Mesenchymal Condensation Research Articles

Selection of Force Sensors for In Situ Measurement of Neotissue Microenvironments.

Mechanical forces are a critical stimulus in both native and engineered tissues. Direct measurement of these microenvironmental forces has been challenging, particularly for cell-dense models. To address this, we previously developed hydrogel-based force sensors that are approximately the size of a cell and can be imaged over time to computationally assess the forces exerted by surrounding cells and matrix. The goal of this project was to identify how the physical characteristics of force sensors impact measurements. Sensors were varied in size, elastic modulus, and surface coating before being included in stem cell suspensions that then spontaneously self-assembled into spheroidal neotissues. Using this model of early mesenchymal condensation, we hypothesized that larger, softer sensors would provide greater sensitivity and precision, whereas protein coatings would influence the directionality of applied forces (tensile vs. compressive). These experiments were conducted using a high-content imaging system that allowed analysis of over a thousand sensors to evaluate the various conditions. Results indicated that measurement fidelity was highest for force sensors that had a diameter >20 µm and modulus ∼0.2 kPa. Extremely soft sensors deformed too much, whereas stiffer sensors deformed too little. Collagen and N-cadherin coatings, which replicated cell-matrix or cell-cell binding, respectively, allowed for tensile forces to be exerted on the sensors, with greater forces being observed for N-cadherin sensors in these highly cellular neotissue constructs. Uncoated sensors were universally compressed due to the lack of cell-sensor adhesion. Disruption of the actin cytoskeleton lessened microenvironmental forces, whereas disruption of microtubules had no measurable effect. Potential future applications of the technology include studies of in situ forces in developing tissues as well as a real-time sensor for monitoring the growth of engineered constructs.

Mutations in fibronectin dysregulate chondrogenesis in skeletal dysplasia.

Fibronectin (FN) is an extracellular matrix glycoprotein essential for the development and function of major vertebrate organ systems. Mutations in FN result in an autosomal dominant skeletal dysplasia termed corner fracture-type spondylometaphyseal dysplasia (SMDCF). The precise pathomechanisms through which mutant FN induces impaired skeletal development remain elusive. Here, we have generated patient-derived induced pluripotent stem cells as a cell culture model for SMDCF to investigate the consequences of FN mutations on mesenchymal stem cells (MSCs) and their differentiation into cartilage-producing chondrocytes. In line with our previous data, FN mutations disrupted protein secretion from MSCs, causing a notable increase in intracellular FN and a significant decrease in extracellular FN levels. Analyses of plasma samples from SMDCF patients also showed reduced FN in circulation. FN and endoplasmic reticulum (ER) protein folding chaperones (BIP, HSP47) accumulated in MSCs within ribosome-covered cytosolic vesicles that emerged from the ER. Massive amounts of these vesicles were not cleared from the cytosol, and a smaller subset showed the presence of lysosomal markers. The accumulation of intracellular FN and ER proteins elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing revealed a specific transcriptomic dysregulation of the patient-derived cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation, reduced chondrogenic markers, and compromised cell proliferation in mutant cells. Moreover, FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGFβ1) expression, crucial for mesenchymal condensation. Exogenous FN or TGFβ1 supplementation effectively improved the MSC condensation and promoted chondrogenesis in FN mutant cells. These findings demonstrate the cellular consequences of FN mutations in SMDCF and explain the molecular pathways involved in the associated altered chondrogenesis.

Wnt5a and Notum Influence the Temporal Dynamics of Cartilaginous Mesenchymal Condensations in Developing Trachea.

The trachea is essential for proper airflow to the lungs for gas exchange. Frequent congenital tracheal malformations affect the cartilage, causing the collapse of the central airway during the respiratory cycle. We have shown that Notum, a Wnt ligand de-acylase that attenuates the canonical branch of the Wnt signaling pathway, is necessary for cartilaginous mesenchymal condensations. In Notum deficient tracheas, chondrogenesis is delayed, and the tracheal lumen is narrowed. It is unknown if Notum attenuates non-canonical Wnt signaling. Notably, we observed premature tracheal chondrogenesis after mesenchymal deletion of the non-canonical Wnt5a ligand. We hypothesize that Notum and Wnt5a are required to mediate the timely formation of mesenchymal condensations, giving rise to the tracheal cartilage. Ex vivo culture of tracheal tissue shows that chemical inhibition of the Wnt non-canonical pathway promotes earlier condensations, while Notum inhibition presents delayed condensations. Furthermore, non-canonical Wnt induction prevents the formation of cartilaginous mesenchymal condensations. On the other hand, cell-cell interactions among chondroblasts increase in the absence of mesenchymal Wnt5a. By performing an unbiased analysis of the gene expression in Wnt5a and Notum deficient tracheas, we detect that mRNA of genes essential for chondrogenesis and extracellular matrix formation are upregulated by E11.5 in Wnt5a mutants. The expression profile supports the premature and delayed chondrogenesis observed in Wnt5a and Notum deficient tracheas, respectively. We conclude that Notum and Wnt5a are necessary for proper tracheal cartilage patterning by coordinating timely chondrogenesis. Thus, these studies shed light on molecular mechanisms underlying congenital anomalies of the trachea.

Odontogenesis-Empowered Extracellular Vesicles Safeguard Donor-Recipient Stem Cell Interplay to Support Tooth Regeneration.

Harnessing the developmental events of mesenchymal condensation to direct postnatal dental stem cell aggregation represents a cutting-edge and promising approach to tooth regeneration. Tooth avulsion is among the most prevalent and serious dental injuries, and odontogenic aggregates assembled by stem cells from human exfoliated deciduous teeth (SHED) have proven effective in revitalizing avulsed teeth after replantation in the clinical trial. However, whether and how SHED aggregates (SA) communicate with recipient components and promote synergistic tissue regeneration to support replanted teeth remains elusive. Here, it is shown that SA-mediated avulsed tooth regeneration involves periodontal restoration and recovery of recipient Gli1+ stem cells, which are mobilized and necessarily contribute to the reestablishment of the tooth-periodontal ligament-bone interface. Mechanistically, the release of extracellular vesicles (EVs) is revealed indispensable for the implanted SA to mobilize recipient Gli1+ cells and regenerate avulsed teeth. Furthermore, SHED aggregates-released EVs (SA-EVs) are featured with odontogenic properties linked to tissue regeneration, which enhance migration, proliferation, and differentiation of Gli1+ cells. Importantly, local application of SA-EVs per se empowers recipient Gli1+ cells and safeguards regeneration of avulsed teeth. Collectively, the findings establish a paradigm in which odontogenesis-featured EVs govern donor-recipient stem cell interplay to achieve tooth regeneration, inspiring cell-free translational regenerative strategies.

Theory and practice of mesenchymal condensation directing tooth development and regeneration

Mesenchymal stem cells, under spatiotemporal regulation of genes and microenvironment, are capable of spontaneously aggregating into dense regions, a phenomenon known as mesenchymal condensation. Mesenchymal condensation is an evolutionarily conserved developmental event that is critical in initiating morphogenesis of teeth and systemic organs. Mesenchymal stem cells hold the intrinsic ability to self-assemble in culture, and the generation of stem cell aggregates based on this property that mimics developmental mesenchymal condensation has become a potent and promising approach in regenerative medicine. This review discusses the mesenchymal condensation principles and its role as well as mechanism in tooth morphogenesis, as well as the engineering strategies for constructing mesenchymal stem cell aggregates and their application experience in tooth regeneration. It aims to start from the perspective of "development-inspired regeneration" and provide insights into understanding stem cell developmental biology and establishing new organ regenerative strategies.

Comparison of knee joint and temporomandibular joint development in pig embryos

Although the knee joint (KNJ) and temporomandibular joint (TMJ) all belong to the synovial joint, there are many differences in developmental origin, joint structure and articular cartilage type. Studies of joint development in embryos have been performed, mainly using poultry and rodents. However, KNJ and TMJ in poultry and rodents differ from those in humans in several ways. Very little work has been done on the embryonic development of KNJ and TMJ in large mammals. Several studies have shown that pigs are ideal animals for embryonic development research. Embryonic day 30 (E30), E35, E45, E55, E75, E90, Postnatal day 0 (P0) and Postnatal day 30 (P30) embryos/fetuses from the pigs were used for this study. The results showed that KNJ develops earlier than TMJ. Only one mesenchymal condensate of KNJ is formed on E30, while two mesenchymal condensates of TMJ are present on E35. All structures of KNJ and TMJ were formed on E45. The growth plate of KNJ begins to develop on E45 and becomes more pronounced from E55 to P30. From E75 to E90, more and more vascular-rich cartilage canals form in the cartilage regions of both joints. The cartilaginous canal of the TMJ divides the condyle into sections along the longitudinal axis of the condyle. This arrangement of cartilaginous canal was not found in the KNJ. The chondrification of KNJ precedes that of TMJ. Ossification of the knee condyle occurs gradually from the middle to the periphery, while that of the TMJ occurs gradually from the base of the mandibular condyle. In the KNJ, the ossification of the articular condyle is evident from P0 to P30, and the growth plate is completely formed on P30. In the TMJ, the cartilage layer of condyle becomes thinner from P0 to P30. There is no growth plate formation in TMJ during its entire development. There is no growth plate formation in the TMJ throughout its development. The condyle may be the developmental center of the TMJ. The chondrocytes and hypertrophic chondrocytes of the growth plate are densely arranged. The condylar chondrocytes of TMJ are scattered, while the hypertrophic chondrocytes are arranged. Embryonic development of KNJ and TMJ in pigs is an important bridge for translating the results of rodent studies to medical applications.

The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling

The development of craniofacial skeletal structures is fascinatingly complex and elucidation of the underlying mechanisms will not only provide novel scientific insights, but also help develop more effective clinical approaches to the treatment and/or prevention of the numerous congenital craniofacial malformations. To this end, we performed a genome-wide analysis of RNA transcription from non-coding regulatory elements by CAGE-sequencing of the facial mesenchyme of human embryos and cross-checked the active enhancers thus identified against genes, identified by GWAS for the normal range human facial appearance. Among the identified active cis-enhancers, several belonged to the components of the PI3/AKT/mTORC1/autophagy pathway. To assess the functional role of this pathway, we manipulated it both genetically and pharmacologically in mice and zebrafish. These experiments revealed that mTORC1 signaling modulates craniofacial shaping at the stage of skeletal mesenchymal condensations, with subsequent fine-tuning during clonal intercalation. This ability of mTORC1 pathway to modulate facial shaping, along with its evolutionary conservation and ability to sense external stimuli, in particular dietary amino acids, indicate that the mTORC1 pathway may play a role in facial phenotypic plasticity. Indeed, the level of protein in the diet of pregnant female mice influenced the activity of mTORC1 in fetal craniofacial structures and altered the size of skeletogenic clones, thus exerting an impact on the local geometry and craniofacial shaping. Overall, our findings indicate that the mTORC1 signaling pathway is involved in the effect of environmental conditions on the shaping of craniofacial structures.

Cochlear aqueduct revisited: A histological study using human fetuses

Background and aimThe cochlear aqueduct (CA) connects between the perilymphatic space of the cochlea and the subarachnoid space in the posterior cranial fossa. The study aimed to examine 1) whether cavitation of the CA occurs on the subarachnoid side or the cochlear side and 2) the growth and/or degeneration of the CA and its concomitant vein. MethodsWe examined paraffin-embedded histological sections from human fetuses: 15 midterm fetuses (crown-rump length or CRL, 39–115 mm) and 12 near-term fetuses (CRL, 225–328 mm). ResultsA linear mesenchymal condensation, i.e., a likely candidate of the CA anlage, was observed without the accompanying vein at 9–10 weeks. The vein appeared until 15 weeks, but it was sometimes distant from the CA. At 10–12 weeks, the subarachnoid space (or the epidural space) near the glossopharyngeal nerve rapidly protruded into the CA anlage and reached the scala tympani, in which cavitation was gradually on-going but without epithelial lining. However, CA cavitation did not to occur in the anlage. At the opening to the scala, the epithelial-like lining of the CA lost its meningeal structure. At near-term, the CA was often narrowed and obliterated. ConclusionThe CA develops from meningeal tissues when the cavitation of the scala begins. The latter cavitation seemed to reduce tissue stiffness leading, to meningeal protrusion. The so-called anlage of CA might be a phylogenetic remnant of the glossopharyngeal nerve branch. A course of cochlear veins appears to be determined by a rule different from the CA development.

An Advanced Bioconjugation Strategy for Covalent Tethering of TGFβ3 with Silk Fibroin Matrices and its Implications in the Chondrogenesis Profile of Human BMSCs and Human Chondrocytes: A Paradigm Shift in Cartilage Tissue Engineering.

The transforming growth factor-β class of cytokines plays a significant role in articular cartilage formation from mesenchymal condensation to chondrogenic differentiation. However, their exogenous addition to the chondrogenic media makes the protocol expensive. It reduces the bioavailability of the cytokine to the cells owing to their burst release. The present study demonstrates an advanced bioconjugation strategy to conjugate transforming growth factor-β3 (TGFβ3) with silk fibroin matrix covalently via a cyanuric chloride coupling reaction. The tethering and change in secondary conformation are confirmed using various spectroscopic analyses. To assess the functionality of the chemically modified silk matrix, human bone marrow-derived mesenchymal stem cells (hBMSCs) and chondrocytes are cultured for 28 days in a chondrogenic differentiation medium. Gene expression and histological analysis reveal enhanced expression of chondrogenic markers with intense Safranin-O and Alcian Blue staining in TGFβ3 conjugated silk matrices than where TGFβ3 is exogenously added to the media for both hBMSCs and chondrocytes. Therefore, this study successfully recapitulates the native niche of TGFβ3 and the role of the silk as a growth factor stabilizer. When cultured over TGFβ3 conjugated silk matrices, hBMSCs display increased proteoglycan secretion and maximum chondrogenic trait with attenuation of chondrocyte hypertrophy over human chondrocytes.

Isthmin-1: A critical regulator of branching morphogenesis and metanephric mesenchyme condensation during early kidney development.

Isthmin-1 (Ism1) was first described to be syn-expressed with Fgf8 inXenopus. However, its biological role has not been elucidated until recent years. Despite of accumulated evidence that Ism1 participates in angiogenesis, tumor invasion, macrophage apoptosis, and glucose metabolism, the cognate receptors for Ism1 remain largely unknown. Ism1 deficiency in mice results in renal agenesis (RA) with a transient loss of Gdnf transcription and impaired mesenchyme condensation at E11.5. Ism1 binds to and activates Integrin α8β1 to positively regulate Gdnf/Ret signaling, thus promoting mesenchyme condensation and ureteric epithelium branching morphogenesis. Here, we propose the hypothesis underlying the mechanism by which Ism1 regulates branching morphogenesis during early kidney development.

Endochondral Repair of Jawbone Defects Using Periosteal Cell Spheroids

Recapitulation of the natural healing process is receiving increasing recognition as a strategy to induce robust tissue regeneration. Endochondral ossification has been recognized as an essential reparative approach in natural jawbone defect healing. However, such an approach has been overlooked in the recent development of cell-based therapeutics for jawbone repair. Therefore, this study aimed to explore a bioinspired stem cell–based strategy for jawbone repair by mimicking the mesenchymal condensation of progenitor cells during the early endochondral ossification process. For this purpose, passage 3 of jawbone periosteum-derived cells (jb-PDCs) was cultured in our previously reported nonadherent microwells (200 µm in diameter, 148 µm in depth, and 100 µm space in between) and self-assembled into spheroids with a diameter of 96.4 ± 5.8 µm after 48 h. Compared to monolayer culture, the jb-PDC spheroids showed a significant reduction of stemness marker expression evidenced by flow cytometry. Furthermore, a significant upregulation of chondrogenic transcription factor SOX9 in both gene and protein levels was observed in the jb-PDC spheroids after 48 h of chondrogenic induction. RNA sequencing and Western blotting analysis further suggested that the enhanced SOX9-mediated chondrogenic differentiation in jb-PDC spheroids was attributed to the activation of the p38 MAPK pathway. Impressively, inhibition of p38 kinase activity significantly attenuated chondrogenic differentiation jb-PDC spheroids, evidenced by a significant decline of SOX9 in both gene and protein levels. Strikingly, the jb-PDC spheroids implanted in 6- to 8-wk-old male C57BL/6 mice with critical-size jawbone defects (1.8 mm in diameter) showed an evident contribution to cartilaginous callus formation after 1 wk, evidenced by histological analysis. Furthermore, micro–computed tomography analysis showed that the jb-PDC spheroids significantly accelerated bone healing after 2 wk in the absence of exogenous growth factors. In sum, the presented findings represent the successful development of cell-based therapeutics to reengineer the endochondral bone repair process and illustrate the potential application to improve bone repair and regeneration in the craniofacial skeleton.

Wnt signaling regulates ion channel expression to promote smooth muscle and cartilage formation in developing mouse trachea.

Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. Tracheobronchomalacia (TBM) and complete tracheal rings (CTR) are disorders affecting the muscle and cartilage of the trachea and bronchi, whose etiology remains poorly understood. We demonstrated that trachealis muscle organization and polarity are disrupted after epithelial ablation of Wntless (Wls), a cargo receptor critical for the Wnt signaling pathway, in developing trachea. The phenotype resembles the anomalous trachealis muscle observed after deletion of ion channel encoding genes in developing mouse trachea. We sought to investigate whether and how the deletion of Wls affects ion channels during tracheal development. We hypothesize that Wnt signaling influences the expression of ion channels to promote trachealis muscle cell assembly and patterning. Deleting Wls in developing trachea causes differential regulation of genes mediating actin binding, cytoskeleton organization, and potassium ion channel activity. Wnt signaling regulates the expression of Kcnj13, Kcnd3, Kcnj8, and Abcc9 as demonstrated by in vitro studies and in vivo analysis in Wnt5a and β-catenin-deficient tracheas. Pharmacological inhibition of potassium ion channels and Wnt signaling impaired contractility of developing trachealis smooth muscle and formation of cartilaginous mesenchymal condensation. Thus, in mice, epithelial-induced Wnt/β-catenin signaling mediates trachealis muscle and cartilage development via modulation of ion channel expression, promoting trachealis muscle architecture, contractility, and cartilaginous extracellular matrix. In turn, ion channel activity may influence tracheal morphogenesis underlying TBM and CTR.NEW & NOTEWORTHY Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. In this study, we focused on the role of ion channels in the differentiation and patterning of the large airways of the developing respiratory tract. We identify a mechanism by which Wnt-beta-catenin signaling controls levels of ion channel-encoding genes to promote tracheal differentiation.

A two-galectin network establishes mesenchymal condensation phenotype in limb development

Previous work showed that Gal-1A and Gal-8, two proteins belonging to the galactoside-binding galectin family, are the earliest determinants of the patterning of the skeletal elements of embryonic chicken limbs, and further, that their experimentally determined interactions in the embryonic limb bud can be interpreted via a reaction–diffusion–adhesion (2GL: two galectin plus ligands) model. Here, we use an ordinary differential equation-based approach to analyze the intrinsic switching modality of the 2GL network and characterize the network behavior independent of the diffusive and adhesive arms of the patterning mechanism. We identify two states: where the concentrations of both the galectins are respectively, negligible, and very high. This bistable switch-like system arises via a saddle–node bifurcation from a monostable state. For the case of mass-action production terms, we provide an explicit Lyapunov function for the system, which shows that it has no periodic solutions. Our model therefore predicts that the galectin network may exist in low expression and high expression states separated in space or time, without any intermediate states. We test these predictions in experiments performed with high density cultures of chick limb mesenchymal cells and observe that cells inside precartilage protocondensations express Gal-1A at a much higher rate than those outside, for which it was negligible. The Gal-1A and -8-based patterning network is therefore sufficient to partition the mesenchymal cell population into two discrete cell states with different developmental (chondrogenic vs. non-chondrogenic) fates. When incorporated into an adhesion and diffusion-enabled framework this system can generate a spatially patterned limb skeleton.

Isthmin-1 (Ism1) modulates renal branching morphogenesis and mesenchyme condensation during early kidney development

The outgrowth of epithelial bud followed by reiterated bifurcations during renal development is driven by the ligand-receptor interactions between the epithelium and the surrounding mesenchyme. Here, by exploring ligand-receptor interactions in E10.5 and E11.5 kidneys by single cell RNA-seq, we find that Isthmin1 (Ism1), a secreted protein, resembles Gdnf expression and modulates kidney branching morphogenesis. Mice deficient for Ism1 exhibit defective ureteric bud bifurcation and impaired metanephric mesenchyme condensation in E11.5 embryos, attributable to the compromised Gdnf/Ret signaling, ultimately leading to renal agenesis and hypoplasia/dysplasia. By HRP-induced proximity labelling, we further identify integrin α8β1 as a receptor of Ism1 in E11.5 kidney and demonstrate that Ism1 promoted cell-cell adhesion through interacting with Integrin α8β1, the receptor whose activation is responsible for Gdnf expression and mesenchyme condensation. Taken together, our work reveals Ism1 as a critical regulator of cell-cell interaction that modulates Gdnf/Ret signaling during early kidney development.

Mesenchymal Stem Cell Aggregation-Released Extracellular Vesicles Induce CD31+ EMCN+ Vessels in Skin Regeneration and Improve Diabetic Wound Healing.

The blood vessel system is essential for skin homeostasis and regeneration. While the heterogeneity of vascular endothelial cells has been emergingly revealed, whether a regeneration-relevant vessel subtype exists in skin remains unknown. Here, a specialized vasculature in skin featured by simultaneous CD31 and EMCN expression contributing to the regeneration process is identified, the decline of which functionally underlies the impaired angiogenesis of diabetic nonhealing wounds. Moreover, enlightened by the developmental process that mesenchymal condensation induces angiogenesis, it is demonstrated that mesenchymal stem/stromal cell aggregates (CAs) provide an efficacious therapy to enhance regrowth of CD31+ EMCN+ vessels in diabetic wounds, which is surprisingly suppressed by pharmacological inhibition of extracellular vesicle (EV) release. It is further shown that CAs promote secretion of angiogenic protein-enriched EVs by proteomic analysis, which directly exert high efficacy in boosting CD31+ EMCN+ vessels and treating nonhealing diabetic wounds. These results add to the current knowledge on skin vasculature and help establish feasible strategies to benefit wound healing under diabetic condition.

Look who's TORking: mTOR-mediated integration of cell status and external signals during limb development and endochondral bone growth.

The balance of cell proliferation and size is key for the control of organ development and repair. Moreover, this balance has to be coordinated within tissues and between tissues to achieve robustness in the organ's pattern and size. The tetrapod limb has been used to study these topics during development and repair, and several conserved pathways have emerged. Among them, mechanistic target of rapamycin (mTOR) signaling, despite being active in several cell types and developmental stages, is one of the least understood in limb development, perhaps because of its multiple potential roles and interactions with other pathways. In the body of this review, we have collated and integrated what is known about the role of mTOR signaling in three aspects of tetrapod limb development: 1) limb outgrowth; 2) chondrocyte differentiation after mesenchymal condensation and 3) endochondral ossification-driven longitudinal bone growth. We conclude that, given its ability to interact with the most common signaling pathways, its presence in multiple cell types, and its ability to influence cell proliferation, size and differentiation, the mTOR pathway is a critical integrator of external stimuli and internal status, coordinating developmental transitions as complex as those taking place during limb development. This suggests that the study of the signaling pathways and transcription factors involved in limb patterning, morphogenesis and growth could benefit from probing the interaction of these pathways with mTOR components.

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates.

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources and are emerging as promising candidates for regenerative medicine, especially for regeneration of the tooth, bone, cartilage, and skin. The self-assembled approach of MSC aggregation, which notably constructs cell clusters mimicking the developing mesenchymal condensation, allows high-density stem cell delivery along with preserved cell-cell interactions and extracellular matrix (ECM) as the microenvironment niche. This method has been shown to enable efficient cell engraftment and survival, thus promoting the optimized application of exogenous MSCs in tissue engineering and safeguarding clinical organ regeneration. This paper provides a detailed protocol for the construction and characterization of self-assembled aggregates based on umbilical cord mesenchymal stem cells (UCMSCs), as well as an example of the cranial bone regenerative application. The implementation of this procedure will help guide the establishment of an efficient MSC transplantation strategy for tissue engineering and regenerative medicine.

Digits in a dish: An in vitro system to assess the molecular genetics of hand/foot development at single-cell resolution.

In vitro models allow for the study of developmental processes outside of the embryo. To gain access to the cells mediating digit and joint development, we identified a unique property of undifferentiated mesenchyme isolated from the distal early autopod to autonomously re-assemble forming multiple autopod structures including: digits, interdigital tissues, joints, muscles and tendons. Single-cell transcriptomic analysis of these developing structures revealed distinct cell clusters that express canonical markers of distal limb development including: Col2a1, Col10a1, and Sp7 (phalanx formation), Thbs2 and Col1a1 (perichondrium), Gdf5, Wnt5a, and Jun (joint interzone), Aldh1a2 and Msx1 (interdigital tissues), Myod1 (muscle progenitors), Prg4 (articular perichondrium/articular cartilage), and Scx and Tnmd (tenocytes/tendons). Analysis of the gene expression patterns for these signature genes indicates that developmental timing and tissue-specific localization were also recapitulated in a manner similar to the initiation and maturation of the developing murine autopod. Finally, the in vitro digit system also recapitulates congenital malformations associated with genetic mutations as in vitro cultures of Hoxa13 mutant mesenchyme produced defects present in Hoxa13 mutant autopods including digit fusions, reduced phalangeal segment numbers, and poor mesenchymal condensation. These findings demonstrate the robustness of the in vitro digit system to recapitulate digit and joint development. As an in vitro model of murine digit and joint development, this innovative system will provide access to the developing limb tissues facilitating studies to discern how digit and articular joint formation is initiated and how undifferentiated mesenchyme is patterned to establish individual digit morphologies. The in vitro digit system also provides a platform to rapidly evaluate treatments aimed at stimulating the repair or regeneration of mammalian digits impacted by congenital malformation, injury, or disease.

Morphological variations of the human spleen: no evidence for a multifocal or lobulated developmental origin.

Adult spleens show extensive morphological variation, with a reported prevalence of 40-98% clefts (also called notches or fissures) on the splenic surface and 10-30% accessory spleens at autopsy. It is hypothesised that both anatomical variants result from a complete or partial failure of multiple splenic primordia to fuse to the main body. According to this hypothesis, fusion of the spleen primordia is completed after birth and spleen morphological variations are often explained as stagnation of spleen development at the foetal stage. We tested this hypothesis by studying early spleen development in embryos, and compared foetal and adult spleen morphology. We assessed 22 embryonic, 17 foetal and 90 adult spleens on the presence of clefts using histology, micro-CT and conventional post-mortem CT-scans, respectively. The spleen primordium was observed as a single mesenchymal condensation in all embryonic specimens. The number of clefts varied from 0 to 6 in foetuses, compared to 0-5 in adults. We found no correlation between foetal age and number of clefts (R2 = 0.004). The independent samples Kolmogorov-Smirnov test showed no significant difference in the total number of clefts between adult and foetal spleens (p = 0.068). We found no morphological evidence for a multifocal origin or a lobulated developmental stage of the human spleen. Our findings show that splenic morphology is highly variable, independent of developmental stage and age. We suggest to abandon the term "persistent foetal lobulation" and to regard splenic clefts, regardless of their number or location, as normal variants.

Collagen hydrogel viscoelasticity regulates MSC chondrogenesis in a ROCK-dependent manner.

Mesenchymal stem cell (MSC) chondrogenesis in three-dimensional (3D) culture involves dynamic changes in cytoskeleton architecture during mesenchymal condensation before morphogenesis. However, the mechanism linking dynamic mechanical properties of matrix to cytoskeletal changes during chondrogenesis remains unclear. Here, we investigated how viscoelasticity, a time-dependent mechanical property of collagen hydrogel, coordinates MSC cytoskeleton changes at different stages of chondrogenesis. The viscoelasticity of collagen hydrogel was modulated by controlling the gelling process without chemical cross-linking. In slower-relaxing hydrogels, although a disordered cortical actin promoted early chondrogenic differentiation, persistent myosin hyperactivation resulted in Rho-associated kinase (ROCK)-dependent apoptosis. Meanwhile, faster-relaxing hydrogels promoted cell-matrix interactions and eventually facilitated long-term chondrogenesis with mitigated myosin hyperactivation and cell apoptosis, similar to the effect of ROCK inhibitors. The current work not only reveals how matrix viscoelasticity coordinates MSC chondrogenesis and survival in a ROCK-dependent manner but also highlights viscoelasticity as a design parameter for biomaterials for chondrogenic 3D culture.