Rapamycin treatment down regulates SMA expression of SKPs.

<p>SKPs were cultured in medium containing either EGF/FGF +/− Rapamycin or 15% FBS +/− Rapamycin for one week. A: SMA expression was quantified by qPCR. Graph shows mean +/− SE of n = 4. B: SKPs were cultured in medium containing either EGF/FGF +/− Rapamycin or 3% FBS +/− Rapamycin for one week. SMA expression and S6 phosphorylation was analyzed by IF staining. Representative images of n = 10 are shown.</p

Pathologic Bladder Microenvironment Attenuates Smooth Muscle Differentiation of Skin Derived Precursor Cells: Implications for Tissue Regeneration

<div><p>Smooth muscle cell containing organs (bladder, heart, blood vessels) are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs) can easily be isolated from adult skin and can be differentiated <i>in vitro</i> into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. <i>In vivo</i>, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from <i>ex vivo</i> stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF) suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain) as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells <i>in vitro</i>, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in <i>in vivo</i> settings. These observations must be considered in drafting any regeneration strategies.</p> </div

SMC differentiation of SKPs correlates with reduced S6 phosphorylation.

<p>A: SKPs were cultured in medium containing 15% FBS for 20 min. S6 phosphorylation was analyzed by IF staining. Representative images on n = 10 are shown. B: SKPs were exposed to conditioned medium from either stretched or non-stretched bladders for 20 min. S6 phosphorylation was analyzed by IF staining. Fluorescent intensity was quantified by image analysis using Volocity software. Graph represents mean +/− SE of n = 50 cells.</p

The microenvironment of stretch injured adult bladders attenuates SMC differentiation of SKPs.

<p>GFP+ SKPs were co-cultured with organoids isolated from stretched or non-stretched adult rat bladders. After one week, SMA expression was analyzed by IF staining and the percentage of GFP+/SMA+ cells was quantified. Inserts show GFP+ cells. Arrows indicate GFP+/SMA+ cells. Graph shows mean +/− SE of n = 100 cells.</p

Bladder stretch induced diffusible factors suppress SMC differentiation of SKPs.

<p>SKPs were cultured in conditioned medium obtained from <i>ex vivo</i> stretched or non-stretched adult bladders. A: SKPs were cultured in medium conditioned by <i>ex vivo</i> stretched or non-stretched bladders for one week. SMA expression was analyzed by qPCR (A) and IF (C), Calponin expression was quantified by qPCR (B). Graphs show mean +/− SE of n = 4. Representative images of n = 10 are shown in C. D: SKPs were differentiated in medium containing 3% FBS or 3% FBS +4 ng/ml FGF2/bFGF for one week. SKPs cultured in medium containing EGF and bFGF were used as control. SMA expression was analyzed by IF staining. Representative images of n = 10 are shown.</p

On-Tissue Localization of Ceramides and Other Sphingolipids by MALDI Mass Spectrometry Imaging

A novel MALDI-FTICR imaging mass spectrometry (MALDI-IMS) workflow is described for on-tissue detection, spatial localization, and structural confirmation of low abundance bioactive ceramides and other sphingolipids. Increasingly, altered or elevated levels of sphingolipids, sphingolipid metabolites, and sphingolipid metabolizing enzymes have been associated with a variety of disorders such as diabetes, obesity, lysosomal storage disorders, and cancer. Ceramide, which serves as a metabolic hub in sphingolipid metabolism, has been linked to cancer signaling pathways and to metabolic regulation with involvement in autophagy, cell-cycle arrest, senescence, and apoptosis. Using kidney tissues from a new Farber disease mouse model in which ceramides of all acyl chain lengths and other sphingolipid metabolites accumulate in tissues, specific ceramides and sphingomyelins were identified by on-tissue isolation and fragmentation, coupled with an on-tissue digestion by ceramidase or sphingomyelinase. Multiple glycosphingolipid species were also detected. The newly generated library of sphingolipid ions was then applied to MALDI-IMS of human lung cancer tissues. Multiple tumor specific ceramide and sphingomyelin species were detected and confirmed by on-tissue enzyme digests and structural confirmation. High-resolution MALDI-IMS in combination with novel on-tissue ceramidase and sphingomyelinase enzyme digestions makes it now possible to rapidly visualize the distribution of bioactive ceramides and sphingomyelin in tissues