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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.

Rest promotes the early differentiation of mouse ESCs *Manuscript Click here to view linked References The pluripotency of ESCs is maintained by coordinated expression of a core regulatory circuit of genes that includes Oct3/4, Sox2 and Nanog. Rest (also called Nrsf) is abundantly expressed in ESCs and is a target of the Oct3/4-Sox2-Nanog regulatory network. However, the functional significance of Rest in the maintenance of pluripotency remains controversial. We have generated Rest conditional knockout and Rest-inducible ES cell lines. Conditional ablation of Rest showed that it is not required for maintenance of pluripotency, but it is involved in the suppression of self-renewal genes during early differentiation of ESCs. In addition, forced expression of REST in ESCs results in rapid differentiation. These results indicate that Rest is not necessary for the maintenance of mouse ESCs, and instead suggest that the Rest transcriptional repressor connects to the Oct3/4-Sox2-Nanog core regulatory circuitry during early ESC differentiation. The transcriptional repressor Rest is a zinc finger protein that binds to a conserved 23 bp motif known as RE1 (repressor element 1, also called NRSE) in a number of genes encoding the fundamental neuronal traits (Chong et al., 1995; vector carrying the floxed last exon of Rest, which encodes the coRest binding site that is essential for the generation of the silencing complex (Andres et al., 1999; Grimes et al., 2000), followed by ires-Gfp to monitor the transcription of the modified allele (Rest 3lox/+, Figure 1A). The transient expression of Cre recombinase generated a Rest floxed ES cell line which lacks a drug selection cassette (Rest 2lox/+). Analyzing the GFP expression allowed us to confirm that Rest is expressed in ESCs (Figure 1B). Rest-/-ESCs were next generated using the floxed Rest ES cell line together with a plasmid expressing Cre recombinase (Figure 1A). After the excision of the floxed Rest gene by the transient transfection of Cre (Rest +/-(1lox)), the second Rest allele was also replaced with the floxed allele (Rest 3lox/-). The transient transfection of Cre into Rest 3lox/-ESCs resulted in the establishment of Rest-/-ESCs that were isogenic to the parental ESCs without any genetic modification except for the Rest alleles. After the recombination of the Rest alleles, the lack of a Rest transcript in such Rest-/-ESCs was confirmed by a Northern blot analysis (Figures 1B, S1A). Consistent with the recombination, a FACS analysis revealed a lack of any GFP signal (F) Forty-eight hrs of the induction of REST causes the ESC differentiation into epithelium-like colonies with a decreased ALP activity. (G) The forced expression of REST in ESCs leads to decreased expression of Nanog, Oct3/4 and Fgf5, whereas it results in increased expression of Gata6. The data are presented as the mean±SD of six independent samples. (H) In vitro differentiation of REST-inducible ESCs into EBs under the absence or presence of doxycycline. The exogenous REST expression results in an increa...

Pannus-like tissue exists in advanced OA cartilage, preferentially in the marginal zone. It expressed IL-1beta and MMP3, which strongly suggests that it contributes to cartilage degradation.

STEM CELLS 2007;25:402-410