The Angiopoietin–Tie2 pathway is a key regulator of postnatal vascular maintenance and remodeling, regulating vascular barrier function and integrity. While the opposing roles of the ligands Angiopoietin-1 (Ang 1) and Angiopoietin-2 (Ang 2) have been recognized for decades, the structural mechanism governing their distinct signaling outputs has only recently been elucidated. As artificial intelligence and protein design continue to develop, emerging evidence suggests that ligand valency and receptor clustering are key determinants of Tie2 pathway activation and endothelial cell function; that is, “form follows function”. This review summarizes the latest discovery in the structural biology and signaling mechanism of the Tie2 pathway using protein design to decode the ligand–receptor interactions. Probing the underlying molecular basis of Tie2 offers new therapeutic opportunities for targeting diseases, featuring vascular dysfunctions such as sepsis, traumatic brain injury, acute respiratory diseases, chronic inflammation, and cancer. This also highlights the next generation of AI-designed protein therapeutics.

Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.

Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.

Over 90% of the U.S. adult population suffers from tooth structure loss due to caries. Most of the mineralized tooth structure is composed of dentin, a material produced and mineralized by ectomesenchyme derived cells known as odontoblasts. Clinicians, scientists, and the general public share the desire to regenerate this missing tooth structure. To bioengineer missing dentin, increased understanding of human tooth development is required. Here we interrogate at the single cell level the signaling interactions that guide human odontoblast and ameloblast development and which determine incisor or molar tooth germ type identity. During human odontoblast development, computational analysis predicts that early FGF and BMP activation followed by later HH signaling is crucial. Here we generate a differentiation protocol based on this sci-RNA-seq analysis to produce mature hiPSC derived odontoblasts in vitro (iOB). Further, we elucidate the critical role of FGF signaling in odontoblast maturation and biomineralization capacity using the de novo designed FGFR1/2c isoform mini binder scaffold C6. Using computational tools, we show on a molecular level how human molar development is delayed compared to incisors. We reveal that enamel knot development is guided by FGF and WNT in incisors and BMP and ROBO in the molars, and that incisor and molar ameloblast development is guided by FGF, EGF and BMP signaling, with tooth type specific intensity of signaling interactions. Dental ectomesenchyme derived cells are the primary source of signaling ligands responsible for both enamel knot and ameloblast development.

Multiple pathologies and non-pathological factors can disrupt the function of the non-regenerative human salivary gland including cancer and cancer therapeutics, autoimmune diseases, infections, pharmaceutical side effects, and traumatic injury. Despite the wide range of pathologies, no therapeutic or regenerative approaches exist to address salivary gland loss, likely due to significant gaps in our understanding of salivary gland development. Moreover, identifying the tissue of origin when diagnosing salivary carcinomas requires an understanding of human fetal development. Using computational tools, we identify developmental branchpoints, a novel stem cell-like population, and key signaling pathways in the human developing salivary glands by analyzing our human fetal single-cell sequencing data. Trajectory and transcriptional analysis suggest that the earliest progenitors yield excretory duct and myoepithelial cells and a transitional population that will yield later ductal cell types. Importantly, this single-cell analysis revealed a previously undescribed population of stem cell-like cells that are derived from SD and expresses high levels of genes associated with stem cell-like function. We have observed these rare cells, not in a single niche location but dispersed within the developing duct at later developmental stages. Our studies introduce new human-specific developmental paradigms for the salivary gland and lay the groundwork for the development of translational human therapeutics.

Tooth enamel secreted by ameloblasts is the hardest material in the human body, acting as a shield protecting the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here we use sci-RNA-seq to establish a spatiotemporal single-cell atlas for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We reveal key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in a novel human ameloblast in vitro differentiation from iPSCs. We furthermore develop a mineralizing enamel organ-like 3D organoid system. These studies pave the way for future regenerative dentistry and therapies toward genetic diseases affecting enamel formation.

The study of the role of epigenetics in regulating disease and degeneration is a constantly expanding field encompassing a broad range of marks from DNA methylation, histone modification, and small RNA regulation that can make rapid changes in gene expression activity in response to extrinsic factors. These modifications are becoming increasingly important in our understanding of degeneration and our pursuit of regenerative therapies. Here, we discuss recent advances in epigenetic regulation in aging and disease and have identified new epigenetic changes that both broaden our understanding of epigenetic misregulation as a driver of degeneration, and we present novel epigenetic editing tools with promising therapeutic and research potential.

It has become clear during recent years that stem cells undergo metabolic remodeling during their activation process. While these metabolic switches take place in pluripotency as well as adult stem cell populations, the rules that govern the switch are not clear. In this review, we summarize some of the transitions in adult and pluripotent cell types and will propose that the key function in this process is the generation of epigenetic metabolites that govern critical epigenetic modifications, and therefore stem cell states.

The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However, the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis, the noncanonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a 2-day window showed significantly enhanced osteogenic yield. Mechanistically, rWNT5a supplementation upregulated protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CamKII) and c-Jun N-terminal kinase (JNK) activity while antagonizing the key effector of canonical Wnt signaling: β-catenin. Conversely, when recombinant WNT3a (rWNT3a) or other positive regulators of β-catenin were employed during this same time window there was a decrease in osteogenic marker expression. However, if rWNT3a was supplemented during a time window following rWNT5a treatment, osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine.

While the involvement of nitric oxide in bone formation, homeostasis and healing has been extensively characterized, its role in directing pluripotent stem cells to the osteogenic lineage has not been described. Yet, the identification of chemical inducers that improve differentiation output to a particular lineage is highly valuable to the development of such cells for the cell-based treatment of osteo-degenerative diseases. This study aimed at investigating the instructive role of nitric oxide (NO) and its synthesizing enzymes on embryonic stem cell (ESC) osteogenic differentiation. Our findings showed that NO levels may support osteogenesis, but that the effect of nitric oxide on osteoblast differentiation may be specific to a particular time phase during the development of osteoblasts in vitro. Endogenously, nitric oxide was specifically secreted by osteogenic cultures during the calcification period. Simultaneously, messenger RNAs for both the endothelial and inducible nitric oxide synthase isoforms (eNOS and iNOS) were upregulated during this late phase development. However, the specific eNOS inhibitor L-N5-(1-Iminoethyl)ornithine dihydrochloride attenuated calcification more so than the specific iNOS inhibitor diphenyleneiodonium. Exogenous stage-specific supplementation of culture medium with the NO donor S-nitroso-N-acetyl-penicillamine increased the percentage of cells differentiating into osteoblasts and enhanced calcification. Our results point to a primary role for eNOS as a pro-osteogenic trigger in ESC differentiation and expand on the variety of supplements that may be used to direct ESC fate to the osteogenic lineage, which will be important in the development of cell-based therapies for osteo-degenerative diseases.

Osteoblasts can be derived from embryonic stem cells (ESCs) by a 30 day differentiation process, whereupon cells spontaneously differentiate upon removal of LIF and respond to exogenously added 1,25α(OH)2 vitamin D3 with enhanced matrix mineralization. However, bone is a load-bearing tissue that has to perform under dynamic pressure changes during daily movement, a capacity that is executed by osteocytes. At present, it is unclear whether ESC-derived osteogenic cultures contain osteocytes and whether these are capable of responding to a relevant cyclic hydrostatic compression stimulus. Here, we show that ESC-osteoblastogenesis is followed by the generation of osteocytes and then mechanically load ESC-derived osteogenic cultures in a compression chamber using a cyclic loading protocol. Following mechanical loading of the cells, iNOS mRNA was upregulated 31-fold, which was consistent with a role for iNOS as an immediate early mechanoresponsive gene. Further analysis of matrix and bone-specific genes suggested a cellular response in favor of matrix remodeling. Immediate iNOS upregulation also correlated with a concomitant increase in Ctnnb1 and Tcf7l2 mRNAs along with increased nuclear TCF transcriptional activity, while the mRNA for the repressive Tcf7l1 was downregulated, providing a possible mechanistic explanation for the noted matrix remodeling. We conclude that ESC-derived osteocytes are capable of responding to relevant mechanical cues, at least such that mimic oscillatory compression stress, which not only provides new basic understanding, but also information that likely will be important for their use in cell-based regenerative therapies.

As the worldwide population grows and life expectancies continue to increase, degenerative diseases of the bones, muscles, and connective tissue are a growing problem for society. Current therapies for osteodegenerative disorders such as hormone replacement therapies, calcium/vitamin D supplements and oral bisphosphonates are often inadequate to stop degeneration and/or have serious negative side effects. Thus, there is an urgent need in the medical community for more effective and safer treatments. Stem cell therapies for osteodegenerative disorders have been rigorously explored over the last decade and are yielding some promising results in animal models and clinical trials. Although much work still needs to be done to ensure the safety and efficacy of these therapies, stem cells represent a new frontier of exciting possibilities for bone and cartilage regeneration.