MATERIALS AND METHODS: Neural induction was carried out with a small molecule cocktail based two-step culture protocol, over a total duration of 14 days. At the 8 and 14 day timepoints, the cells were analyzed for expression of neural markers with immunocytochemistry, qRT-PCR and Western Blot. The Fluo 4-AM calcium flux assay was also performed after a further 14 days of neural maturation.
RESULTS: More pronounced morphological changes characteristic of the neural lineage (i.e. neuritogenesis) were observed in all three cell types treated with small molecules, as compared to the untreated controls. This was corroborated by the immunocytochemistry, qRT-PCR and western blot data, which showed upregulated expression of several early and mature neural markers in all three cell types treated with small molecules, versus the corresponding untreated controls. Finally, the Fluo-4 AM calcium flux assay showed consistently higher calcium transient (F/Fo) peaks for the small molecule-treated versus untreated control groups.
CONCLUSIONS: Small molecules can enhance the neurogenic differentiation of DPSCs, SCAPs and GMSCs, which offer much potential for therapeutic applications.
METHODS: The hESCs were differentiated into neural stem cells (NSCs), and NSC-DECM was extracted from confluent monolayers of NSCs through treatment with deionized water. DFSCs seeded on NSC-DECM, Geltrex, and tissue culture polystyrene (TCPS) were subjected to neural induction during a period of 21 days. Expression of early/intermediate (Musashi1, PAX6, NSE, and βIII-tubulin) and mature/late (NGN2, NeuN, NFM, and MASH1) neural markers by DFSCs was analyzed at the 7-, 14-, and 21-day time points with quantitative real-time polymerase chain reaction. Immunocytochemistry for detection of βIII-tubulin, PAX6, and NGN2 expression by DFSCs on day 7 of neural induction was also carried out.
RESULTS: Quantitative RT-PCR showed that expression of PAX6, Musashi1, βIII-tubulin, NSE, NGN2, and NFM by DFSCs was enhanced on NSC-DECM versus either the Geltrex or TCPS groups. Immunocytochemistry showed that DFSCs in the NSC-DECM group displayed more intense staining for βIII-tubulin, PAX6, and NGN2 expression, together with more neurite outgrowths and elongated morphology, as compared with either Geltrex or TCPS.
CONCLUSIONS: DECM derived from neurogenesis of hESCs can enhance the neurogenic potential of DFSCs.
METHODS: A subcohort of 201 children with behavioural outcome measures was identified within a longitudinal, Australian birth-cohort study. The faecal microbiota were analysed at 1, 6, and 12 months of age. Behavioural outcomes were measured at 2 years of age.
FINDINGS: In an unselected birth cohort, we found a clear association between decreased normalised abundance of Prevotella in faecal samples collected at 12 months of age and increased behavioural problems at 2 years, in particular Internalizing Problem scores. This association appeared independent of multiple potentially confounding variables, including maternal mental health. Recent exposure to antibiotics was the best predictor of decreased Prevotella.
INTERPRETATION: Our findings demonstrate a strong association between the composition of the gut microbiota in infancy and subsequent behavioural outcomes; and support the importance of responsible use of antibiotics during early life.
FUNDING: This study was funded by the National Health and Medical Research Council of Australia (1082307, 1147980, 1129813), The Murdoch Children's Research Institute, Barwon Health, Deakin University, Perpetual Trustees, and The Shepherd Foundation. The funders had no involvement in the data collection, analysis or interpretation, trial design, recruitment or any other aspect pertinent to the study.