One of the subjects partially instrumented to illustrate the electrode positioning (for details on the skeletal landmarks, see Material & Methods).

<p>One of the subjects partially instrumented to illustrate the electrode positioning (for details on the skeletal landmarks, see Material & Methods).</p

Additional file 2: of Kinematic adaptions to induced short-term pelvic limb lameness in trotting dogs

Kinematic results for the pelvic limbs. For further explanation, see Additional file 1. (DOC 332 kb

Additional file 1: of Kinematic adaptions to induced short-term pelvic limb lameness in trotting dogs

Kinematic results for the thoracic limbs. Mean ± standard deviation (Mean ± SD in °) of the limb, segment and joint angles for all dogs. Kinematic values for the limbs are: angle at touch-down (TD), lift-off (LO) and mid-stance (mid-stance). Kinematic values for the segments and joints are: angle at touch-down (TD) and lift-off (LO) as well as minimum (min), maximum (max) and amplitude (i.e. range of motion, ROM) during stance (ST) and swing (SW) phases. Mean SDs (mSD in °; i.e. SDs from the 10 strides per dog averaged for all dogs) illustrate the relatively low intraindividual variation compared with the interindividual variation (SD of Mean ± SD) and particularly compared with the angular difference between sound and lame trotting (Diff Mean ± SD in °). Note that this mean Diff was calculated by, first, subtracting the lame from the sound values per dog and, second, averaging these angular differences for all dogs (i.e. mean Diff represents the angular changes associated with lame locomotion). Positive Diff values indicate that the angle was greater during sound than lame trotting; negative values indicate the reverse. Significant differences between sound and lame trotting for each limb (I) as well as significant differences between the angular differences of the two limbs (II) at: * P < 0.05, ** P < 0.01, *** P < 0.001. For definition of angles, see Fig. 1 in [16]. (DOC 349 kb

Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles

<div><p>The biological relevance of extracellular vesicles (EV) in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC) also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced <i>Arabidopsis thaliana</i>-DNA (<i>A</i>.<i>t</i>.-DNA) as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR) techniques we detected high copy numbers of <i>A</i>.<i>t</i>.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified <i>A</i>.<i>t</i>.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of <i>A</i>.<i>t</i>.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.</p></div

Detection of <i>A</i>.<i>t</i>.-DNA in EV-recipient cells with SYBR Green-based qPCR, TOPO^® TA Cloning and sequencing.

<p>Shown is the total number of primary PCR replicates carried out with 1 μg DNA/PCR using complete isolated DNA of each single tissue flask, therefrom resulting nested PCR replicates, the number of positive samples in SYBR Green nested PCR, the No. of colonies which underwent sequencing and No. of colonies with the correct sequence (sequencing positive). In total, three different EV preparations were applied in this experiment. For each EV sample, 2x T25 of recipient cells were incubated with unmanipulated EV (-) and 1x T25 with EV after DNaseI (+) treatment. The nomenclature C(-)16 stands for: EV sample C without DNase treatment C(-), qPCR No. 16 carried out with 1 μg DNA per reaction.</p

<i>Arabidopsis thaliana (A</i>.<i>t</i>.<i>)</i> virus production and transfer.

<p>(a) <i>A</i>.<i>t</i>.-DNA was cloned into the LeGO-V2-wpre plasmid vector containing Venus-fluorescence protein for detection. Primers for subsequent primary and nested <i>A</i>.<i>t</i>.-PCR shown with arrows were located within the <i>A</i>.<i>t</i>.-sequence giving rise to products of 387 bp and 106 bp respectively. (b, c) hMSC were transduced with LeGO-V2-wpre-<i>A</i>.<i>t</i>. virus supernatant. Shown is a hMSC culture 8 days after transduction (x40) detecting green cells (b) in a near confluent culture (c, phase contrast). (d, e) Recipient hMSC were incubated for 2 weeks with EV purified from hMSC-<i>A</i>.<i>t</i>. culture supernatant. Shown are Venus-positive cells (d) in the recipient culture after incubation with EV without (3 left images) or with DNase digestion (most right image) and their respective phase contrast pictures (e) (magnification x200).</p

Detection of <i>A</i>.<i>t</i>.-sequences in recipient cells using SYBR Green-based qPCR.

<p>(a) Standard dilutions of <i>A</i>.<i>t</i>.-DNA in duplicates with 10.000–10 copies/PCR reaction show a linear dependency whereas 1 copy/PCR was located below the detection limit. (b) Three to four replicates of DNA isolations from EV without DNase treatment (G(-); EV from harvest G without DNAse treatment) showed high abundant <i>A</i>.<i>t</i>.-sequences with Ct = 16 whereas those with DNase treatment (G(+)) showed much lower <i>A</i>.<i>t</i>.-DNA amounts with Ct near the detection limit. As comparison, positive standard with 100 copies/PCR was plotted. (c) Several replicates of the sample C(-)16 (EV from harvest C without DNase treatment, PCR run No. 16 carried out with 1μg DNA per reaction) were detected with Ct of ≥ 33. As comparison, positive standard with 10 copies/PCR was plotted. (d) Melting temperatures (Tm) of samples in (c) show the replicates with one high and several lower peaks with the correct Tm. The blue curves correspond to the positive standard of 10 copies/PCR reaction. Two exemplary arrows for sample C(-)16 in (c) and (d) point to lime and red colored probes with high and low Tm peaks, respectively.</p

Detection of <i>A</i>.<i>t</i>.-sequences in recipient cells using TaqMan-based qPCR.

<p>(a) Standard dilutions of <i>A</i>.<i>t</i>.-DNA in quadruplicates with 1.000–10 copies/PCR show a linear dependency. (b) Eightfold replicates of two different DNA isolations from EV without (G(-)) DNase treatment showed high abundant <i>A</i>.<i>t</i>.-sequences with Ct = 13 and 18 whereas those with DNase treatment (G(+)) showed much lower <i>A</i>.<i>t</i>.-DNA amounts. As comparison, positive standard with 100 copies/PCR was plotted. (c) Several replicates of the sample C(-)16 were detected with Ct of ≥ 40. As comparison, positive standard with 10 copies/PCR was plotted. All negative controls did not give rise to signals at any time (not shown).</p

Characterization of EV.

<p>Human MSC were cultured on ibidi μ-slides, fixed and analyzed using electron microscopy. Shown is a part of cell membrane of a hMSC releasing EV (a). After ultracentrifugation of the supernatant, EV were resuspended in small volumes, sucked into carbo-tubes, fixed and analyzed using electron microscopy. Patches (b) and single EV (c) of 50–1,000 nm were detected. Ten μg of characteristic EV-proteins (CD81, HSP70, CD9 and CD63, GAPDH as housekeeper) were analyzed by Western blot (d). For quantification of EV using flow cytometry, size beads ranging from 0.2–2 μm were used to define the EV analysis area P1 (e) and impurities of 0.1μm filtered PBS in P1 (f). Purified EV in P1 were quantified using counting beads excluding the particles contained in filtered PBS. Total EV amounts per harvest (samples A-N from three individual donors) blotted against the protein content of each EV harvest revealed interindividual differences in protein cargo but reproducibility within one donor culture after repeated EV harvests (g). To investigate the underestimation of EV due to “swarm detection” in flow cytometry, 6 EV harvests were measured with NanoSight revealing ca. 1,000 fold higher concentration (401 ± 290) with a mode size of 146 ± 7.7 nm (h).</p

Detection of Venus-fluorescence and <i>A</i>.<i>t</i>.-sequences in recipient cells after passaging.

<p>(a) 2x10⁵ hMSC were seeded into T25, incubated overnight to reach adherence (d0) and fed with EV derived from <i>A</i>.<i>t</i>.-hMSC cultures for 2 weeks. (b) Venus-positive cells were detected in 2 of 4 flasks (d14). One flask with 7 positive cells was passaged into 4xT25 flasks. (c) 7 days later (d21), one flask contained 10 Venus-positive cells. This culture was expanded again into 4xT25. (d) Venus-positive cells at d28 were evident in 2 flasks out of 4 with 13 cells in one flask and 1 cell in the second flask. Exemplarily, one positive MSC spot with corresponding phase-contrast for each time point is shown. Magnification x 100. (e, f) DNA of the flask with 13 Venus-positive cells was pretested in nested SYBR Green-based qPCR. Out of 10 primary reaction tubes, 4 were positive in the nested qPCR tested in 8 replicates (tubes 2, 4, 7 and 8; not shown) and were retested in TaqMan-based qPCR (e) and ddPCR (f). Shown are the results for positive control (pc, 10 copies/PCR reaction; 4 replicates in TaqMan-based qPCR and 2 replicates in ddPCR), negative control (nc, untransduced hMSC; 8 replicates in TaqMan-based qPCR and 2 replicates in ddPCR), and tube 2 and 4 (16 replicates in TaqMan-based qPCR and ddPCR).</p