Investigating the renogenic potential of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a multipotent cell population which have been described to exert renoprotective and regenerative effects in experimental models of kidney injury. In addition, it was recently shown that human MSCs are able to contribute to the development of both renal tubules and glomeruli. These results suggest that MSCs might be potential candidates for stem cell-based de novo renal tissue generation. The current study was aimed at re-evaluating the renogenic capacity of mouse and human bone marrow-derived MSCs. In order to elucidate the renogenic potential of MSCs, a novel method of embryonic kidney culture was used that is based on disaggregation of mouse kidney rudiments and their subsequent re-aggregation in the presence of cells from different origins to form kidney chimeras. Initially, MSCs did show expression of some genes involved in renal development; however, neither mouse nor human cells expressed important renal development genes, such as Wt1 and Pax2. Accordingly, MSCs were demonstrated to have low renogenic potential in the chimeric kidney model as they did not engraft into ureteric buds, the precursors of collecting duct system, and were only occasionally found in the condensing metanephric mesenchyme, which gives rise to nephrons. In addition, the incorporation of MSCs into embryonic kidneys had some detrimental effect on metanephric development. This effect was mediated through a paracrine action of the cells, as conditioned medium derived from mouse MSCs was demonstrate to reduce ureteric bud branching in in vitro kidney rudiment culture. On the contrary, mouse neonatal kidney cells did engraft into the condensing mesenchyme of chimeric kidneys and were subsequently found in some developing nephron-like structures. Regarding the potential of mouse embryonic stem cells to contribute to renal development in the re-aggregated kidney chimeras, the cells were found to some extent in both the condensing mesenchyme and the laminin-positive tubular compartment of chimeric kidneys, possibly the ureteric buds. No negative effect on kidney development was observed using the neonatal kidney cells as well as the embryonic stem cells. Ultimately it has been shown that the pre-conditioning of mouse MSCs with medium derived from mouse neonatal kidney cells facilitated the engraftment of MSCs into condensing mesenchyme of chimeric kidneys. It also prevented the negative action of MSCs on kidney development confirmed in the in vitro kidney rudiment culture. MSCs were demonstrated to up-regulate GDNF expression upon the pre-conditioning which is important factor for outgrowth and branching of ureteric buds. In conclusion, although pre-conditioning of the MSCs with medium derived from kidney cells was able to improve considerably the renogenic potential of the cells in the chimeric kidney, MSCs demonstrate a relatively low renogenic potential and for this reason are not good candidates for regenerative approaches aimed at recapitulation of nephrogenesis

Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures

Engineering kidneys from simple cell suspensions:an exercise in self-organization

Increasing numbers of people approaching and living with end-stage renal disease and failure of the supply of transplantable kidneys to keep pace has created an urgent need for alternative sources of new organs. One possibility is tissue engineering of new organs from stem cells. Adult kidneys are arguably too large and anatomically complex for direct construction, but engineering immature kidneys, transplanting them, and allowing them to mature within the host may be more feasible. In this review, we describe a technique that begins with a suspension of renogenic stem cells and promotes these cells’ self-organization into organ rudiments very similar to foetal kidneys, with a collecting duct tree, nephrons, corticomedullary zonation and extended loops of Henle. The engineered rudiments vascularize when transplanted to appropriate vessel-rich sites in bird eggs or adult animals, and show preliminary evidence for physiological function. We hope that this approach might one day be the basis of a clinically useful technique for renal replacement therapy

Investigating the renogenic potential of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a multipotent cell population which have been described to exert renoprotective and regenerative effects in experimental models of kidney injury. In addition, it was recently shown that human MSCs are able to contribute to the development of both renal tubules and glomeruli. These results suggest that MSCs might be potential candidates for stem cell-based de novo renal tissue generation. The current study was aimed at re-evaluating the renogenic capacity of mouse and human bone marrow-derived MSCs. In order to elucidate the renogenic potential of MSCs, a novel method of embryonic kidney culture was used that is based on disaggregation of mouse kidney rudiments and their subsequent re-aggregation in the presence of cells from different origins to form kidney chimeras. Initially, MSCs did show expression of some genes involved in renal development; however, neither mouse nor human cells expressed important renal development genes, such as Wt1 and Pax2. Accordingly, MSCs were demonstrated to have low renogenic potential in the chimeric kidney model as they did not engraft into ureteric buds, the precursors of collecting duct system, and were only occasionally found in the condensing metanephric mesenchyme, which gives rise to nephrons. In addition, the incorporation of MSCs into embryonic kidneys had some detrimental effect on metanephric development. This effect was mediated through a paracrine action of the cells, as conditioned medium derived from mouse MSCs was demonstrate to reduce ureteric bud branching in in vitro kidney rudiment culture. On the contrary, mouse neonatal kidney cells did engraft into the condensing mesenchyme of chimeric kidneys and were subsequently found in some developing nephron-like structures. Regarding the potential of mouse embryonic stem cells to contribute to renal development in the re-aggregated kidney chimeras, the cells were found to some extent in both the condensing mesenchyme and the laminin-positive tubular compartment of chimeric kidneys, possibly the ureteric buds. No negative effect on kidney development was observed using the neonatal kidney cells as well as the embryonic stem cells. Ultimately it has been shown that the pre-conditioning of mouse MSCs with medium derived from mouse neonatal kidney cells facilitated the engraftment of MSCs into condensing mesenchyme of chimeric kidneys. It also prevented the negative action of MSCs on kidney development confirmed in the in vitro kidney rudiment culture. MSCs were demonstrated to up-regulate GDNF expression upon the pre-conditioning which is important factor for outgrowth and branching of ureteric buds. In conclusion, although pre-conditioning of the MSCs with medium derived from kidney cells was able to improve considerably the renogenic potential of the cells in the chimeric kidney, MSCs demonstrate a relatively low renogenic potential and for this reason are not good candidates for regenerative approaches aimed at recapitulation of nephrogenesis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Using Fluorescence Recovery After Photobleaching to Study Gap Junctional Communication In Vitro.

Fluorescence recovery after photobleaching (FRAP) is a microscopy-based technique to study the movement of fluorescent molecules inside a cell. Although initially developed to investigate intracellular mobility, FRAP can be also used to measure intercellular dynamics. This chapter describes how to perform FRAP experiment to study gap junctional communication in living cells. The procedures described here can be carried out with a laser-scanning confocal microscope and any in vitro cultured cells known to communicate via gap junctions. In addition, the method can be easily adjusted to measure gap junction function in 3D cell cultures as well as ex vivo tissue

Boundary regularity of mass-minimizing integral currents and a question of Almgren

This short note is the announcement of a forthcoming work in which we prove a first general boundary regularity result for area-minimizing currents in higher codimension, without any geometric assumption on the boundary, except that it is an embedded submanifold of a Riemannian manifold, with a mild amount of smoothness ($C^3, a_0$ for a positive $a_0$ suffices). Our theorem allows to answer a question posed by Almgren at the end of his Big Regularity Paper. In this note we discuss the ideas of the proof and we also announce a theorem which shows that the boundary regularity is in general weaker that the interior regularity. Moreover we remark an interesting elementary byproduct on boundary monotonicity formulae

Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures.</p