Differenzierung von Stammzellen aus Fettgewebe in epitheliale Tubuluszellen

Mesenchymale Stammzellen (MSC) rücken in der regenerativen Medizin und im Tissue Engineering immer mehr in den Vordergrund. Im Gegensatz zu embryonalen Stammzellen bergen sie keine ethischen Probleme und sind leicht zu isolieren. Die ursprünglich aus Knochenmark gewonnenen MSC können inzwischen aus vielen verschiedenen Quellen wie Nabelschnurblut [Kern et al. 2006], Dentalgewebe [Huang et al., 2009], Plazenta [Huang et al., 2009], Haut [Salvolini et al., 2009] und aus Fettgewebe isoliert werden [Zuk et al., 2001]. Der Immunphänotyp variiert zwischen den aus verschiedenen Quellen gewonnenen MSC nur gering. Die Gewinnung aus Fettgewebe hat den Vorteil, dass eine minimal invasive Prozedur und eine hohe Ausbeute zusammenkommen. Die Stammzellen aus Fettgewebe (ASC) können in der Therapie eingesetzt werden und zu der Regeneration von Geweben nach Verletzungen beitragen [Wong et al., 2008; Poulsom et al., 2001]. Der genaue Mechanismus mit dem die Stammzellen wirken ist allerdings noch nicht geklärt. Sowohl die Integration der MSCs in das Gewebe als auch ein rein parakriner Einfluss wurden nachgewiesen. Klar ist nur, dass ein positiver Effekt von einer Therapie mit MSC ausgeht [Mizuno et al., 2009]. In vivo sind Zellen verschiedenen Einflüssen ausgesetzt, die den Zustand einer Zelle bestimmen. Lösliche Faktoren wie Wachstumsfaktoren, Hormone oder Vitamine wirken dabei ebenso wie die extrazelluläre Matrix und Zell-Zell-Kontakte auf die Zelle ein, die mit Wachstum, Zellform, Differenzierung oder Ähnlichem antwortet. In meiner Arbeit wurden daher drei unterschiedliche Ansätze für die in vitro Differenzierung von ASC in epitheliale Tubuluszellen untersucht: (1) die Wirkung von löslichen Faktoren, die dem Medium zugesetzt wurden, (2) der Einfluss der extrazellulären Matrix aus zum einen Tubuluszellen und zum anderen Matrigel und (3) die Co-Kultur, bei der auch direkter Zell-Zell-Kontakt untersucht wurde. Damit, und mit einer Kombination der einzelnen Bereiche, sollte das natürliche Umfeld der Tubuluszellen simuliert werden und epitheliale Differenzierung initiieren. Die Zugabe von ATRA, ActA und BMP-7 führte zu einer Differenzierung in die epitheliale Richtung, während die extrazelluläre Matrix aus Tubuluszellen nicht dafür ausreichte. Matrigel hingegen, konnte besonders in der Verbindung mit konditioniertem Medium eine Differenzierung induzieren. Die indirekte Co-Kultur über Membraneinsätze, über die u. a. der parakrine Einfluss der Tubuluszellen untersucht werden sollte, führte zu morphologischen Veränderungen der ASC, die aber nicht mit den hier verwendeten epithelialen Markern nachgewiesen werden konnte. Der direkte Zell-Zell-Kontakt zeigte eine Reduktion des Oberflächen Markers CD90 verbunden mit einer Erhöhung der Expression von CK18. Die Differenzierung von ASC in epitheliale Zellen ist also auf drei verschiedenen Wegen möglich. Zwischen verschiedenen Isolationen von ASC traten hohe Schwankungen bezüglich der Expression von Oberflächenmarkern und Proliferation auf, was auch einen Einfluss auf die Differenzierung haben könnte. Ein Grund dafür könnte die Heterogenität von ASC sein. Zur Reduzierung dieser wurden daher ein Waschschritt eine Stunde nach Kulturbeginn und eine immunomagnetische Isolation mit CD49a, CD90, CD105 oder CD271 durchgeführt. Die immunomagnetische Aufreinigung führte nur zu einer leichten Verbesserung der Heterogenität, aber zu einer sehr geringen Zellausbeute. Für den Waschschritt konnte gezeigt werden, dass die Expression der Stammzellmarker Nestin, oct4 und sall1 signifikant erhöht und Desmin und smA Expression reduziert wurden, was auf eine Reduktion der Heterogenität hindeutete. Der zusätzliche Waschschritt kann also schnell und unkompliziert die Heterogenität der ASC reduzieren. Die Differenzierung von ASC in epitheliale Tubuluszellen durch den Einfluss von Zell-Zell-Kontakten zeigt vielversprechende Ansätze und sollte weiter verfolgt werden. Eine verlängerte Kulturdauer sollte dabei angestrebt werden, da auch die adipogene Differenzierung zumeist erst nach 14 bis 21 Tagen nachweisbar war. Dafür müsste die Markierung mit CellTracker länger nachweisbar sein. Eine Inhibierung der Proliferation könnte die Grundlage dazu liefern. Um den Stand der Differenzierung in die epitheliale Richtung nachzuweisen, könnten andere epitheliale Marker, Ionenkanäle, die erst spät in den Tubuluszellen angelegt werden, und funktionelle Mechanismen untersucht werden. Das Zusammenspiel der verschiedenen Einflüsse auf die Zelle könnte ebenfalls noch genauer untersucht werden. Für die Regenerative Medizin ist es aus Gründen der GMP sinnvoller, die Zellen nur mit dem Zusatz an löslichen Faktoren zu differenzieren. Der Ansatz mit ATRA, ActA und BMP-7 scheint sehr vielversprechend zu sein und könnte in dieser Hinsicht weiter ausgebaut werden.In regenerative medicine and tissue engineering, mesenchymal stem cells (MSC) get more and more in the focus. Originally obtained from bone marrow, MSC are now derived from different tissues, showing only small differences in immunophenotype or differentiation potential. Fatty tissue is easily accessable with a minimal invasive procedure, leading to a high amount of MSC. These adipose derived stem cells (ASC) have the same marker panel, differentiation capability and adherence to plastik as bone marrow derived MSC. In earlier studies it was believed, that MSC can only differentiate in celltypes of mesodermal origin as adipocytes, osteoblasts and chondrocytes. Recently, those limitations were disproved as the differentiation into hepatocytes [Banas et.al., 2007; Lange et al., 2006], neural [Krampera et al., 2006], endothelial [Cao et al., 2005], and epithelial cells [Brzoska et al., 2005] was shown. Acute kidney injury is mostly regulated by self-repair, but in some cases patients do not recover. Regenerative medicine using ASC might be a solution for the treatment of kidney injuries. It could already be proven that the use of stem cells can positively influence regeneration [Chen et al., 2004; Tögel et al., 2005]. In vivo a cell is defined through many different influences like a multitude of soluble factors (growth factors, cytokines, hormones and vitamins), as well as the extracellular matrix and cell-cell-contact. My thesis investigates therefore three different ways, which might possibly lead to an epithelial differentiation of ASC: (1) the addition of growth factors and cytokines to the culture medium, (2) the influence of extracellular matrix both derived from tubular cells and Matrigel, and (3) indirect and direct cell-cell-contact. It could be shown that a combination of all-trans retinoic acid, activin A and BMP-7 significally enhanced the expression of the epithelial markers cytokeratin 18 and zona occludens 1, while the extracellular matrix derived from tubular cells did not change this markers on ASC. However, for the differentiation into epithelial cells, direct cell contact showed also a good prospect to induce differentiation of ASC. Meanwhile, culture on Matrigel might change the ASC into epithelial direction depending on the cells, which display a great heterogeneity coming from different donors and thus different isolations. Since ASC are a heterogeneous mixture of cells, which could influence the differentiation potential, an attempt was made to reduce the heterogeneity by an additional washing step and immunomagnetic isolation by CD49a, CD90, CD105 or CD271. A washing step after one hour proved to reduce the heterogeneity of ASC and was more effective than immunomagnetic isolation with a much higher cell yield

Regulation of mRNA translation by MID1: a common mechanism of expanded CAG repeat RNAs

Expansion of CAG repeats, which code for the disease-causing polyglutamine protein, is a common feature in polyglutamine diseases. RNA-mediated mechanisms that contribute to neuropathology in polyglutamine diseases are important. RNA-toxicity describes a phenomenon by which the mutant CAG repeat RNA recruits RNA-binding proteins, thereby leading to aberrant function. For example the MID1 protein binds to mutant huntingtin (HTT) RNA, which is linked to Huntington's disease (HD), at its CAG repeat region and induces protein synthesis of mutant protein. But is this mechanism specific to HD or is it a common mechanism in CAG repeat expansion disorders? To answer this question, we have analyzed the interaction between MID1 and three other CAG repeat mRNAs, Ataxin2 (ATXN2), Ataxin3 (ATXN3), and Ataxin7 (ATXN7), that all differ in the sequence flanking the CAG repeat. We show that ATXN2, ATXN3, and ATXN7 bind to MID1 in a CAG repeat length-dependent manner. Furthermore, we show that functionally, in line with what we have previously observed for HTT, the binding of MID1 to ATXN2, ATXN3, and ATXN7 mRNA induces protein synthesis in a repeat length-dependent manner. Our data suggest that regulation of protein translation by the MID1 complex is a common mechanism for CAG repeat containing mRNAs

Growth characteristics of human adenoviruses on porcine cell lines

Human adenoviruses (hAdV) have been recognized as a highly prevalent virus family causing severe disease in immunocompromised patients. In xenotransplantation the xenograft therefore will be exposed to these viruses, which in case of its infection might contribute to posttransplant complications. To evaluate the susceptibility of porcine cells for hAdV, we infected the porcine cell line POEK with seven serotypes representing all six hAdV species. Additionally, a second porcine cell line (ST) was infected with two serotypes. Viral replication of serotypes varied: porcine cells were fully permissive for serotypes 1, 4 and 17, semi-permissive for 11 and 21, and non-permissive for 31 and 40. Furthermore, we demonstrated the interaction of serotype 1 with the porcine homologue of the coxsackie-adenovirus receptor, the receptor used by many hAdV serotypes for cell attachment. Thus, various adenovirus types of different hAdV species may be capable of infecting different porcine tissue types

The Anti-Diabetic Drug Metformin Reduces BACE1 Protein Level by Interfering with the MID1 Complex

<div><p>Alzheimer’s disease (AD), the most common form of dementia in the elderly, is characterized by two neuropathological hallmarks: senile plaques, which are composed of Aβ peptides, and neurofibrillary tangles, which are composed of hyperphosphorylated TAU protein. Diabetic patients with dysregulated insulin signalling are at increased risk of developing AD. Further, several animal models of diabetes show increased Aβ expression and hyperphosphorylated tau. As we have shown recently, the anti-diabetic drug metformin is capable of dephosphorylating tau at AD-relevant phospho-sites. Here, we investigated the effect of metformin on the main amyloidogenic enzyme BACE1 and, thus, on the production of Aβ peptides, the second pathological hallmark of AD. We find similar results in cultures of primary neurons, a human cell line model of AD and <i>in vivo</i> in mice. We show that treatment with metformin decreases BACE1 protein expression by interfering with an mRNA-protein complex that contains the ubiquitin ligase MID1, thereby reducing BACE1 activity. Together with our previous findings these results indicate that metformin may target both pathological hallmarks of AD and may be of therapeutic value for treating and/or preventing AD.</p></div

BACE1 cleavage products are reduced after metformin treatment.

<p>SH-SY5Y-APP^swe cells were treated with increasing concentrations of metformin for 24 hours and protein levels were analyzed on western blots using CTF-specific antibodies. Graphs show quantification of western blots, mean values +/− SEM. n = 4 per group, * = p<0.05.</p

BACE1 translation is regulated by pS6 and inhibited by metformin.

<p>(A) Metformin decreases phosphorylation of S6. Primary neurons were treated with increasing concentrations of metformin for 24 hours and phosphorylation of S6 was analyzed on western blots using pS6-, total S6- or β-actin-specific antibodies. Graphs show quantification of western blots, mean values +/− SEM. n = 5 per group, * = p<0.05. (B) Two different <i>in vitro</i> transcribed, biotinylated BACE1-mRNA fragments containing a putative MIDAS structural motif were incubated with cell lysates that were transfected with MID1-FLAG. RNAs were immobilized on streptavidin-coated magnetic beads. After extensive washing RNA-bound proteins were analyzed by western blotting using antibodies detecting MID1-FLAG, S6K, and S6. As a negative control an experiment with RNAse treatment was performed. (C) RNA immunoprecipitation. Primary neurons were transfected with MID1-FLAG and incubated with or without metformin. Afterwards MID1-mRNPs were purified by immunoprecipitation and MID1-bound mRNAs were analyzed for the presence of BACE1 mRNA using realtime-PCR. Columns represent mean values +/− SEM. n = 4 (D) Kinase inhibitors involved in regulating S6 phosphorylation decrease BACE1 protein levels to a similar extent as metformin. Primary neurons were treated with 2.5 mM metformin, or 1 µM Temsirolimus (an mTOR inhibitor), or 2.5 µM compound C (an AMPK inhibitor), or 2.5 µM DG2 (an S6K1 inhibitor), or 1 µM HWT (a PI3K inhibitor) 24 hours, after which BACE1 protein levels were analyzed on western blots using BACE1- or β-actin-specific antibodies.</p

Metformin reduces BACE1 protein levels <i>in vivo</i>.

<p>Wild type mice were treated for 2 weeks with 5/l metformin in the drinking water (≈0.03 M). Brain lysates of these mice were analyzed on western blots detecting BACE1, pS6, total S6, GAPDH and β-actin. Bars represent mean values +/− SEM, (n = 5 per group, *p<0.05).</p

Metformin treatment reduces BACE activity.

<p>Primary neurons were treated with or without 2.5β-secretase activity assay. (A) The reaction was followed over a time-course of 120 min. Lines represent mean values of fluorescent signal (relative fluorescence units (RFU)). (B) Data at time point 120 min of the assay from (A) is shown in a graph. Columns represent mean values +/− SEM. n = 3 per group, p<0.05.</p

Metformin decreases BACE1 protein level.

<p>(A) Primary neurons were treated with increasing concentrations of metformin for 24 hours and BACE1 protein levels were analyzed on western blots using BACE1-, TACE- or β-actin-specific antibodies. Graphs show quantification of western blots, mean values +/− SEM. n = 7 per group, * = p<0.05. (B, C) Relative BACE1 and TACE mRNA expression was measured in cells treated as in (A) by means of real-time PCR. Columns represent mean values +/− SEM. n = 8.</p

Hypothetical model of the MID1 regulatory complex.

<p>A schematic representation of the MID1-PP2A complex with S6K and S6 is shown. MID1 binds to PP2A via its4 regulatory subunit, causing degradation of PP2A and increased phosphorylation of TAU. The MID1 complex also binds to the BACE1 mRNA. By reducing the activity of PP2A and thereby promoting phosphorylation, S6K gets activated and phosphorylates S6. Activated pS6 can then stimulate S6-dependent translation. After metformin treatment, the MID1-complex disassembles and, due to increased PP2A activity, S6-dependent translation of the BACE1 mRNA is reduced and phosphorylation of TAU decreases.</p