Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells

<p>Abstract</p> <p>Background</p> <p>Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity.</p> <p>Results</p> <p>After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G₁phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation.</p> <p>Conclusion</p> <p>Physiologic oxygen tension during <it>in vitro </it>culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.</p

Tumour stromal cells derived from paediatric malignancies display MSC-like properties and impair NK cell cytotoxicity

<p>Abstract</p> <p>Background</p> <p>Tumour growth and metastatic infiltration are favoured by several components of the tumour microenvironment. Bone marrow-derived multipotent mesenchymal stromal cells (MSC) are known to contribute to the tumour stroma. When isolated from healthy bone marrow, MSC exert potent antiproliferative effects on immune effector cells. Due to phenotypic and morphological similarities of MSC and tumour stromal cells (TStrC), we speculated that immunotherapeutic approaches may be hampered if TStrC may still exhibit immunomodulatory properties of MSC.</p> <p>Methods</p> <p>In order to compare immunomodulatory properties of MSC and tumour stromal cells (TStrC), we established and analyzed TStrC cultures from eleven paediatric tumours and MSC preparations from bone marrow aspirates. Immunophenotyping, proliferation assays and NK cell cytotoxicity assays were employed to address the issue.</p> <p>Results</p> <p>While TStrC differed from MSC in terms of plasticity, they shared surface expression of CD105, CD73 and other markers used for MSC characterization. Furthermore, TStrC displayed a strong antiproliferative effect on peripheral blood mononuclear cells (PBMC) in coculture experiments similar to MSC. NK cell cytotoxicity was significantly impaired after co-culture with TStrC and expression of the activating NK cell receptors NKp44 and NKp46 was reduced.</p> <p>Conclusions</p> <p>Our data show that TStrC and MSC share important phenotypic and functional characteristics. The inhibitory effect of TStrC on PBMC and especially on NK cells may facilitate the immune evasion of paediatric tumours.</p

Plasticity of human multipotent mesenchymal stromal cells and their preparation for regenerative application in paediatric patients

Humane multipotente mesenchymale Stromazellen (MSC) können in verschiedene Zelltypen differenzieren. Zusätzlich besitzen sie auch immunmodulatorische Eigenschaften und sekretorische Fähigkeiten. In dieser Arbeit konnte gezeigt werden, dass MSC nicht nur aus Knochenmark (KM-MSC), sondern auch aus den Papillen von noch retinierten Weisheitszähnen (Z-MSC) isoliert und propagiert werden können. Z-MSC unterscheiden sich nicht wesentlich von KM-MSC hinsichtlich Morphologie, Proliferation, Immun­phänotyp und Differenzierung. Geringe Unterschiede waren lediglich im Zusammenhang mit der adipogenen Differenzierungs­kapazität der Z-MSC und bei der Expression des osteogenen Markers Bone sialoprotein (BSP) festzustellen. Dies veranschaulichte, dass Zahnpapillen leicht für die Gewinnung von MSC genutzt werden können. Ein weiterer Teil dieser Arbeit beschäftigte sich mit der regenerations­medizinischen, lokalen Anwendung von MSC bei steroidinduzierten avaskulären Osteonekrosen (AVN) des Knies. Es wurden unter anderem in vitro Experimente durchgeführt, die den Einfluss von Hypoxie und IFNgamma auf MSC bezüglich des osteogenen Differenzierungs­potenzials und der Sekretion des angiogenetischen Faktors VEGF analysierten. Die Ergebnisse zeigten die uneingeschränkte osteogene Differenzierung von MSC bei reduziertem Sauerstoff­partialdruck von 3% und die Sekretion von VEGF bei zusätzlicher O2-Reduktion auf 1%. Daraufhin sollten MSC therapeutisch eingesetzt werden. An einer kleinen Kohorte von fünf pädiatrischen AVN-Patienten, denen expandierte autologe MSC während der konventionellen Therapie der Core Decompression in die nekrotische Läsion injiziert worden waren, konnte Verträglichkeit und Sicherheit des Transplantats demonstriert werden. Bei einem mittleren Follow-up von 29 Monaten konnte bei allen Patienten Schmerz reduziert, Beweglichkeit verbessert und die Knochen­regeneration radiologisch beobachtet werden. In der vorliegenden Arbeit konnte zu dem an einem Patienten mit akuter Graft-versus-host disease (GvHD) das Engraftment und die Verteilung in den Organen von systemisch applizierten MSC untersucht werden. Durch Detektion der HLA-Disparität von Empfänger und Spender-MSC konnten immunhistologisch transfundierte MSC in Leber und Teilen des Darms dargestellt werden. Damit verbunden, wurden erste Experimente zur Modifikation der Glykanstruktur von MSC unternommen, die auf die Verbesserung der Migrationseigenschaften zielen.Human multipotent mesenchymal stromal cells (MSC) may differentiate into several different cell types. In addition, MSC display immunomodulatory and secretory properties. This work has shown that MSC can be isolated not only from bone marrow aspirates, but also from the papilla dentalis of retained wisdom teeth (PD-MSC). With regards to morphology, proliferation, immunophenotype and differentiation characteristics, BM-MSC and PD-MSC do not differ considerably from each other. Only slight differences in association with the adipogenic differentiation capacity of PD-MSC and the expression of the osteogenic differentiation marker bone sialo protein (BSP) were identified. These results illustrate that papilla dentalis may be easily be used as a source for MSC extraction. A further section of this study focused on the regenerative aspects of the local administration of MSC for the treatment of steroid-induced avascular osteonecrosis (AVN) of the knee. Among others in vitro experiments were carried out to analyze relevant aspects such as the influence of hypoxia and IFNgamma on the osteogenic differentiation potential of MSC and the secretion of angiogenic factor VEGF. Results showed no effect on osteogenic differentiation of MSC under oxygen tension of 3% and no effect on secretion of VEGF at further reduced oxygen levels of 1%. These results lead us to use MSC for therapeutic treatment. A small cohort of five patients with AVN was treated by instillation of expanded autologous MSC to the site of necrotic lesion during conventional core decompression surgery. Application of MSC in this group demonstrated safety and feasibility. Within the median follow-up of 29 months pain was reduced and mobility was improved for all patients. Radiological findings showed regeneration of the treated bone. Lastly, this work evaluated the engraftment and distribution of systemically applied MSC in different tissues of a patient treated for acute graft-versus-host disease (GvHD). Through detection of HLA-disparity between recipient and donor MSC the existence of transfused MSC in liver, colon and small intestine could be shown immunohistologically. In association with this, preliminary experiments were performed to modify particular MSC glycan structures with the aim of improving homing capacity