Dermal papilla cells induce keratinocyte tubulogenesis in culture.

The ability of dermal papilla (DP) cells to induce hair growth was reported in many studies. However, early stages of hair follicle development and signals that govern this process are poorly understood. Therefore, an in vitro model may be a convenient system to study epithelial-mesenchymal interactions and early stages of epidermal morphogenesis, especially in humans. To investigate the role of DP cells in epidermal morphogenesis we modified the method of isolation of DP cells from hair follicle of human scalp and developed the three-dimensional model of epidermal morphogenesis. Isolated DP cells were able to differentiate in adipogenic and osteogenic directions and retained activity of alkaline phosphatase (AP) for seven passages in culture. DP cells were able to induce tubule-like structures in three-dimensional model in vitro and to reorganize collagen matrix. Prolonged cultivation of DP cells has been a big problem because of the loss of hair follicle-inducing ability and growth activity after several passages. To solve this problem we immortalized DP cells by the transfection of the human telomerase reverse transcriptase cDNA (hTERT). Immortalized DP-hTERT cells retained AP activity and demonstrated low ability to osteogenic differentiation. The conditioned medium collected from actively proliferated cells as well as DP-hTERT cells themselves were capable to induce tubulogenesis after prolonged keratinocyte cultivation.

Dynein is a motor for nuclear rotation while vimentin IFs is a "brake".

The positioning of the nucleus is achieved by two interconnected processes, anchoring and migration, both of which are controlled by cytoskeleton structures. Rotation is a special type of nuclear motility in many cell types, but its significance remains unclear. We used a vimentin-null cell line, MFT-16, which shows extensive nuclear rotation to study the phenomenon in detail. By selective disruption of cytoskeletal structures and video-microscopic analysis, nuclear rotation was a microtubule-dependent process that F-actin partially impedes. The dynein-dynactin complex is responsible and inhibiting this motor by expression of a dominant negative mutant of its component P-150 completely stops it. Nuclear rotation is powered by dynein associated with the nuclear envelope along stationary microtubules, centrosomes remaining immobile. We confirmed that vimentin IFs inhibit nuclear rotation, and variant proteins of the mutated wild type gene for vimentin that lacked considerable fragments of the N- and C-terminal domains restored nuclear anchoring. Immunochemical analysis showed that these mutated IFs also bound plectin, arguing for a key role of this cytolinker protein in nuclear anchoring. It is proposed that this versatile machinery guarantees not only rotation and the correct location of a nucleus, but also its orientation in a cell.

Sparse plating increases the heterogeneity of proliferative potential of fibroblasts.

High heterogeneity of proliferative potential in the cultures of diploid human fibroblasts was reported in many studies. It was generally believed that the heterogeneity of proliferative potential of human fibroblasts reflects the unevenness of their senescence. However we show here that immortalized (telomerized) human fibroblasts obey the same rule. Up to 50% of these cells rapidly ceased to proliferate when plated at low density in contrast to usual conditions of mass culture where at least 98% of these cells keep on proliferating. Initially, we proposed that the appearance of non-dividing or slow-dividing cells in low-density cell culture experiments could be caused by cell damage due to the experimental setup. Indeed, lowering of oxygen level and addition of conditioned medium improved colony formation, but there were a large number of non-proliferating cells (13-20%). When we sparsely plated cells on a feeder layer of cells of certain density, the portion of non-proliferating cells decreased to 2%, i.e. became the same as in mass culture. Thus, the heterogeneity of proliferative potential is partially a result of the adverse effect of low cell density.