Organogenesis in the embryo involves cell differentiation and organization events that are unique to each tissue and organ and are susceptible to developmental toxicants. Animal models are the gold standard for identifying putative teratogens, but the limited throughput of developmental toxicological studies in animals coupled with the limited concordance between animal and human teratogenicity motivates a different approach. In vitro organoid models can mimic the cellular architecture and phenotype of many tissues and organs, and the three-dimensional (3D) architecture of organoids presents an opportunity to study developmental human toxicology. Common themes during development like the involvement of epithelial-mesenchymal transition and tissue fusion present an opportunity to develop in vitro models to study cell and tissue morphogenesis. We previously described organoids composed of human stem and progenitor cells that recapitulated the cellular features of palate fusion, and here we further characterized the model by examining pharmacological inhibitors targeting known palatogenesis and epithelial morphogenesis pathways as well as twelve cleft palate teratogens identified from rodent models. Organoid survival was dependent on signaling through EGF, IGF, HGF, and FGF pathways, and organoid fusion was disrupted by inhibition of BMP signaling. We observed concordance between the effects of EGF, FGF, and BMP inhibitors on organoid fusion and epithelial cell migration in vitro, suggesting that organoid fusion is dependent on epithelial morphogenesis. Three of the twelve putative cleft palate teratogens studied here significantly disrupted in vitro fusion, including theophylline, triamcinolone, and valproic acid. Tributyltin chloride and all-trans retinoic acid (ATRA) were cytotoxic to fusing organoids. The study herein demonstrates the utility of the in vitro fusion assay for identifying chemicals that disrupt human organoid survival and morphogenesis in a scalable format amenable to toxicology screening. This dataset is associated with the following publication: Belair, D., C. Wolf, S. Moorefield, C. Wood, C. Becker, and B. Abbott. A Three-Dimensional Organoid Culture Model to Assess the Influence of Chemicals on Morphogenetic Fusion.. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 394-408, (2018).

Cleft palate (CP) is a common birth defect, occurring in an estimated 1 in 1000 births worldwide. The secondary palate is formed by paired palatal shelves that grow toward each other, appose, attach and fuse. CP can result from disruption of any of these processes. The palatal shelves basically consist of a mesenchymal tissue core covered with a layer of epithelial cells. One of the mechanisms that can cause CP is failure of fusion, i.e., failure to remove the epithelial seam between the palatal shelves to allow the mesenchyme to merge and form a continuous palate. This process requires complex interactions between mesenchymal and epithelial cells, and signaling components such as growth factors. Epidermal growth factor (EGF) plays an important role in palate growth and differentiation, while it may impede fusion. We developed a 3D organotypic model using human mesenchymal and epithelial stem cells to mimic human embryonic palatal shelves, and tested its functional relevance by monitoring the effects of human EGF (hEGF) on proliferation and fusion. Spheroids were generated from human umbilical-derived mesenchymal stem cells (hMSCs) directed down an osteogenic lineage by culture medium and evaluated for osteogenic differentiation. Heterotypic spheroids, or organoids, were constructed by coating hMSC spheroids with MaxGel™ extracellular matrix solution followed by a layer of human progenitor epithelial keratinocytes (hPEK). Organoids were incubated in co-culture medium with or without hEGF and assessed for cell proliferation and spheroid pairs were assessed for time to fusion. Osteogenic differentiation in hMSC spheroids was highest by day 13. hEGF delayed fusion of heterotypic organoids after 12 and 18 hours of contact. hEGF increased proliferation in organoids at 4 ng/ml, and proliferation was detected in hPEKs alone on microcarrier beads, suggesting a potential mechanism for delayed fusion by hEGF. Our results show that this model of human palatal fusion consisting of a core of differentiated hMSCs with a hPEK outer layer appropriately mimics the morphology of the developing human palate and responds to hEGF as expected. Future studies will focus on using the organoid model to evaluate the effects of teratogenic chemicals on palatal fusion, and validating the results. This dataset is associated with the following publication: Wolf, C., D. Belair, C. Becker, K. Das, J. Schmid, and B. Abbott. Development of an organotypic stem cell model for the study of human embryonic palatal fusion. BIRTH DEFECTS RESEARCH PART B: DEVELOPMENTAL AND REPRODUCTIVE TOXICOLOGY. John Wiley & Sons, Ltd., Indianapolis, IN, USA, 1322-1334, (2018).

Morphogenetic events are driven by cell-generated physical forces and complex cellular dynamics. To improve our capacity to predict developmental effects from cellular alterations, we built a multi-cellular agent-based model in CompuCell3D that recapitulates the cellular networks and collective cell behavior underlying growth and fusion of the mammalian secondary palate. The model incorporated multiple signaling pathways (TGF?, BMP, FGF, EGF, SHH) in a biological framework to recapitulate morphogenetic events from palatal outgrowth through midline fusion. It effectively simulated higher-level phenotypes (e.g., midline contact, medial edge seam (MES) breakdown, mesenchymal confluence, fusion defects) in response to genetic or environmental perturbations. Perturbation analysis of various control features revealed model functionality with respect to cell signaling systems and feedback loops for growth and fusion, diverse individual cell behaviors and collective cellular behavior leading to physical contact and midline fusion, and quantitative analysis of the TGF/EGF switch that controls MES breakdown – a key event in morphogenetic fusion. The virtual palate model was then executed with theoretical chemical perturbation scenarios to simulate switch behavior leading to a disruption of fusion following chronic (e.g., dioxin) and acute (e.g., retinoic acid, hydrocortisone) toxicant exposures. This computer model adds to similar systems models toward a ‘virtual embryo’ for simulation and quantitative prediction of adverse developmental outcomes following genetic perturbation and/or environmental. This dataset is associated with the following publication: Hutson, S., M. Leung, N. Baker, R. Spencer, and T. Knudsen. (CHEMICAL RESEARCH IN TOXICOLOGY) Computational Model of Secondary Palate Fusion and Disruption. CHEMICAL RESEARCH IN TOXICOLOGY. American Chemical Society, Washington, DC, USA, 30(4): 965-979, (2017).

Effects of chemicals on palate organoid fusion. Portions of this dataset are inaccessible because: Dataset upload. For questions, contact data lead. They can be accessed through the following means: Dataset is attached. Format: Various file formats. This dataset is associated with the following publication: Wolf, C., H. Ftizpatrick, C. Becker, J. Smith, and C. Wood. An improved multicellular human organoid model for the study of chemical effects on palatal fusion. Birth Defects Research. John Wiley & Sons, Inc., Hoboken, NJ, USA, 115(16): 1513-1533, (2023).

Background Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts) into enucleated M I or M II oocytes. Results Analysis of the cell cycle stages revealed that 91.2 ± 0.2% of confluent cells were at the G0/G1 phase and 54.1 ± 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5%) for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5%) blastocysts were obtained and this was significantly (P < 0.05) lower than that (7.6%) of embryos produced by transferring confluent cells into M II oocytes. No reconstructed embryos developed to the blastocyst stage when nocodazole-treated cells were used as donors. Conclusions Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells.

The below table includes a smaller list of data that was analyzed by dChip and filtered by pvalue such that a file with about 4600 genes was obtained which allowed for ease of use from 40,000 genes. Experiment Overall Design: The total RNA was extracted from 2T3 pre-osteoblast cells exposed to static or simulated microgravity (Rotating Wall Vessel) conditions. The RNA was then sent to Affymetrix microarray core facility at Baylor College of Medicine (Houston TX) for microarray analysis.