Distinct differentiation trajectories leave lasting impacts on gene regulation and function of V2a interneurons.

During development, early regionalization segregates lineages and directs diverse cell fates. Sometimes, however, distinct progenitors produce analogous cell types. For example, V2a neurons, are excitatory interneurons that emerge from different anteroposterior progenitors.

CDX2 dose-dependently influences the gene regulatory network underlying human extraembryonic mesoderm development.

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.

TBXT dose sensitivity and the decoupling of nascent mesoderm specification from EMT progression in 2D human gastruloids.

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.

CDX2 dose-dependently influences the gene regulatory network underlying human extraembryonic mesoderm development.

Proper regulation of gene dosage is critical for the development of the early embryo and the extraembryonic tissues that support it. Specifically, loss of leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. In this study, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development.

Human spinal interneurons repair the injured spinal cord through synaptic integration.

Advances in cell therapy offer promise for some of the most devastating neural injuries, including spinal cord injury (SCI). Endogenous VSX2-expressing spinal V2a interneurons have been implicated as a key component in plasticity and therapeutically driven recovery post-SCI. While transplantation of generic V2a neurons may have therapeutic value, generation of human spinal V2a neurons with rostro-caudal specificity and assessment of their functional synaptic integration with the injured spinal cord has been elusive.

TBXT dose sensitivity and the decoupling of nascent mesoderm specification from EMT progression in 2D human gastruloids.

In the nascent mesoderm, levels of Brachyury (TBXT) expression must be precisely regulated to ensure cells exit the primitive streak and pattern the anterior-posterior axis, but how this varying dosage informs morphogenesis is not well understood. In this study, we define the transcriptional consequences of TBXT dose reduction during early human gastrulation using human induced pluripotent stem cell (hiPSC)-based models of gastrulation and mesoderm differentiation. Multiomic single-nucleus RNA and single-nucleus ATAC sequencing of 2D gastruloids comprised of WT, TBXT heterozygous (TBXT-Het), or TBXT null (TBXT-KO) hiPSCs reveal that varying TBXT dosage does not compromise a cell's ability to differentiate into nascent mesoderm, but that the loss of TBXT significantly delays the temporal progression of the epithelial to mesenchymal transition (EMT).

Hepatitis C virus infects and perturbs liver stem cells.

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.

Loss of TJP1 disrupts gastrulation patterning and increases differentiation toward the germ cell lineage in human pluripotent stem cells.

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).

Diseased, differentiated and difficult: Strategies for improved engineering of neurological systems.

The rapidly growing field of cellular engineering is enabling scientists to more effectively create 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.

Engineering multicellular living systems-a Keystone Symposia report.

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.

Self-Assembled Heterotypic Cardiac Spheroids from Human Pluripotent Stem 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.

Modelling T-cell immunity against hepatitis C virus with liver organoids in a microfluidic coculture system.

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 animal models are limited and 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.

Co-emergence of cardiac and gut tissues promotes cardiomyocyte maturation within human iPSC-derived organoids.

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.

Allele-Specific Gene Editing Rescues Pathology in a Human Model of Charcot-Marie-Tooth Disease Type 2E.

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.

Passive Clearing and 3D Lightsheet Imaging of the Intact and Injured Spinal Cord in Mice.

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.

Bioengineered optogenetic model of human neuromuscular junction.

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.

Developmental lineage of human pluripotent stem cell-derived cardiac fibroblasts affects their functional phenotype.

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.

Axial elongation of caudalized human organoids mimics aspects of neural tube development.

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.

Deep neural net tracking of human pluripotent stem cells reveals intrinsic behaviors directing morphogenesis.

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.

SARS-CoV-2 infection of human iPSC-derived cardiac cells reflects cytopathic features in hearts of patients with COVID-19.

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.

Spinal Interneurons as Gatekeepers to Neuroplasticity after Injury or Disease.

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.

Dimethyloxalylglycine, a small molecule, synergistically increases the homing and angiogenic properties of human mesenchymal stromal cells when cultured as 3D spheroids.

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 ) or to (ii) a hypoxic-mimetic small molecule, dimethyloxalylglycine (DMOG), with cells cultured at 21% O .

Engineering the Spatiotemporal Mosaic Self-Patterning of Pluripotent Stem Cells.

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.