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Three-dimensional pluripotent stem cell (PSC) cultures have the ability to undergo differentiation, self-organization, and morphogenesis to yield complex, in vitro tissue models that recapitulate key elements of native tissues. These tissue models offer a system for studying mechanisms of tissue development, investigating disease mechanisms, and performing drug screening. It remains challenging, however, to standardize PSC aggregate differentiation and morphogenesis methods due to heterogeneity stemming from biological and environmental sources. It is also difficult to monitor and assess large numbers of individual samples longitudinally throughout culture using typical batch-based culture methods. To address these challenges, we have developed a microfluidic platform for culture, longitudinal monitoring, and phenotypic analysis of individual stem cell aggregates. This platform uses a hydrodynamic loading principle to capture pre-formed stem cell aggregates in independent traps. We demonstrated that multi-day culture of aggregates in this platform reduces heterogeneity in phenotypic parameters such as size and morphology. Additionally, we showed that culture and analysis steps can be performed sequentially in the same platform, enabling correlation of multiple modes of analysis for individual samples. We anticipate this platform being applied to improve abilities for phenotypic analysis of PSC aggregate tissues and to facilitate research in standardizing culture systems in order to dually increase the yield and reduce the heterogeneity of PSC-derived tissues.

Osteogenesis of mesenchymal stem cells (MSC) can be regulated by the mechanical environment. MSCs grown in 3D spheroids (mesenspheres) have preserved multi-lineage potential, improved differentiation efficiency, and exhibit enhanced osteogenic gene expression and matrix composition in comparison to MSCs grown in 2D culture. Within 3D mesenspheres, mechanical cues are primarily in the form of cell-cell contraction, mediated by adhesion junctions, and as such adhesion junctions are likely to play an important role in the osteogenic differentiation of mesenspheres. However the precise role of N- and OB-cadherin on the biomechanical behaviour of mesenspheres remains unknown. Here we have mechanically tested mesenspheres cultured in suspension using parallel plate compression to assess the influence of N-cadherin and OB-cadherin adhesion junctions on the viscoelastic properties of the mesenspheres during osteogenesis. Our results demonstrate that N-cadherin and OB-cadherin have different effects on mesensphere viscoelastic behaviour and osteogenesis. When OB-cadherin was silenced, the viscosity, initial and long term Young's moduli and actin stress fibre formation of the mesenspheres increased in comparison to N-cadherin silenced mesenspheres and mesenspheres treated with a scrambled siRNA (Scram) at day 2. Additionally, the increased viscoelastic material properties correlate with evidence of calcification at an earlier time point (day 7) of OB-cadherin silenced mesenspheres but not Scram. Interestingly, both N-cadherin and OB-cadherin silenced mesenspheres had higher BSP2 expression than Scram at day 14. Taken together, these results indicate that N-cadherin and OB-cadherin both influence mesensphere biomechanics and osteogenesis, but play different roles.

In the nascent mesoderm, TBXT expression must be precisely regulated to ensure that cells exit the primitive streak and pattern the anterior-posterior axis, but how varying dosage informs morphogenesis is not well understood. In this study, we define the transcriptional consequences of TBXT dosage reduction during early human gastrulation using human induced pluripotent stem cell models of gastrulation and mesoderm differentiation. Multi-omic single-nucleus RNA and single-nucleus ATAC sequencing of 2D gastruloids comprising wild-type, TBXT heterozygous or TBXT null human induced pluripotent stem cells reveal that varying TBXT dosage does not compromise the ability of a cell to differentiate into nascent mesoderm, but instead directly influences the temporal progression of the epithelial-to-mesenchymal transition with wild type transitioning first, followed by TBXT heterozygous and then TBXT null. By differentiating cells into nascent mesoderm in a monolayer format, we further illustrate that TBXT dosage directly impacts the persistence of junctional proteins and cell-cell adhesions. These results demonstrate that epithelial-to-mesenchymal transition progression can be decoupled from the acquisition of mesodermal identity in the early gastrula and shed light on the mechanisms underlying human embryogenesis., Competing Interests: Competing interests B.G.B. is a founder, shareholder and advisor of Tenaya Therapeutics and is an advisor for Silver Creek Pharmaceuticals. I.M.-V. is the founder and Chief Executive Officer of Vitra Labs, Inc. T.C.M. is an employee at Genentech and is an advisor for Vitra Labs. The work presented here is not related to the interests of these commercial entities., (© 2024. Published by The Company of Biologists Ltd.)

Loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT), heterozygous (CDX2-Het), and homozygous null CDX2 (CDX2-KO) genotypes, differentiating these cells in a 2D gastruloid model, and subjecting these cells to single-nucleus RNA and ATAC sequencing, we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile, suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo, we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together, these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo., Competing Interests: Competing interests B.G.B. is a founder, shareholder, and advisor of Tenaya Therapeutics and is an advisor for Silver Creek Pharmaceuticals. T.C.M. is an employee at Genentech and is an advisor for Vitra Labs. The work presented here is not related to the interests of these commercial entities., (© 2024. Published by The Company of Biologists Ltd.)

Importance: The hepatitis C virus (HCV) causes liver disease, affecting millions. Even though we have effective antivirals that cure HCV, they cannot stop terminal liver disease. We used an adult stem cell-derived liver organoid system to understand how HCV infection leads to the progression of terminal liver disease. Here, we show that HCV maintains low-grade infections in liver organoids for the first time. HCV infection in liver organoids leads to transcriptional reprogramming causing cancer cell development and altered immune response. Our finding shows how HCV infection in liver organoids mimics HCV infection and patient pathogenesis. These results reveal that HCV infection in liver organoids contributes to liver disease progression., Competing Interests: The authors declare no conflict of interest.

Biological patterning events that occur early in development establish proper tissue morphogenesis. Identifying the mechanisms that guide these patterning events is necessary in order to understand the molecular drivers of development and disease and to build tissues in vitro. In this study, we use an in vitro model of gastrulation to study the role of tight junctions and apical/basolateral polarity in modulating bone morphogenic protein-4 (BMP4) signaling and gastrulation-associated patterning in colonies of human pluripotent stem cells (hPSCs). Disrupting tight junctions via knockdown (KD) of the scaffolding tight junction protein-1 (TJP1, also known as ZO1) allows BMP4 to robustly and ubiquitously activate pSMAD1/5 signaling over time, resulting in loss of the patterning phenotype and marked differentiation bias of pluripotent stem cells to primordial germ cell-like cells (PGCLCs). These findings give important insights into how signaling events are regulated and lead to spatial emergence of diverse cell types in vitro., Competing Interests: Declaration of interests I.V. is the founder and Chief Executive Officer of Vitra Labs, Inc. K.T. is on the scientific advisory board of I Peace, Inc., without salary. S.Y. is a scientific advisor to iPS Academia Japan, Orizuru Therapeutics, and Altos Labs without salary. T.C.M. is Vice President of Cell Engineering at SanaX. I.V. and T.C.M. are inventors on a patent filing by the Gladstone Institutes and the University of California, San Francisco, relating to high-throughput generation of primordial germ cell-like cells (PGCLCs)., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation"., (© 2022 New York Academy of Sciences.)

The rapidly growing field of cellular engineering is enabling scientists to more effectively create in vitro models of disease and develop specific cell types that can be used to repair damaged tissue. In particular, the engineering of neurons and other components of the nervous system is at the forefront of this field. The methods used to engineer neural cells can be largely divided into systems that undergo directed differentiation through exogenous stimulation (i.e., via small molecules, arguably following developmental pathways) and those that undergo induced differentiation via protein overexpression (i.e., genetically induced and activated; arguably bypassing developmental pathways). Here, we highlight the differences between directed differentiation and induced differentiation strategies, how they can complement one another to generate specific cell phenotypes, and impacts of each strategy on downstream applications. Continued research in this nascent field will lead to the development of improved models of neurological circuits and novel treatments for those living with neurological injury and disease., Competing Interests: TM was employed by the company Sana Biotechnology, Inc. The remaining 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 Elder, Fattahi, McDevitt and Zholudeva.)

Hepatitis C virus (HCV) remains a global public health challenge with an estimated 71 million people chronically infected, with surges in new cases and no effective vaccine. New methods are needed to study the human immune response to HCV since in vivo animal models are limited and in vitro cancer cell models often show dysregulated immune and proliferative responses. Here, we developed a CD8 + T cell and adult stem cell liver organoid system using a microfluidic chip to coculture 3D human liver organoids embedded in extracellular matrix with HLA-matched primary human T cells in suspension. We then employed automated phase contrast and immunofluorescence imaging to monitor T cell invasion and morphological changes in the liver organoids. This microfluidic coculture system supports targeted killing of liver organoids when pulsed with a peptide specific for HCV non-structural protein 3 (NS3) (KLVALGINAV) in the presence of patient-derived CD8 + T cells specific for KLVALGINAV. This demonstrates the novel potential of the coculture system to molecularly study adaptive immune responses to HCV in an in vitro setting using primary human cells.

Engineered cardiac tissue models aim to recapitulate the multicellular composition of the native myocardium by incorporating multiple tissue-relevant cell populations. Here, we describe the process of generating self-assembled cardiac microtissue spheroids comprised of heterotypic cardiac cell types. The absence of exogenous extracellular matrix (ECM) or scaffolding makes microtissue assembly dependent upon intercellular adhesion interactions over cell-ECM interactions, analogous to early development. Therefore, this approach creates a 3D platform to study how multicellular heterotypic interactions impact tissue structure, function, and phenotype., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

During embryogenesis, paracrine signaling between tissues in close proximity contributes to the determination of their respective cell fate(s) and development into functional organs. Organoids are in vitro models that mimic organ formation and cellular heterogeneity, but lack the paracrine input of surrounding tissues. Here, we describe a human multilineage iPSC-derived organoid that recapitulates cooperative cardiac and gut development and maturation, with extensive cellular and structural complexity in both tissues. We demonstrate that the presence of endoderm tissue (gut/intestine) in the organoids contributed to the development of cardiac tissue features characteristic of stages after heart tube formation, including cardiomyocyte expansion, compartmentalization, enrichment of atrial/nodal cells, myocardial compaction, and fetal-like functional maturation. Overall, this study demonstrates the ability to generate and mature cooperative tissues originating from different germ lineages within a single organoid model, an advance that will further the examination of multi-tissue interactions during development, physiological maturation, and disease., Competing Interests: Declaration of interests T.C.M. is a consultant for Tenaya Therapeutics and B.G.B. is a co-founder. Both authors own equity in Tenaya Therapeutics. The other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users. Here we describe a standardized method for engineering an isogenic, patient specific human neuromuscular junction (NMJ) that allows for automated quantification of NMJ function to diagnose disease using a small sample of blood serum and evaluate new therapeutic modalities. By combining tissue engineering, optogenetics, microfabrication, optoelectronics and video processing, we created a novel platform for the precise investigation of the development and degeneration of human NMJ. We demonstrate the utility of this platform for the detection and diagnosis of myasthenia gravis, an antibody-mediated autoimmune disease that disrupts the NMJ function., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cardiac fibroblasts (CFBs) support heart function by secreting extracellular matrix (ECM) and paracrine factors, respond to stress associated with injury and disease, and therefore are an increasingly important therapeutic target. We describe how developmental lineage of human pluripotent stem cell-derived CFBs, epicardial (EpiC-FB), and second heart field (SHF-FB) impacts transcriptional and functional properties. Both EpiC-FBs and SHF-FBs exhibited CFB transcriptional programs and improved calcium handling in human pluripotent stem cell-derived cardiac tissues. We identified differences including in composition of ECM synthesized, secretion of growth and differentiation factors, and myofibroblast activation potential, with EpiC-FBs exhibiting higher stress-induced activation potential akin to myofibroblasts and SHF-FBs demonstrating higher calcification and mineralization potential. These phenotypic differences suggest that EpiC-FBs have utility in modeling fibrotic diseases while SHF-FBs are a promising source of cells for regenerative therapies. This work directly contrasts regional and developmental specificity of CFBs and informs CFB in vitro model selection., (© 2021 Federation of American Societies for Experimental Biology.)

Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene ( NEFL , CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases., Competing Interests: BC is a founder of Tenaya Therapeutics (https://www.tenayatherapeutics.com/), a company focused on finding treatments for heart failure, including genetic cardiomyopathies. BC holds equity in Tenaya, and Tenaya provides research support for heart failure related research. The remaining 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 © 2021 Feliciano, Wu, Watry, Marley, Ramadoss, Ghanim, Liu, Zholudeva, McDevitt, Saporta, Conklin and Judge.)

The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here, we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI., 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 © 2021 McCreedy, Jalufka, Platt, Min, Kirchhoff, Pritchard, Reid, Manlapaz, Mihaly, Butts, Iyer, Sakiyama-Elbert, Crone and McDevitt.)

Axial elongation of the neural tube is crucial during mammalian embryogenesis for anterior-posterior body axis establishment and subsequent spinal cord development, but these processes cannot be interrogated directly in humans as they occur post-implantation. Here, we report an organoid model of neural tube extension derived from human pluripotent stem cell (hPSC) aggregates that have been caudalized with Wnt agonism, enabling them to recapitulate aspects of the morphological and temporal gene expression patterns of neural tube development. Elongating organoids consist largely of neuroepithelial compartments and contain TBXT+SOX2+ neuro-mesodermal progenitors in addition to PAX6+NES+ neural progenitors. A critical threshold of Wnt agonism stimulated singular axial extensions while maintaining multiple cell lineages, such that organoids displayed regionalized anterior-to-posterior HOX gene expression with hindbrain (HOXB1) regions spatially distinct from brachial (HOXC6) and thoracic (HOXB9) regions. CRISPR interference-mediated silencing of TBXT, a Wnt pathway target, increased neuroepithelial compartmentalization, abrogated HOX expression and disrupted uniaxial elongation. Together, these results demonstrate the potent capacity of caudalized hPSC organoids to undergo axial elongation in a manner that can be used to dissect the cellular organization and patterning decisions that dictate early human nervous system development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Lineage tracing is a powerful tool in developmental biology to interrogate the evolution of tissue formation, but the dense, three-dimensional nature of tissue limits the assembly of individual cell trajectories into complete reconstructions of development. Human induced pluripotent stem cells (hiPSCs) can recapitulate aspects of developmental processes, providing an in vitro platform to assess the dynamic collective behaviors directing tissue morphogenesis. Here, we trained an ensemble of neural networks to track individual hiPSCs in time-lapse microscopy, generating longitudinal measures of cell and cellular neighborhood properties on timescales from minutes to days. Our analysis reveals that, while individual cell parameters are not strongly affected by pluripotency maintenance conditions or morphogenic cues, regional changes in cell behavior predict cell fate and colony organization. By generating complete multicellular reconstructions of hiPSC behavior, our tracking pipeline enables fine-grained understanding of morphogenesis by elucidating the role of regional behavior in early tissue formation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Strategies aiming at increasing the survival and paracrine activity of human mesenchymal stromal cells (MSCs) are of utmost importance to achieve the full therapeutic potential of these cells. Herein, we propose both physical and biochemical strategies to enhance the survival, homing, angiogenic, and immunomodulatory properties of MSCs in vitro. To that purpose, we compared the effect of exposing either 2D monolayer or 3D spheroids of MSCs to (i) hypoxia (2% O 2 ) or to (ii) a hypoxic-mimetic small molecule, dimethyloxalylglycine (DMOG), with cells cultured at 21% O 2 . 3D-cultured MSC spheroids evidenced higher survival upon exposure to oxidative stress and expressed higher levels of factors involved in tissue repair processes, namely tumor necrosis factor-stimulated gene-6, matrix metalloproteinase-2, and vascular endothelial growth factor. MSCs cultured as 3D spheroids and further exposed to hypoxia or hypoxic-mimetic conditions provided by DMOG synergistically favored the expression of the cell surface marker C-X-C chemokine receptor type-4, involved in homing processes to injured tissues, and adhesion to extracellular matrix components as fibronectin. These results highlight the role of ex vivo preconditioning approaches, presenting a novel strategy that combine biochemical stimuli with 3D spheroid organization of MSCs to maximize their tissue regeneration potential., (© 2021 Wiley-VCH GmbH.)

Although coronavirus disease 2019 (COVID-19) causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human induced pluripotent stem cell (iPSC)-derived heart cells to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural genes corroborates adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and nuclear disruption. Human autopsy specimens from patients with COVID-19 reflected similar alterations, particularly sarcomeric fragmentation. These notable cytopathic features in cardiomyocytes provide insights into SARS-CoV-2-induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise concerns about the long-term consequences of COVID-19 in asymptomatic and severe cases., (Copyright © 2021 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 License 4.0 (CC BY).)

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Spinal interneurons are important facilitators and modulators of motor, sensory, and autonomic functions in the intact CNS. This heterogeneous population of neurons is now widely appreciated to be a key component of plasticity and recovery. This review highlights our current understanding of spinal interneuron heterogeneity, their contribution to control and modulation of motor and sensory functions, and how this role might change after traumatic spinal cord injury. We also offer a perspective for how treatments can optimize the contribution of interneurons to functional improvement., (Copyright © 2021 the authors.)

Pluripotent stem cells (PSCs) possess the ability to self-organize into complex tissue-like structures; however, the genetic mechanisms and multicellular dynamics that direct such patterning are difficult to control. Here, we pair live imaging with controlled induction of gene knockdown by CRISPR interference (CRISPRi) to generate changes within subpopulations of human PSCs, allowing for control over organization and analysis of emergent behaviors. Specifically, we use forced aggregation of mixtures of cells with and without an inducible CRISPRi system to knockdown molecular regulators of tissue symmetry. We then track the resulting multicellular organization through fluorescence live imaging concurrent with the induction of knockdown. Overall, this technique allows for controlled initiation of symmetry breaking by CRISPRi to produce changes in cellular behavior that can be tracked over time within high-density pluripotent stem cell colonies.

Microfluidic technologies provide many advantages for studying differentiation of three-dimensional (3D) stem cell aggregates, including the ability to control the culture microenvironment, isolate individual aggregates for longitudinal tracking, and perform imaging-based assays. However, applying microfluidics to studying mechanisms of stem cell differentiation requires an understanding of how microfluidic culture conditions impact cell phenotypes. Conventional cell culture techniques cannot directly be applied to the microscale, as microscale culture varies from macroscale culture in multiple aspects. Therefore, the objective of this work was to explore key parameters in microfluidic culture of 3D stem cell aggregates and to understand how these parameters influence stem cell behavior and differentiation. These studies were done in the context of differentiation of embryonic stem cells (ESCs) to motor neurons (MNs). We assessed how media exchange frequency modulates the biochemical microenvironment, including availability of exogenous factors (e.g. nutrients, small molecule additives) and cell-secreted molecules, and thereby impacts differentiation. The results of these studies provide guidance on how key characteristics of 3D cell cultures can be considered when designing microfluidic culture parameters. We demonstrate that discontinuous perfusion is effective at supporting stem cell aggregate growth. We find that there is a balance between the frequency of media exchange, which is needed to ensure that cells are not nutrient-limited, and the need to allow accumulation of cell-secreted factors to promote differentiation. Finally, we show how microfluidic device geometries can influence transport of biomolecules and potentially promote asymmetric spatial differentiation. These findings are instructive for future work in designing devices and experiments for culture of cell aggregates.

Native cardiac tissue is composed of heterogeneous cell populations that work cooperatively for proper tissue function; thus, engineered tissue models have moved toward incorporating multiple cardiac cell types in an effort to recapitulate native multicellular composition and organization. Cardiac tissue models composed of stem cell-derived cardiomyocytes (CMs) require inclusion of non-myocytes to promote stable tissue formation, yet the specific contributions of the supporting non-myocyte population on the parenchymal CMs and cardiac microtissues have to be fully dissected. This gap can be partly attributed to limitations in technologies able to accurately study the individual cellular structure and function that comprise intact three-dimensional (3D) tissues. The ability to interrogate the cell-cell interactions in 3D tissue constructs has been restricted by conventional optical imaging techniques that fail to adequately penetrate multicellular microtissues with sufficient spatial resolution. Light sheet fluorescence microscopy (LSFM) overcomes these constraints to enable single-cell resolution structural and functional imaging of intact cardiac microtissues. Multicellular spatial distribution analysis of heterotypic cardiac cell populations revealed that CMs and cardiac fibroblasts were randomly distributed throughout 3D microtissues. Furthermore, calcium imaging of live cardiac microtissues enabled single-cell detection of CM calcium activity, which showed that functional heterogeneity correlated with spatial location within the tissues. This study demonstrates that LSFM can be utilized to determine single-cell spatial and functional interactions of multiple cell types within intact 3D engineered microtissues, thereby facilitating the determination of structure-function relationships at both tissue-level and single-cell resolution. Impact statement The ability to achieve single-cell resolution by advanced three-dimensional light imaging techniques enables exquisite new investigation of multicellular analyses in native and engineered tissues. In this study, light sheet fluorescence microscopy was used to define structure-function relationships of distinct cell types in engineered cardiac microtissues by determining heterotypic cell distributions and interactions throughout the tissues as well as by assessing regional differences in calcium handing functional properties at the individual cardiomyocyte level.

Supraphysiologic doses of bone morphogenetic protein-2 (BMP-2) are used clinically to promote bone formation in fracture nonunions, large bone defects, and spinal fusion. However, abnormal bone formation (i.e., heterotopic ossification) caused by rapid BMP-2 release from conventional collagen sponge scaffolds is a serious complication. We leveraged the strong affinity interactions between heparin microparticles (HMPs) and BMP-2 to improve protein delivery to bone defects. We first developed a computational model to investigate BMP-2-HMP interactions and demonstrated improved in vivo BMP-2 retention using HMPs. We then evaluated BMP-2-loaded HMPs as a treatment strategy for healing critically sized femoral defects in a rat model that displays heterotopic ossification with clinical BMP-2 doses (0.12 mg/kg body weight). HMPs increased BMP-2 retention in vivo, improving spatial localization of bone formation in large bone defects and reducing heterotopic ossification. Thus, HMPs provide a promising opportunity to improve the safety profile of scaffold-based BMP-2 delivery., (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).)

Cell-derived matrices (CDMs) provide an exogenous source of human extracellular matrix (ECM), with applications as cell delivery vehicles, substrate coatings for cell attachment and differentiation, and as biomaterial scaffolds. However, commercial application of CDMs has been hindered due to the prolonged culture time required for sufficient ECM accumulation. One approach to increasing matrix deposition in vitro is macromolecular crowding (MMC), which is a biophysical phenomenon that limits the diffusion of ECM precursor proteins, resulting in increased ECM accumulation at the cell layer. Hyaluronic acid (HA), a natural MMC highly expressed in vivo during fetal development, has been shown to play a role in ECM production, but has not been investigated as a macromolecule for increasing cell-mediated ECM deposition in vitro. In the current study, we hypothesized that HA can act as a MMC, and increase cell-mediated ECM production. Human dermal fibroblasts were cultured for 3, 7, or 14 days with 0%, 0.05%, or 0.5% high molecular weight HA. Ficoll 70/400 was used as a positive control. SDS-PAGE, Sircol, and hydroxyproline assays indicated that 0.05% HA-treated cultures had significantly higher mean collagen deposition at 14 days, whereas Ficoll 70/400-treated cultures had significantly lower collagen production compared to the HA and untreated controls. However, fluorescent immunostaining of ECM proteins and quantification of mean gray values did not indicate statistically significant differences in ECM production in HA or Ficoll 70/400-treated cultures compared to untreated controls. Raman imaging (a marker-free spectral imaging method) indicated that HA increased ECM deposition in human dermal fibroblasts. These results are consistent with decreases in CDM stiffness observed in Ficoll 70/400-treated cultures by atomic force microscopy. Overall, these results indicate that there are macromolecule- and cell type- dependent effects on matrix assembly, turnover, and stiffness in cell-derived matrices. STATEMENT OF SIGNIFICANCE: Cell-derived matrices (CDMs) are versatile biomaterials with many regenerative medicine applications, including as cell and drug delivery vehicles and scaffolds for wound healing and tissue regeneration. While CDMs have several advantages, their commercialization has been limited due to the prolonged culture time required to achieve CDM synthesis in vitro. In this study, we explored the use of hyaluronic acid (HA) as a macromolecular crowder in human fibroblast cell cultures to support production of CDM biomaterials. Successful application of macromolecular crowding will allow development of human cell-derived, xeno-free biomaterials that re-capitulate the native human tissue microenvironment., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Human pluripotent stem cells (hPSCs) have the intrinsic ability to self-organize into complex multicellular organoids that recapitulate many aspects of tissue development. However, robustly directing morphogenesis of hPSC-derived organoids requires novel approaches to accurately control self-directed pattern formation. Here, we combined genetic engineering with computational modeling, machine learning, and mathematical pattern optimization to create a data-driven approach to control hPSC self-organization by knock down of genes previously shown to affect stem cell colony organization, CDH1 and ROCK1. Computational replication of the in vitro system in silico using an extended cellular Potts model enabled machine learning-driven optimization of parameters that yielded emergence of desired patterns. Furthermore, in vitro the predicted experimental parameters quantitatively recapitulated the in silico patterns. These results demonstrate that morphogenic dynamics can be accurately predicted through model-driven exploration of hPSC behaviors via machine learning, thereby enabling spatial control of multicellular patterning to engineer human organoids and tissues. A record of this paper's Transparent Peer Review process is included in the Supplemental Information., (Copyright © 2019 Elsevier Inc. All rights reserved.)

V2a interneurons are located in the hindbrain and spinal cord, where they provide rhythmic input to major motor control centers. Many of the phenotypic properties and functions of excitatory V2a interneurons have yet to be fully defined. Definition of these properties could lead to novel regenerative therapies for traumatic injuries and drug targets for chronic degenerative diseases. Here we describe how to produce V2a interneurons from mouse and human pluripotent stem cells (PSCs), as well as strategies to characterize and mature the cells for further analysis. The described protocols are based on a sequence of small-molecule treatments that induce differentiation of PSCs into V2a interneurons. We also include a detailed description of how to phenotypically characterize, mature, and freeze the cells. The mouse and human protocols are similar in regard to the sequence of small molecules used but differ slightly in the concentrations and durations necessary for induction. With the protocols described, scientists can expect to obtain V2a interneurons with purities of ~75% (mouse) in 7 d and ~50% (human) in 20 d.

Impact Statement: Understanding the relationship between parenchymal and supporting cell populations is paramount to recapitulate the multicellular complexity of native tissues. Incorporation of stromal cells is widely recognized to be necessary for the stable formation of stem cell-derived cardiac tissues; yet, the types of stromal cells used have varied widely. This study systematically characterized several stromal populations and found that stromal phenotype and morphology was highly variable depending on cell source and exerted differential impacts on cardiac tissue function and induced pluripotent stem cell-cardiomyocyte phenotype. Therefore, the choice of supporting stromal population can differentially impact the phenotypic or functional performance of engineered cardiac tissues.

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

In the original version of this Article, an incorrect DOI number was provided in the Code Availability statement regarding the deposition of the computational model. The correct DOI is 10.5281/zenodo.1413539. This error has been corrected in both the PDF and HTML versions of the Article.

Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent stem cells, organoid biology, organ-on-chip assays, and 3-D bioprinting have all contributed to a heightened interest in the design, assembly, and manufacture of living systems with a broad range of potential uses. This white paper summarizes the state of the emerging field of "multi-cellular engineered living systems," which are composed of interacting cell populations. Recent accomplishments are described, focusing on current and potential applications, as well as barriers to future advances, and the outlook for longer term benefits and potential ethical issues that need to be considered.

Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our in vitro system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference. Mosaic knockdown of ROCK1 or CDH1 resulted in differential patterning within hiPSC colonies due to cellular self-organization, while retaining an epithelial pluripotent phenotype. Knockdown induction stimulates a transient wave of differential gene expression within the mixed populations that stabilized in coordination with observed self-organization. Mosaic patterning enables genetic interrogation of emergent multicellular properties, which can facilitate better understanding of the molecular pathways that regulate symmetry-breaking during morphogenesis., Competing Interests: AL, DJ, PS, JM, FM, VW No competing interests declared, MM Currently an employee of Tenaya Therapeutics. BC On the Scientific Advisory Boards of: CODA Therapeutics, making therapies related to pain relief; Cellogy Inc., a cell dynamics measurement company; and Scientist.com, a scientific services company. Also a founder of Tenaya Therapeutics (https://www.tenayatherapeutics.com/), a company focused on finding treatments for heart failure, including the use of CRISPR interference to interrogate genetic cardiomyopathies. Holds equity in Tenaya, and Tenaya provides research support for heart failure-related research. TM A consultant for Tenaya Therapeutics., (© 2018, Libby et al.)

The initiation of heterogeneity within a population of phenotypically identical progenitors is a critical event for the onset of morphogenesis and differentiation patterning. Gap junction communication within multicellular systems produces complex networks of intercellular connectivity that result in heterogeneous distributions of intracellular signaling molecules. In this study, we investigate emergent systems-level behavior of the intercellular network within embryonic stem cell (ESC) populations and corresponding spatial organization during early neural differentiation. An agent-based model incorporates experimentally-determined parameters to yield complex transport networks for delivery of pro-differentiation cues between neighboring cells, reproducing the morphogenic trajectories during retinoic acid-accelerated mouse ESC differentiation. Furthermore, the model correctly predicts the delayed differentiation and preserved spatial features of the morphogenic trajectory that occurs in response to intercellular perturbation. These findings suggest an integral role of gap junction communication in the temporal coordination of emergent patterning during early differentiation and neural commitment of pluripotent stem cells.

Hydrogels present versatile platforms for the encapsulation and delivery of proteins and cells for regenerative medicine applications. However, differences in hydrogel cross-linking density, polymer weight content, and affinity for proteins all contribute to diverse diffusion rates of proteins through hydrogel networks. Here, we describe a simple method to accurately measure protein diffusion through hydrogels, within a few hours and without the use of large amounts of protein. We tracked the diffusion of several proteins of varying molecular weights along the axial direction of capillary tubes filled with alginate, collagen, or poly(ethylene glycol) hydrogels. The rate of protein diffusion decreased with increasing molecular weight. A computational model of protein diffusion through capillary tubes was also created to predict and verify experimental protein diffusion coefficients. This in vitro capillary tube-based method of measuring protein diffusion represents a simple strategy to interrogate protein diffusion through natural and synthetic hydrogels and aid in the design of better biomaterial-based delivery vehicles that can effectively modulate protein release.

The highly proliferative and pluripotent characteristics of embryonic stem cells engender great promise for tissue engineering and regenerative medicine, but the rapid identification and isolation of target cell phenotypes remains challenging. Therefore, the objectives of this study were to characterize cell mechanics as a function of differentiation and to employ differences in cell stiffness to select population subsets with distinct mechanical, morphological, and biological properties. Biomechanical analysis with atomic force microscopy revealed that embryonic stem cells stiffened within one day of differentiation induced by leukemia inhibitory factor removal, with a lagging but pronounced change from spherical to spindle-shaped cell morphology. A microfluidic device was then employed to sort a differentially labeled mixture of pluripotent and differentiating cells based on stiffness, resulting in pluripotent cell enrichment in the soft device outlet. Furthermore, sorting an unlabeled population of partially differentiated cells produced a subset of "soft" cells that was enriched for the pluripotent phenotype, as assessed by post-sort characterization of cell mechanics, morphology, and gene expression. The results of this study indicate that intrinsic cell mechanical properties might serve as a basis for efficient, high-throughput, and label-free isolation of pluripotent stem cells, which will facilitate a greater biological understanding of pluripotency and advance the potential of pluripotent stem cell differentiated progeny as cell sources for tissue engineering and regenerative medicine.

Protein delivery is often used in tissue engineering applications to control differentiation processes, but is limited by protein instability and cost. An alternative approach is to control the cellular microenvironment through biomaterial-mediated sequestration of cell-secreted proteins important to differentiation. Thus, we utilized heparin-based microparticles to modulate cellular differentiation via protein sequestration in an in vitro model system of endochondral ossification. Heparin and poly(ethylene-glycol) (PEG; a low-binding material control)-based microparticles were incorporated into ATDC5 cell spheroids or incubated with ATDC5 cells in transwell culture. Reduced differentiation was observed in the heparin microparticle group as compared to PEG and no microparticle-containing groups. To determine if observed changes were due to sequestration of cell-secreted protein, the proteins sequestered by heparin microparticles were analyzed using SDS-PAGE and mass spectrometry. It was found that heparin microparticles bound insulin-like growth factor binding proteins (IGFBP)-3 and 5. When incubated with a small-molecule inhibitor of IGFBPs, NBI 31772, a similar delay in differentiation of ATDC5 cells was observed. These results indicate that heparin microparticles modulated chondrocytic differentiation in this system via sequestration of cell-secreted protein, a technique that could be beneficial in the future as a means to control cellular differentiation processes., Statement of Significance: In this work, we present a proof-of-principle set of experiments in which heparin-based microparticles are shown to modulate cellular differentiation through binding of cell-secreted protein. Unlike existing systems that rely on expensive protein with limited half-lives to elicit changes in cellular behavior, this technique focuses on temporal modulation of cell-generated proteins. This technique also provides a biomaterials-based method that can be used to further identify sequestered proteins of interest. Thus, this work indicates that glycosaminoglycan-based biomaterial approaches could be used as substitutes or additions to traditional methods for modulating and identifying the cell-secreted proteins involved in directing cellular behavior., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

This study proposes to use alginate encapsulation as a strategy to assess the paracrine activity of 3D- and 2D-cultured human bone marrow mesenchymal stem/stromal cells (BM MSC) in the setting of wound repair and regeneration processes. A side-by-side comparison of MSC culture in three different 3D configurations (spheroids, encapsulated spheroids and encapsulated single cells) versus 2D monolayer cell culture is presented. The results reveal enhanced resistance to oxidative stress and paracrine potential of 3D spheroid-organized BM MSC. MSC spheroids (148±2μm diameter) encapsulated in alginate microbeads evidence increased angiogenic and chemotactic potential relatively to encapsulated single cells, as supported by higher secreted levels of angiogenic factors and by functional assays showing the capability of encapsulated MSC to promote formation of tubelike structures and migration of fibroblasts into a wounded area. In addition, a higher expression of the anti-inflammatory factor tumor necrosis factor alpha-induced protein 6 (TSG-6) was demonstrated by RT-PCR for encapsulated and non-encapsulated spheroids. Culture of spheroids within an alginate matrix maintains low aggregation levels below 5% and favors resistance to oxidative stress. These are important factors towards the establishment of more standardized and controlled systems, crucial to explore the paracrine effects of 3D-cultured MSC in therapeutic settings., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Gene delivery to primary human cells is a technology of critical interest to both life science research and therapeutic applications. However, poor efficiencies in gene transfer and undesirable safety profiles remain key limitations in advancing this technology. Here, we describe a materials-based approach whereby application of a bioresorbable mineral coating improves microparticle-based transfection of plasmid DNA lipoplexes in several primary human cell types. In the presence of these mineral-coated microparticles (MCMs), we observed up to 4-fold increases in transfection efficiency with simultaneous reductions in cytotoxicity. We identified mechanisms by which MCMs improve transfection, as well as coating compositions that improve transfection in three-dimensional cell constructs. The approach afforded efficient transfection in primary human fibroblasts as well as mesenchymal and embryonic stem cells for both two- and three-dimensional transfection strategies. This MCM-based transfection is an advancement in gene delivery technology, as it represents a non-viral approach that enables highly efficient, localized transfection and allows for transfection of three-dimensional cell constructs.

Bone morphogenetic protein-2 (BMP-2) is an osteoinductive growth factor used clinically to induce bone regeneration and fusion. Some complications associated with BMP-2 treatment have been attributed to rapid release of BMP-2 from conventional collagen scaffolds, motivating the development of tunable sustained-release strategies. We incorporated BMP-2-binding heparin microparticles (HMPs) into a hydrogel scaffold to improve spatiotemporal control of BMP-2 delivery to large bone defects. HMPs pre-loaded with BMP-2 were mixed into alginate hydrogels and compared to hydrogels containing BMP-2 alone. BMP-2 release from scaffolds in vitro, BMP-2 retention within injury sites in vivo, and bone regeneration in a critically sized femoral defect were evaluated. Compared to hydrogel delivery alone, BMP-2-loaded HMPs reduced BMP-2 release in vitro and increased early BMP-2 retention in the bone defect. BMP-2-loaded HMPs induced bone formation at both ectopic and orthotopic sites; however, the volume of induced bone was lower for defects treated with BMP-2-loaded HMPs compared to hydrogel delivery. To better understand the effect of HMPs on BMP-2 release kinetics, a computational model was developed to predict BMP-2 release from constructs in vivo. The model suggested that HMPs limited BMP-2 release into surrounding tissues, and that changing the HMP density could modulate BMP-2 release. Taken together, these experimental and computational results suggest the importance of achieving a balance of BMP-2 retention within the bone defect and BMP-2 release into surrounding soft tissues. HMP delivery of BMP-2 may provide a method of tuning BMP-2 release in vivo that can be further investigated to improve current methods of bone regeneration., Statement of Significance: The development of effective biomaterials for sustained protein delivery is a crucial component of tissue engineering strategies. However, in most applications, including bone repair, the optimal balance between protein presentation in the injury site and protein release into the surrounding tissues is unknown. Herein, we introduced heparin microparticles (HMPs) into a tissue engineered construct to increase in vivo retention of bone morphogenetic protein-2 (BMP-2) and enhance healing in femoral defects. Although HMPs induced bone regeneration, no increase in bone volume was observed, leading to further experimental and computational analysis of the effect of HMP-BMP-2 interactions on protein retention and release. Ultimately, this work provides insight into designing tunable protein-material interactions and their implications for controlling BMP-2 delivery., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Osteogenesis of mesenchymal stem cells (MSC) can be regulated by the mechanical environment. MSCs grown in 3D spheroids (mesenspheres) have preserved multi-lineage potential, improved differentiation efficiency, and exhibit enhanced osteogenic gene expression and matrix composition in comparison to MSCs grown in 2D culture. Within 3D mesenspheres, mechanical cues are primarily in the form of cell-cell contraction, mediated by adhesion junctions, and as such adhesion junctions are likely to play an important role in the osteogenic differentiation of mesenspheres. However the precise role of N- and OB-cadherin on the biomechanical behaviour of mesenspheres remains unknown. Here we have mechanically tested mesenspheres cultured in suspension using parallel plate compression to assess the influence of N-cadherin and OB-cadherin adhesion junctions on the viscoelastic properties of the mesenspheres during osteogenesis. Our results demonstrate that N-cadherin and OB-cadherin have different effects on mesensphere viscoelastic behaviour and osteogenesis. When OB-cadherin was silenced, the viscosity, initial and long term Young's moduli and actin stress fibre formation of the mesenspheres increased in comparison to N-cadherin silenced mesenspheres and mesenspheres treated with a scrambled siRNA (Scram) at day 2. Additionally, the increased viscoelastic material properties correlate with evidence of calcification at an earlier time point (day 7) of OB-cadherin silenced mesenspheres but not Scram. Interestingly, both N-cadherin and OB-cadherin silenced mesenspheres had higher BSP2 expression than Scram at day 14. Taken together, these results indicate that N-cadherin and OB-cadherin both influence mesensphere biomechanics and osteogenesis, but play different roles., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tissue engineering strategies involving the in vivo delivery of recombinant growth factors are often limited by the inability of biomaterials to spatially control diffusion of the delivered protein within the site of interest. The poor spatiotemporal control provided by porous collagen sponges, which are used for the clinical delivery of bone morphogenetic protein-2 (BMP-2) for bone regeneration, has necessitated the use of supraphysiological protein doses, leading to inflammation and heterotopic ossification. This study describes a novel tissue engineering strategy to spatially control rapid BMP-2 diffusion from collagen sponges in vivo by creating a high-affinity BMP-2 sink around the collagen sponge. We designed an electrospun poly-ɛ-caprolactone nanofiber mesh containing physically entrapped heparin microparticles, which have been previously demonstrated to bind and retain large amounts of BMP-2. Nanofiber meshes containing 0.05 and 0.10 mg of microparticles/cm 2 demonstrated increased BMP-2 binding and decreased BMP-2 release in vitro compared with meshes without microparticles. However, when microparticle-containing meshes were used in vivo to limit the diffusion of BMP-2 delivered by using collagen sponges in a rat femoral defect, no differences in heterotopic ossification or biomechanical properties were observed. Further investigation revealed that, although BMP-2 binding to heparin microparticles was rapid, the presence of serum components attenuated microparticle-BMP-2 binding and increased BMP-2 release in vitro. These observations provide a plausible explanation for the results observed in vivo and suggest that competitive protein binding in vivo may hinder the ability of affinity-based biomaterials to modulate growth factor delivery.

The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however, the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here, we report the directed differentiation of CHX10 + V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid, sonic hedgehog, and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ∼25% CHX10 + cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time, CHX10 + cells expressed neuronal markers [neurofilament, NeuN, and vesicular glutamate transporter 2 (VGlut2)], and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10 + cells within the differentiated population, which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice, hPSC-derived V2a cultures survived at the site of injection, coexpressed NeuN and VGlut2, extended neurites >5 mm, and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI., Competing Interests: The authors declare no conflict of interest.