No abstract

The purpose of this study was to characterize neural crest-derived cells within the adult murine iris. The iris was isolated from P0-Cre ⁄ Floxed-EGFP transgenic (TG) mice. The isolated iris cells formed EGFP-positive spheres on non-adhesive culture plates. Immunostaining showed that these EGFP-positive spheres expressed neural crest markers including Sox10 and p75NTR, and these cells showing in vitro sphere-forming ability were originally resided in the iris stroma (IS), in vivo. Real-time RT-PCR showed that the EGFP-positive spheres expressed significantly higher levels of the neural crest markers than EGFP-negative spheres and bone marrow-derived mesenchymal stem cells. Furthermore, the iris stromal sphere had capability to differentiate into various cell lineages including smooth muscle and cartilage. These data indicate that neural crest-derived multipotent cells can be isolated from the murine IS and expanded in sphere culture.

No abstract

Extrinsic and intrinsic regulators are responsible for the tight control of hematopoietic stem cells (HSCs), which differentiate into all blood cell lineages. To understand the fundamental basis of HSC biology, we focused on differentially expressed genes (DEGs) in long-term and short-term HSCs, which are closely related in terms of cell development but substantially differ in their stem cell capacity. To analyze the transcriptional regulation of the DEGs identified in the novel transcriptome profiles obtained by our RNA-seq analysis, we developed a computational method to model the linear relationship between gene expression and the features of putative regulatory elements. The transcriptional regulation modes characterized here suggest the importance of transcription factors (TFs) that are expressed at steady state or at low levels. Remarkably, we found that 24 differentially expressed TFs targeting 21 putative TF-binding sites contributed significantly to transcriptional regulation. These TFs tended to be modulated by other nondifferentially expressed TFs, suggesting that HSCs can achieve flexible and rapid responses via the control of nondifferentially expressed TFs through a highly complex regulatory network. Our novel transcriptome profiles and new method are powerful tools for studying the mechanistic basis of cell fate decisions.

Background/Aims: Stevens-Johnson syndrome (SJS), ocular cicatricial pemphigoid (OCP) and alkali burns are associated with chronic, severe inflammation of the ocular surface that occasionally lead to corneal stem cell deficiencies. The corneal stroma in these diseases has not been studied comprehensively. The purpose of this study was to determine whether the keratocytes in the stroma were normal and whether the stroma remained inflamed in the chronic phase of these diseases. Methods: Five pathological corneas, two with SJS, two with OCP and one with an alkali burn were examined. Corneal specimens were obtained during lamellar keratoplasty and the histological sections were immunostained with antibodies against CD34 and several cell surface antigens. The level of expression of proteoglycans (lumican, keratocan, biglycan) and chemokines (monocyte chemoattractant protein 1, macrophage inflammatory protein (MIP) 1a, MIP1b) were examined by quantitative real-time RT-PCR. Results: The number of CD34-positive cells in the stroma was decreased and the expression level of biglycan increased in all of the pathological corneas. The numbers of CD45-positive and CD14-positive cells were increased in four of the five pathological corneas. The expression level of MIP1a and MIP1b were markedly increased in all of the pathological corneas. Conclusions: These findings indicate that the keratocytes are abnormal and inflammation is still present in the corneal stroma in the chronic phase of SJS, OCP and alkali burns.Stevens-Johnson syndrome (SJS) and ocular cicatricial pemphigoid (OCP) of the cornea occasionally result in total limbal stem cell deficiency that often leads to severe ocular complications, which do not respond to classic treatment either in the acute phase or in the chronic phase.1-4 The outcome of surgery for these diseases is also poor, 5 suggesting that the prolonged inflammation and severe dry eye or abnormal epithelial differentiation is not conducive to good surgical results. 6 Severe alkali burns of the cornea also result in limbal stem cell deficiency and poor prognosis in some cases.7-10 Compared with immunologically driven diseases such as SJS, however, the operative outcome is reported to be better especially when an autograft limbal transplantation is performed. 11-13In the chronic phase of SJS and chemical injury, inflammatory cells infiltrate into the conjunctivalised corneal epithelium.14 The outer layer of the corneal stroma under a pannus is occasionally invaded by blood vessels and opacities develop in these areas, but the pathological alterations in these areas have not been studied in detail. We hypothesised that abnormalities of keratocytes and persistent inflammation exist in the corneal stroma during the chronic phase of ocular surface diseases. To determine the pathological state of corneal stroma of eyes with severe ocular surface diseases covered by a pannus in the chronic phase, we examined five pathological corneas. If our hypothesis is correct, it should be possible to alter the e...