Tat-interactive protein 60 (Tip60) is a member of the MYST family, proteins of which are related by an atypical histone acetyltransferase (HAT) domain. Although Tip60 has been implicated in cellular activities including DNA repair, apoptosis, and transcriptional regulation, its function during embryonic development is unknown. We ablated the Tip60 gene (Htatip) from the mouse by replacing exons 1-9 with a neomycin resistance cassette. Development and reproduction of wild-type and heterozygous animals were normal. However, homozygous ablation of the Tip60 gene caused embryolethality near the blastocyst stage of development, as evidenced by inability of cells in Tip60-null blastocysts to hatch and survive in culture. Monitoring cell proliferation and death by detecting EdU-substituted DNA and TUNEL labeling revealed suppression of cell proliferation concomitant with increased cell death as Tip60-null cells attempted to hatch from blastocysts. These findings indicate that Tip60 is essential for cellular survival during the blastocyst-gastrula transition of embryogenesis.
This article serves three purposes. We summarize current knowledge of the origin and characteristics of EPI-NCSC, review their application in a mouse model of spinal cord injury, and we present new data that highlight aspects of pluripotency of EPI-NCSC. EPI-NCSC are multipotent stem cells, which are derived from the embryonic neural crest and are located in the bulge of hair follicles. EPI-NCSC can undergo self-renewal and they are able to generate all major neural crest derivatives, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. Despite their ectodermal origin, neural crest cells can also generate cell types that typically are derived from mesoderm. We were therefore interested in exploring aspects of EPI-NCSC pluripotency. We here show that EPI-NCSC can fuse with adult skeletal muscle fibers and that incorporated EPI-NCSC nuclei are functional. Furthermore, we show that adult skeletal muscle represents an environment conducive to long-term survival of neurogenic EPI-NCSC. Genes used to create induced pluripotent stem (iPS) cells are present in our EPI-NCSC longSAGE gene expression library. Here we have corroborated this notion by real-time PCR. Our results show similarities in the expression of Myc, Klf4, Sox2 and Lin28 genes between EPI-NCSC and embryonic stem cells (ESC). In contrast there were major differences in Nanog and Pou5f1 (Oct-4) expression levels between EPI-NCSC and ESC, possibly explaining why EPI-NCSC are not tumorigenic. Overall, as embryonic remnants in an adult location EPI-NCSC show several attractive characteristics for future cell replacement therapy and/or biomedical engineering: Due to their ability to migrate, EPI-NCSC can be isolated as a highly pure population of multipotent stem cells by minimally-invasive procedures. The cells can be expanded in vitro into millions of stem cells/progenitors and they share some characteristics with pluripotent stem cells without being tumorigenic. Since the patients' own EPI-NCSC could be used for autologous transplantation, this would avoid graft rejection.
EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.
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