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Seizures are made up of the coordinated activity of networks of neurons, suggesting that control of neurons in the pathologic circuits of epilepsy could allow for control of the disease. Optogenetics has been effective at stopping seizure-like activity in non-human disease models by increasing inhibitory tone or decreasing excitation, although this effect has not been shown in human brain tissue. Many of the genetic means for achieving channelrhodopsin expression in non-human models are not possible in humans, and vector-mediated methods are susceptible to species-specific tropism that may affect translational potential. Here we demonstrate adeno-associated virus-mediated, optogenetic reductions in network firing rates of human hippocampal slices recorded on high-density microelectrode arrays under several hyperactivity-provoking conditions. This platform can serve to bridge the gap between human and animal studies by exploring genetic interventions on network activity in human brain tissue., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Harnessing adeno-associated virus (AAV) vectors for therapeutic gene delivery has emerged as a progressively promising strategy to treat disorders of both the central nervous system (CNS) and peripheral nervous system (PNS), and there are many ongoing clinical trials. However, unique physiological and molecular characteristics of the CNS and PNS pose obstacles to efficient vector delivery, ranging from the blood-brain barrier to the diverse nature of nervous system disorders. Engineering novel AAV capsids may help overcome these ongoing challenges and maximize therapeutic transgene delivery. This article discusses strategies for innovative AAV capsid development, highlighting recent advances. Notably, advances in next generation sequencing and machine learning have sparked new approaches for capsid investigation and engineering. Furthermore, we outline future directions and additional challenges in AAV-mediated gene therapy in the CNS and PNS., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: David Schaffer reports financial support was provided by NIH 1U01MH130700 and 1R01NS126397. David V Schaffer has patent #BERK-482 issued to The Regents of the University of California. David V Schaffer has patent #UCRBK21332 issued to The Regents of the University of California. David V Schaffer has patent #BERK-365 issued to The Regents of the University of California. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

While extracellular matrix (ECM) stress relaxation is increasingly appreciated to regulate stem cell fate commitment and other behaviors, much remains unknown about how cells process stress-relaxation cues in tissue-like three-dimensional (3D) geometries versus traditional 2D cell culture. Here, we develop an oligonucleotide-crosslinked hyaluronic acid-based ECM platform with tunable stress relaxation properties capable of use in either 2D or 3D. Strikingly, stress relaxation favors neural stem cell (NSC) neurogenesis in 3D but suppresses it in 2D. RNA sequencing and functional studies implicate the membrane-associated protein spectrin as a key 3D-specific transducer of stress-relaxation cues. Confining stress drives spectrin's recruitment to the F-actin cytoskeleton, where it mechanically reinforces the cortex and potentiates mechanotransductive signaling. Increased spectrin expression is also accompanied by increased expression of the transcription factor EGR1, which we previously showed mediates NSC stiffness-dependent lineage commitment in 3D. Our work highlights spectrin as an important molecular sensor and transducer of 3D stress-relaxation cues.

Adult neural stem cells (NSCs) reside in the dentate gyrus of the hippocampus, and their capacity to generate neurons and glia plays a role in learning and memory. In addition, neurodegenerative diseases are known to be caused by a loss of neurons and glial cells, resulting in a need to better understand stem cell fate commitment processes. We previously showed that NSC fate commitment toward a neuronal or glial lineage is strongly influenced by extracellular matrix stiffness, a property of elastic materials. However, tissues in vivo are not purely elastic and have varying degrees of viscous character. Relatively little is known about how the viscoelastic properties of the substrate impact NSC fate commitment. Here, we introduce a polyacrylamide-based cell culture platform that incorporates mismatched DNA oligonucleotide-based cross-links as well as covalent cross-links. This platform allows for tunable viscous stress relaxation properties via variation in the number of mismatched base pairs. We find that NSCs exhibit increased astrocytic differentiation as the degree of stress relaxation is increased. Furthermore, culturing NSCs on increasingly stress-relaxing substrates impacts cytoskeletal dynamics by decreasing intracellular actin flow rates and stimulating cyclic activation of the mechanosensitive protein RhoA. Additionally, inhibition of motor-clutch model components such as myosin II and focal adhesion kinase partially or completely reverts cells to lineage distributions observed on elastic substrates. Collectively, our results introduce a unique system for controlling matrix stress relaxation properties and offer insight into how NSCs integrate viscoelastic cues to direct fate commitment., Competing Interests: Competing interests statement:The authors declare no competing interest.

The behavior of stem cells is regulated by mechanical cues in their niche that continuously vary due to extracellular matrix (ECM) remodeling, pulsated mechanical stress exerted by blood flow, and/or cell migration. However, it is still unclear how dynamics of mechanical cues influence stem cell lineage commitment, especially in a 3D microenvironment where mechanosensing differs from that in a 2D microenvironment. In the present study, we investigated how temporally varying mechanical signaling regulates expression of the early growth response 1 gene (Egr1), which we recently discovered to be a 3D matrix-specific mediator of mechanosensitive neural stem cell (NSC) lineage commitment. Specifically, we temporally controlled the activity of Ras homolog family member A (RhoA), which is known to have a central role in mechanotransduction, using our previously developed Arabidopsis thaliana cryptochrome-2-based optoactivation system. Interestingly, pulsed RhoA activation induced Egr1 upregulation in stiff 3D gels only, whereas static light stimulation induced an increase in Egr1 expression across a wide range of 3D gel stiffnesses. Actin assembly inhibition limited Egr1 upregulation upon RhoA activation, implying that RhoA signaling requires an actin-involved process to upregulate Egr1. Consistently, static-light RhoA activation rather than pulsed-light activation restricted neurogenesis in soft gels. Our findings indicate that the dynamics of RhoA activation influence Egr1-mediated stem cell fate within 3D matrices in a matrix stiffness-dependent manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Gene delivery vehicles based on adeno-associated viruses (AAVs) are enabling increasing success in human clinical trials, and they offer the promise of treating a broad spectrum of both genetic and non-genetic disorders. However, delivery efficiency and targeting must be improved to enable safe and effective therapies. In recent years, considerable effort has been invested in creating AAV variants with improved delivery, and computational approaches have been increasingly harnessed for AAV engineering. In this review, we discuss how computationally designed AAV libraries are enabling directed evolution. Specifically, we highlight approaches that harness sequences outputted by next-generation sequencing (NGS) coupled with machine learning (ML) to generate new functional AAV capsids and related regulatory elements, pushing the frontier of what vector engineering and gene therapy may achieve., Competing Interests: Declaration of interests D.V.S. and J.L. are inventors on patents related to viral vector-directed evolution and engineered AAV variants., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Manufacturing sufficient adeno-associated virus (AAV) to meet current and projected clinical needs is a significant hurdle to the growing gene therapy industry. The recently discovered membrane-associated accessory protein (MAAP) is encoded by an alternative open reading frame in the AAV cap gene that is found in all presently reported natural serotypes. Recent evidence has emerged supporting a functional role of MAAP in AAV egress, although the underlying mechanisms of MAAP function remain unknown. Here, we show that inactivation of MAAP from AAV2 by a single point mutation that is silent in the VP1 open reading frame (ORF) (AAV2-ΔMAAP) decreased exosome-associated and secreted vector genome production. We hypothesized that novel MAAP variants could be evolved to increase AAV production and thus subjected a library encoding over 1 × 10 6 MAAP protein variants to five rounds of packaging selection into the AAV2-ΔMAAP capsid. Between each successive packaging round, we observed a progressive increase in both overall titer and ratio of secreted vector genomes conferred by the bulk-selected MAAP library population. Next-generation sequencing uncovered enriched mutational features, and a resulting selected MAAP variant containing missense mutations and a frameshifted C-terminal domain increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids., Competing Interests: Declaration of interests A.J.S., H.L., and D.V.S. are inventors on a patent related to MAAP variants for increased production of recombinant AAV., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Incubators and accelerators catalyze the launch of life science startups and have evolved from simple facilities to vibrant ecosystems offering research infrastructure, programs, and funding. Analysis of financing activities indicates the outperformance of incubator companies relative to accelerators in fundraising, mergers and acquisitions (M&As), and initial public offerings (IPOs), attributed to extended interactions with investors and peers., Competing Interests: Declaration of interests W.M., R.S., and R.C. are co-founders of BEVC Management, LLC, a venture capital investment firm focused on early-stage companies. D.V.S. is executive director of QB3, an inventor on numerous patents related to gene delivery and stem cell technologies, and co-founder of 4D Molecular Therapeutics and Axent Biosciences., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Adeno-associated viruses (AAVs) hold tremendous promise as delivery vectors for gene therapies. AAVs have been successfully engineered-for instance, for more efficient and/or cell-specific delivery to numerous tissues-by creating large, diverse starting libraries and selecting for desired properties. However, these starting libraries often contain a high proportion of variants unable to assemble or package their genomes, a prerequisite for any gene delivery goal. Here, we present and showcase a machine learning (ML) method for designing AAV peptide insertion libraries that achieve fivefold higher packaging fitness than the standard NNK library with negligible reduction in diversity. To demonstrate our ML-designed library's utility for downstream engineering goals, we show that it yields approximately 10-fold more successful variants than the NNK library after selection for infection of human brain tissue, leading to a promising glial-specific variant. Moreover, our design approach can be applied to other types of libraries for AAV and beyond.

In embryonic stem cell (ESC) models for early development, spatially and temporally varying patterns of signaling and cell types emerge spontaneously. However, mechanistic insight into this dynamic self-organization is limited by a lack of methods for spatiotemporal control of signaling, and the relevance of signal dynamics and cell-to-cell variability to pattern emergence remains unknown. Here, we combine optogenetic stimulation, imaging and transcriptomic approaches to study self-organization of human ESCs (hESC) in two-dimensional (2D) culture. Morphogen dynamics were controlled via optogenetic activation of canonical Wnt/β-catenin signaling (optoWnt), which drove broad transcriptional changes and mesendoderm differentiation at high efficiency (>99% cells). When activated within cell subpopulations, optoWnt induced cell self-organization into distinct epithelial and mesenchymal domains, mediated by changes in cell migration, an epithelial to mesenchymal-like transition and TGFβ signaling. Furthermore, we demonstrate that such optogenetic control of cell subpopulations can be used to uncover signaling feedback mechanisms between neighboring cell types. These findings reveal that cell-to-cell variability in Wnt signaling is sufficient to generate tissue-scale patterning and establish a hESC model system for investigating feedback mechanisms relevant to early human embryogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

During the intricate process by which cells give rise to tissues, embryonic and adult stem cells are exposed to diverse mechanical signals from the extracellular matrix (ECM) that influence their fate. Cells can sense these cues in part through dynamic generation of protrusions, modulated and controlled by cyclic activation of Rho GTPases. However, it remains unclear how extracellular mechanical signals regulate Rho GTPase activation dynamics and how such rapid, transient activation dynamics are integrated to yield long-term, irreversible cell fate decisions. Here, we report that ECM stiffness cues alter not only the magnitude but also the temporal frequency of RhoA and Cdc42 activation in adult neural stem cells (NSCs). Using optogenetics to control the frequency of RhoA and Cdc42 activation, we further demonstrate that these dynamics are functionally significant, where high- vs. low-frequency activation of RhoA and Cdc42 drives astrocytic vs. neuronal differentiation, respectively. In addition, high-frequency Rho GTPase activation induces sustained phosphorylation of the TGFβ pathway effector SMAD1, which in turn drives the astrocytic differentiation. By contrast, under low-frequency Rho GTPase stimulation, cells fail to accumulate SMAD1 phosphorylation and instead undergo neurogenesis. Our findings reveal the temporal patterning of Rho GTPase signaling and the resulting accumulation of an SMAD1 signal as a critical mechanism through which ECM stiffness cues regulate NSC fate.

During differentiation, neural stem cells (NSCs) encounter diverse cues from their niche, including not only biophysical cues from the extracellular matrix (ECM) but also cell-cell communication. However, it is still poorly understood how these cues cumulatively regulate mechanosensitive NSC fate commitment, especially in 3D matrices that better mimic in vivo systems. Here, we develop a click chemistry-based 3D hydrogel material system to fully decouple cell-cell and cell-ECM interactions by functionalizing small peptides: the HAVDI motif from N-cadherin and RGD motif from fibronectin. The hydrogel is engineered to range in stiffness from 75 Pa to 600 Pa. Interestingly, HAVDI-mediated interaction shows increased neurogenesis, except for the softest gel (75 Pa). Moreover, the HAVDI ligation attenuates the mechanosensing state of NSCs, exhibiting restricted cytoskeletal formation and RhoA signaling. Given that mechanosensitive neurogenesis has been reported to be regulated by cytoskeletal formation, our finding suggests that the enhanced neurogenesis in the HAVDI-modified gel may be highly associated with the HAVDI interaction-mediated attenuation of mechanosensing. Furthermore, NSCs in the HAVDI gel shows higher β-catenin activity, which has been known to promote neurogenesis. Our findings provide critical insights into how mechanosensitive NSC fate commitment is regulated as a consequence of diverse interactions in 3D microenvironments.

Short-time Fourier transforms with short segment lengths are typically used to analyze single ion charge detection mass spectrometry (CDMS) data either to overcome effects of frequency shifts that may occur during the trapping period or to more precisely determine the time at which an ion changes mass or charge, or enters an unstable orbit. The short segment lengths can lead to scalloping loss unless a large number of zero-fills are used, making computational time a significant factor in real-time analysis of data. Apodization specific fitting leads to a 9-fold reduction in computation time compared to zero-filling to a similar extent of accuracy. This makes possible real-time data analysis using a standard desktop computer. Rectangular apodization leads to higher resolution than the more commonly used Gaussian or Hann apodization and makes it possible to separate ions with similar frequencies, a significant advantage for experiments in which the masses of many individual ions are measured simultaneously. Equally important is a >20% increase in S/N obtained with rectangular apodization compared to Gaussian or Hann, which directly translates to a corresponding improvement in accuracy of both charge measurements and ion energy measurements that rely on the amplitudes of the fundamental and harmonic frequencies. Combined with computing the fast Fourier transform in a lower-level language, this fitting procedure eliminates computational barriers and should enable real-time processing of CDMS data on a laptop computer.

The balance between stem cell renewal and differentiation is determined by the interplay between intrinsic cellular controls and extrinsic factors presented by the microenvironment, or 'niche'. Previous studies on cultured human epidermis have utilised suspension culture and restricted cell spreading to investigate regulation of differentiation in single keratinocytes. However, keratinocytes are typically adherent to neighbouring cells in vivo. We therefore developed experimental models to investigate the combined effects of cell-ECM adhesion and cell-cell contact. We utilized lipid-modified oligonucleotides to form clusters of keratinocytes which were subsequently placed in suspension to induce terminal differentiation. In this experimental model cell-cell contact had no effect on suspension-induced differentiation of keratinocytes. We next developed a high-throughput platform for robust geometrical confinement of keratinocytes to hexagonal ECM-coated islands permitting direct cell-cell contact between single cells. As in the case of circular islands, differentiation was stimulated on the smallest single hexagonal islands. However, the percentage of involucrin-positive cells on small bowtie islands was significantly lower than on single islands, demonstrating that cell-cell contact reduced differentiation in response to decreased substrate adhesion. None of the small bowtie islands contained two involucrin-positive cells. Rather, if one cell was involucrin-positive the other was involucrin-negative. This suggests that there is intrinsic asymmetry in the effect of cell-cell contact in decreasing differentiation. Thus, our reductionist approaches provide new insights into the effect of the niche on keratinocyte differentiation. STATEMENT OF SIGNIFICANCE: Stem cell behaviour is regulated by a combination of external signals, including the nature of the adhesive substrate and cell-cell interactions. An understanding of how different signals are integrated creates the possibility of developing new biomaterials to promote tissue regeneration and broaden our understanding of skin diseases such as eczema and psoriasis, in which stem cell proliferation and differentiation are perturbed. In this study we have applied two methods to engineer intercellular adhesion of human epidermal stem cells, one involving lipid-modified DNA and the other involving hexagonal micropatterns. We show that the effect of cell-cell adhesion depends on cell-substrate adhesion and uncover evidence that two cells in equivalent environments can nevertheless behave differently., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Human pluripotent stem cells (hPSCs) are an exciting and promising source to enable cell replacement therapies for a variety of unmet medical needs. Though hPSCs can be successfully derived into numerous physiologically relevant cell types, effective translation to the clinic is limited by challenges in scalable production of high-quality cells, cellular immaturity following the differentiation process, and the use of animal-derived components in culture. To address these limitations, we have developed a fully defined, reproducible, and tunable thermoreversible polymer for high-quality, scalable 3D cell production. Our reproducible synthesis method enables precise control of gelation temperature (24°C-32°C), hydrogel stiffness (100-4000 Pa), and the prevention of any unintended covalent crosslinking. After material optimization, we demonstrated hPSC expansion, pluripotency maintenance, and differentiation into numerous lineages within the hydrogel. Overall, this 3D thermoreversible hydrogel platform has broad applications in scalable, high-quality cell production to overcome the biomanufacturing burden of stem cell therapy., Competing Interests: H.J.J., S.C., and D.V.S. are co-inventors on related intellectual property. H.J.J., R.J.M., and D.V.S. are co-founders of a stem cell therapy company., (© 2022 The Authors.)

Instrumental resolution of Fourier transform-charge detection mass spectrometry instruments with electrostatic ion trap detection of individual ions depends on the precision with which ion energy is determined. Energy can be selected using ion optic filters or from harmonic amplitude ratios (HARs) that provide Fellgett's advantage and eliminate the necessity of ion transmission loss to improve resolution. Unlike the ion energy-filtering method, the resolution of the HAR method increases with charge (improved S / N ) and thus with mass. An analysis of the HAR method with current instrumentation indicates that higher resolution can be obtained with the HAR method than the best resolution demonstrated for instruments with energy-selective optics for ions in the low MDa range and above. However, this gain is typically unrealized because the resolution obtainable with molecular systems in this mass range is limited by sample heterogeneity. This phenomenon is illustrated with both tobacco mosaic virus (0.6-2.7 MDa) and AAV9 (3.7-4.7 MDa) samples where mass spectral resolution is limited by the sample, including salt adducts, and not by instrument resolution. Nevertheless, the ratio of full to empty AAV9 capsids and the included genome mass can be accurately obtained in a few minutes from 1× PBS buffer solution and an elution buffer containing 300+ mM nonvolatile content despite extensive adduction and lower resolution. Empty and full capsids adduct similarly indicating that salts encrust the complexes during late stages of droplet evaporation and that mass shifts can be calibrated in order to obtain accurate analyte masses even from highly salty solutions.

While extracellular matrix (ECM) mechanics strongly regulate stem cell commitment, the field's mechanistic understanding of this phenomenon largely derives from simplified two-dimensional (2D) culture substrates. Here, we found a 3D matrix-specific mechanoresponsive mechanism for neural stem cell (NSC) differentiation. NSC lineage commitment in 3D is maximally stiffness sensitive in the range of 0.1 to 1.2 kPa, a narrower and more brain-mimetic range than we had previously identified in 2D (0.75 to 75 kPa). Transcriptomics revealed stiffness-dependent up-regulation of early growth response 1 ( Egr1 ) in 3D but not in 2D. Egr1 knockdown enhanced neurogenesis in stiff ECMs by driving β-catenin nuclear localization and activity in 3D, but not in 2D. Mechanical modeling and experimental studies under osmotic pressure indicate that stiff 3D ECMs are likely to stimulate Egr1 via increases in confining stress during cell volumetric growth. To our knowledge, Egr1 represents the first 3D-specific stem cell mechanoregulatory factor.

Cells reside in a dynamic microenvironment that presents them with regulatory signals that vary in time, space, and amplitude. The cell, in turn, interprets these signals and accordingly initiates downstream processes including cell proliferation, differentiation, migration, and self-organization. Conventional approaches to perturb and investigate signaling pathways (e.g., agonist/antagonist addition, overexpression, silencing, knockouts) are often binary perturbations that do not offer precise control over signaling levels, and/or provide limited spatial or temporal control. In contrast, optogenetics leverages light-sensitive proteins to control cellular signaling dynamics and target gene expression and, by virtue of precise hardware control over illumination, offers the capacity to interrogate how spatiotemporally varying signals modulate gene regulatory networks and cellular behaviors. Recent studies have employed various optogenetic systems in stem cell, embryonic, and somatic cell patterning studies, which have addressed fundamental questions of how cell-cell communication, subcellular protein localization, and signal integration affect cell fate. Other efforts have explored how alteration of signaling dynamics may contribute to neurological diseases and have in the process created physiologically relevant models that could inform new therapeutic strategies. In this review, we focus on emerging applications within the expanding field of optogenetics to study gene regulation, cell signaling, neurodevelopment, and neurological disorders, and we comment on current limitations and future directions for the growth of the field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhu, Johnson, Chen and Schaffer.)

Competing Interests: Competing interests The authors declare no competing interests.

Background: Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging., Methods: Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to simultaneously quantify and rank efficiency of competing AAV vectors across all cell types in the same animal., Results: To demonstrate proof-of-concept for the scAAVengr workflow, we quantified - with cell-type resolution - the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant identified using this pipeline, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. scAAVengr was then used to identify top-performing AAV variants in mouse brain, heart, and liver following systemic injection., Conclusions: These results validate scAAVengr as a powerful method for development of AAV vectors., Funding: This work was supported by funding from the Ford Foundation, NEI/NIH, Research to Prevent Blindness, Foundation Fighting Blindness, UPMC Immune Transplant and Therapy Center, and the Van Sloun fund for canine genetic research., Competing Interests: BÖ, MJ, MK, ST, JH, SJ, ZX, VD, SI, LP, GA No competing interests declared, MV is an inventor on AAV capsid variants (US patent IDs: 10,214,785, 10,745,453). MV has also received royalty payments from UC Berkeley. The author has no other competing interests to declare, JS has served as a consultant (with no consulting fee) for Pixium Vision, GenSight Biologics and SparingVision. Personal financial interests: Pixium Vision, GenSight Biologics, Prophesee and Chronolife, SparingVision, SHARPEYE, Vegavect, Newsight Therapeutics. The author has no other competing interests to declare, DS is named as an inventor on patent applications on AAV capsid variants (U.S. Patent Applications No. 16/315,032, 16/486,681). DS is also a co-founder of 4D Molecular Therapeutics, and DS performs consultancy and owns stock options in this company. The author has no other competing interests to declare, AP has received an honorarium from the University of Rhode Island, and has applied for patents on specific Nuclear-Anchored Independent Labeling System (PCT/US2020/038520 and PCT/US2020/038528). The author has no other competing interests to declare, JF is an inventor on patent application on AAV capsid variants (U.S. Patent Application No. 16/315,032, 16/486,681). The author has no other competing interests to declare, WB is an inventor on patent application on AAV capsid variants(16/315,032). The author has no other competing interests to declare, WS is an inventor on a patent application for methods of AAV capsid development (PCT/US2019/068489). The author has no other competing interests to declare, LB is named as an inventor on patent applications on AAV capsid variants and AAV screening methods (U.S. Patent Applications No. 16/315,032, 16/486,681, PCT/US2019/068489). LB has consulted on AAV-mediated gene therapy for Vedere Therapeutics, and is a named founder of Vegavect and Newsight Therapeutics. The author has no other competing interests to declare, (© 2021, Öztürk et al.)

We describe a genome-wide screening strategy to identify target genes whose modulation increases the capacity of a cell to produce recombinant adeno-associated viral (AAV) vector. Specifically, a single-guide RNA (sgRNA) library for a CRISPR-based genome-wide transcriptional activation screen was inserted into an AAV vector, and iterative rounds of viral infection and rescue in HEK293 producer cells enabled the enrichment of sgRNAs targeting genes whose upregulation increased AAV production. Numerous gain-of-function targets were identified, including spindle and kinetochore associated complex subunit 2 (SKA2) and inositol 1, 4, 5-trisphosphate receptor interacting protein (ITPRIP). Furthermore, individual or combinatorial modulation of these targets in stable producer cell lines increased vector genomic replication and loading into AAV virions, resulting in up to a 3.8-fold increase in AAV manufacturing capacity. Our study offers an efficient approach to engineer viral vector producer cell lines and enhances our understanding of the roles of SKA2 and ITPRIP in AAV packaging., Competing Interests: C.R.B., D.S.O., and D.V.S. are inventors on patents related to cell lines for increased production of AAV. D.V.S. is a co-founder of 4D Molecular Therapeutics., (© 2021 The Authors.)

Biophysical cues in the extracellular matrix (ECM) regulate cell behavior in a complex, nonlinear, and interdependent manner. To quantify these important regulatory relationships and gain a comprehensive understanding of mechanotransduction, there is a need for high-throughput matrix platforms that enable parallel culture and analysis of cells in various matrix conditions. Here we describe a multiwell hyaluronic acid (HA) platform in which cells are cultured on combinatorial arrays of hydrogels spanning a range of elasticities and adhesivities. Our strategy utilizes orthogonal photopatterning of stiffness and adhesivity gradients, with the stiffness gradient implemented by a programmable light illumination system. The resulting platform allows individual treatment and analysis of each matrix environment while eliminating contributions of haptotaxis and durotaxis. In human mesenchymal stem cells, our platform recapitulates expected relationships between matrix stiffness, adhesivity, and cell mechanosensing. We further applied the platform to show that as integrin ligand density falls, cell adhesion and migration depend more strongly on CD44-mediated interactions with the HA backbone. We anticipate that our system could bear great value for mechanistic discovery and screening where matrix mechanics and adhesivity are expected to influence phenotype.

The past several years have witnessed significant advances in the development of therapeutic gene delivery for neurological disorders of the central nervous system (CNS). In particular, genome-wide sequencing analysis has deepened our understanding of mutations that underlie many monogenic disorders, which in turn has contributed to clinical advances involving adeno-associated virus (AAV) vector delivery of replacement genes to treat recessive disorders. Moreover, gene therapy has been further bolstered with advances in genome editing tools that allow researchers to silence, repair, and amend endogenous genes. However, despite strong preclinical and clinical progress, challenges remain, including delivery and safety. Here, we discuss advances in AAV engineering, recent developments in cargo design, and translation of these technologies towards clinical progress., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Human pluripotent stem cells harbor an unlimited capacity to generate therapeutically relevant cells for applications in regenerative medicine. However, to utilize these cells in the clinic, scalable culture systems that activate defined receptors and signaling pathways to sustain stem cell self-renewal are required; and synthetic materials offer considerable promise to meet these needs. De novo development of materials that target novel pathways has been stymied by a limited understanding of critical receptor interactions maintaining pluripotency. Here, we identify peptide agonists for the human pluripotent stem cell (hPSC) laminin receptor and pluripotency regulator, α 6 -integrin, through unbiased, library-based panning strategies. Biophysical characterization of adhesion suggests that identified peptides bind hPSCs through α 6 -integrin with sub-μM dissociation constants similar to laminin. By harnessing a high-throughput microculture platform, we developed predictive guidelines for presenting these integrin-targeting peptides alongside canonical binding motifs at optimal stoichiometries to generate nascent culture surfaces. Finally, when presented as self-assembled monolayers, predicted peptide combinations supported hPSC expansion, highlighting how unbiased screens can accelerate the discovery of targeted biomaterials.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

The progressively deeper understanding of mechanisms underlying stem cell fate decisions has enabled parallel advances in basic biology-such as the generation of organoid models that can further one's basic understanding of human development and disease-and in clinical translation-including stem cell based therapies to treat human disease. Both of these applications rely on tight control of the stem cell microenvironment to properly modulate cell fate, and materials that can be engineered to interface with cells in a controlled and tunable manner have therefore emerged as valuable tools for guiding stem cell growth and differentiation. With a focus on the central nervous system (CNS), a broad range of material solutions that have been engineered to overcome various hurdles in constructing advanced organoid models and developing effective stem cell therapeutics is reviewed. Finally, regulatory aspects of combined material-cell approaches for CNS therapies are considered., Competing Interests: Conflict of Interest D.V.S. and R.J.M. are inventors on patents related to stem cell manufacturing, and D.V.S., R.J.M., R.G.S., and H.J.J. are co-founders of a company that works on stem cell manufacturing.

Stem cells undergo differentiation in complex and dynamic environments wherein instructive signals fluctuate on various timescales. Thus, cells must be equipped to properly respond to the timing of signals, for example, to distinguish sustained signaling from transient noise. However, how stem cells respond to dynamic variations in differentiation cues is not well characterized. Here, we use optogenetic activation of β-catenin signaling to probe the dynamic responses of differentiating adult neural stem cells (NSCs). We discover that, while elevated, sustained β-catenin activation sequentially promotes proliferation and differentiation, transient β-catenin induces apoptosis. Genetic perturbations revealed that the neurogenic/apoptotic fate switch was mediated through cell-cycle regulation by Growth Arrest and DNA Damage 45 gamma (Gadd45γ). Our results thus reveal a role for β-catenin dynamics in NSC fate decisions and may suggest a role for signal timing to minimize cell-fate errors, analogous to kinetic proofreading of stem-cell differentiation., Competing Interests: The authors declare no competing interest.

Optogenetic modulation of protein interactions enables spatiotemporal control of cellular signaling dynamics in a variety of biological systems. However, light patterning by standard microscopes is limited by their complexity, sample throughput, and cost. To address the need for low-cost, user-friendly, and high-throughput photopatterning, we have engineered devices for light activation at variable amplitudes (LAVA). This protocol describes the assembly of LAVA devices, which enable spatial and temporal control of optogenetic stimulation and cellular signaling dynamics in multiwell cell culture plates. For complete details on the use and execution of this protocol, please refer to Repina et al. (2020)., Competing Interests: N.A.R., T.M., and D.V.S. are co-inventors on related intellectual property., (© 2020 The Authors.)

The field of optogenetics uses genetically encoded photoswitches to modulate biological phenomena with high spatiotemporal resolution. We report a set of rationally designed optogenetic photoswitches that use the photolyase homology region of A. thaliana cryptochrome 2 (Cry2PHR) as a building block and exhibit highly efficient and tunable clustering in a blue-light dependent manner. CL6mN (Cry2-mCherry-LRP6c with N mutated PPPAP motifs) proteins were designed by mutating and/or truncating five crucial PPP(S/T)P motifs near the C-terminus of the optogenetic Wnt activator Cry2-mCherry-LRP6c, thus eliminating its Wnt activity. Light-induced CL6mN clusters have significantly greater dissociation half-lives than clusters of wild-type Cry2PHR. Moreover, the dissociation half-lives can be tuned by varying the number of PPPAP motifs, with the half-life increasing as much as 6-fold for a variant with five motifs (CL6m5) relative to Cry2PHR. Finally, we demonstrate the compatibility of CL6mN with previously reported Cry2-based photoswitches by optogenetically activating RhoA in mammalian cells.

Efforts to identify mutations that underlie inherited genetic diseases combined with strides in the development of gene therapy vectors over the last three decades have culminated in the approval of several adeno-associated virus (AAV)-based gene therapies. Genetic diseases that manifest in the lung such as cystic fibrosis (CF) and surfactant deficiencies, however, have so far proven to be elusive targets. Early clinical trials in CF using AAV serotype 2 (AAV2) achieved safety, but not efficacy endpoints; however, importantly, these studies provided critical information on barriers that need to be surmounted to translate AAV lung gene therapy toward clinical success. Bolstered with an improved understanding of AAV biology and more clinically relevant lung models, next-generation molecular biology and bioinformatics approaches have given rise to novel AAV capsid variants that offer improvements in transduction efficiency, immunological profile, and the ability to circumvent physical barriers in the lung such as mucus. This review discusses the principal limiting barriers to clinical success in lung gene therapy and focuses on novel engineered AAV capsid variants that have been developed to overcome those challenges.

The emergence of several cell therapy candidates in the clinic is an encouraging sign for human diseases/disorders that currently have no effective treatment; however, scalable production of these cell therapies has become a bottleneck. To overcome this barrier, three-dimensional (3D) cell culture strategies have been considered for enhanced cell production. Here, we demonstrate a high-throughput 3D culture platform used to systematically screen 1200 culture conditions with varying doses, durations, dynamics, and combinations of signaling cues to derive oligodendrocyte progenitor cells and midbrain dopaminergic neurons from human pluripotent stem cells (hPSCs). Statistical models of the robust dataset reveal previously unidentified patterns about cell competence to Wnt, retinoic acid, and sonic hedgehog signals, and their interactions, which may offer insights into the combinatorial roles these signals play in human central nervous system development. These insights can be harnessed to optimize production of hPSC-derived cell replacement therapies for a range of neurological indications., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

New technologies to target nucleotide diversification in vivo are promising enabling strategies to perform directed evolution for engineering applications and forward genetics for addressing biological questions. Recently, we reported EvolvR-a system that employs CRISPR-guided Cas9 nickases fused to nick-translating, error-prone DNA polymerases to diversify targeted genomic loci-in E. coli . As CRISPR-Cas9 has shown activity across diverse cell types, EvolvR has the potential to be ported into other organisms, including eukaryotes, if nick-translating polymerases can be active across species. Here, we implement and characterize EvolvR's function in Saccharomyces cerevisiae , representing a key first step to enable EvolvR-mediated mutagenesis in eukaryotes. This advance will be useful for mutagenesis of user-defined loci in the yeast chromosomes for both engineering and basic research applications, and it furthermore provides a platform to develop the EvolvR technology for performance in higher eukaryotes.

Genome editing has the potential to treat an extensive range of incurable monogenic and complex diseases. In particular, advances in sequence-specific nuclease technologies have dramatically accelerated the development of therapeutic genome editing strategies that are based on either the knockout of disease-causing genes or the repair of endogenous mutated genes. These technologies are progressing into human clinical trials. However, challenges remain before the therapeutic potential of genome editing can be fully realized. Delivery technologies that have serendipitously been developed over the past couple decades in the protein and nucleic acid delivery fields have been crucial to genome editing success to date, including adeno-associated viral and lentiviral vectors for gene therapy and lipid nanoparticle and other non-viral vectors for nucleic acid and protein delivery. However, the efficiency and tissue targeting capabilities of these vehicles must be further improved. In addition, the genome editing enzymes themselves need to be optimized, and challenges regarding their editing efficiency, specificity and immunogenicity must be addressed. Emerging protein engineering and synthetic chemistry approaches can offer solutions and enable the development of safe and efficacious clinical genome editing.

Spatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics affect cell behavior. Here, we show that optogenetic Wnt/β-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). By patterning human embryonic stem cell (hESC) cultures with varying light intensities, LAVA devices enabled dose-responsive control of optoWnt activation and Brachyury expression. Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models the embryonic presentation of Wnt signals in vitro. LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology., Competing Interests: Declaration of Interests N.A.R., T.M., and D.V.S. are co-inventors on related intellectual property., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Efficient adeno-associated virus-mediated (AAV-mediated) gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply directed evolution - guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library - to create AAV viral capsids with the capability to deliver genes to the outer retina in primates. A replication-incompetent library, produced via providing rep in trans, was created to mitigate risk of AAV propagation. Six rounds of in vivo selection with this library in primates - involving intravitreal library administration, recovery of genomes from outer retina, and extensive next-generation sequencing of each round - resulted in vectors with redirected tropism to the outer retina and increased gene delivery efficiency to retinal cells. These viral vectors expand the toolbox of vectors available for primate retina, and they may enable less invasive delivery of therapeutic genes to patients, potentially offering retina-wide infection at a similar dosage to vectors currently in clinical use.

Fragment antigen-binding domains (Fabs) from anti-Frizzled and anti-LRP6 monoclonal antibodies were conjugated using SpyTag-SpyCatcher chemistry via a one-pot reaction. The resulting synthetic heterodimeric agonist outperformed the natural ligand, Wnt-3a, in activating canonical Wnt signaling in mammalian cells. This approach should be broadly applicable to activate receptor-mediated cellular signaling.

Elucidating how the spatial organization of extrinsic signals modulates cell behavior and drives biological processes remains largely unexplored because of challenges in controlling spatial patterning of multiple microenvironmental cues in vitro. Here, we describe a high-throughput method that directs simultaneous assembly of multiple cell types and solid-phase ligands across length scales within minutes. Our method involves lithographically defining hierarchical patterns of unique DNA oligonucleotides to which complementary strands, attached to cells and ligands-of-interest, hybridize. Highlighting our method's power, we investigated how the spatial presentation of self-renewal ligand fibroblast growth factor-2 (FGF-2) and differentiation signal ephrin-B2 instruct single adult neural stem cell (NSC) fate. We found that NSCs have a strong spatial bias toward FGF-2 and identified an unexpected subpopulation exhibiting high neuronal differentiation despite spatially occupying patterned FGF-2 regions. Overall, our broadly applicable, DNA-directed approach enables mechanistic insight into how tissues encode regulatory information through the spatial presentation of heterogeneous signals., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Mechanical cues regulate the function of a broad range of stem cells in culture and in tissue. For example, soft substrates promote the neuronal differentiation of neural stem cells (NSCs) by suppressing cytoskeletal contractility. However, the mechanisms that link cytoskeletal signaling to the transcriptional regulatory processes that ultimately govern stiffness-dependent NSC fate commitment are not fully understood. Here, we show that Angiomotin (AMOT), which can bind both F-actin and the neurosuppressive transcriptional coactivator Yes-associated protein (YAP), is critical for mechanotransduction in NSCs. On soft substrates, loss of AMOT substantially reduces neurogenesis, whereas on stiff substrates, loss of AMOT negates the rescue of neurogenesis normally induced by pharmacologic inhibition of myosin activity. Furthermore, overexpression of a phospho-mimetic S175E AMOT mutant, which has been established to enhance AMOT-YAP binding, increases β-catenin activity and rescues neurogenesis on stiff substrates. Together, our data identify AMOT as an important intermediate signal transducer that allows NSCs to sense and respond to extracellular stiffness cues.

Interfacing gene delivery vehicles with biomaterials has the potential to play a key role in diversifying gene transfer capabilities, including localized, patterned, and controlled delivery. However, strategies for modifying biomaterials to interact with delivery vectors must be redesigned whenever new delivery vehicles and applications are explored. We have developed a vector-independent biomaterial platform capable of interacting with various adeno-associated viral (AAV) serotypes. A water-soluble, cysteine-tagged, recombinant protein version of the recently discovered multi-AAV serotype receptor (AAVR), referred to as cys-AAVR, was conjugated to maleimide-displaying polycaprolactone (PCL) materials using click chemistry. The resulting cys-AAVR-PCL system bound to a broad range of therapeutically relevant AAV serotypes, thereby providing a platform capable of modulating the delivery of all AAV serotypes. Intramuscular injection of cys-AAVR-PCL microspheres with bound AAV vectors resulted in localized and sustained gene delivery as well as reduced spread to off-target organs compared to a vector solution. This cys-AAVR-PCL system is thus an effective approach for biomaterial-based AAV gene delivery for a broad range of therapeutic applications., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Transcription factors (TFs) are potent proteins that control gene expression and can thereby drive cell fate decisions. Fluorescent reporters have been broadly knocked into endogenous TF loci to investigate the biological roles of these factors; however, the sensitivity of such analyses in human pluripotent stem cells (hPSCs) is often compromised by low TF expression levels and/or reporter silencing. Complementarily, we report an inducible and quantitative reporter platform based on the Cre-LoxP recombination system that enables robust, quantifiable, and continuous monitoring of live hPSCs and their progeny to investigate the roles of TFs during human development and disease. Stem Cells 2019;37:1556-1566., (©AlphaMed Press 2019.)

Huntington's disease (HD) is a currently incurable and, ultimately, fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion within exon 1 of the huntingtin (HTT) gene, which results in the production of a mutant protein that forms inclusions and selectively destroys neurons in the striatum and other adjacent structures. The RNA-guided Cas9 endonuclease from CRISPR-Cas9 systems is a versatile technology for inducing DNA double-strand breaks that can stimulate the introduction of frameshift-inducing mutations and permanently disable mutant gene function. Here, we show that the Cas9 nuclease from Staphylococcus aureus, a small Cas9 ortholog that can be packaged alongside a single guide RNA into a single adeno-associated virus (AAV) vector, can be used to disrupt the expression of the mutant HTT gene in the R6/2 mouse model of HD following its in vivo delivery to the striatum. Specifically, we found that CRISPR-Cas9-mediated disruption of the mutant HTT gene resulted in a ∼50% decrease in neuronal inclusions and significantly improved lifespan and certain motor deficits. These results thus illustrate the potential for CRISPR-Cas9 technology to treat HD and other autosomal dominant neurodegenerative disorders caused by a trinucleotide repeat expansion via in vivo genome editing., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Stem cells with the potential for neural differentiation are a promising therapeutic avenue both for treating neurological disease and as a system to advance our fundamental understanding of disease biology in vitro. Precisely controlled extracellular environments that recapitulate critical aspects of embryonic development or the adult stem cell niche are necessary to ensure effective differentiation into the desired cell type. Biomaterials in particular have enabled new avenues for directing stem cell differentiation through the precise presentation of biochemical and biophysical cues. Furthermore, as translation of stem cell technologies necessitates the need for scalable cultures, biomaterials will continue to be valuable tools for guiding stem cell behavior in scalable, complex, three-dimensional cultures. In this review, we highlight the critical signals that guide neurogenesis and how biomaterials can be used to control and direct the neural differentiation of pluripotent and adult stem cells. In addition, we discuss recent new technologies that are further advancing material-based regulation of stem cells. Finally, we highlight the current state of the field and how next-generation biomaterials can enable scalable stem cell culture for cell replacement therapies as well as emerging advanced tissue models for studying tissue morphogenesis and disease pathology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species., (Copyright © 2019. Published by Elsevier Inc.)

Identification of conditions for guided and specific differentiation of human stem cell and progenitor cells is important for continued development and engineering of in vitro cell culture systems for use in regenerative medicine, drug discovery, and human toxicology. Three-dimensional (3D) and organotypic cell culture models have been used increasingly for in vitro cell culture because they may better model endogenous tissue environments. However, detailed studies of stem cell differentiation within 3D cultures remain limited, particularly with respect to high-throughput screening. Herein, we demonstrate the use of a microarray chip-based platform to screen, in high-throughput, individual and paired effects of 12 soluble factors on the neuronal differentiation of a human neural progenitor cell line (ReNcell VM) encapsulated in microscale 3D Matrigel cultures. Dose-response analysis of selected combinations from the initial combinatorial screen revealed that the combined treatment of all-trans retinoic acid (RA) with the glycogen synthase kinase 3 inhibitor CHIR-99021 (CHIR) enhances neurogenesis while simultaneously decreases astrocyte differentiation, whereas the combined treatment of brain-derived neurotrophic factor and the small azide neuropathiazol enhances the differentiation into neurons and astrocytes. Subtype specification analysis of RA- and CHIR-differentiated cultures revealed that enhanced neurogenesis was not biased toward a specific neuronal subtype. Together, these results demonstrate a high-throughput screening platform for rapid evaluation of differentiation conditions in a 3D environment, which will aid the development and application of 3D stem cell culture models., (© 2018 Wiley Periodicals, Inc.)

Advancing our knowledge of how neural stem cell (NSC) behavior in the adult hippocampus is regulated has implications for elucidating basic mechanisms of learning and memory as well as for neurodegenerative disease therapy. To date, numerous biochemical cues from the endogenous hippocampal NSC niche have been identified as modulators of NSC quiescence, proliferation, and differentiation; however, the complex repertoire of signaling factors within stem cell niches raises the question of how cues act in combination with one another to influence NSC physiology. To help overcome experimental bottlenecks in studying this question, we adapted a high-throughput microculture system, with over 500 distinct microenvironments, to conduct a systematic combinatorial screen of key signaling cues and collect high-content phenotype data on endpoint NSC populations. This novel application of the platform consumed only 0.2% of reagent volumes used in conventional 96-well plates, and resulted in the discovery of numerous statistically significant interactions among key endogenous signals. Antagonistic relationships between fibroblast growth factor 2, transforming growth factor β (TGF-β), and Wnt-3a were found to impact NSC proliferation and differentiation, whereas a synergistic relationship between Wnt-3a and Ephrin-B2 on neuronal differentiation and maturation was found. Furthermore, TGF-β and bone morphogenetic protein 4 combined with Wnt-3a and Ephrin-B2 resulted in a coordinated effect on neuronal differentiation and maturation. Overall, this study offers candidates for further elucidation of significant mechanisms guiding NSC fate choice and contributes strategies for enhancing control over stem cell-based therapies for neurodegenerative diseases., (© 2018 Wiley Periodicals, Inc.)