Isolation of stage-specific spermatogenic cells by dynamic histone incorporation and removal in spermatogenesis.

Due to the lack of an efficient in vitro spermatogenesis system, studies on mammalian spermatogenesis require the isolation of specific germ cell populations for further analyses. BSA gradient and elutriation have been used for several decades to purify testicular germ cells; more recently, flow cytometric cell sorting has become popular. Although each method has its advantages and disadvantages and is used depending on the purpose of the experiment, reliance on flow cytometric cell sorting is expected to be more prevalent because fewer cells can be managed. However, the currently used flow cytometric cell sorting method for testicular germ cells relies on karyotypic differences via DNA staining. Thus, it remains challenging to separate post-meiotic haploid cells (spermatids) according to their differentiation stage despite significant variations in morphology and chromatin state. In this study, we developed a method for finely separating testicular germ cells using VC mice carrying fluorescently tagged histones. This method enables the separation of spermatogonia, spermatocytes, and spermatids based on the intensity of histone fluorescence and cell size. Combined with a DNA staining dye, this method separates spermatids after elongation according to each spermiogenic stage. Although the necessity for a specific transgenic mouse line is less versatile, this method is expected to be helpful for the isolation of testicular germ cell populations because it is highly reproducible and independent of complex cell sorter settings and staining conditions.

CD82 is a marker to isolate ß cell precursors from human iPS cells and plays a role for the maturation of ß cells.

Generation of pancreatic ß cells from pluripotent stem cells is a key technology to develop cell therapy for insulin-dependent diabetes and considerable efforts have been made to produce ß cells. However, due to multiple and lengthy differentiation steps, production of ß cells is often unstable. It is also desirable to eliminate undifferentiated cells to avoid potential risks of tumorigenesis. To isolate ß cell precursors from late stage pancreatic endocrine progenitor (EP) cells derived from iPS cells, we have identified CD82, a member of the tetraspanin family. CD82+ cells at the EP stage differentiated into endocrine cells more efficiently than CD82- EP stage cells. We also show that CD82+ cells in human islets secreted insulin more efficiently than CD82- cells. Furthermore, knockdown of CD82 expression by siRNA or inhibition of CD82 by monoclonal antibodies in NGN3+ cells suppressed the function of ß cells with glucose-stimulated insulin secretion, suggesting that CD82 plays a role in maturation of EP cells to ß cells.

Sall1 transiently marks undifferentiated heart precursors and regulates their fate.

Cardiac progenitor cells (CPCs) are a crucial source of cells in cardiac development and regeneration. However, reported CPCs are heterogeneous, and no gene has been identified to transiently mark undifferentiated CPCs throughout heart development. Here we show that Spalt-like gene 1 (Sall1), a zing-finger transcription factor, is expressed in undifferentiated CPCs giving rise to both left and right ventricles. Sall1 was transiently expressed in precardiac mesoderm contributing to the first heart field (left ventricle precursors) but not in the field itself. Similarly, Sall1 expression was maintained in the second heart field (outflow tract/right ventricle precursors) but not in cardiac cells. In vitro, high levels of Sall1 at mesodermal stages enhanced cardiomyogenesis, whereas its continued expression suppressed cardiac differentiation. Our study demonstrates that Sall1 marks CPCs in an undifferentiated state and regulates cardiac differentiation. These findings provide fundamental insights into CPC maintenance, which can be instrumental for CPC-based regenerative medicine.

Possible pathogenic role of Th17 cells for atopic dermatitis.

The critical role of IL-17 has recently been reported in a variety of conditions. Since IL-17 deeply participates in the pathogenesis of psoriasis and keratinocyte production of certain cytokines, the involvement of T helper cell 17 (Th17) in atopic dermatitis (AD) is an issue to be elucidated. To evaluate the participation of Th17 cells in AD, we successfully detected circulating lymphocytes intracellularly positive for IL-17 by flow cytometry, and the IL-17+ cell population was found exclusively in CD3+CD4+ T cells. The percentage of Th17 cells was increased in peripheral blood of AD patients and associated with severity of AD. There was a significant correlation between the percentages of IL-17+ and IFN-gamma+ cells, although percentage of Th17 cells was not closely related to Th1/Th2 balance. Immunohistochemically, IL-17+ cells infiltrated in the papillary dermis of atopic eczema more markedly in the acute than chronic lesions. Finally, IL-17 stimulated keratinocytes to produce GM-CSF, TNF-alpha, IL-8, CXCL10, and VEGF. A marked synergistic effect between IL-17 and IL-22 was observed on IL-8 production. The number of Th17 cells is increased in the peripheral blood and acute lesional skin of AD. Th17 cells may exaggerate atopic eczema.

Enhanced T-cell response to mosquito extracts by NK cells in hypersensitivity to mosquito bites associated with EBV infection and NK cell lymphocytosis.

Hypersensitivity to mosquito bites is characterized by severe systemic as well as local symptoms, and associated with chronic active EBV infection and NK cell lymphocytosis. In this HEN disease, we investigated the response of PBMC to MSG extracts. PBMC were taken from three defined cases of HEN disease, three borderline cases, five individuals with simple exaggerated reactions to mosquito bites without systemic symptoms (simple responders), and eight healthy donors. PBMC, or purified CD4+, CD8+ or CD56+ cells, were cultured with MSG extracts prepared from each of five mosquito species to examine their proliferation and cytokine secretion. The patients with HEN disease had high stimulation indices with variations in responses to the extracts from Aedes albopictus, Aedes aegypti, Anopheles sinensis and Culex pipiens pallens. However, a non-Japan-habitant species Anopheles stephensi did not stimulate the patients' PBMC. Some borderline or simple responders showed moderate proliferation, and healthy donors had no reactive PBMC. In HEN disease, both CD56+ NK cells (producing IFN-gamma) and CD4+ Th0 cells (producing IL-4 and IFN-gamma) were increased in the blood. CD4+ cells, but not CD56+ NK cells or CD8+ cells, propagated in response to MSG extracts. However, this response of CD4+ cells and their IL-4 production were strongly enhanced by coexisting CD56+ cells. We suggest that the CD4+ T cell serving as the primary responder to MSG antigen and the NK cell functioning as the enhancer are both pathogenic in the development of HMB.