Effects of the Extracts from Fruit and Stem of Camellia japonica on Induced Pluripotency and Wound Healing.

Small molecules that improve reprogramming, stem cell properties, and regeneration can be widely applied in regenerative medicine. Natural plant extracts represent an abundant and valuable source of bioactive small molecules for drug discovery. Natural products themselves or direct derivatives of them have continued to provide small molecules that have entered clinical trials, such as anticancer and antimicrobial drugs. Here, we tested 3695 extracts from native plants to examine whether they can improve induced pluripotent stem cell (iPSC) generation using genetically homogeneous secondary mouse embryonic fibroblasts (MEFs) harboring doxycycline (dox)-inducible reprograming transgenes. Among the tested extracts, extracts from the fruit and stem of Camellia japonica (CJ) enhanced mouse and human iPSC generation and promoted efficient wound healing in an in vivo mouse wound model. CJ is one of the best-known species of the genus Camellia that belongs to the Theaceae family. Our findings identified the natural plant extracts from the fruit and stem of CJ as novel regulators capable of enhancing cellular reprogramming and wound healing, providing a useful supplement in the development of a more efficient and safer method to produce clinical-grade iPSCs and therapeutics.

ESRP1-Induced CD44 v3 Is Important for Controlling Pluripotency in Human Pluripotent Stem Cells.

The importance of alternative splicing (AS) events in pluripotency regulation has been highlighted by the determination of different roles and contributions of different splice isoforms of pluripotency-related genes and by the identification of distinct pluripotency-related splicing factors. In particular, epithelial splicing regulatory protein 1 (ESRP1) has been characterized as an essential splicing factor required for the regulation of human pluripotency and differentiation. Nevertheless, a detailed molecular characterization of ESRP1 (mRNA splice variants 1-6) in human pluripotency is lacking. In this study, we determined that ESRP1 splice variants are differentially expressed in undifferentiated and differentiated human pluripotent stem cells (PSCs). Undifferentiated human PSCs predominantly expressed the ESRP1 v1, v4, and v5, and their expression was downregulated upon differentiation. Ectopic expression of ESRP1 v1, v4, or v5 enhanced the pluripotent reprogramming of human fibroblasts and restored the ESRP1 knockdown-mediated reduction of reprogramming efficiency. Notably, undifferentiated human PSCs expressed the cell surface protein CD44 variant 3 (CD44 v3), and isoform switching from CD44 v3 to CD44 variant 6 (CD44 v6) occurred upon differentiation. Importantly, the human PSC-specific ESRP1 variants influenced CD44 v3 expression. CD44 knockdown or inhibition of binding of CD44 with its major ligand, hyaluronan, significantly induced the loss of human PSC pluripotency and the reduction of reprogramming efficiency. Our results demonstrate that the effect of ESRP1 and CD44 on human PSC pluripotency is isoform-dependent and that ESRP1-induced CD44 v3 is functionally associated with human PSC pluripotency control. Stem Cells 2018;36:1525-1534.

Zebrafish knockout of Down syndrome gene, DYRK1A, shows social impairments relevant to autism.

BACKGROUND: DYRK1A maps to the Down syndrome critical region at 21q22. Mutations in this kinase-encoding gene have been reported to cause microcephaly associated with either intellectual disability or autism in humans. Intellectual disability accompanied by microcephaly was recapitulated in a murine model by overexpressing Dyrk1a which mimicked Down syndrome phenotypes. However, given embryonic lethality in homozygous knockout (KO) mice, no murine model studies could present sufficient evidence to link Dyrk1a dysfunction with autism. To understand the molecular mechanisms underlying microcephaly and autism spectrum disorders (ASD), we established an in vivo dyrk1aa KO model using zebrafish. METHODS: We identified a patient with a mutation in the DYRK1A gene using microarray analysis. Circumventing the barrier of murine model studies, we generated a dyrk1aa KO zebrafish using transcription activator-like effector nuclease (TALEN)-mediated genome editing. For social behavioral tests, we have established a social interaction test, shoaling assay, and group behavior assay. For molecular analysis, we examined the neuronal activity in specific brain regions of dyrk1aa KO zebrafish through in situ hybridization with various probes including c-fos and crh which are the molecular markers for stress response. RESULTS: Microarray detected an intragenic microdeletion of DYRK1A in an individual with microcephaly and autism. From behavioral tests of social interaction and group behavior, dyrk1aa KO zebrafish exhibited social impairments that reproduce human phenotypes of autism in a vertebrate animal model. Social impairment in dyrk1aa KO zebrafish was further confirmed by molecular analysis of c-fos and crh expression. Transcriptional expression of c-fos and crh was lower than that of wild type fish in specific hypothalamic regions, suggesting that KO fish brains are less activated by social context. CONCLUSIONS: In this study, we established a zebrafish model to validate a candidate gene for autism in a vertebrate animal. These results illustrate the functional deficiency of DYRK1A as an underlying disease mechanism for autism. We also propose simple social behavioral assays as a tool for the broader study of autism candidate genes.