Mesenchymal Stem Cells Promote Oligodendroglial Differentiation in Hippocampal Slice Cultures

We have previously shown that soluble factors derived from mesenchymal stem cells (MSCs) induce oligodendrogenic fate and differentiation in adult rat neural progenitors (NPCs) in vitro. Here, we investigated if this pro-oligodendrogenic effect is maintained after cells have been transplanted onto rat hippocampal slice cultures, a CNS-organotypic environment. We first tested whether NPCs, that were pre-differentiated in vitro by MSC-derived conditioned medium, would generate oligodendrocytes after transplantation. This approach resulted in the loss of grafted NPCs, suggesting that oligodendroglial pre-differentiated cells could not integrate in the tissue and therefore did not survive grafting. However, when NPCs together with MSCs were transplanted in situ into hippocampal slice cultures, the grafted NPCs survived and the majority of them differentiated into oligodendrocytes. In contrast to the prevalent oligodendroglial differentiation in case of the NPC/MSC co-transplantation, naive NPCs transplanted in the absence of MSCs differentiated predominantly into astrocytes. In summary, the pro-oligodendrogenic activity of MSCs was maintained only after co-transplantation into hippocampal slice cultures. Therefore, in the otherwise astrogenic milieu, MSCs established an oligodendrogenic niche for transplanted NPCs, and thus, co-transplantation of MSCs with NPCs might provide an attractive approach to re-myelinate the various regions of the diseased CNS. Copyright (C) 2009 S. Karger AG, Base

K+ Channel Inhibition Differentially Regulates Migration of Intestinal Epithelial Cells in Inflamed vs. Non-Inflamed Conditions in a PI3K/Akt-Mediated Manner

Background Potassium channels have been shown to determine wound healing in different tissues, but their role in intestinal epithelial restitution-the rapid closure of superficial wounds by intestinal epithelial cells (IEC)-remains unclear. Methods In this study, the regulation of IEC migration by potassium channel modulation was explored with and without additional epidermal growth factor (EGF) under baseline and interferon-gamma (IFN-gamma)-pretreated conditions in scratch assays and Boyden chamber assays using the intestinal epithelial cell lines IEC-18 and HT-29. To identify possibly involved subcellular pathways, Western Blot (WB)-analysis of ERK and Akt phosphorylation was conducted and PI3K and ERK inhibitors were used in scratch assays. Furthermore, mRNA-levels of the potassium channel KCNN4 were determined in IEC from patients suffering from inflammatory bowel diseases (IBD). Results Inhibition of Ca2+-dependent potassium channels significantly increased intestinal epithelial restitution, which could not be further promoted by additional EGF. In contrast, inhibition of KCNN4 after pretreatment with IFN-gamma led to decreased or unaffected migration. This effect was abolished by EGF. Changes in Akt, but not in ERK phosphorylation strongly correlated with these findings and PI3K but not ERK inhibition abrogated the effect of KCNN4 inhibition. Levels of KCNN4 mRNA were higher in samples from IBD patients compared with controls. Conclusions Taken together, we demonstrate that inhibition of KCNN4 differentially regulates IEC migration in IFN-gamma-pretreated vs. non pretreated conditions. Moreover, our data propose that the PI3K signaling cascade is responsible for this differential regulation. Therefore, we present a cellular model that contributes new aspects to epithelial barrier dysfunction in chronic intestinal inflammation, resulting in propagation of inflammation and symptoms like ulcers or diarrhea

Intrinsic and extrinsic determinants of central nervous system axon outgrowth into alginate-​based anisotropic hydrogels

Appropriate target reinnervation and functional recovery after spinal cord injury depend on longitudinally directed regrowth of injured axons. Anisotropic alginate-based capillary hydrogels (ACH) support peripheral nervous system derived axon growth, which is accompanied by glial supporting cell migration into the ACH. The aim of the present study was to analyze central nervous system (CNS) derived (entorhinal cortex, spinal cord slice cultures) axon regrowth into ACH containing linearly aligned capillaries of defined capillary sizes without and with gelatin constituent. Anisotropic ACH were prepared by ionotropic gel formation using Ba2+, Cu2+, Sr2+, or Zn2+ ions resulting in gels with average capillary diameters of 11, 13, 29, and 89 μm, respectively. Postnatal rat entorhinal cortex or spinal cord slice cultures were placed on top of 500 μm thick ACH. Seven days later axon growth and astroglial migration into the ACH were determined. Axon density within capillaries correlated positively with increasing capillary diameters, whereas longitudinally oriented axon outgrowth diminished with increasing capillary diameter. Axons growing into the hydrogels were always accompanied by astrocytes strongly suggesting that respective cells are required to mediate CNS axon elongation into ACH. Overall, midsize capillary diameter ACH appeared to be the best compromise between axon density and orientation. Taken together, ACH promote CNS axon ingrowth, which is determined by the capillary diameter and migration of slice culture derived astroglia into the hydrogel

Increasing capillary diameter and the incorporation of gelatin enhance axon outgrowth in alginate-based anisotropic hydrogels

Substantial recovery of function following peripheral and central nervous system (CNS) injury critically depends on longitudinally directed axon regeneration across the injury site, which requires a mech. guidance providing scaffold. We have previously shown that anisotropic alginate-based hydrogels with a defined capillary diam. (25 μm), which form via a self-organizing process driven by unidirectional diffusion of divalent cations into sodium alginate sols, promoted longitudinally oriented elongation of CNS axons in vitro and in vivo. In the present study the influence of various capillary diams. and the incorporation of gelatin to promote directed axon outgrowth and Schwann cell migration were assessed in a dorsal root ganglion outgrowth assay in vitro. Superimposing an alginate sol with Cu2+, Sr2+, or Zn2+ ion contg. solns. allowed the creation of hydrogels with capillaries 18, 25 and 55 μm in diam., resp. Axon outgrowth and Schwann cell migration were analyzed in terms of axon length/d. and Schwann cell d. within the capillary structures. Axon ingrowth into capillary hydrogels, which was always accompanied by Schwann cells, was enhanced with increasing capillary diam. The incorporation of gelatin did not influence overall axon d., but promoted the length of axon outgrowth within the hydrogels. The longitudinal orientation of axons decreased in wider capillaries, which suggests that medium-sized capillaries are the optimal substrate to elicit substantial axon growth and longitudinal orientation after axon injury

Indoxyl 3-sulfate inhibits maturation and activation of human monocyte-derived dendritic cells

Indole is produced from L-tryptophan by commensal bacteria and further metabolized to indoxyl 3-sulfate (I3S) in the liver. Physiologic concentrations of I3S are related to a lower risk to develop graft versus host disease in allogeneic stem cell transplanted patients pointing towards an immunoregulatory function of I3S. Here we investigated the impact of I3S on the maturation of human monocyte-derived dendritic cells (DCs). Even pathophysiologic concentrations of I3S did not affect viability of mature DCs, but I3S decreased the expression of co-stimulatory molecules such as CD80 and CD86 on mature DCs. Furthermore, I3S inhibited IL-12 and IL-6 secretion by mature DCs while IL-10 was significantly upregulated. Co-culture of I3S-treated mature DCs with allogeneic T cells revealed no alteration in T cell proliferation. However, interferon gamma and TNF production of T cells was suppressed. As I3S exerted no direct effect on T cells, the defect in T cell activation was mediated by I3S-treated mature DCs. Our study suggests an anti-inflammatory and tolerizing effect of I3S on human DCs

Restitution of scratch-wounded IEC-18 monolayers is marked by migration of adjacent cells.

<p>A: representative sequence of images taken every hour (from top to bottom) beginning directly after mechanical wounding of a confluent IEC-18 monolayer. The approximate location of the initial wound margin (top picture) is indicated by a green dashed line and also displayed in subsequent images. Scale bar: 100μm. B: Representative images of migrating cells sending ahead filopodia (red arrows) and pseudopodia (orange arrows). C: Exemplary sequence of processes at the wound margin. Two adjacent cells are edged in red and shown at the indicated time points. Over time they migrate into the denuded area undergoing considerable morphological changes. D: Exemplary sequence of events within the cell sheet distant from the wound. Two representative cells are edged in red and show only bare movements or changes in shape over six hours.</p

Oligodendrogenesis of adult neural progenitors: differential effects of ciliary neurotrophic factor and mesenchymal stem cell derived factors

The oligodendrogenic program of progenitor cells in the adult CNS follows a sequential process of progenitor proliferation, fate choice, determination, differentiation, maturation and survival. Previously, we described a soluble activity derived from mesenchymal stem cells that induces oligodendrogenesis in adult neural progenitor cells. Here, we hypothesized that ciliary neurotrophic factor might be a candidate for this activity, since (i) it is expressed by mesenchymal stem cells and (ii) it can promote oligodendrogenesis during development. Along the course of the study, we found differential effects by ciliary neurotrophic factor and by the mesenchymal stem cells-derived activity on neural progenitors. While the mesenchymal stem cells-derived activity induced oligodendrogenesis at the expense of astrogenesis and promoted oligodendroglial differentiation/maturation, the effect of ciliary neurotrophic factor was restricted to the latter one. This was reflected at the levels of the cell fate determinants Olig1, Olig2, Id2, and the oligodendroglia-maturation transcription factor GTX/Nkx6.2. Finally, experiments using blocking antibodies excluded ciliary neurotrophic factor to be the mesenchymal stem cell-derived oligodendroglial activity. In summary, this work provides evidence for differential effects of ciliary neurotrophic factor and mesenchymal stem cells-derived activity on oligodendrogenesis of adult neural progenitor cells