Engineering large-scale hiPSC-derived vessel-integrated muscle-like lattices for enhanced volumetric muscle regeneration.

Engineering biomimetic tissue implants with human induced pluripotent stem cells (hiPSCs) holds promise for repairing volumetric tissue loss. However, these implants face challenges in regenerative capability, survival, and geometric scalability at large-scale injury sites. Here, we present scalable vessel-integrated muscle-like lattices (VMLs), containing dense and aligned hiPSC-derived myofibers alongside passively perfusable vessel-like microchannels inside an endomysium-like supporting matrix using an embedded multimaterial bioprinting technology. The contractile and millimeter-long myofibers are created in mechanically tailored and nanofibrous extracellular matrix-based hydrogels. Incorporating vessel-like lattice enhances myofiber maturation in vitro and guides host vessel invasion in vivo, improving implant integration. Consequently, we demonstrate successful de novo muscle formation and muscle function restoration through a combinatorial effect between improved graft-host integration and its increased release of paracrine factors within volumetric muscle loss injury models. The proposed modular bioprinting technology enables scaling up to centimeter-sized prevascularized hiPSC-derived muscle tissues with custom geometries for next-generation muscle regenerative therapies.

The universe is asymmetric, the mouse brain too.

Hemispheric brain asymmetry is a basic organizational principle of the human brain and has been implicated in various psychiatric conditions, including autism spectrum disorder. Brain asymmetry is not a uniquely human feature and is observed in other species such as the mouse. Yet, asymmetry patterns are generally nuanced, and substantial sample sizes are required to detect these patterns. In this pre-registered study, we use a mouse dataset from the Province of Ontario Neurodevelopmental Network, which comprises structural MRI data from over 2000 mice, including genetic models for autism spectrum disorder, to reveal the scope and magnitude of hemispheric asymmetry in the mouse. Our findings demonstrate the presence of robust hemispheric asymmetry in the mouse brain, such as larger right hemispheric volumes towards the anterior pole and larger left hemispheric volumes toward the posterior pole, opposite to what has been shown in humans. This suggests the existence of species-specific traits. Further clustering analysis identified distinct asymmetry patterns in autism spectrum disorder models, a phenomenon that is also seen in atypically developing participants. Our study shows potential for the use of mouse models to understand the biological bases of typical and atypical brain asymmetry but also warrants caution as asymmetry patterns seem to differ between humans and mice.

Control of epiblast cell fate by mechanical cues.

In amniotes, embryonic tissues originate from multipotent epiblast cells, arranged in a mosaic of presumptive territories. How these domains fated to specific lineages become segregated during body formation remains poorly understood. Using single cell RNA sequencing analysis and lineage tracing in the chicken embryo, we identify epiblast cells contributing descendants to the neural tube, somites and lateral plate after completion of gastrulation. We show that intercalation after cell division generates important movements of epiblast cells which lead to their relocation to different presumptive territories, explaining this broad spectrum of fates. This tissue remodeling phase is transient, being soon followed by the establishment of boundaries restricting cell movements therefore defining the presumptive territories of the epiblast. Finally, we find that the epiblast faces distinct mechanical constraints along the antero-posterior axis, leading to cell fate alterations when challenged. Together, we demonstrate the critical role of mechanical cues in epiblast fate determination.

A human pluripotent stem cell-based somitogenesis model using microfluidics.

Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated biochemical and biomechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Herein, we develop a human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on the PSM tissues cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and the PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the biochemical and biomechanical events that guide somite formation.

Mapping mouse axial progenitor dynamics in vitro.

In this issue of Developmental Cell, Bolondi et al. systematically assesses neuro-mesodermal progenitor (NMP) dynamics by combining a mouse stem-cell-based embryo model with molecular recording of single cells, shedding light on the dynamics of neural tube and paraxial mesoderm formation during mammalian development.

Engineering Stem Cell Fate Controlling Biomaterials to Develop Muscle Connective Tissue Layered Myofibers.

Skeletal muscle connective tissue (MCT) surrounds myofiber bundles to provide structural support, produce force transduction from tendons, and regulate satellite cell differentiation during muscle regeneration. Engineered muscle tissue composed of myofibers layered within MCT has not yet been developed. Herein, a bioengineering strategy to create MCT-layered myofibers through the development of stem cell fate-controlling biomaterials that achieve both myogenesis and fibroblast differentiation in a locally controlled manner at the single construct is introduced. The reciprocal role of transforming growth factor-beta 1 (TGF-ß1) and its inhibitor as well as 3D matrix stiffness to achieve co-differentiation of MCT fibroblasts and myofibers from a human-induced pluripotent stem cell (hiPSC)-derived paraxial mesoderm is studied. To avoid myogenic inhibition, TGF-ß1 is conjugated on the gelatin-based hydrogel to control the fibroblasts' populations locally; the TGF-ß1 degrades after 2 weeks, resulting in increased MCT-specific extracellular matrix (ECM) production. The locations of myofibers and fibroblasts are precisely controlled by using photolithography and co-axial wet spinning techniques, which results in the formation of MCT-layered functional myofibers in 3D constructs. This advanced engineering strategy is envisioned as a possible method for obtaining biomimetic human muscle grafts for various biomedical applications.

Cellular and molecular control of vertebrate somitogenesis.

Segmentation is a fundamental feature of the vertebrate body plan. This metameric organization is first implemented by somitogenesis in the early embryo, when paired epithelial blocks called somites are rhythmically formed to flank the neural tube. Recent advances in in vitro models have offered new opportunities to elucidate the mechanisms that underlie somitogenesis. Notably, models derived from human pluripotent stem cells introduced an efficient proxy for studying this process during human development. In this Review, we summarize the current understanding of somitogenesis gained from both in vivo studies and in vitro studies. We deconstruct the spatiotemporal dynamics of somitogenesis into four distinct modules: dynamic events in the presomitic mesoderm, segmental determination, somite anteroposterior polarity patterning, and epithelial morphogenesis. We first focus on the segmentation clock, as well as signalling and metabolic gradients along the tissue, before discussing the clock and wavefront and other models that account for segmental determination. We then detail the molecular and cellular mechanisms of anteroposterior polarity patterning and somite epithelialization.

Direct reprogramming of non-limb fibroblasts to cells with properties of limb progenitors.

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors.

Modeling Human Paraxial Mesoderm Development with Pluripotent Stem Cells.

Paraxial mesoderm in the early embryo is segmented into epithelial blocks called somites that establish the metameric organization of the vertebrate body plan. Somites are sequentially formed from head to tail in a rhythmic manner controlled by an oscillating gene regulatory network known as the segmentation clock. We know very little about this important process during human development due to limited access to human embryos and ethical concerns. To bypass these difficulties, model systems derived from human pluripotent stem cells have been established. Here, we detail three protocols modeling different aspects of human paraxial mesoderm development in vitro: a 2D cell monolayer system recapitulating dynamics of the human segmentation clock, a 3D organoid system called "somitoid" supporting the simultaneous formation of somite-like structures, and another organoid system called "segmentoid" reconstituting in vivo-like hallmarks of somitogenesis. Together, these complementary model systems provide an excellent platform to decode somitogenesis and advance human developmental biology.

A human pluripotent stem cell-based somitogenesis model using microfluidics.

Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated bio-chemical and -mechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Here we report a new human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on PSM cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the bio-chemical and -mechanical events that guide somite formation.

The Clockwork Embryo: Mechanisms Regulating Developmental Rate.

Organismal development requires the reproducible unfolding of an ordered sequence of discrete steps (cell fate determination, migration, tissue folding, etc.) in both time and space. Here, we review the mechanisms that grant temporal specificity to developmental steps, including molecular clocks and timers. Individual timing mechanisms must be coordinated with each other to maintain the overall developmental sequence. However, phenotypic novelties can also arise through the modification of temporal patterns over the course of evolution. Two main types of variation in temporal patterning characterize interspecies differences in developmental time: allochrony, where the overall developmental sequence is either accelerated or slowed down while maintaining the relative duration of individual steps, and heterochrony, where the duration of specific developmental steps is altered relative to the rest. New advances in in vitro modeling of mammalian development using stem cells have recently enabled the revival of mechanistic studies of allochrony and heterochrony. In both cases, differences in the rate of basic cellular functions such as splicing, translation, protein degradation, and metabolism seem to underlie differences in developmental time. In the coming years, these studies should identify the genetic differences that drive divergence in developmental time between species.

Reconstructing human brown fat developmental trajectory in vitro.

Brown adipocytes (BAs) represent a specialized cell type that is able to uncouple nutrient catabolism from ATP generation to dissipate energy as heat. In humans, the brown fat tissue is composed of discrete depots found throughout the neck and trunk region. BAs originate from a precursor common to skeletal muscle, but their developmental trajectory remains poorly understood. Here, we used single-cell RNA sequencing to characterize the development of interscapular brown fat in mice. Our analysis identified a transient stage of BA differentiation characterized by the expression of the transcription factor GATA6. We show that recapitulating the sequence of signaling cues identified in mice can lead to efficient differentiation of BAs in vitro from human pluripotent stem cells. These precursors can in turn be efficiently converted into functional BAs that can respond to signals mimicking adrenergic stimuli by increasing their metabolism, resulting in heat production.

Chemical QuantArray: A Quantitative Tool for Mass Spectrometry Imaging.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is a powerful analytical technique that provides spatially preserved detection and quantification of analytes in tissue specimens. However, clinical translation still requires improved throughput, precision, and accuracy. To accomplish this, we created "Chemical QuantArray", a gelatin tissue microarray (TMA) mold filled with serial dilutions of isotopically labeled endogenous metabolite standards. The mold is then cryo-sectioned onto a tissue homogenate to produce calibration curves. To improve precision and accuracy, we automatically remove pixels outside of each TMA well and investigated several intensity normalizations, including the utilization of a second stable isotope internal standard (IS). Chemical QuantArray enables the quantification of several endogenous metabolites over a wide dynamic range and significantly improve over current approaches. The technique reduces the space needed on the MALDI slides for calibration standards by approximately 80%. Furthermore, removal of empty pixels and normalization to an internal standard or matrix peak provided precision (<20% RSD) and accuracy (<20% DEV). Finally, we demonstrate the applicability of Chemical QuantArray by quantifying multiple purine metabolites in 14 clinical tumor specimens using a single MALDI slide. Chemical QuantArray improves the analytical characteristics and practical feasibility of MALDI-MSI metabolite quantification in clinical and translational applications.

Direct force measurement and loading on developing tissues in intact avian embryos.

Developmental morphogenesis is driven by tissue stresses acting on tissue rheology. Direct measurements of forces in small tissues (100 µm-1 mm) in situ, such as in early embryos, require high spatial precision and minimal invasiveness. Here, we introduce a control-based approach, tissue force microscopy (TiFM), that integrates a mechanical cantilever probe and live imaging with closed-loop feedback control of mechanical loading in early chicken embryos. By testing previously qualitatively characterized force-producing tissues in the elongating body axis, we show that TiFM quantitatively captures stress dynamics with high sensitivity. TiFM also provides the means to apply stable, minimally invasive and physiologically relevant loads to drive tissue deformation and to follow the resulting morphogenetic progression associated with large-scale cell movements. Together, TiFM allows us to control tissue force measurement and manipulation in small developing embryos, and promises to contribute to the quantitative understanding of complex multi-tissue mechanics during development.

Metabolic regulation of species-specific developmental rates.

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1-3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.

Reconstruction and deconstruction of human somitogenesis in vitro.

The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system1-4. How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial 'salt and pepper' expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development.