In vitro investigation of renal epithelial injury suggests that primary cilium length is regulated by hypoxia-inducible mechanisms.

Primary cilia are non-motile sensory organelles that project from cells in many tissues. The role of renal primary cilium-based signalling in regulating epithelial cell proliferation and differentiation is highlighted by studies showing that defects of the cilium lead to epithelial de-differentiation, over proliferation and polycystic kidney disease. Recent studies show that renal primary cilia may also play a role in controlling epithelial differentiation during renal repair. After injury, renal cilium length increases dramatically and then undergoes a normalization that coincides with structural and functional repair in both human patients and mouse models of renal injury. These changes in cilium length are likely to modulate cilium-based signalling, but the injury-related factors that influence renal primary cilium length have yet to be determined. Here, we investigated the effect of three factors commonly associated with renal injury on renal cilium length in an in vitro setting. MDCK (Madin Darby canine kidney) cell cultures bearing primary cilia were treated with BSA to simulate albuminuria, cobalt chloride to simulate hypoxia and the inflammation-related cytokine tumour necrosis factor α. Primary cilium length was only increased in cultures treated with cobalt chloride. Our results suggest a role for hypoxia and the induction of HIF-1α (hypoxia-inducible factor 1α) in increasing renal primary cilium length following renal injury.

Density gradients for the isolation of germ cells from embryoid bodies.

In previous reports, the isolation of embryonic stem cell (ESC)-derived germ cells utilized fluorescent protein knock-in cell lines that could be sorted by flow cytometry. The present study aimed to isolate putative germ stem cells from embryoid bodies (EB) using Percoll and Nycodenz density gradients. The optimal ESC concentration to establish EB was identified as 15,000 cells per 30 mul drop and the optimal culture time to obtain the highest number of germ cells within EB was identified as 120 h, with over 25% of cells confirmed as germ cells for the specific cell line used. Germ cells were isolated from 120-hourold EB by Percoll and Nycodenz density gradients, while isolation of primordial germ cells from genital ridges of embryonic day 12.5 fetuses was used as a control. Putative germ cells were isolated from EB at proportions of 80.3 +/- 3.0% (SEM) and 75.8 +/- 0.9% for Percoll and Nycodenz respectively. Primordial germ cells were isolated from genital ridges at rates of 89.7 +/- 2.7% and 89.5 +/- 0.9% respectively. Although the purity of the isolated germ cells was similar between the two gradients, more germ cells with higher viability were obtained with the Percoll gradient.

Mouse Germ Cell Development in-vivo and in-vitro.

In mammalian development, primordial germ cells (PGCs) represent the initial population of cells that are committed to the germ cell lineage. PGCs segregate early in development, triggered by signals from the extra-embryonic ectoderm. They are distinguished from surrounding cells by their unique gene expression patterns. Some of the more common genes used to identify them are Blimp1, Oct3/4, Fragilis, Stella, c-Kit, Mvh, Dazl and Gcna1. These genes are involved in regulating their migration and differentiation, and in maintaining the pluripotency of these cells.Recent research has demonstrated the possibility of obtaining PGCs, and subsequently, mature germ cells from a starting population of embryonic stem cells (ESCs) in culture. This phenomenon has been investigated using a variety of methods, and ESC lines of both mouse and human origin. Embryonic stem cells can differentiate into germ cells of both the male and female phenotype and in one case has resulted in the birth of live pups from the fertilization of oocytes with ESC derived sperm. This finding leads to the prospect of using ESC derived germ cells as a treatment for sterility. This review outlines the evolvement of germ cells from ESCs in vitro in relation to in vivo events.