The Serotonin Receptor Subtype 5b Specifically Interacts with Serotonin Receptor Subtype 1A

Previously, we described the dysregulation of serotonin (5-HT) receptor subtype 5b (5-ht5b) in a mouse model of Rett syndrome (RTT). 5-ht5b has not been extensively studied, so we set out to characterize it in more detail. Unlike common cell surface receptors, 5-ht5b displays no membrane expression, while receptor clusters are located in endosomes. This unusual subcellular localization is at least in part controlled by glycosylation of the N-terminus, with 5-ht5b possessing fewer glycosylation sites than related receptors. We analyzed whether the localization to endosomes has any functional relevance and found that 5-ht5b receptors can specifically interact with 5-HT1A receptors and retain them in endosomal compartments. This interaction reduces 5-HT1A surface expression and is mediated by interactions between the fourth and fifth trans-membrane domain (TMD). This possibly represents a mechanism by which 5-ht5b receptors regulate the activity of other 5-HT receptor

Functional properties of equine adipose-derived mesenchymal stromal cells cultured with equine platelet lysate

Successful translation of multipotent mesenchymal stromal cell (MSC)-based therapies into clinical reality relies on adequate cell production procedures. These should be available not only for human MSC, but also for MSC from animal species relevant to preclinical research and veterinary medicine. The cell culture medium supplementation is one of the critical aspects in MSC production. Therefore, we previously established a scalable protocol for the production of buffy-coat based equine platelet lysate (ePL). This ePL proved to be a suitable alternative to fetal bovine serum (FBS) for equine adipose-derived (AD-) MSC culture so far, as it supported AD-MSC proliferation and basic characteristics. The aim of the current study was to further analyze the functional properties of equine AD-MSC cultured with the same ePL, focusing on cell fitness, genetic stability and pro-angiogenic potency. All experiments were performed with AD-MSC from n = 5 horses, which were cultured either in medium supplemented with 10% FBS, 10% ePL or 2.5% ePL. AD-MSC cultured with 2.5% ePL, which previously showed decreased proliferation potential, displayed higher apoptosis but lower senescence levels as compared to 10% ePL medium (p < 0.05). Non-clonal chromosomal aberrations occurred in 8% of equine AD-MSC cultivated with FBS and only in 4.8% of equine AD-MSC cultivated with 10% ePL. Clonal aberrations in the AD-MSC were neither observed in FBS nor in 10% ePL medium. Analysis of AD-MSC and endothelial cells in an indirect co-culture revealed that the ePL supported the pro-angiogenic effects of AD-MSC. In the 10% ePL group, more vascular endothelial growth factor (VEGF-A) was released and highest VEGF-A concentrations were reached in the presence of ePL and co-cultured cells (p < 0.05). Correspondingly, AD-MSC expressed the VEGF receptor-2 at higher levels in the presence of ePL (p < 0.05). Finally, AD-MSC and 10% ePL together promoted the growth of endothelial cells and induced the formation of vessel-like structures in two of the samples. These data further substantiate that buffy-coat-based ePL is a valuable supplement for equine AD-MSC culture media. The ePL does not only support stable equine AD-MSC characteristics as demonstrated before, but it also enhances their functional properties

Intercellular Transport of Oct4 in Mammalian Cells: A Basic Principle to Expand a Stem Cell Niche?

Background: The octamer-binding transcription factor 4 (Oct4) was originally described as a marker of embryonic stem cells. Recently, the role of Oct4 as a key regulator in pluripotency was shown by its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. While artificial induction of pluripotency using transcription factors is possible in mammalian cell culture, it remains unknown whether a potential natural transfer mechanism might be of functional relevance in vivo. The stem cell based regeneration of deer antlers is a unique model for rapid and complete tissue regeneration in mammals and therefore most suitable to study such mechanisms. Here, the transfer of pluripotency factors from resident stem cell niche cells to differentiated cells could recruit more stem cells and start rapid tissue regeneration. Methodology/Principal Findings: We report on the ability of STRO-1 + deer antlerogenic mesenchymal stem cells (DaMSCs) to transport Oct4 via direct cell-to-cell connections. Upon cultivation in stem cell expansion medium, we observed nuclear Oct4 expression in nearly all cells. A number of these cells exhibit Oct4 expression not only in the nucleus, but also with perinuclear localisation and within far-ranging intercellular connections. Furthermore, many cells showed intercellular connections containing both F-actin and a-tubulin and through which transport could be observed. To proof that intercellular Oct4-transfer has functional consequences in recipient cells we used a co-culture approach with STRO-1 + DaMSCs and a murine embryonic fibroblast indicator cell line (Oct4-GFP MEF). In this cell line a reporter gene (GFP) unde

Localization and Characterization of STRO-1+ Cells in the Deer Pedicle and Regenerating Antler

The annual regeneration of deer antlers is a unique developmental event in mammals, which as a rule possess only a very limited capacity to regenerate lost appendages. Studying antler regeneration can therefore provide a deeper insight into the mechanisms that prevent limb regeneration in humans and other mammals, and, with regard to medical treatments, may possibly even show ways how to overcome these limitations. Traditionally, antler regeneration has been characterized as a process involving the formation of a blastema from de-differentiated cells. More recently it has, however, been hypothesized that antler regeneration is a stem cell-based process. Thus far, direct evidence for the presence of stem cells in primary or regenerating antlers was lacking. Here we demonstrate the presence of cells positive for the mesenchymal stem cell marker STRO-1 in the chondrogenic growth zone and the perivascular tissue of the cartilaginous zone in primary and regenerating antlers as well as in the pedicle of fallow deer (Dama dama). In addition, cells positive for the stem cell/progenitor cell markers STRO-1, CD133 and CD271 (LNGFR) were isolated from the growth zones of regenerating fallow deer antlers as well as the pedicle periosteum and cultivated for extended periods of time. We found evidence that STRO-1+ cells isolated from the different locations are able to differentiate in vitro along the osteogenic and adipogenic lineages. Our results support the view that the annual process of antler regeneration might depend on the periodic activation of mesenchymal progenitor cells located in the pedicle periosteum. The findings of the present study indicate that not only limited tissue regeneration, but also extensive appendage regeneration in a postnatal mammal can occur as a stem cell-based process

Mesenchymal Stromal Cells Adapt to Chronic Tendon Disease Environment with an Initial Reduction in Matrix Remodeling

Tendon lesions are common sporting injuries in humans and horses alike. The healing process of acute tendon lesions frequently results in fibrosis and chronic disease. In horses, local mesenchymal stromal cell (MSC) injection is an accepted therapeutic strategy with positive influence on acute lesions. Concerning the use of MSCs in chronic tendon disease, data are scarce but suggest less therapeutic benefit. However, it has been shown that MSCs can have a positive effect on fibrotic tissue. Therefore, we aimed to elucidate the interplay of MSCs and healthy or chronically diseased tendon matrix. Equine MSCs were cultured either as cell aggregates or on scaffolds from healthy or diseased equine tendons. Higher expression of tendon-related matrix genes and tissue inhibitors of metalloproteinases (TIMPs) was found in aggregate cultures. However, the tenogenic transcription factor scleraxis was upregulated on healthy and diseased tendon scaffolds. Matrix metalloproteinase (MMPs) expression and activity were highest in healthy scaffold cultures but showed a strong transient decrease in diseased scaffold cultures. The release of glycosaminoglycan and collagen was also higher in scaffold cultures, even more so in those with tendon disease. This study points to an early suppression of MSC matrix remodeling activity by diseased tendon matrix, while tenogenic differentiation remained unaffected

Mixed culture of Oct4-GFP MEF cells and STRO-1⁺ DaMSCs.

<p>(<b>a</b>) Colony of Oct4-GFP MEF cells without any GFP expression. (<b>b</b>) Individual Oct4-GFP MEF cell after 24 hours of pre-cultivation in stem cell expansion medium on the eve of co-cultivation. (<b>c</b>) The same Oct4-GFP MEF cell after 24 hours of co-cultivation with STRO-1⁺ DaMSCs (interacting DaMSCs are outside of the display window). GFP expression (pseudo-coloured green) is visible within the cytoplasm. (<b>d</b>) Mixed culture after 96 hours of co-cultivation. More GFP⁺ cells interacting with GFP⁻ cells are visible. (<b>e</b>) Cell with distinct GFP expression after 120 hours of co-cultivation. At that time GFP⁺ cells interact continuously with GFP⁻ cells and are well integrated into the forming multilayer. (<b>f</b>) After 144 hours of co-cultivation some cells exhibit widely distributed intracellular GFP expression. (a,b,c,f = phase contrast pictures; d,e = varel contrast pictures; a–e = pictures of living cultures, f = fixed cells).</p

Transport of Oct4 fusion protein via cytoplasmic connections.

<p>(<b>a–d</b>) Time series pictures of a transfected STRO-1⁺ DaMSC. The GFP fluorescence of the Oct4 fusion protein is visible within the cytoplasm as well as inside the membrane tubes. Membrane dilatations (gondolas) filled with Oct4 fusion proteins are moving away from the cellular body. Starting points are marked with white dashed lines. Scale bar = 20 µm.</p

RT-PCR analyses of Oct4 expression in STRO-1⁺ cells.

<p>STRO-1⁺ cells derived from cultured pedicle periost (pp) of regenerating fallow deer antlers, growth zone (gz) of regenerating red deer antlers and human bone marrow (bm) from iliac crest biopsies were used as source for cDNA. PCR on these samples yielded bands representing the 3′-end of exon1 (335 bp). The bands were extracted and sequenced, demonstrating a very high homology of Oct4 of deer and human origin.</p

Cytoskeletal elements of STRO-1⁺ DaMSCs and long-distance cell-to-cell connections (TNTs).

<p>(<b>a,b</b>) Different cell-to-cell connections between STRO-1⁺ cells. Intercellular connections are able to bridge long distances even across neighbouring cells (Phalloidin/Tubulin staining, merged images). (<b>b</b>) Thick tubes (diameter >0.4 µm, example is marked by red arrow) contain F-actin and α-tubulin, whereas thin tubes (diameter <0.4 µm, example is marked by green arrow) contain only F-actin. (<b>c</b>) Negative control, staining without Tubulin antibody. (<b>d–f</b>) Scanning electron microscope pictures (SEM) of a mixed culture of antlerogenic cells. Cells form long connections across neighbouring cells (<b>d,e</b>) and very high magnification (<b>f</b>) proves that the surface of TNTs exhibit small appendages; long-distance connections are also possible between morphologically distinguishable cell types (<b>d</b>). (<b>h–m</b>) Multichannel pictures of Phalloidin (<b>h,k</b>) and Tubulin (<b>i,l</b>) stained DaMSCs demonstrate that their intercellular connections continuously consist of F-actin but only partially of microtubules. The visible spindle apparatus (<b>i</b>, detail enlargement) point to an initiating cell division and provides evidence that the used α-tubulin antibody is also efficient in DaMSCs. (<b>j,m</b>). Merged Images.</p