No abstract

Defined sets of transcriptional factors can reprogram human somatic cells to induced pluripotent stem (iPS) cells. However, many types of human cells are not easily accessible to minimally invasive procedures. Here we evaluated dental pulp cells (DPCs) as an optimal source of iPS cells, since they are easily obtained from extracted teeth and can be expanded under simple culture conditions. From all 6 DPC lines tested with the conventional 3 or 4 reprogramming factors, iPS cells were effectively established from 5 DPC lines. Furthermore, determination of the HLA types of 107 DPC lines revealed 2 lines homozygous for all 3 HLA loci and showed that if an iPS bank is established from these initial pools, the bank will cover approximately 20% of the Japanese population with a perfect match. Analysis of these data demonstrates the promising potential of DPC collections as a source of iPS cell banks for use in regenerative medicine.

A cancer stem cell population in malignant brain tumors takes an essential part in brain tumor initiation, growth, and recurrence. Growth factors, such as epidermal growth factor, fibroblast growth factor-2, vascular endothelial growth factor, platelet-derived growth factor, and hepatocyte growth factor, are shown to support the proliferation of neural stem cells and also may play key roles in gliomagenesis. However, the responsible growth factor(s), which controls maintenance of brain tumor stem cells, is not yet uncovered. We have established three cancer stem cell lines from human gliomas. These cells were immunoreactive with the neuronal progenitor markers, nestin and CD133, and established tumors that closely resembled the features of original tumor upon transplantation into mouse brain. Three cell lines retained their self-renewal ability and proliferation only in the presence of epidermal growth factor (>2.5 ng/ml). In sharp contrast, other growth factors, including fibroblast growth factor-2, failed to support maintenance of these cells. The tyrosine kinase inhibitors of epidermal growth factor signaling (AG1478 and gefitinib) suppressed the proliferation and self-renewal of these cells. Gefitinib inhibited phosphorylation of epidermal growth factor receptor as well as Akt kinase and extracellular signal-regulated kinase 1/2. Flow cytometric analysis revealed that epidermal growth factor concentration-dependently increased the population of CD133-positive cells. Gefitinib significantly reduced CD133-positive fractions and also induced their apoptosis. These results indicate that maintenance of human brain tumor stem cells absolutely requires epidermal growth factor and that tyrosine kinase inhibitors of epidermal growth factor signaling potentially inhibit proliferation and induce apoptosis of these cells.

Unlike the thoroughly investigated melanocyte population in the hair follicle of the epidermis, the growth and differentiation requirements of the melanocytes in the eye, harderian gland and inner ear -the so-called non-cutaneous melanocytes -remain unclear. In this study, we investigated the in vitro and in vivo effects of the factors that regulate melanocyte development on the stem cells or the precursors of these non-cutaneous melanocytes. In general, a reduction in KIT receptor tyrosine kinase signaling leads to disordered melanocyte development. However, melanocytes in the eye, ear and harderian gland were revealed to be less sensitive to KIT signaling than cutaneous melanocytes. Instead, melanocytes in the eye and harderian gland were stimulated more effectively by endothelin 3 (ET3) or hepatocyte growth factor (HGF) signals than by KIT signaling, and the precursors of these melanocytes expressed the lowest amount of KIT. The growth and differentiation of these non-cutaneous melanocytes were specifically inhibited by antagonists for ET3 and HGF. In transgenic mice induced to express ET3 or HGF in their skin and epithelial tissues from human cytokeratin 14 promoters, the survival and differentiation of non-cutaneous and dermal melanocytes, but not epidermal melanocytes, were enhanced, apparently irrespective of KIT signaling. These results provide a molecular basis for the clear discrimination between non-cutaneous or dermal melanocytes and epidermal melanocytes, a difference that might be important in the pathogenesis of melanocyte-related diseases and melanomas.

SUMMARYRest (RE1-silencing transcription factor, also called Nrsf) is involved in the maintenance of the undifferentiated state of neuronal stem/progenitor cells in vitro by preventing precocious expression of neuronal genes. However, the function of Rest during neurogenesis in vivo remains to be elucidated because of the early embryonic lethal phenotype of conventional Rest knockout mice. In the present study, we have generated Rest conditional knockout mice, which allow the effect of genetic ablation of Rest during embryonic neurogenesis to be examined in vivo. We show that Rest plays a role in suppressing the expression of neuronal genes in cultured neuronal cells in vitro, as well as in non-neuronal cells outside of the central nervous system, but that it is dispensable for embryonic neurogenesis in vivo. Our findings highlight the significance of extrinsic signals for the proper intrinsic regulation of neuronal gene expression levels in the specification of cell fate during embryonic neurogenesis in vivo.