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Tissue Engineering and Regenerative Medicine

Functional and Sustainable 3D Human Neural Network Models from Pluripotent Stem Cells William Cantley, Chuang Du, Selene Lomoio, Thomas DePalma, Emily Peirent, Dominic Kleinknecht, Martin Hunter, Min Tang-Schomer, Giuseppina Tesco, and David L Kaplan ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 01 Oct 2018 Downloaded from http://pubs.acs.org on October 1, 2018

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ACS Biomaterials Science & Engineering

Functional and Sustainable 3D Human Neural Network Models from Pluripotent Stem Cells

William L. Cantley1, Chuang Du2, Selene Lomoio3, Thomas DePalma2¶, Emily Peirent2¶, Dominic Kleinknecht2, Martin Hunter2, Min D. Tang-Schomer4, Giuseppina Tesco3, David L. Kaplan2*

1

Department of Cell, Molecular and Developmental Biology, Sackler School, Tufts University, 136

Harrison Ave, Boston MA, 02111, United States of America 2

Department of Biomedical Engineering, Tufts University, 200 College Ave, Medford MA,

02155,United States of America 3

Department of Neuroscience, Sackler School, Tufts University, 136 Harrison Ave, Boston MA, 02111,

United States of America 4

The Jackson Laboratory, 10 Discovery Drive, Farmington CT, 06032, United States of America

*

Corresponding Author

Email: [email protected] (DK)

¶

These authors contributed equally to this work

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Abstract: Three-dimensional (3D) in vitro cell and tissue culture models, particularly for the central nervous system, allow for the exploration of mechanisms of organ development, cellular interactions, and disease progression within defined environments. Here we describe the development and characterization of human 3D tissue models that promote the differentiation and long-term survival of functional neural networks. This work builds upon previous work in which primary rodent neurons were successfully grown in a similar 3D system.1 The model was adapted to human induced pluripotent stem cells (hiPSCs) allowing for a more direct exploration of the human-condition. These tissue cultures show diverse cell populations including neurons and astroglial cells interacting in 3D, and exhibit spontaneous neural activity confirmed through electrophysiological recordings and calcium imaging over at least 9 months. This approach allows for the direct integration of pluripotent stem cells into the 3D construct bypassing early neural differentiation steps (embryoid bodies and neural rosettes). The streamlined process, in combination with the longevity of the cultures, provides a system that can be manipulated to support a variety of experimental applications, including the study of network development, maturation, plasticity and/or degeneration. This tissue model has been tested with stem cells derived from healthy individuals as well as Alzheimer’s and Parkinson’s disease patients. We observed similar growth and gene expression which indicates the feasibility of generating patientderived brain tissue models to uncover early-stage biomarkers of the disease state supporting earlier diagnosis, improving understanding of disease progression, and with additional model development, the potential use for investigating drug targets in neurodegenerative diseases.

Keywords: Human Stem Cell, Neural Differentiation, 3D Tissue Engineering, Long-Term Culture


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Introduction

Inaccessibility to live human brain has limited studies of brain functions and disorders. With billions of neurons interconnected by trillions of synapses, parsing out accurate and specific information can prove difficult which results in an incomplete picture of its plasticity and function. Commonly used brain imaging techniques, such as fMRI and EEG, are unable to attain single-cell level of resolution even when used in combination. More involved techniques, such as the implantation of neurotrophic electrodes,2,3 are able to provide information at the single-cell level, but require invasive procedures. This inherent limitation demonstrated the need for alternative models to be used in the study and experimentation of neural cell functions in the context of complex 3D brain-related niches are required to advance our collective understanding.

Much of the knowledge surrounding the human brain has been gained through the use of animal (in vivo) models. While animal studies continue to significantly help in understanding brain development and function, they do not fully mimic human conditions of complexity, constitution 4 or disease states.5 Using animals as the sole preclinical test model has also come under increasing scrutiny relating to their validity as a model for pharmacologic testing.6-8 While animals may be the best available option, less than 10% of experimental drugs pass phase 1 studies.9 Considering the high attrition rate of drug candidates throughout the testing pipeline, especially for neurological diseases, there is compelling need for better pre-clinical model of the human brain.

Tissue explants in the form of brain slices or hanging drop cultures have been used as alternatives to in vivo systems, but these culture methods have limitations in sample acquisition, damage to tissue during harvesting, limited time in culture and inaccessibility to experimental intervention as they are often closed systems.10 There have been improvements in culture methods to increase the


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survival of organotypic neural tissue, such as microfluidic devices.11 but even with these advances, human neurological tissues are rarely removed from a healthy patient and even diseased tissue is often only available post-mortem. Regardless of the source, culturing primary neurons is problematic due to their post-mitotic nature, which limits culture expansion.

The discovery of pluripotent stem cells (PSCs) and the technology of the reprograming somatic cells into induced PSCs (iPSCs) have alleviated the problem of human neuronal cell sources.12,13 These pluripotent cells can be exponentially expanded, and when under appropriate conditions can be directed to differentiate into specific cell types, including neurons. Early work has shown that stem cells can be directed towards different cell fates by modifying matrix stiffness, developing osteogenic, myogenic and neurogenic cell types, respectively as the stiffness decreases.14-17 There have been multiple advances supporting the use of pluripotent cells to differentiate into functioning neurons in 3D, including human and murine cerebral organoids18,19 and human cortical spheroids.20 These approaches use the embryoid body stage of hiPSC differentiation to create developmental brain models.18-20 Grown free-floating in spinning bioreactors, under minimal direction the spheres formed organized structures that mimicked those found in the human brain with varying levels of consistency. The structures developed by these cells in an undirected physical environment demonstrated an ingrained ability to self-organize, but the dense nature of these approaches can limit the real-time accessibility to the neural network.

Numerous approaches have been investigated as the work to engineer biologically relevant 3D neural networks progresses. This work includes the use of hydrogels,21 porous polystyrenes,22 and fibers23 to name a few. While each approach has advantages (tissue-like hydrogels, stable porous scaffolds, and organized fibers), we took an approach that utilized the structural integrity and longterm culture support of a porous scaffold and combined it with the native ECM-like environment of


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hydrogel which has been previously shown to approximate the stiffness of brain tissue.1 The fibroin scaffold provides a stabile, biocompatible and modifiable backbone whose porous nature supports improved nutrient diffusion and network formation, while the collagen-I hydrogel provides more native cell-matrix interactions and 3D architecture to support long-term culture lengths required to model neural development and eventual degeneration.

Recent progress in 3D tissue engineering has started bridging the gap between the accessibility of in vitro models and in vivo accuracy. In vitro, engineered models of human brain tissue can provide stable long-term systems to mimic brain tissue structure and function to support a wide range of neurological studies. In order to achieve this goal, it is necessary to generate cultures that support the growth of biologically relevant cells. The goal of this study was to develop human iPSCscomprised 3D neural cultures as a platform to explore network formation and function over extended time frames, as well as the study of neurological diseases. A complex 3D human neuralnetwork model containing various neural subtypes and supporting astrocytes provides a unique advantage for long-term and functional cultures. Such systems should provide new and useful approaches for the study of neurodegenerative diseases, chronic drug effects and insight into the mechanisms of aging. Further, the extended time frames with sustained structure and function permit facile imaging and analytical assessments.

Materials and Methods Cells: The hiPSC lines ND41866*C, GM24666*A, and ND35367*F were obtained from the Coriell Biorepository (Camden, NJ, USA), the YZ1 hiPSC line was obtained from Dr. Giuseppina Tesco courtesy of the University of Connecticut-Wesleyan Stem Cell Core (UCSCC, Farmington, CT, USA).


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hiPSC Maintenance: The hiPSCs were generated as described (ND41866,24 GM24666,25 ND35367,26 YZ127) and were maintained in feeder-free conditions as previously described.28) In brief, plates were coated using Matrigel hESC-qualified Matrix (Corning, Corning, NY, USA) and DMEM/F12 media (Thermo Fisher, Cambridge, MA, USA). The coated plates were stored at 4oC and were warmed to 37oC prior to use. The cells were maintained with daily media changes using mTeSR1 (Stem Cell Technologies, Vancouver, BC, Canada) and were passaged every 5-7 days using ReLeSR passaging reagent. (Stem Cell Technologies, Vancouver, BC, Canada)

Silk Preparation: Silk protein was processed from Bombyx mori cocoons as described previously.29 In brief, silk cocoons were cut into fragments and boiled for 30 min in a 0.02M Na2CO3 solution. The dried fibroin was solubilized in a 9.3M LiBr solution, which was then dialyzed away in deionized water. The silk solution was then diluted to 6% weight/volume in diH2O for scaffold preparation.

Scaffold Preparation: The solubilized silk was then used to generate the porous scaffolds via salt leaching, as explained previously30 using 500-600um NaCl crystals to generate a sponge-like structure. Scaffolds (2mm tall cylinder with 6mm outer diameter and a 2 mm central window (Fig 1&2C)) were then coated with poly-L-ornithine (20ug/mL in 1x DPBS) (Sigma-Aldrich, St. Louis, MO, USA) for at least 2 hours at room temperature, and then washed 2x with DPBS, 1x with DMEM/F12, followed by a 2-hour (minimum) laminin coating (20ug/mL in DMEM/F12) (Roche, Basel, Switzerland). The coated sponges were stored under sterile conditions at 4°C until use.

Scaffold Seeding: hiPSCs were treated with ReLeSR (Stem Cell Technologies, Vancouver, BC, Canada) to specifically remove undifferentiated portions of the colonies. The resulting stem cell cluster suspension was collected in a 15mL conical tube and a subset (500µL) was trypsinized and counted in order to obtain a cell concentration. The cell cluster suspension was then gently spun


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down and resuspended to generate a stock of 5x106 cells/mL. 5.0x105 of these cell clusters were then added to the scaffold (100μL of 5.0x106 cells/mL) and incubated for 24 hours in a 96 well plate. The media (100µL) was changed daily for 5 days following seeding, moving the scaffolds to fresh wells with each change. After allowing 5 days of cellular expansion the scaffolds were then moved to a fresh well and filled with 100μL of a cold collagen-type I rat-tail solution (3.0mg/mL) mixed with 10x PBS and 1N NaOH (88:10:2). The collagen-filled scaffolds were then incubated at 37oC until the gelation occurred (~30 minutes). Once solidified, the filled scaffolds were then transferred to a 24-well plate and flooded with 1mL of Final Neuro Medium (FNM) (Neurobasal Medium, Antibiotic-Antimycotic, Glutamax, with B-27 supplement) with medium changes every four days.

Two-Dimensional Culture: hiPSCs were treated with ReLeSR (Stem Cell Technologies, Vancouver, BC, Canada) to specifically remove undifferentiated portions of the colonies. 5.0x105 of these cell clusters were then seeded onto PLO/Laminin coated 6 well plates. The maintenance media was changed daily for 5 days following seeding. After 5 days of stem cell maintenance and expansion the medium was changed to Final Neuro Medium (FNM) (Neurobasal Medium, Antibiotic-Antimycotic, Glutamax, with B-27 supplement) with medium changes every four days.

Nucleic Acid Isolation: At the time of desired analysis full scaffolds were snap frozen and stored at 80oC. These frozen scaffolds were then homogenized using a liquid nitrogen-chilled Spectrum Bessman Tissue Pulverizer (Fisher Scientific, Hampton, New Hampshire, USA). The homogenized sample was run through a Qiashredder column and then purified using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany). DNA was quantified using Quant-iT PicoGreen dsDNA Assay kit (Thermo-Fisher, Cambridge, MA, USA). Genomic DNA was removed


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from the isolated RNA using TURBO DNA-free kit (Thermo-Fisher, Cambridge, MA, USA), which was then quantified using a NanoDrop 2000 Spectrophotometer (Thermo-Fisher, Cambridge, MA, USA)

rt-PCR: cDNA was generated using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Cambridge, MA, USA). This cDNA was used for qPCR using the Taqman system (Fast Advanced Mastermix, Table S1 for full list of probes) (Thermo Fisher, Cambridge, MA, USA). qPCR was conducted utilizing a BioRad CFX96 RT-PCR system (BioRad, Hercules, CA, USA) with the specific hiPSC maintenance cultures used as controls. All samples were run at a miniumum of biological triplicates (each an average of technical triplicates). If a value never crossed the threshold the value of the last cycle run was used for ddCT calculations. The housekeeping gene used was 18S and the controls were always the hiPSC maintenance cultures for the specific pluripotent line (Fig S1).

Immunocytochemistry: Cultures were fixed in 4% PFA (Santa Cruz, Dallas, TX, USA) for 10-20 minutes at room temperature, washed with 1x PBS, and then blocked with goat serum blocking solution (Goat Serum (15mL), TritonX-100 (Sigma Aldrich, St. Louis, MO, USA), Sodium Azide (5mL), in 1x PBS (fill to 250mL)) for 1 hour. Primary antibodies (Beta-III-tubulin (B3T - ab78078) (Abcam, Cambridge, UK), Glial Fibrillary Acidic Protein (GFAP - ab7260) (Abcam, Cambridge, UK), Neurofilament Heavy chain (NFH - ab5539) (Sigma Aldrich, St. Louis, MO, USA) were diluted in the blocking solution at 1:500, and incubated at 4oC overnight. Cultures were washed 3x with 1xPBS followed by treatment with secondary antibodies diluted 1:250 in the blocking solution and allowed to interact overnight at 4oC. Finally, the cultures were washed 3x with 1xPBS and then NucBlue (Thermo Fisher, Cambridge, MA, USA) was used for nuclei staining.

Confocal Imaging and Analysis: A Leica SP8 Confocal microscope (Leica, Wetzlar, GER) was used to collect the source image stacks. ImageJ was used to quantify B3T density and to perform intensity


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measurements for calcium imaging. Imaging was primarily performed in the bulk of the scaffold unless window was specified.

Extracellular Field Potential Recordings (EFP): Scaffolds were recorded in a bath consisting of NaCl 140mM, KCl 2.8mM, CaCl2 2mM, MgCl2 2mM, HEPES 10mM, and D-glucose 10mM, with pH adjusted to 7.4 with NaOH. The sharp glass microelectrodes were filled with extracellular solution and had a resistance of 60-80 megaohmsThe recording electrode was placed near the edge of the central window. Fast field potential changes (spikes) were recorded with the NPI amplifier at a bandwidth of 0.3-10 kHz and were further amplified with an A-M Systems Differential AC amplifier (Model 1700) to a combined total gain of 10,000x. The signals were digitized at 10 KHz by a Molecular Devices digitizer (Digidata 1550) using a Dell Optiplex GX620 computer with pClamp 10 software (Molecular Devices). Electrical stimulations (40 V, 1 ms pulses) of the cultured tissues were generated via a Grass S44 Stimulator, passed through a Grass Stimulus Isolation Unit (SIU5), and delivered through a platinum parallel bipolar electrode (FHC PBSB0875) with a distance of 800 μm between two tips positioned near the recording electrode. Activity inhibitors were used to confirm neuronal activity by use of glutamate receptor blockers (6-cyano-7-nitroquinoxaline-2,3-dione; CNQX (50 μM), (2R)-amino-5-phosphonopentanoate; AP5 (250 μM), GABA antagonist (-) Bicuculline (100μM) acetylcholine antagonists to both nicotinic and muscarinic Mecamylamine hydrochloride (10μM) and Scopolamine hydrobromide (50μM) respectively, as well as the neuronal sodium channel blocker tetrodotoxin (TTX, 5uM). After recording responses with electrical stimulation, the procedure was repeated immediately after the addition of the specific receptor blockers and then a third time with addition of TTX. The responses of the post-TTX recordings were subtracted from the pre-TTX recordings to identify neural component of the stimulation response and to ascertain the presence of specific neural subtypes as described in more detail previously.31


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Calcium imaging: Fluo-4 (Invitrogen, Carlsbad, CA, USA) was used as described in the manufacturer’s manual. In brief the fluo-4 was dissolved in Puronic-F127 (Life Technologies, Carlsbad, CA, USA) at a volume of 1uL/ug. The resuspended fluo-4 was then diluted in artificial cerebrospinal fluid (NaCl: 140mM, KCl: 2.8mM, CaCl2: 2mM, MgCl2: 2mM, HEPES: 10mM, Glucose: 10mM, pH7.4) 1:1000. The cells were incubated in the Fluo-4 solution for 60 min at 37oC. After the incubation the cells were washed once with PBS and supplied with fresh artificial cerebrospinal fluid and imaged for 3 minutes in a climate-controlled chamber at 5% CO2 and 37oC on a Keyence BZ-X700 (Itasca, IL, USA). Recordings were also taken using a Leica SP8 confocal microscope(Leica, Wetzlar, GER). Some samples were excited with MDNI-caged glutamate (Tocris, Bristol, UK) after a baseline recording was obtained, as previously described.32 The resulting recordings were analyzed using ImageJ to identify regions of interest and track fluorescent intensity over time. Drift was corrected using ImageJ Image Stabiilizer,33 dF/F was calculated as previously described.34,35

Statistics: Prism 5 by GraphPad Software was used to perform statistics. To assess significance in DNA content within the cultures a two-way ANOVA (variables: initial seeding density, time point) was performed with a Bonferroni post-hoc test on group averages. This same analysis was used to analyze the qPCR data. Student T-Test was used to determine significance in the LFP data (Fig 6A&B). P values are denoted as (*) P

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