Chondrogenic differentiation of human amniotic fluid stem cells

Im Jahr 2003 schaffte die Entdeckung von Stammzellen im Fruchtwasser ein neues und sehr vielversprechendes Gebiet im Bereich der Stammzellenforschung. Vor dieser Zeit wurden diese Zellen nur innerhalb der Pränatal Diagnostik verwenden um vererbbare Defekte des ungeborenen Kindes aufzudecken. Die Zellen innerhalb des Fruchtwassers können leicht währen der Amniozentese gewonnen. Stammzellen innerhalb des Fruchtwassers sind ckit positiv und besitzen das Potential in Zellen aller drei Keimblätter zu differenzieren (Entoderm, Mesoderm und Ektoderm). Im Vergleich zu embryonalen Stammzellen gibt es keine ethischen Probleme bezüglich der Quelle dieser Zellen. Andere Vorteile sind ein geringes Risiko gegenüber Tumorbildung, Selbsterneuerung, Pluropotenzität und außerdem wachsen sie ohne „feeder layers“. 2006 wurden Protokolle erschaffen in denen Fruchtwasserstammzellen in neuronale, hepatische und osteogenische Zelllinien differenziert wurden konnten . The mechanistic target of rapamcin (mTOR) ist ein Signalweg der Prozesse wie z.B. Stoffwechsel , Zellzyklus, Wachstum, Translation, Ribosombiogenese und den Transport von Nährstoffen reguliert. Er besteht zumindest aus zwei Komplexen (mTORC1 und mTORC2) welche sich in ihrer Zusammensetzung und ihren Substraten unterscheiden. In unseren Studien konzentrierten wir uns auf das Differenzierungspotential von Fruchtwasserstammzellen in Chondrozyten. Diese Zellen sind an der Entwicklung vongefäßlose Knorpel oder lange Knochen beteiligt. In diesen Schritten werden einige Marker aktiviert (z.B.Sox9) um die Umwandlung von mesenchymalen Vorläuferzellen in Chondrozyten zu regulieren. Außerdem produzieren Chondrozyten eine extrazelluläre Matrix die aus Kollagenen und Glycosaminoglycanen zusammengesetzt ist. Weltweit leiden mehr als 50% der Leute die über 50 Jahre alt sind an degenerativen Krankheiten wie Osteoarthritis welche durch einen Verlust an Chondrozyten und der von ihnen gebildeten extrazellulären Matrix gekennzeichnet ist. Die Erschaffung von Chondrozyten aus Fruchtwasserstammzellen könnte eine Möglichkeit sein die Lebensqualität dieser Patienten zu erhöhen. Anhand einer Pelletbildung haben wir erfolgreich magnetisch-getrennte monoklonale Fruchtwasserstammzellen in Chondrozyten umgewandelt. Western Blot Analysen und die Messung schwefelhaltiger Glycosaminoglycane bestätigten die Bildung von Chondrozyten. Der mTOR Signalweg hat eine wichtige Rolle in diesem Prozess. Western Blot Auswertungen charakteristischer Marker für Chondrozyten und Glycosaminoglycan Messungen zeigten auf, dass mTOR in den neu erschaffenen Chonrozyten aktiviert ist und dass ein Blocken des mTOR Weges durch seine Hemmstoffe den Differenzierungsprozess beeinflusst.In 2003, the discovery of human amniotic fluid stem cells (hAFSC) initiated a new and very promising field in stem cell research.Prior to this, these cells were only used for prenatal diagnosis for detecting inherited defects in the unborn child. The cells within the amniotic fluid can be easily obtained by amniocentesis. Stem cells within the amniotic fluid are ckit positive and harbor the potential to differentiate into cells of all three embryonic germ layers (entoderm, mesoderm and ectoderm). Compared to embryonic stem cells, no ethical issues arise because of the source of these cells. Other advantages are: low risk for tumor formation, self renewal, pluropotency and growth without the need of feeder layers. In 2006, protocols were established for the differentiation of hAFSC into cells of neuronal, hepatic and osteogenic lineage .The mechanistic target of Rapamycin (mTOR) is a pathway that regulates processes such as metabolism, cell cycle, proliferation, translation, ribosome biogenesis and nutrient transport. It consists at least of two complexes (mTORC1 and mTORC2) which differ in their assembly and substrates. In our studies we focused on the differentiation potential of hAFSC into chondrocytes. These cells are involved in the development of avascular cartilage and long bones. In these processes several markers are activated (e.g.Sox9) to regulate the conversion from mesenchymal progenitor cells into chondrocytes. Additionally, chondrocytes produce an extracellular matrix composed of Collagens and Glycosaminoglycans. Worldwide more than 50% of people who are over 50 years old suffer from degenerative diseases like osteoarthritis which is characterized by a loss of chondrocytes and their produced extracellular matrix. Generation of chondrocytes out off hAFSC could be an option to treat these diseases and to increase the life quality of patients. We successfully differentiated magnetic beat sorted monoclonal hAFSC via a pellet formation protocol into chondrocytes. Western blot analyzes and sulfated Glucosaminoglycan assays confirmed the generation of chondrocytes. The mTOR pathway plays an important role in this converting process. Western blotting for characteristic chondrogenic markers and Glycosaminoglycan measurements reveal that in the new generated chondrocytes the mTOR pathway is activated and that blocking of mTOR by its inhibitors influences the differentiation process

mTORC1 is essential for early steps during Schwann cell differentiation of amniotic fluid stem cells and regulates lipogenic gene expression.

Schwann cell development is hallmarked by the induction of a lipogenic profile. Here we used amniotic fluid stem (AFS) cells and focused on the mechanisms occurring during early steps of differentiation along the Schwann cell lineage. Therefore, we initiated Schwann cell differentiation in AFS cells and monitored as well as modulated the activity of the mechanistic target of rapamycin (mTOR) pathway, the major regulator of anabolic processes. Our results show that mTOR complex 1 (mTORC1) activity is essential for glial marker expression and expression of Sterol Regulatory Element-Binding Protein (SREBP) target genes. Moreover, SREBP target gene activation by statin treatment promoted lipogenic gene expression, induced mTORC1 activation and stimulated Schwann cell differentiation. To investigate mTORC1 downstream signaling we expressed a mutant S6K1, which subsequently induced the expression of the Schwann cell marker S100b, but did not affect lipogenic gene expression. This suggests that S6K1 dependent and independent pathways downstream of mTORC1 drive AFS cells to early Schwann cell differentiation and lipogenic gene expression. In conclusion our results propose that future strategies for peripheral nervous system regeneration will depend on ways to efficiently induce the mTORC1 pathway

Rapamycin Maintains the Chondrocytic Phenotype and Interferes with Inflammatory Cytokine Induced Processes

Osteoarthritis (OA) is hallmarked by a progressive degradation of articular cartilage. Besides risk factors including trauma, obesity or genetic predisposition, inflammation has a major impact on the development of this chronic disease. During the course of inflammation, cytokines such as tumor necrosis factor-alpha(TNF-α) and interleukin (IL)-1β are secreted by activated chondrocytes as well as synovial cells and stimulate the production of other inflammatory cytokines and matrix degrading enzymes. The mTORC1 inhibitor rapamycin is a clinical approved immunosuppressant and several studies also verified its chondroprotective effects in OA. However, the effect of blocking the mechanistic target of rapamycin complex (mTORC)1 on the inflammatory status within OA is not well studied. Therefore, we aimed to investigate if inhibition of mTORC1 by rapamycin can preserve and sustain chondrocytes in an inflammatory environment. Patient-derived chondrocytes were cultured in media supplemented with or without the mTORC1 inhibitor rapamycin. To establish an inflammatory environment, either TNF-α or IL-1β was added to the media (=OA-model). The chondroprotective and anti-inflammatory effects of rapamycin were evaluated using sulfated glycosaminoglycan (sGAG) release assay, Caspase 3/7 activity assay, lactate dehydrogenase (LDH) assay and quantitative real time polymerase chain reaction (PCR). Blocking mTORC1 by rapamycin reduced the release and therefore degradation of sGAGs, which are components of the extracellular matrix secreted by chondrocytes. Furthermore, blocking mTORC1 in OA chondrocytes resulted in an enhanced expression of the main chondrogenic markers. Rapamycin was able to protect chondrocytes from cell death in an OA-model shown by reduced Caspase 3/7 activity and diminished LDH release. Furthermore, inhibition of mTORC1 preserved the chondrogenic phenotype of OA chondrocytes, but also reduced inflammatory processes within the OA-model. This study highlights that blocking mTORC1 is a new and promising approach for treating OA. Low side effects make rapamycin an attractive implementation to existing therapeutic strategies. We showed that rapamycin’s chondroprotective property might be due to an interference with IL-1β triggered inflammatory processes

Effectiveness of outpatient geriatric rehabilitation after inpatient geriatric rehabilitation or hospitalisation: a systematic review and meta-analysis

BACKGROUND: Due to the increasing number of older people with multi-morbidity, the demand for outpatient geriatric rehabilitation (OGR) will also increase. OBJECTIVE: To assess the effects of OGR on the primary outcome functional performance (FP) and secondary outcomes: length of in-patient stay, re-admission rate, patients' and caregivers' quality of life, mortality and cost-effectiveness. We also aim to describe the organisation and content of OGR. METHODS: Systematic review and meta-analysis. Five databases were queried from inception to July 2022. We selected randomised controlled trials written in English, focusing on multidisciplinary interventions related to OGR, included participants aged ≥65 and reported one of the main outcomes. A meta-analysis was performed on FP, patients' quality of life, length of stay and re-admissions. The structural, procedural and environmental aspects of OGR were systematically mapped. RESULTS: We selected 24 studies involving 3,405 participants. The meta-analysis showed no significant effect on the primary outcome FP (activity). It demonstrated a significant effect of OGR on shortening length of in-patient stay (P = 0.03, MD = -2.41 days, 95%CI: [-4.61-0.22]). Frequently used elements of OGR are: inpatient start of OGR with an interdisciplinary rehabilitation team, close cooperation with primary care, an OGR coordinator, individual goal setting and education for both patient and caregiver. CONCLUSION: This review showed that OGR is as effective as usual care on FP activity. It shows low certainty of evidence for OGR being effective in reducing the length of inpatient stay. Further research is needed on the various frequently used elements of OGR

Human monoclonal amniotic fluid stem cells can be differentiated into a early Schwann cell phenotype.

<p>(A) AFS cells are small cells with omnidirectional protruding filopodia and upon differentiation to Schwann-like cells, at day 15 of treatment, cells exhibited an increase in cellular volume and an elongated cell morphology. Scale bar represents 50 µm. (B) Immunofluorescence staining of AFS cells differentiated for 15 days (dAFS) compared to undifferentiated AFS cells (AFS) and MCM1 neural crest-derived cells (control), for the Schwann cell markers NGFR, GFAP and S100b (labeled in red, nuclei labeled in green). Purity of cells is indicated as percent positive cells versus total amount of cells ± S.D. Scale bar represents 10 µm. (C) Quantitative RT-PCR of cDNA derived from AFS cells and from AFS cells subjected to Schwann cell differentiation after different time points was performed. Results are shown as fold change expression of respective genes compared to undifferentiated AFS cells. The results are expressed as means ± SEM of three independent experiments. P<0.05 for * vs undifferentiated AFS cells.</p

Scheme for the applied differentiation protocol.

<p>In order to initiate human AFS cell differentiation to a Schwann cell phenotype AFS cells were first treated in serum free α-MEM with 1 mM β-mercaptoethanol (Diff. I) for 24 hours. Afterwards cells were incubated in α-MEM supplemented with 10% fetal bovine serum and 35 ng/ml retinoic acid (Diff. II) for 72 hours. Subsequently, cells were cultured in α-MEM containing 10% fetal bovine serum supplemented with 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor, 5 mM forskolin, 5 ng/mL platelet-derived growth factor-AA and 200 ng/mL recombinant human heregulin-beta1 (Diff. III) until day 15 of differentiation. Media was changed every 3 days, indicated by arrows. Pharmacologic (pharm.) treatment, consisting of rapamycin or statin, was applied together with Diff. III media.</p

Model of mTORC1 involvement in Schwann cell differentiation.

<p>Rapamycin blocks mTORC1 and results in the down regulation of Schwann cell markers (e.g.: S100b) and in the down regulation of lipogenic genes (e.g.: LDLR, HMGCR). Our data indicates that S6K1 regulates the expression of S100b, but not of LDLR and HMGCR.</p