Characterization of human mesenchymal stem cells by the appearance of integrins and functional analysis of collagen I-binding integrins

Introduction: Human mesenchymal stem cells (hMSC) are easily obtainable from bone mar-row and possess the ability to differentiate into osteoblasts. Therefore, they have been sug-gested as a suitable source for bone regeneration. HMSC are equipped with a variety of in-tegrins that mediate essential cell-matrix interactions. Collagen I represent approximately 90% of the bone protein content. Cell attachment to collagen I is mediated by three members of the integrin receptor family named a1b1, a2b1 and a11b1 integrins. The main aim of this doctoral thesis was to investigate the basal expression of those integrins in hMSC and to func-tionally analyze the knockdown effect of a single collagen I-binding integrin on hMSC behav-ior in vitro. Materials and methods: HMSC were cultured on collagen I-coated surface. A lentiviral trans-fer of a1-, a2- and a11-specific shRNA was applied for downregulation of the corresponding integrin mRNA. Quantitative PCR and western blot analysis were used to assess the basal ex-pression, knockdown efficiency and integrin compensation. Colorimetric adhesion assay was used for estimation of the extent of cells attachment. HMSC spreading and migration was ob-served by time lapse experiments. JC-1 staining was used for investigation of the initiation of apoptosis. Results: Quantitative PCR were used to assess the basal expression of collagen I-binding integrins in three hMSC donors. We found that these integrins are differently expressed as integrin a11 had the highest and integrin a2 the lowest expression. Next, we applied lentiviral delivery of target-specific short hairpin RNA (shRNA) in order to knockdown each of the collagen I-binding integrins and compared them to the hMSC transduced with a sequence against a non-human gene abbreviated as shRNA control. We achieved significant downregulation (> 80%) of the collagen I-binding integrin mRNA and protein. Subsequently to the transduction, we did not noticed pronounce morphological cell changes, however, a clear decrease of a2- and a11-knockdown hMSC numbers was observed during cultivation. Using a quantitative adhesion assay, we estimated that 120 min after plating only 30% of integrin a11-deficent cells were able to attach to collagen I. In contrast, at the same time point, 70% of integrin a2-knockdown hMSC were attached while integrin a1- and shRNA control hMSC have already reached 100% cell adhesion. Furthermore, a time lapse-based investigation showed that integrin a1- and shRNA control hMSC need approximately 35 min to fully spread on collagen I. In contrast, integrin a2- and a11-knockdown hMSC took approximately double more time for spreading in comparison to shRNA control hMSC. Additionally, we analyzed the migration capability of the four different hMSC lines. The average path which integrin a1- and shRNA control hMSC passed was approximately 170 µm with mean speed of 11.5 µm/h. In parallel integrin a2 and a11-deficient hMSC migrated to a distance of approximately 70 µm with a velocity of 5 µm/h. Since it was observed a lost of a2- and a11-deficient hMSC, next we performed JC-1 staining that visualizes mitochondrial leakage, a hallmark of apoptosis. The majority of integrin a2- and a11-knockdown hMSC exhibited mitochondrial leakage whereas integrin a1- and shRNA control hMSC showed intact mitochondria. Finally, we used quantitative PCR to investigate whether there were compensatory effects between the three integrin receptors. We detect that knockdown of integrin a1 led to upregulation of a2 and a11. Similarly, when integrin a2 was downregulated, integrin a1 and a11 expression increased. Interestingly, knockdown of integrin a11 caused only a slight increase in integrin a1 but not in a2 expression. We also observed that upon osteogenic stimulation, integrin a2 and a11-deficient hMSC further reduced in number and did not mineralize the matrix even on a single cell level. Moreover, our preliminary investigation in hMSC-derived from osteoporosis suffering patients showed a tremendous downregulation of integrin a2. Conclusions: Our results strongly suggested that integrins a2b1 and a11b1 mediate an indis-pensible signaling for hMSC. Once these receptors were ablated from cell surface, hMSC re-duced their spreading, adhesion, migration and survival rates. Our integrin knockdown mod-els can be used for further investigations and understanding of the integrins a2b1 and a11b1 importance and signaling in hMSC and hOB since we observed a strong downregulation of integrin a2 expression in osteoporosis.Einleitung: Human mesenchymale Stammzellen (hMSC) können auf einfache Weise aus dem Knochenmark gewonnen werden und haben die Fähigkeit, sich in Osteoblasten zu differenzieren. Daher scheinen sie eine geeignete Quelle für die Regeneration von Knochen zu sein. HMSC enthalten eine Vielzahl an Integrinen, die essenzielle Zell-Matrix Wechselwirkungen vermitteln. Kollagen I macht ungefähr 90% des Proteingehalts im Knochen aus. Die Anhaftung der Zelle an Kollagen I wird durch drei Mitglieder der Integrin Rezeptor Familie, den Integrinen a1b1, a2b1 und a11b1, vermittelt. Das Ziel dieser Doktorarbeit war, die basale Expression dieser Inetgrine in hMSC zu untersuchen sowie eine funktionelle Analyse des Knockdown Effektes eines einzelnen Kollagen I-bindenden Integrins auf das hMSC Verhalten in vitro durchzuführen. Material und Methoden: HMSC wurden auf Kollagen I beschichteten Oberflächen kultiviert. Mittels lentiviralen Gentransfers mit a1-, a2- und a11- spezifischer shRNA (short hairpin RNA) wurde für die entsprechende Integrin mRNA herunterreguliert. Quantitative PCR und Westernblot dienten dazu, die basale Expression, die Knockdown Effizienz und kompensatorische Effekte zu erfassen. Durch den Einsatz eines kolorimetrischen Adhäsionsassays wurde das Ausmaß der Zelladhäsion ermittelt. Die Ausbreitung und Migration der hMSC wurde durch Zeitraffermikroskopie beobachtet. Eine JC-1 Färbung wurde zur Untersuchung des Apoptoseverhaltens verwendet. Ergebnisse: Mittels quantitativer PCR wurde die basale Expression von Kollagen I-bindenden Integrinen von drei hMSC Spendern ermittelt. Wir haben herausgefunden, dass diese Integrine in unterschiedlichem Maß exprimiert werden. Integrin a11 zeigte die stärkste und Integrin a2 die schwächste Expression. Um einen Knockdown in den jeweiligen Kollagen I bindenden Integrinen zu erreichen, wurde ein lentiviraler Transfer mit shRNA durchgeführt. Als Kontrolle dienten hMSC, welche mit einer Sequenz gegen ein nicht humanes Gen transduziert wurden, welche als shRNA Kontrolle abgekürzt wurde. Wir erzielten eine signifikaten Herunterregulierung (> 80%) der Integrin-mRNA und -Proteine. In Folge der Transduktion konnten wir keine ausgeprägten morphologischen Veränderungen feststellen, jedoch stellten wir während der Kultivierung einen eindeutigen Rückgang der Zellzahl von a2- und a11-Knockdown hMSC fest. Im quantitativen Adhäsionsassay zeigte sich, dass 120 Minuten nach dem Ausplattieren nur 30% der a11-defizienten Zellen fähig waren, an eine mit Kollagen I beschichteten Oberfläche zu adhärieren. Dagegen waren nach der gleichen Zeitspanne 70% der a2-Knockdown hMSC an Kollagen I und bereits 100% der a1- Knockdown und shRNA Kontroll hMSC adhärent. Des weiteren stellte sich bei einer Zeitraffermikroskopie heraus, dass a1-Knockdown und shRNA Kontroll hMSC ungefähr 35 Minuten benötigten, um sich auf Kollagen I völlig auszubreiten. Dagegen brauchten a2- und a11-Knockdown hMSC ungefähr die doppelte Zeit zum Ausbreiten. Zusätzlich untersuchten wir die Migrationsfähigkeit der Zellen. Die durchschnittliche Strecke, die a1- und shRNA Kontroll hMSC zurücklegten, betrug ca. 170 µm mit einer mittleren Geschwindigkeit von 11.5 µm/h, während a2 and a11-defiziente hMSC ca. 70 µm weit wanderten mit einer Geschwindigkeit von 5 µm/h. Da ein Verlust an a2- and a11-defizienten hMSC zu verzeichnen war, führten wir zunächst eine JC-1 Färbung durch, mittels derer mitochondrische Schäden als Kennzeichen von Apoptose sichtbar gemacht werden können. Die Mehrzahl der a2- and a11-Knockdown hMSC wiesen mitochondrische Schäden auf, während a1- und shRNA Kontroll hMSC intakte Mitochondrien hatten. Zuletzt untersuchten wir mögliche kompensatorische Effekte zwischen den drei Rezeptoren mittels quantitativer PCR. Wir konnten feststellten, dass der Knockdown des a1 Integrins zu einer Hochregulierung von a2 and a11 führte. Ein ähnliches Ergebnis zeigte sich bei der Herunterregulierung von Integrin a2, die eine erhöhte Expression der a1 and a11 Integrine hervorrief. Interessanterweise bewirkte der Knockdown des a11 Integrins einen leichten Anstieg der a1 aber nicht der a2 Integrin Expression. Wir haben zudem festgestellt, dass sich die Zellzahl der Intergrin a2 und a11-defiziente hMSC nach osteogener Stimulierung weiter reduzierte und die Matrix auch auf Einzelzellebene nicht mineralisiert war. Außerdem zeigte unsere vorausgegange Untersuchung mit hMSC von Osteoporosepatienten eine enorme Herunterregulierung von Integrin a2. Zusammenfassung: Unsere Ergebnisse deuten daraufhin, dass a2b1 und a11b1 Integrine unentbehrliche Signale in hMSC vermitteln. Sobald den Zellen diese Rezeptoren fehlten, wurden die Ausbreitung, die Adhäsion, die Migration und die Überlebensraten reduziert. Insbesondere durch unsere Feststellung der Herunterregulierung der Intergin a2 Expression in Osteoporosepatienten, können unsere Intergin Kockdown Modelle in weiterführenden Untersuchungen eingesetzt werden, um das Verständnis über die a2b1 und a11b1 Integrin Signalwege in hMSC und hOB auszudehnen

Cortico-ocular coupling in the service of episodic memory formation

Encoding of visual information is a necessary requirement for most types of episodic memories. In search for a neural signature of memory formation, amplitude modulation of neural activity has been repeatedly shown to correlate with and suggested to be functionally involved in successful memory encoding. We here report a complementary view on why and how brain activity relates to memory, indicating a functional role of cortico-ocular interactions for episodic memory formation. Recording simultaneous magnetoencephalography and eye tracking in 35 human participants, we demonstrate that gaze variability and amplitude modulations of alpha/beta oscillations (10–20 Hz) in visual cortex covary and predict subsequent memory performance between and within participants. Amplitude variation during pre-stimulus baseline was associated with gaze direction variability, echoing the co-variation observed during scene encoding. We conclude that encoding of visual information engages unison coupling between oculomotor and visual areas in the service of memory formation

Cortico-ocular coupling in the service of episodic memory formation

Encoding of visual information is a necessary requirement for most types of episodic memories. In search for a neural signature of memory formation, amplitude modulation of neural activity has been repeatedly shown to correlate with and suggested to be functionally involved in successful memory encoding. We here report a complementary view on why and how brain activity relates to memory, indicating a functional role of cortico-ocular interactions for episodic memory formation. Recording simultaneous magnetoencephalography and eye tracking in 35 human participants, we demonstrate that gaze variability and amplitude modulations of alpha/beta oscillations (10–20 Hz) in visual cortex covary and predict subsequent memory performance between and within participants. Amplitude variation during pre-stimulus baseline was associated with gaze direction variability, echoing the co-variation observed during scene encoding. We conclude that encoding of visual information engages unison coupling between oculomotor and visual areas in the service of memory formation.publishe

Saccades are locked to the phase of alpha oscillations during natural reading

We saccade 3 to 5 times per second when reading. However, little is known about the neuronal mechanisms coordinating the oculomotor and visual systems during such rapid processing. Here, we ask if brain oscillations play a role in the temporal coordination of the visuomotor integration. We simultaneously acquired MEG and eye-tracking data while participants read sentences silently. Every sentence was embedded with a target word of either high or low lexical frequency. Our key finding demonstrated that saccade onsets were locked to the phase of alpha oscillations (8 to 13 Hz), and in particular, for saccades towards low frequency words. Source modelling demonstrated that the alpha oscillations to which the saccades were locked, were generated in the right-visual motor cortex (BA 7). Our findings suggest that the alpha oscillations serve to time the processing between the oculomotor and visual systems during natural reading, and that this coordination becomes more pronounced for demanding words

Decomposing the role of alpha oscillations during brain maturation

Childhood and adolescence are critical stages of the human lifespan, in which fundamental neural reorganizational processes take place. A substantial body of literature investigated accompanying neurophysiological changes, focusing on the most dominant feature of the human EEG signal: the alpha oscillation. Recent developments in EEG signal-processing show that conventional measures of alpha power are confounded by various factors and need to be decomposed into periodic and aperiodic components, which represent distinct underlying brain mechanisms. It is therefore unclear how each part of the signal changes during brain maturation. Using multivariate Bayesian generalized linear models, we examined aperiodic and periodic parameters of alpha activity in the largest openly available pediatric dataset (N=2529, age 5-22 years) and replicated these findings in a preregistered analysis of an independent validation sample (N=369, age 6-22 years). First, the welldocumented age-related decrease in total alpha power was replicated. However, when controlling for the aperiodic signal component, our findings provided strong evidence for an age-related increase in the aperiodic-adjusted alpha power. As reported in previous studies, also relative alpha power revealed a maturational increase, yet indicating an underestimation of the underlying relationship between periodic alpha power and brain maturation. The aperiodic intercept and slope decreased with increasing age and were highly correlated with total alpha power. Consequently, earlier interpretations on age-related changes of total alpha power need to be reconsidered, as elimination of active synapses rather links to decreases in the aperiodic intercept. Instead, analyses of diffusion tensor imaging data indicate that the maturational increase in aperiodic-adjusted alpha power is related to increased thalamocortical connectivity. Functionally, our results suggest that increased thalamic control of cortical alpha power is linked to improved attentional performance during brain maturation

Brain areas associated with visual spatial attention display topographic organization during auditory spatial attention

Spatially selective modulation of alpha power (8–14 Hz) is a robust finding in electrophysiological studies of visual attention, and has been recently generalized to auditory spatial attention. This modulation pattern is interpreted as reflecting a top-down mechanism for suppressing distracting input from unattended directions of sound origin. The present study on auditory spatial attention extends this interpretation by demonstrating that alpha power modulation is closely linked to oculomotor action. We designed an auditory paradigm in which participants were required to attend to upcoming sounds from one of 24 loudspeakers arranged in a circular array around the head. Maintaining the location of an auditory cue was associated with a topographically modulated distribution of posterior alpha power resembling the findings known from visual attention. Multivariate analyses allowed the prediction of the sound location in the horizontal plane. Importantly, this prediction was also possible, when derived from signals capturing saccadic activity. A control experiment on auditory spatial attention confirmed that, in absence of any visual/auditory input, lateralization of alpha power is linked to the lateralized direction of gaze. Attending to an auditory target engages oculomotor and visual cortical areas in a topographic manner akin to the retinotopic organization associated with visual attention

FEF-controlled alpha delay activity precedes stimulus-induced gamma-band activity in visual cortex

Recent findings in the visual system of nonhuman primates have demonstrated an important role of gamma-band activity (40–100 Hz) in the feedforward flow of sensory information, whereas feedback control appears to be established dynamically by oscillations in the alpha (8–13 Hz) and beta (13–18 Hz) bands (van Kerkoerle et al., 2014; Bastos et al., 2015). It is not clear, however, how alpha oscillations are controlled and how they interact with the flow of visual information mediated by gamma-band activity. Using noninvasive human MEG recordings in subjects performing a visuospatial attention task, we show that fluctuations in alpha power during a delay period in a spatial attention task preceded subsequent stimulus-driven gamma-band activity. Importantly, these interactions correlated with behavioral performance. Using Granger analysis, we further show that the right frontal-eye field (rFEF) exerted feedback control of the visual alpha oscillations. Our findings suggest that alpha oscillations controlled by the FEF route cortical information flow by modulating gamma-band activity

Neuromagnetic Indication of Dysfunctional Emotion Regulation in Affective Disorders

Dysfunctional emotion regulation is often reported in affective disorders, but it is unclear whether this dysfunction concerns initial processing of emotional input or regulation of resulting emotion. The present study addressed these aspects in 27 depressive and 15 borderline personality disorder patients and 28 healthy controls who were instructed to either passively view unpleasant and neutral pictures or downregulate emotional responses by reappraisal, while neuromagnetic brain activity was measured. All three groups showed more early response to unpleasant than to neutral pictures, whereas patients failed to show subsequent activity suppression under instructions to down-regulate. This deficient emotion regulation was evident primarily in those subjects reporting high childhood adversity. Results support intact emotional input processing but impaired emotion regulation in affective disorders and indicate a moderating influence of early life stress