Bettina Wilm

The serosal mesothelium is a major source of smooth muscle cells of the gut vasculature

Most internal organs are situated in a coelomic cavity and are covered by a mesothelium. During heart development, epicardial cells (a mesothelium) move to and over the heart, undergo epithelial-mesenchymal transition (EMT), and subsequently differentiate into endothelial and vascular smooth muscle cells. This is thought to be a unique process in blood vessel formation. Still, structural and developmental similarities between the heart and gut led us to test the hypothesis that a conserved or related mechanism may regulate blood vessel development to the gut, which, similar to the heart, is housed in a coelomic cavity. By using a combination of molecular genetics, vital dye fate mapping, organ culture and immunohistochemistry, we demonstrate that the serosal mesothelium is the major source of vasculogenic cells in developing mouse gut. Our studies show that the gut is initially devoid of a mesothelium but that serosal mesothelial cells expressing the Wilms tumor protein (Wt1) move to and over the gut. Subsequently, a subset of these cells undergoes EMT and migrates throughout the gut. Using Wt1-Cre genetic lineage marking of serosal cells and their progeny, we demonstrate that these cells differentiate to smooth muscle of all major blood vessels in the mesenteries and gut. Our data reveal a conserved mechanism in blood vessel formation to coelomic organs, and have major implications for our understanding of vertebrate organogenesis and vascular deficiencies of the gut.

Mesothelium contributes to vascular smooth muscle and mesenchyme during lung development

During mouse development, the sophisticated vascular network of the lung is established from embryonic day (E)≈10.5 and continues to develop postnatally. This network is composed of endothelial cells enclosed by vascular smooth muscle, pericytes, and other mesenchymal cells. Recent in vivo lineage labeling studies in the developing heart and intestine suggest that some of the vascular smooth muscle cells arise from the surface mesothelium. In the developing lung, the Wilms tumor 1 gene (Wt1) is expressed only in the mesothelial cells. Therefore, we lineage-labeled the mesothelium in vivo by using a Wt1-Cre transgene in combination with either Rosa26RlacZ, Rosa26RCAG-hPLAP, or Rosa26REYFP reporter alleles. In all three cases, cells derived from lineage-labeled mesothelium are found inside the lung and as smooth muscle actin (SMA) and PDGF receptor-beta positive cells in the walls of pulmonary blood vessels. To corroborate this finding, we used 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate, succinimidyl ester “mixed isomers” (CCFSE) dye to label mesothelial cells on the surface of the embryonic lung. Over the course of 72-h culture, dye-labeled cells also appear within the lung mesenchyme. Together, our data provide evidence that mesothelial cells serve as a source of vascular smooth muscle cells in the developing lung and suggest that a conserved mechanism applies to the development of blood vessels in all coelomic organs.

ERK5: structure, regulation and function.

Extracellular signal-regulated kinase 5 (ERK5), also termed big mitogen-activated protein kinase-1 (BMK1), is the most recently identified member of the mitogen-activated protein kinase (MAPK) family and consists of an amino-terminal kinase domain, with a relatively large carboxy-terminal of unique structure and function that makes it distinct from other MAPK members. It is ubiquitously expressed in numerous tissues and is activated by a variety of extracellular stimuli, such as cellular stresses and growth factors, to regulate processes such as cell proliferation and differentiation. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade plays a critical role in cardiovascular development and vascular integrity. Recent data points to a potential role in pathological conditions such as cancer and tumour angiogenesis. This review focuses on the physiological and pathological role of ERK5, the regulation of this kinase and the recent development of small molecule inhibitors of the ERK5 signalling cascade.

CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis

Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5 −/− cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5 −/− cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.

Differentiation of podocyte and proximal tubule-like cells from a mouse kidney-derived stem cell line.

In this study we have shown that the papilla of the mouse kidney contains a population of Pax2+ cells that are detectable from the early postnatal period through to adulthood. Lineage analysis suggests that some of these Pax2+ cells are derived from the metanephric mesenchyme, a population of progenitor cells that gives rise to the nephrons during kidney organogenesis. Here we describe a method for isolating and culturing the Pax2+ population, and demonstrate that some cells within this population are multipotent stem cells, as they are clonogenic and appear to undergo unlimited self-renewal. Further, under appropriate culture conditions, these stem cells can differentiate to generate renal cell types, such as podocyte- and proximal tubule-like cells, and are also able to generate nonrenal cell types, such as adipocytes and osteocytes. The availability of a kidney-derived multipotent stem cell line with the potential to generate podocytes and proximal tubule cells in culture will expedite progress in understanding the biology of these important renal cell types, and will be a useful tool in toxicological studies and drug discovery.

Serosal mesothelium retains vasculogenic potential

Mesothelia comprise the epithelial covering of coelomic organs and line the cavities in which they are housed. Mesothelia contribute to the vasculature of the heart and the intestinal tract by developmental processes of epithelial–mesenchymal transition (EMT), migration, and differentiation into endothelial cells, vascular smooth muscle cells, and pericytes. Here, we establish a novel in vitro system to analyze the differentiative potential of mesothelia. Using explants from serosal mesothelium (the mesothelial covering of the gut), we demonstrate that much of the developmental program observed in embryonic mesothelia is retained in the adult structure. Namely, processes of epithelial spreading, EMT, and differentiation into smooth muscle cells from these cells are observed. Interestingly, we were unable to stimulate endothelial cell differentiation using serum or various signaling factors. Taken together, these data reveal that differentiated serosal cells retain vasculogenic potential and provide a generalizable model for future studies on the developmental and differentiative capacity of the mesothelial cell type. Developmental Dynamics 236:2973–2979, 2007.

Measures of kidney function by minimally invasive techniques correlate with histological glomerular damage in SCID mice with adriamycin-induced nephropathy

Maximising the use of preclinical murine models of progressive kidney disease as test beds for therapies ideally requires kidney function to be measured repeatedly in a safe, minimally invasive manner. To date, most studies of murine nephropathy depend on unreliable markers of renal physiological function, exemplified by measuring blood levels of creatinine and urea, and on various end points necessitating sacrifice of experimental animals to assess histological damage, thus counteracting the principles of Replacement, Refinement and Reduction. Here, we applied two novel minimally invasive techniques to measure kidney function in SCID mice with adriamycin-induced nephropathy. We employed i) a transcutaneous device that measures the half-life of intravenously administered FITC-sinistrin, a molecule cleared by glomerular filtration; and ii) multispectral optoacoustic tomography, a photoacoustic imaging device that directly visualises the clearance of the near infrared dye, IRDye 800CW carboxylate. Measurements with either technique showed a significant impairment of renal function in experimental animals versus controls, with significant correlations with the proportion of scarred glomeruli five weeks after induction of injury. These technologies provide clinically relevant functional data and should be widely adopted for testing the efficacies of novel therapies. Moreover, their use will also lead to a reduction in experimental animal numbers.

Development of embryonic stem cells in recombinant kidneys

Embryonic stem cells (ESC) are self-renewing and can generate all cell types during normal development. Previous studies have begun to explore fates of ESCs and their mesodermal derivatives after injection into explanted intact metanephric kidneys and neonatal kidneys maturing in vivo. Here, we exploited a recently described recombinant organ culture model, mixing fluorescent quantum dot labeled mouse exogenous cells with host metanephric cells. We compared abilities of undifferentiated ESCs with ESC-derived mesodermal or non-mesodermal cells to contribute to tissue compartments within recombinant, chimeric metanephroi. ESC-derived mesodermal cells downregulated Oct4, a marker of undifferentiated cells, and, as assessed by locations of quantum dots, contributed to Wilms’ tumor 1-expressing forming nephrons, synaptopodin-expressing glomeruli, and organic ion-transporting tubular epithelia. Similar results were observed when labeled native metanephric cells were recombined with host cells. In striking contrast, non-mesodermal ESC-derived cells strongly inhibited growth of embryonic kidneys, while undifferentiated ESCs predominantly formed Oct4 expressing colonies between forming nephrons and glomeruli. These findings clarify the conclusion that ESC-derived mesodermal cells have functional nephrogenic potential, supporting the idea that they could potentially replace damaged epithelia in diseased kidneys. On the other hand, undifferentiated ESCs and non-mesodermal precursors derived from ESCs would appear to be less suitable materials for use in kidney cell therapies.

Preventing Plasmon Coupling between Gold Nanorods Improves the Sensitivity of Photoacoustic Detection of Labeled Stem Cells in Vivo.

Gold nanorods are excellent contrast agents for imaging technologies which rely on near-infrared absorption such as photoacoustic imaging. For cell tracking applications, the cells of interest are labeled with the contrast agent prior to injection. However, after uptake into cells by endocytosis, the confinement and high concentration in endosomes leads to plasmon band broadening and reduced absorbance. This would limit the potential of multispectral optoacoustic tomography in terms of spectral processing and, consequently, sensitivity. Here, we show that steric hindrance provided by silica coating of the nanorods leads to the preservation of their spectral properties and improved photoacoustic sensitivity. This strategy allowed the detection and monitoring of as few as 2 × 10(4) mesenchymal stem cells in mice over a period of 15 days with a high spatial resolution. Importantly, the silica-coated nanorods did not affect the viability or differentiation potential of the transplanted mesenchymal stem cells.

Integration potential of mouse and human bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a multipotent cell population which has been described to exert renoprotective and regenerative effects in experimental models of kidney injury. Several lines of evidence indicate that MSCs also have the ability to contribute to nephrogenesis, suggesting that the cells can be employed in stem cell-based applications aimed at de novo renal tissue generation. In this study we re-evaluate the capacity of mouse and human bone marrow-derived MSCs to contribute to the development of renal tissue using a novel method of embryonic kidney culture. Although MSCs show expression of some genes involved in renal development, their contribution to nephrogenesis is very limited in comparison to other stem cell types tested. Furthermore, we found that both mouse and human MSCs have a detrimental effect on the ex vivo development of mouse embryonic kidney, this effect being mediated through a paracrine action. Stimulation with conditioned medium from a mouse renal progenitor population increases the ability of mouse MSCs to integrate into developing renal tissue and prevents the negative effects on kidney development, but does not appear to enhance their ability to undergo nephrogenesis.