An improved multicellular human organoid model for the study of chemical effects on palatal fusion.

BACKGROUND: Tissue fusion is a mechanism involved in the development of the heart, iris, genital tubercle, neural tube, and palate during embryogenesis. Failed fusion of the palatal shelves could result in cleft palate (CP), a common birth defect. Organotypic models constructed of human cells offer an opportunity to investigate developmental processes in the human. Previously, our laboratory developed an organoid model of the human palate that contains human mesenchyme and epithelial progenitor cells to study the effects of chemicals on fusion. METHODS: Here, we developed an organoid model more representative of the embryonic palate that includes three cell types: mesenchyme, endothelial, and epithelial cells. We measured fusion by a decrease in epithelial cells at the contact point between the organoids and compared the effects of CP teratogens on fusion and toxicity in the previous and current organoid models. We further tested additional suspect teratogens in our new model. RESULTS: We found that the three-cell-type model is more sensitive to fusion inhibition by valproic acid and inhibitors of FGF, BMP, and TGFßRI/II. In this new model, we tested other suspect CP teratogens and found that nocodazole, topiramate, and Y27632 inhibit fusion at concentrations that do not induce toxicity. CONCLUSION: This sensitive human three-cell-type organotypic model accurately evaluates chemicals for cleft palate teratogenicity.

A Three-Dimensional Organoid Culture Model to Assess the Influence of Chemicals on Morphogenetic Fusion.

Embryologic development 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 three-dimensional (3D) morphogenesis of developing tissues and can thus be useful tools for studying developmental toxicology. Common themes during development like the involvement of epithelial-mesenchymal transition and tissue fusion present an opportunity to develop in vitro organoid models that capture key morphogenesis events that occur in the embryo. 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 12 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 12 putative cleft palate teratogens studied here (theophylline, triamcinolone, and valproic acid) significantly disrupted in vitro organoid fusion, while tributyltin chloride and all-trans retinoic acid were cytotoxic to fusing organoids. The study herein demonstrates the utility of the in vitro fusion assay for identifying chemicals that disrupt human organoid morphogenesis in a scalable format amenable to toxicology screening.

Development of an organotypic stem cell model for the study of human embryonic palatal fusion.

BACKGROUND: Cleft palate (CP) is a common birth defect, occurring in an estimated 1 in 1,000 births worldwide. The secondary palate is formed by paired palatal shelves, consisting of a mesenchymal core with an outer layer of epithelial cells that grow toward each other, attach, and fuse. One of the mechanisms that can cause CP is failure of fusion, that is, failure to remove the epithelial seam between the palatal shelves to allow the mesenchyme confluence. Epidermal growth factor (EGF) plays an important role in palate growth and differentiation, while it may impede fusion. METHODS: We developed a 3D organotypic model using human mesenchymal and epithelial stem cells to mimic human embryonic palatal shelves, and tested 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. Heterotypic spheroids, or organoids, were constructed by coating hMSC spheroids with extracellular matrix solution followed by a layer of human progenitor epithelial keratinocytes (hPEKs). Organoids were incubated in co-culture medium with or without hEGF and assessed for cell proliferation and time to fusion. RESULTS: Osteogenic differentiation in hMSC spheroids was highest by Day 13. hEGF delayed fusion of organoids after 12 and 18 hr of contact. hEGF increased proliferation in organoids at 4 ng/ml, and proliferation was detected in hPEKs alone. CONCLUSION: Our results show that this model of human palatal fusion appropriately mimics the morphology of the developing human palate and responds to hEGF as expected.

The prevalence of undiagnosed thyroid disease in patients with symptomatic vocal fold paresis.

OBJECTIVE: Vocal fold paresis has a multifactorial etiology and is idiopathic in many individuals. The incidence of thyroid-related neuropathy in the larynx has not been previously described. The purpose of this study was to evaluate the prevalence of previously undiagnosed thyroid disease in patients with laryngeal neuropathy and to compare this prevalence with that in a cohort of patients with a neurotologic neuropathy. STUDY DESIGN AND SETTING: Case series with chart review; tertiary care, otolaryngology practice. SUBJECTS AND METHODS: Charts of 308 consecutive patients with dysphonia and vocal fold paresis and 333 consecutive patients with sensorineural hearing loss, who presented for evaluation during a 3-year period, were reviewed. RESULTS: One hundred forty-six of 308 (47.4%) patients with vocal fold paresis were diagnosed with concurrent thyroid disease, whereas 55 of 333 (16.5%) patients with sensorineural hearing loss were diagnosed with concurrent thyroid disease (P<0.001, Pearson chi-square = 92.896; degrees of freedom = 5). Thyroid diagnoses among those with vocal fold paresis included benign growths (29.9%), thyroiditis (7.8%), hyperthyroidism (4.5%), hypothyroidism (3.6%), and thyroid malignancy (1.6%). CONCLUSIONS: Thyroid abnormalities are more prevalent in patients with dysphonia and vocal fold paresis than in patients with symptomatic sensorineural hearing loss, suggesting a greater association between previously undiagnosed thyroid abnormalities and laryngeal neuropathy than that between neurotologic neuropathy and thyroid disease.

Developing a neurology training program in Honduras: a joint project of neurologists in Honduras and the World Federation of Neurology.

One of the major barriers to the provision of quality care for patients with neurological disorders in developing countries is a low ratio of neurologists per inhabitants, the World Health Organization recommends one neurologist per 100,000. In 1998 Honduras had one neurologist per 325,000 inhabitants and all the neurologists were trained outside the country. The Education Committee of the World Federation of Neurology (WFN), in collaboration with the Postgraduate Direction of the National Autonomous University of Honduras, the Honduran Neurological Association, and the Honduran Secretary of Health helped establish the country's first Neurology Training Program in 1998. This program was established using a problem- and epidemiological-oriented methodology with oversight by an external WFN review board. By 2006 the program has resulted in a 31% increase in the national neurologist ratio per inhabitant, significantly improved the quality of patient care and promoted research in the neurosciences. The Honduras Neurology Training Program has provided a valuable model for other developing countries with similar needs for neurological care. Based on this Honduras experience, members of the Education Committee of the WFN have established guidelines for neurology training programs in developing countries.

A profile of children with disabilities receiving SSI: highlights from the National Survey of SSI Children and Families.

This article provides a nationally representative profile of noninstitutionalized children 0 to 17 years of age who were receiving support from the Supplemental Security Income (SSI) program because of a disability. To assess the role of the SSI program in providing assistance to low-income children with disabilities and their families, it is important to obtain detailed information on demographic characteristics, income and assets, health and disabilities, and health care utilization. Yet administrative records of the Social Security Administration do not contain many of the relevant data items, and the records provide only an incomplete picture of the family relationships affecting the lives of children with disabilities. The National Survey of SSI Children and Families fills this gap. This summary article is based on survey interviews conducted between July 2001 and June 2002 and provides some highlights characterizing children with disabilities who were receiving SSI and their families. Most children receiving SSI (hereafter referred to as "SSI children") lived in a family headed by a single mother, and less than one in three lived with both parents. A very high proportion, about half, were living in a household with at least one other individual reported to have had a disability. About 70 percent of children received some kind of special education. SSI support was the most important source of family income, with earnings a close second. On average, SSI payments accounted for nearly half of the income for the children's families, and earnings accounted for almost 40 percent. When all sources of family income were considered, slightly more than half (54 percent) of SSI children lived in families above the poverty threshold, a notable fact given that the federal SSI program guarantees only a subpoverty level of income. However, beyond these averages there was substantial variation, with some children living in families with income well below the poverty threshold and others having income well over 200 percent of the poverty threshold. About one-third of SSI children lived in families owning a home, two-thirds lived with parents or guardians with at least one car, and about 40 percent lived with parents or guardians with zero liquid assets. Less than 4 percent lived with adults who owned stocks, mutual funds, notes, certificates of deposit, or savings bonds. The Social Security Administration's administrative records contain only a limited amount of information about disability diagnoses. The National Survey of SSI Children and Families supplements those records with data from an array of questions on functional limitations, self-reported health, and the perceived severity of disabilities. The data suggest that a great degree of variation in severity exists within the childhood caseload, as reflected in reports of the presence or absence of six functional limitations, perceived overall health status, and perceived impact of disability on the child's ability to do things. Overall, 36 percent of the children were reported to have had disabilities that affected their abilities to do things "a great deal," and for 21 percent their difficulties had very little or no impact. Physical disabilities were most common among children aged 0 to 5, and mental disabilities dominated the picture for the other two age groups: 6 to 12 and 13 to 17. Virtually all SSI children are covered by some form of health insurance, with Medicaid being by far the most common source of health insurance coverage. Just as in the case of the severity of disabilities, substantial variation was reported in health care utilization among SSI children. Almost 30 percent of children had two or fewer doctor visits during the 12 months preceding the interview, and close to 50 percent had five or more doctor visits. About four-fifths of the children had no reported hospitalizations or surgeries during the previous year. More than 40 percent of the children visited an emergency room during the previous year, most of them more than once. Importantly, no out-of-pocket costs associated with medical care were reported for more than two-thirds of the children, and only about 3 percent had annual expenses exceeding $1,000 for physical and mental health care. This finding suggests that SSI payments are not used to cover medical expenses for the overwhelming majority of children. The use of supportive therapies varied widely among SSI children: more than half reported having used physical, occupational, or speech therapy; only 8 percent used respite care for the parents or other family members. An analysis of the perception of the survey respondents shows that more than one-third of children had unmet needs for mental health counseling services, and about three-quarters of families had unmet needs for respite care. In several service categories, the proportion perceived to have had unmet service needs was around 10 percent or less. In the dominant service category of physical, occupational, and speech therapy, only 11 percent perceived to have had unmet service needs.