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. 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.

Diverse progenitor cells preserve salivary gland ductal architecture after radiation-induced damage.

The ductal system of the salivary gland has long been postulated to be resistant to radiation-induced damage, a common side effect incurred by head and neck cancer patients receiving radiotherapy. Yet, whether the ducts are capable of regenerating after genotoxic injury, or whether damage to ductal cells induces lineage plasticity, as has been reported in other organ systems, remains unknown. Here, using the murine salivary gland, we show that two ductal progenitor populations, marked exclusively by KRT14 and KIT, maintain non-overlapping ductal compartments after radiation exposure but do so through distinct cellular mechanisms. KRT14+ progenitor cells are fast-cycling cells that proliferate in response to radiation-induced damage in a sustained manner and divide asymmetrically to produce differentiated cells of the larger granulated ducts. Conversely, KIT+ intercalated duct cells are long-lived progenitors for the intercalated ducts that undergo few cell divisions either during homeostasis or after gamma radiation, thus maintaining ductal architecture with slow rates of cell turnover. Together, these data illustrate the regenerative capacity of the salivary ducts and highlight the heterogeneity in the damage responses used by salivary progenitor cells to maintain tissue architecture.

Sustained fertility from first-wave follicle oocytes that pause their growth.

Ovulation results from the cyclical recruitment of non-renewing, quiescent oocytes for growth. Therefore, the primordial follicles that are established during development from an oocyte encapsulated by granulosa cells are thought to comprise the lifelong ovarian reserve 1-4. However, using oocyte lineage tracing in mice, we observed that a subset of oocytes recruited for growth in the first juvenile wave remain paused for many months before continuing growth, ovulation, fertilization and development into healthy offspring. This small subset of genetically-labeled fetal oocytes, labeled with Sycp3-CreERT2, is distinguished by earlier entry and slower dynamics of meiotic prophase I. While labeled oocytes were initially found in both primordial follicles and growing follicles of the first wave, they disappeared from primordial follicles by puberty. Unexpectedly, these first-wave labeled growing oocytes persisted throughout reproductive lifespan and contributed to offspring at a steady rate beyond 12 months of age, suggesting that follicles can pause mid-growth for extended periods then successfully resume. These results challenge the conclusion from lineage tracing of granulosa cells that first-wave follicles make a limited contribution to fertility5 and furthermore suggest that growth-paused oocytes comprise a second and previously unrecognized ovarian reserve.

A Roadmap for Three-Dimensional Analysis of the Intact Mouse Ovary.

Recent advances in tissue clearing methodologies have enabled three-dimensional (3D) visualization of the ovary and, consequently, in-depth exploration of the dynamic changes occurring at the single-cell level. Here we describe methods for whole-mount immunofluorescence, clearing, imaging, and analysis of whole ovarian tissue in 3D throughout murine development and aging.

Generation of Schwann cell derived melanocytes from hPSCs identifies pro-metastatic factors in melanoma.

The neural crest (NC) is highly multipotent and generates diverse lineages in the developing embryo. However, spatiotemporally distinct NC populations display differences in fate potential, such as increased gliogenic and parasympathetic potential from later migrating, nerve-associated Schwann cell precursors (SCPs). Interestingly, while melanogenic potential is shared by both early migrating NC and SCPs, differences in melanocyte identity resulting from differentiation through these temporally distinct progenitors have not been determined. Here, we leverage a human pluripotent stem cell (hPSC) model of NC temporal patterning to comprehensively characterize human NC heterogeneity, fate bias, and lineage development. We captured the transition of NC differentiation between temporally and transcriptionally distinct melanogenic progenitors and identified modules of candidate transcription factor and signaling activity associated with this transition. For the first time, we established a protocol for the directed differentiation of melanocytes from hPSCs through a SCP intermediate, termed trajectory 2 (T2) melanocytes. Leveraging an existing protocol for differentiating early NC-derived melanocytes, termed trajectory 1 (T1), we performed the first comprehensive comparison of transcriptional and functional differences between these distinct melanocyte populations, revealing differences in pigmentation and unique expression of transcription factors, ligands, receptors and surface markers. We found a significant link between the T2 melanocyte transcriptional signature and decreased survival in melanoma patients in the cancer genome atlas (TCGA). We performed an in vivo CRISPRi screen of T1 and T2 melanocyte signature genes in a human melanoma cell line and discovered several T2-specific markers that promote lung metastasis in mice. We further demonstrated that one of these factors, SNRPB, regulates the splicing of transcripts involved in metastasis relevant functions such as migration, cell adhesion and proliferation. Overall, this study identifies distinct developmental trajectories as a source of diversity in melanocytes and implicates the unique molecular signature of SCP-derived melanocytes in metastatic melanoma.

A synthetic tear protein resolves dry eye through promoting corneal nerve regeneration.

Corneal architecture is essential for vision and is greatly perturbed by the absence of tears due to the highly prevalent disorder dry eye. With no regenerative therapies available, pathological alterations of the ocular surface in response to dryness, including persistent epithelial defects and poor wound healing, result in lifelong morbidity. Here, using a mouse model of aqueous-deficient dry eye, we reveal that topical application of the synthetic tear protein Lacripep reverses the pathological outcomes of dry eye through restoring the extensive network of corneal nerves that are essential for tear secretion, barrier function, epithelial homeostasis, and wound healing. Intriguingly, the restorative effects of Lacripep occur despite extensive immune cell infiltration, suggesting tissue reinnervation and regeneration can be achieved under chronic inflammatory conditions. In summary, our data highlight Lacripep as a first-in-class regenerative therapy for returning the cornea to a near homeostatic state in individuals who suffer from dry eye.

Long-term functional regeneration of radiation-damaged salivary glands through delivery of a neurogenic hydrogel.

Salivary gland acinar cells are severely depleted after radiotherapy for head and neck cancer, leading to loss of saliva and extensive oro-digestive complications. With no regenerative therapies available, organ dysfunction is irreversible. Here, using the adult murine system, we demonstrate that radiation-damaged salivary glands can be functionally regenerated via sustained delivery of the neurogenic muscarinic receptor agonist cevimeline. We show that endogenous gland repair coincides with increased nerve activity and acinar cell division that is limited to the first week after radiation, with extensive acinar cell degeneration, dysfunction, and cholinergic denervation occurring thereafter. However, we found that mimicking cholinergic muscarinic input via sustained local delivery of a cevimeline-alginate hydrogel was sufficient to regenerate innervated acini and retain physiological saliva secretion at nonirradiated levels over the long term (>3 months). Thus, we reveal a previously unknown regenerative approach for restoring epithelial organ structure and function that has extensive implications for human patients.

Neuronal-epithelial cross-talk drives acinar specification via NRG1-ERBB3-mTORC2 signaling.

Acinar cells are the principal secretory units of multiple exocrine organs. A single-cell, layered, lumenized acinus forms from a large cohort of epithelial progenitors that must initiate and coordinate three cellular programs of acinar specification, namely, lineage progression, secretion, and polarization. Despite this well-known outcome, the mechanism(s) that regulate these complex programs are unknown. Here, we demonstrate that neuronal-epithelial cross-talk drives acinar specification through neuregulin (NRG1)-ERBB3-mTORC2 signaling. Using single-cell and global RNA sequencing of developing murine salivary glands, we identified NRG1-ERBB3 to precisely overlap with acinar specification during gland development. Genetic deletion of Erbb3 prevented cell lineage progression and the establishment of lumenized, secretory acini. Conversely, NRG1 treatment of isolated epithelia was sufficient to recapitulate the development of secretory acini. Mechanistically, we found that NRG1-ERBB3 regulates each developmental program through an mTORC2 signaling pathway. Thus, we reveal that a neuronal-epithelial (NRG1/ERBB3/mTORC2) mechanism orchestrates the creation of functional acini.

Functional Specialization of Human Salivary Glands and Origins of Proteins Intrinsic to Human Saliva.

Salivary proteins are essential for maintaining health in the oral cavity and proximal digestive tract, and they serve as potential diagnostic markers for monitoring human health and disease. However, their precise organ origins remain unclear. Through transcriptomic analysis of major adult and fetal salivary glands and integration with the saliva proteome, the blood plasma proteome, and transcriptomes of 28+ organs, we link human saliva proteins to their source, identify salivary-gland-specific genes, and uncover fetal- and adult-specific gene repertoires. Our results also provide insights into the degree of gene retention during gland maturation and suggest that functional diversity among adult gland types is driven by specific dosage combinations of hundreds of transcriptional regulators rather than by a few gland-specific factors. Finally, we demonstrate the heterogeneity of the human acinar cell lineage. Our results pave the way for future investigations into glandular biology and pathology, as well as saliva's use as a diagnostic fluid.