured in 2D and day 12 NSCs cultured in alginate hydrogel tube (3D) were prepared with RNeasy mini kit (cat # 74104 QIAGEN). Libraries were prepared with TruSeq Stranded mRNA Library Prep Kit and sequenced with Illumina NextSeq 500. 20 million 75 bp paired-end reads were generated for each sample. Methods for the data processing, heatmap generating, PCA analysis, differential gene expression analysis have been described in previous publication57. Statistical Analysis. The data are presented as the mean ± standard deviation (SD) from three independent experiments. Unpaired t-test was used to compare two groups and one-way ANOVA was used to to compare more than two groups. GraphPad Prism 7 10 ACS Paragon Plus Environment

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(GraphPad Software, Inc., La Jolla, CA) was used for analysis. Details of statistical analysis can be found in our previous publication57. Data Availability. The sequencing data can be assessed in Gene Expression Omnibus (GEO) with number GSE99776 and GSE109683.

RESULTS Starting materials Two hPSCs, the H9 hESCs and iPSCs made from human fibroblast (Fib-iPSCs)58, were used for this study. They were maintained on Matrigel-coated plates in the chemical defined Essential 8 (E8) medium and formed compact colonies typical to hPSCs (Figure S1a, e). Majority (e.g. >95%) of the cells expressed pluripotency makers, OCT4 and NANOG (Figure S1b, f). In the in vitro embryoid body (EB) assay, they formed all three germ layer cells, such the NESTIN+ ectodermal, α-SMA+ mesodermal and HNF-3β+ endodermal cells (Figure S1c, g). They generated teratomas containing all three germ layer tissues in immunodeficient mice (Figure S1d, h). These data show our starting materials are high quality hPSCs.

Overview of producing hPSC-NSCs with alginate hydrogel tubes A micro-extruder was used for processing cells into alginate hydrogel tubes (or AlgTubes) (Figure 1a). The detailed method for designing and fabricating the extruder has been described in our previous publication57. To process hydrogel tubes, a cell suspension and an alginate solution is pumped into a custom-made micro-extruder with two syringe pumps (Figure 1a). They form coaxial core-shell flows that flow through a nozzle and into 11 ACS Paragon Plus Environment


a 100 mM CaCl2 buffer. The Ca2+ ions instantly crosslink the shell alginate solution into an alginate hydrogel tube (Figure 1b and Movie S). Subsequently, cells are grown in the hydrogel tubes that are suspended in cell culture medium. The tubes protect cells from hydrodynamic stresses and limit the radial diameter of the cell mass less than 400 μm to ensure efficient mass transport (Figure 1c). In the tubes, single hPSCs grow into small spheroids on day 5 and fill the tubes around 9 days to form fibrous hPSC masses. To generate massive numbers of hPSCs, the day 9 cell masses can be released by dissolving the hydrogel tubes with 0.5mM EDTA solution (5 minutes at room temperature), and dissociated into single cells with Accutase and processed into new hydrogel tubes for a second round of expansion. Once the targeted cell number is reached, hPSCs can be differentiated into NSCs within 7 days (Figure 1d).

As we previously described57, the inner and outer diameter, and shell thickness of the hydrogel tubes can be precisely controlled through adjusting the nozzle diameter of the micro-extruder, the flow rates of the cell suspension and alginate solution (Figure S2a-c) following the equations shown in Figure S2d. Our following studies showed alginate hydrogel tubes are highly efficient for the scalable manufacturing of hPSC-NSCs.

Expanding hPSCs in alginate hydrogel tubes We previously showed hPSCs could be expanded in alginate hydrogel tubes with extremely high viability, growth rate and yield57. Our studies in this report confirmed these findings. Single hPSCs first formed small clusters within 24 hours. These clusters grew in size as cells proliferated and filled the hydrogel tubes in about 9 days, generating


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monodispersed (in diameter) fibrous cell masses (Figure S3a, b, c). During the culturing, very few dead cells were detected by live/dead cell staining (Figure S3d). When seeded at 1.0x106 cells/mL, hPSCs expanded ~28, ~145, ~480 fold to yield ~28x, 145x, 480x106 cells/mL of microspace on day 5, 7 and 9, respectively (Figure S3e, f). Immunostaining showed majority of the cells expressed pluripotency markers such as OCT4, NANOG, SSEA4 and ALP (Figure S3g). Flow cytometry analysis found >95% of cells expressed these pluripotency markers (Figure S3h, i). These data show the robustness of using alginate hydrogel tubes for hPSC expansion.

Differentiating hPSCs into NSCs in 2D cultures Literature research has shown hPSCs could be efficiently differentiated into NSCs through inhibiting the SMAD signaling using small molecules in 2D cultures61. We successfully repeated these findings. hPSCs were first plated on Matrigel-coated plate at the density of 2.0x106 cells per well overnight. To differentiate hPSCs into NSCs, bFGFs and TGFβs were removed from the E8 hPSC expansion medium and two small molecules LDN193189 (100 nM) and SB431542 (10 µM), which inhibit the SMAD signaling, were added to the medium (Figure 2a). Medium was changed daily. During the 7-day differentiation, very few dead cells were found (Figure 2b, g). Immunostaining analyses showed majority of day 8 cells were positive for NSC markers PAX6, NESTIN, FOXG1 and SOX1 (Figure 2c, h). Flow cytometry analysis found 93% cells were PAX6+/NESTIN+ (Figure 2d, i). OCT4+/ NANOG+ undifferentiated hPSCs were not detectable on day 8 (Figure 2e, j). When these NSCs were passaged to Matrigel-coated plates and cultured for additional 19 days in neural differentiation medium, ~90% of the



final cells were TUJ1+ neurons. Among them, ~60% were TBR1+ neurons (Figure 2f, k, l). After 7 days differentiation, ~8.8x106 cells per well were generated (Figure 2m). Thus about 4.4 NSCs were produced per input hPSC. These results confirmed that high purity and quality NSCs could be made from hPSCs through inhibiting SMAD signaling with small molecules. Importantly, both the hPSC expansion medium and the neural differentiation medium are chemically-defined. Similar results were obtained using H9 hPSCs and Fib-iPSCs. We termed NSCs made in 2D culturing as 2D-NSCs.

Differentiating hPSCs into NSCs in alginate hydrogel tubes We then studied whether hPSCs could be differentiated into NSCs in alginate hydrogel tubes. hPSCs were first expanded in alginate hydrogel tubes for 5 days to form spheroids, followed by 7 days neural differentiation using the same differentiation medium as 2D cultures (Figure 3a). Very few dead cells were found as shown by phase image and the live/dead cell staining (Figure 3b-d). On day 12, about 4.8x108 cells were produced per milliliter of the microspace in the hydrogel tubes (Figure 3e). Immunostaining and confocal imaging showed majority of day 12 cells were positive for NSC markers PAX6, NESTIN, FOXG1 and SOX1 (Figure 3f, g). Flow cytometry analysis found 94.4% of the cells were PAX6+ /NESTIN+ (Figure 3h). Similar results were obtained using H9 hESCs and Fib-iPSCs as starting cells (Figure S4). Our results showed the differentiation efficiencies were very similar in AlgTubes and 2D cultures (Figure 2 vs Figure 3). We termed NSCs made in alginate hydrogel tubes as 3D-NSCs. qRT-PCR data showed both the 3D-NSCs and 2D-NSCs expressed very low levels of pluripotency marker OCT4 and NANOG, indicating the high efficiency of NSC differentiation (Figure 3i). qRT-PCR


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analyses also showed 3D-NSCs had slightly higher expression levels for some NSC markers (Figure 3j, k), indicating 3D-NSCs and 2D-NSCs had difference, at lease in gene expressions.

To test if the 3D-NSCs have the capability to become both neurons and glial cells, we released the NSCs from the hydrogel tubes and cultured them on Matrigel-coated plates in either neural differentiation medium or glial cell differentiation medium. When cultured 19 days in neural differentiation medium, ~90% of the cells became TUJ1+ neurons. Among them, ~60% were TBR1+ neurons (Figure 4a-c). Some of these neurons expressed the post-mitotic neuron marker NeuN and formed synapses (Figure 4d). 3DNSCs could also be differentiated into GFAP+ astrocyte in vitro (Figure 4e). When day 12 3D-NSCs were transplanted to rat brains, 3D-NSCs survived well and matured into TUJ1+ neurons at 30 days post-transplantation (Figure 4f). No tumors or abnormal brain structures were found. These results showed 3D-NSCs were able to mature both in vitro and in vivo and were safe in vivo. We also cryopreserved hPSC-NSCs. After recovering, the cryopreserved hPSC-NSCs grew well on 2D plates and expressed NSC markers, PAX6 and NESTIN at passage 1 and 5 (Figure 4g, h).

Global gene expressions of 2D-NSCs and 3D-NSCs To systematically study if 2D-NSCs and 3D-NSCs are different, we studied the genomewide gene expression using RNA-sequencing. We sequenced the H9s, 3D-NSCs and 2D-NSCs derived from H9s (3 biological replicates for each). Hierarchical clustering analysis showed 3D-NSCs and 2D-NSCs clustered closely and were very different from



H9s (Figure 5a). The genome-wide gene expression profile correlation coefficients between 3D-NSCs and 2D-NSCs were >0.81, showing that they had similar global gene expression (Figure 5c, d). However, principal component analysis (PCA) indicated 2DNSCs and 3D-NSCs had some differences in gene expression (Figure 5b), which drove us to perform detailed differential gene expression analysis.

Differential gene expression analysis identified 1769 genes upregulated in 3D-NSCs, and 1491 genes upregulated in 2D-NSCs (Data S1). The top 10 GO terms upregulated in 3DNSCs were related to nucleic acid biosynthesis and metabolic process, as well as transcription (Figure 6a, b). The top 10 GO terms upregulated in 2D-NSCs were related to protein transport and mRNA catabolic process. Since 2D-NSCs and 3D-NSCs were made in two and three dimensions, we compared the dimension-related genes in details and found: (a) 3D-NSCs had higher expression of ECM genes including collagen (COL22A1, COL19A1, COL6A5); laminin (LAMC3); integrin (ITGA4, ITGB8, ITGA9 and ITGA2); proteases (MMP11); and other ECM components (EFEMP1 and FBLN1) (Figure 6c-g); 2D-NSCs had higher expression of ECM genes including collagen (COL9A3, COL5A3, COL1A1, COL20A1, COL15A1, COL2A1, COL11A1, COL12A1, COL18A1, COL14A1, COL21A1 and COL3A1); laminin (LAMA5, LAMC2 and LAMB1); integrin (ITGAL, ITGA11, ITGA8, ITGB4 and ITGB5); proteases (TIMP3, MMP19, MMP23B, MMP17 and TIMP1); and other ECM components (HSPG2 and NID1) (Figure 6c-g). (b) 3D-NSCs had higher expression of genes for glycolysis including ALDOB (Figure 6h); 2D-NSCs had higher expression of genes for glycolysis including PGK1, HK2, ALDOC, GPI, ALDOA, PGK2, ENO1, PGM1, TPI1, ENO2, PGM2, PFKL and GCK (Figure 6h). (c)


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3D-NSCs had higher expression of genes for NOTCH signaling including NOTCH4 and LFNG, and NOTCH target including HES5, HES1 and LFNG (Figure 6i); 2D-NSCs had higher expression of genes for NOTCH signaling including JAG1, NOTCH2NL and DLL3, and NOTCH target including JAG1 and HEY1 (Figure 6i). (d) 3D-NSCs had higher expression of genes for growth factors and cytokines including S100A6, PTN, FGF13, EGF and BDNF (Figure 6j, k); 2D-NSCs had higher expression of genes for growth factors and cytokines including VEGFA, BMP4, GPI, ARTN and BMP2 (Figure 6j, k). (e) 3D-NSCs had higher expression of genes for cell motility and migration including NRCAM, NEUROG2, NDN and DCX (Figure 6l); 2D-NSCs had higher expression of genes for cell motility and migration including VEGFA, FLNA and SLIT2 (Figure 6l). Whether and how these differences in gene expression influence the NSCs’ functions should be studied in the future, especially with relevant diseases models. We did not find significant differences in genes related to NSCs identity, neural differentiation, cell senescence, apoptosis, cell death, DNA damage, stress and oncogene (Figure S5b-d and Figure S6a-e).

A prototype bioreactor for scalable NSC production Using alginate hydrogel tubes, we built a prototype bioreactor to demonstrate the scalable production of hPSC-NSCs (Figure 7a, b). On day 0, single hPSCs and alginate hydrogel tubes were processed into the bioreactor. Cells were cultured in E8 medium for 5 days, followed by additional 7 days of NSC differentiation (Figure 7c). Phase image and live/dead cell staining showed no or undetectable dead cells (Figure 7d, e). Immunostaining and flow cytometry analysis showed 93.3% of the day 12 cells were



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NSCs (Figure 7f, g). Immunostaining and confocal imaging showed majority of day 12 cells were positive for NSC markers FOXG1 and SOX1, but no undifferentiated OCT4+/NANOG+ hPSCs (Figure 7h, i). When the bioreactor-NSCs released from alginate hydrogel tubes were cultured on Matrigel-coated plates for additional 19 days in neural differentiation medium, similar percentages of TUJ1+/TBR1+ neurons were generated to 3D-NSCs made using tissue culture plates (Figure 7j, k). qRT-PCR analysis showed 3D-NSCs made using the bioreactor had similar expression levels of NSC markers to 3D-NSCs made using tissue culture plates (Figure 7l). These results showed the bioreactor itself did not alter the NSC differentiation. This bioreactor can be further scaled up for large-scale hPSC-NSC production in the future.

DISCUSSION

Currently, hPSC-NSCs are made either in 2D culture61 or as EBs in 3D suspension culture62. Both have difficulty to produce large numbers of high quality hPSC-NSCs. Both methods provide culturing microenvironments that are very different from the cells’ in vivo 3D microenvironments63–67. 2D culturing is also labor-, space- and reagent-consuming, and considered not suitable for preparing cells in large scales (e.g. ≤109)48. In 3D suspension culturing, hPSCs in suspension culturing suffer from severe cellular agglomeration55,56,68. The agglomeration leads to heterogeneity in cell aggregate size, and impaired mass transport in the large cell aggregates, which leads to low culture efficiency55,56,68. Agitating (stirring or shaking) the culture can reduce the agglomeration and

enhance

mass transport55,56,68.

However,

agitation

generates complicate

hydrodynamic conditions (including medium flow direction, velocity, pressure, shear force 18 ACS Paragon Plus Environment

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and chemical environment) that are spatially and temporally varied, which results in critical stress in some locations (e.g. near vessel wall and impeller tip) that induce significant cell death49–53,55,56,68,69. This leads to low cell expansion rate and volumetric yield. Additionally, the hydrodynamic conditions are very sensitive to the agitation rate, medium viscosity and bioreactor design such as the impeller geometry, size & position, vessel geometry & size, positions of probes for pH, temperature, oxygen68,69. They are currently not well understood and hard to control55,56,68,69. These knowledge and technology gaps lead to large culture variations between batches as well as difficulty in scaling up. For instance, in a study to make cardiomyocytes from hPSCs in six different batches (100 mL culture volume) using 3D suspension culturing, the final cell yield varied from 40 million to 125 million cells and the product purity varied from 28% to 88% 70,71. In the same study, when the culture volume was increased from ~100 mL to ~1,000 mL, the yield and differentiation efficiency were significantly altered, and re-optimization was required70,71. To our best knowledge, the largest culture volume demonstrated to date is 95%), high purity (>90%) and high yield (~5x108 cells/mL of microspace). The high cell viability, expansion, and yield makes this technology very attractive for industry-scale cell production. For instance, our modeling shows it requires ~104811 liters of total culture volume, 11 passaging operations, and 55 days to produce ~1.2x1014 hPSC-NSCs (from ~108 hPSC seeds) using stirred-tank bioreactors (Figure S7a, c), which is technically and economically challenging. As aforementioned, culturing hPSCs for >10 liters is still a challenge68,72. The same production can be done with 320 liters of alginate hydrogel tubes in 27 days and 1 passaging (Figure S7b, c). The reductions in culture volume, time, and passaging make the production technically feasible. They also lead to enormous cuts overall production cost. For our calculation, we assume that cells have 4-fold expansion in 4 days 20 ACS Paragon Plus Environment

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in each passage in stirred-tank bioreactors, and have 1000-fold expansion in 10 days per passage in alginate hydrogel tubes, and hPSCs are seeded at 5.0x105 cells/mL, and have 80% passaging efficiency (i.e., % of cells remaining viable after one passaging) (Figure S7c). These numbers are based on data from my lab and others50,51,72,73.

CONCLUSION Through using simple hydrogel materials to create uniform and cell-friendly microenvironments, the alginate hydrogel cell culture system produced 250 times more cells per culture volume than the current-state-of-the-art. This scalable bioprocess can be used to produce large numbers of hPSC-NSCs for various biomedical applications in the future. We believe it will significantly advance the application of hPSC-NSCs for treating various neurological disorders.



Associated content Supporting information Supplementary materials on characterizing the starting hPSCs (Figure S1), controlling the diameter and shell thickness of alginate hydrogel tubes (Figure S2), expanding hPSCs in alginate hydrogel tubes (Figure S3), differentiating Fib-iPSCs into NSCs in alginate hydrogel tubes (Figure S4), comparing gene expression between 3D-NSCs and 2D-NSCs (Figure S5, S6), comparing two bioprocesses for manufacturing ~10 14 hPSCNSCs from ~108 hPSCs seeding (Figure S7), antibodies and real time PCR primers used in this study (Table S1, S2), comparing GO terms between 3D-NSCs and 2D-NSCs (Data S1), as well as processing alginate hydrogel tubes (Movie S).

Author Information Corresponding Author E-mail: [email protected] Phone: (+1)-402-472-5313 ORCID Yuguo Lei: 0000-0002-7682-6912 Author Contributions: Y.L. and H.L conceived the idea and designed the study. H.L., Q.L. and O.W. performed experiments and analyzed data. Q.D., K.L. and C.Z analyzed RNA sequencing data. C.E. contributed to confocal images. Y.L. and H.L. wrote the manuscript. Z.W revised the manuscript.

Acknowledgements:


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The Morrison Microscopy Core Research Facility at University of Nebraska, Lincoln for confocal microscope imaging. The Flow Cytometry core of University of Nebraska, Lincoln for flow cytometry. The UNMC deep sequencing core for RNA sequencing.

Competing financial interests: No

Abbreviations: hPSC, human pluripotent stem cell hESC, human embryonic stem cell iPSC, induced pluripotent stem cell NSC, neural stem cell hPSC-NSC, human pluripotent stem cell derived neural stem cell 2D-NSC, two dimensional derived neural stem cell 3D-NSC, three dimensional derived neural stem cell EB, embryonic body cGMP, current Good Manufacturing Practice qRT-PCR, quantitative real time polymerase chain reaction PCA, principal component analysis



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Figure Legends Figure 1. Overview of culturing cells in alginate hydrogel tubes. (a, b) A microextruder is built for processing cells into microscale alginate hydrogel tubes. A cell suspension and an alginate solution is pumped into the central channel and side channel of the micro-extruder, respectively, to form coaxial core-shell flows that are extruded through the nozzle into a CaCl2 buffer. The shell alginate solution is instantly crosslinked by Ca2+ ions to form an alginate hydrogel tube. (c) Subsequently, cells are suspended and cultured in the alginate hydrogel tubes that are suspended in the cell culture medium in a culture vessel. The hydrogel tubes protect cells from hydrodynamic stresses and confine the cell mass

A hydrogel-based bioprocess for scalable ... - ACS Publications

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