Application of adipose-derived stromal cells in fat grafting: Basic science and literature review

Autologous fat is considered the ideal material for soft‑tissue augmentation in plastic and reconstructive surgery. The primary drawback of autologous fat grafting is the high resorption rate. The isolation of mesenchymal stem cells from adipose tissue inevitably led to research focusing on the study of combined transplantation of autologous fat and adipose derived stem cells (ADSCs) and introduced the theory of ʻcell‑assisted lipotransferʼ. Transplantation of ADSCs is a promising strategy, due to the high proliferative capacity of stem cells, their potential to induce paracrine signalling and ability to differentiate into adipocytes and vascular cells. The current study examined the literature for clinical and experimental studies on cell‑assisted lipotransfer to assess the efficacy of this novel technique when compared with traditional fat grafting. A total of 30 studies were included in the present review. The current study demonstrates that cell‑assisted lipotransfer has improved efficacy compared with conventional fat grafting. Despite relatively positive outcomes, further investigation is required to establish a consensus in cell‑assisted lipotransfer

Cell adhesion molecules and intracellular proteins that facilitate the interactions between neuronal and glial cells of the nervous system: Design of therapeutic approaches for spinal cord injury

Schwann cells (SC) are the glial cells of the peripheral nervous system (PNS) that contribute to the development, survival and function of neuronal cells as well as to peripheral nerve regeneration after injury. Furthermore, there is experimental evidence that SC could have beneficial applications in clinical transplantation for the repair of CNS after trauma. Yet SC do not integrate well into the injured tissue of the CNS. This study focuses on specific molecules for the development of combinatorial approaches for genetic manipulation of SC in order to overcome this disadvantage and become suitable for therapeutic transplantation in the injured CNS. In the first part, retroviruses were used as molecular vehicles for the expression of cell adhesion molecules Ll or PSA-NCAM by SC. In vivo experiments of transplantation of these genetically modified SC followed in a model of mouse spinal cord injury. The injured animals were subjected in behavioral study of their hind limb locomotor function and it was shown that the animals that received PSA-NCAM-SC transplants and to a lesser extent the animals that received Ll-SC exhibited improved motor function, which by means of immunohistochemistry was attributed to higher myelination and regeneration of the damaged nerve fibers. In the second part of this study, BM88 protein was evaluated as a potent candidate for genetic modification of SC prior to their transplantation in the injured CNS. Primary results showed that BM88, a neuronal protein expressed only by neurons in the CNS, is also expressed by SC in the PNS. This was confirmed by immunofluorescence on normal or injured sciatic nerve specimens and also on primary cultured SC. Given the role of BM88 protein in neuronal cells of the CNS, where it drives neural precursors to cell cycle exit and differentiation, overexpression experiments using lentiviruses were performed in primary cultured SC, which showed that BM88 overexpression drives not only overexpressing cells but also neighboring wild type SC to reduced proliferation, possibly in an autocrine/paracrine manner. No differences in differentiation were observed.Τα κύτταρα Schwann (κ.S.) στο Περιφερικό Νευρικό Σύστημα (ΠΝΣ) συμβάλλουν στην επιβίωση, την ανάπτυξη και τη λειτουργία των νευρικών κυττάρων και την επιδιόρθωση των περιφερικών νεύρων μετά από τραυματισμό. Επίσης πειραματικά δεδομένα τους δίνουν προοπτικές κλινικής εφαρμογής σε περιπτώσεις τραυματισμού του ΚΝΣ. Ωστόσο τα κ.S. δεν ενσωματώνονται ικανοποιητικά μέσα στο τραυματισμένο ΚΝΣ. Η εργασία εστιάζει στη μελέτη μορίων για την ανάπτυξη τεχνικών γενετικής τροποποίησης κ.S., με στόχο την προσπέλαση των εμποδίων αυτών και τη μεταμόσχευση τους για τη θεραπεία του τραυματισμένου ΚΝΣ. Στο πρώτο μέρος χρησιμοποιήθηκαν ρετροϊοί για την έκφραση της Ll και της PSA-NCAM. Ακολούθησαν in vivo πειράματα μεταμόσχευσης των γενετικά τροποποιημένων κ.S. σε μοντέλο τραυματισμού του νωτιαίου μυελού ποντικού. Τα πειραματόζωα υποβλήθηκαν σε μελέτη της κινητικής λειτουργίας των πίσω άκρων τους, όπου εκείνα που προσέλαβαν μοσχεύματα PSA-NCAM-κ.S., και λιγότερο εκείνα που προσέλαβαν Ll-κ.S. παρουσιάζουν βελτιωμένη κινητικότητα σε σύγκριση με αρνητικούς μάρτυρες, η οποία με μέσα ανοσοϊστοχημείας αποδόθηκε στη συνεισφορά των μεταμοσχευμένων κυττάρων στην αναγέννηση και τη μυελινοποίηση των τραυματισμένων νευραξόνων. Στη συνέχεια μελετήθηκε η πρωτεΐνη ΒΜ88 ως υποψήφια για προσεγγίσεις μεταμόσχευσης γενετικά τροποποιημένων κ.S. στο τραυματισμένο ΚΝΣ. Αρχικά ανακαλύφθηκε ότι τα κ.S. εκφράζουν την πρωτεΐνη ΒΜ88 η οποία στο ΚΝΣ εκφράζεται μόνο από νευρικά κύτταρα και επάγει την έξοδο των νευροβλαστικών κυττάρων από τον κυτταρικό κύκλο. Με ανοσοϊστοχημεία μελετήθηκε η έκφραση της πρωτεΐνης στα κ.S. του ισχιακού νεύρου, in vivo και in vitro ενώ επίσης πραγματοποιήθηκαν πειράματα υπερέκφρασης της πρωτεΐνης ΒΜ88 με τη χρήση λεντιιών, όπου φάνηκε ότι προκαλεί καταστολή του πολλαπλασιασμού των ίδιων των κ.S. που την υπερεκφράζουν, αλλά και των γειτονικών/μη μετασχηματισμένων κ.S., πιθανά μέσω αυτοκρινούς/παρακρινούς μηχανισμού. Δε φάνηκε επίδραση της ΒΜ88 στη μυελινοποίηση

Brain Infection by Group B Streptococcus Induces Inflammation and Affects Neurogenesis in the Adult Mouse Hippocampus

Central nervous system infections caused by pathogens crossing the blood–brain barrier are extremely damaging and trigger cellular alterations and neuroinflammation. Bacterial brain infection, in particular, is a major cause of hippocampal neuronal degeneration. Hippocampal neurogenesis, a continuous multistep process occurring throughout life in the adult brain, could compensate for such neuronal loss. However, the high rates of cognitive and other sequelae from bacterial meningitis/encephalitis suggest that endogenous repair mechanisms might be severely affected. In the current study, we used Group B Streptococcus (GBS) strain NEM316, to establish an adult mouse model of brain infection and determine its impact on adult neurogenesis. Experimental encephalitis elicited neurological deficits and death, induced inflammation, and affected neurogenesis in the dentate gyrus of the adult hippocampus by suppressing the proliferation of progenitor cells and the generation of newborn neurons. These effects were specifically associated with hippocampal neurogenesis while subventricular zone neurogenesis was not affected. Overall, our data provide new insights regarding the effect of GBS infection on adult brain neurogenesis

Embryonic cultured cortical neurons express Cend1, RanBPM and Dyrk1B.

<p>(<i>a</i>) Immunofluorescence labeling and confocal analysis of cortical neurons from E16.5 mouse embryos cultured for 8 days <i>in </i><i>vitro</i>. Cultures were double-labeled for the neuronal marker betaIII-tubulin (Tuj1 antibody; red) and Cend1, RanBPM or Dyrk1B (green), respectively, as indicated. The panels on the right show at higher magnification the areas marked by white rectangles in the merged figures. Scale bar: 20 μm. (<i>b</i>) Immunoblot analysis of cortical neuron lysates cultured for 8 days <i>in </i><i>vitro</i>, confirming the expression of Cend1, RanBPM and Dyrk1B. </p

(<i>a</i>, <i>b</i>) Interactions between Cend1, RanBPM and Dyrk1B affect cyclin D1 protein levels.

<p>(<i>a</i>) Single, double or triple transient transfections with Cend1, RanBPM and Dyrk1B were performed in Neuro 2a cells, as indicated, in order to examine cyclin D1 levels. Cells were collected 16 h post-transfection and lysates were subjected to Western blot analysis (60 μg cell lysate per lane) using the indicated palette of antibodies. (<i>b</i>) Quantification of cyclin D1 protein levels normalized relative to β-tubulin was performed using the Image J software. **Student’s t-test, p<0.01, n= 10. Error bars represent SEM. Note that in double transfections with Cend1 or Dyrk1B, RanBPM caused a marked increase in cyclin D1, while in triple transfected Neuro 2a cells cyclin D1 dropped again in similar levels to those in Dyrk1B single transfected cells. (<i>c</i>, <i>d</i>) <b>Cend1 segregates Dyrk1B in the nucleus in the presence of RanBPM</b>. Double (<i>c</i>) and triple (<i>d</i>) immunofluorescence labeling and confocal analysis of Neuro 2a cells transiently transfected with RanBPM and Dyrk1B (<i>c</i>) or triple transfected with RanBPM, Dyrk1B and Cend1 (<i>d</i>), 16 h post-transfection. (<i>c</i>) RanBPM (green) facilitates Dyrk1B (red) translocation in the cytoplasm in double-transfected cells (arrows, <i>i</i>-iv). Nuclei were visualized with TO-PRO-3 (Blue, <i>iii</i>). (<i>d</i>) In triple transfected cells with RanBPM (green), Cend1 (red) and Dyrk1B (blue), Dyrk1B remains in the nucleus (arrows <i>iii</i>, iv).</p

Cend1, RanBPM and Dyrk1B kinase are expressed in mouse brain and form complexes.

<p>(<i>a</i>) Mouse brain homogenate (60 μg of protein per lane) was subject to SDS-electrophoresis and immunoblotting with antibodies to Cend1 (lane 1), Dyrk1A (lane 2), Dyrk1B (lane 3) and RanBPM (lane 4). In lane 1 the 23kD band corresponding to the single polypeptide chain of Cend1 is seen as well as residual disulfide bond-linked 46kD homodimer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082172#B24" target="_blank">24</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082172#B25" target="_blank">25</a>]. (<i>b</i>) HEK293T cells were either not transfected (CTL) or transiently transfected with Cend1, RanBPM, Dyrk1B and GFP at the indicated combinations. Cells were harvested and RanBPM was immunoprecipitated from cell lysates using anti-FLAG antibody. Co-immunoprecipitated proteins were identified by Western blot using the indicated palette of antibodies. In the left panel, shown is the total cell lysate (input) with the different protein bands detected in each case. In the right panel, shown are the proteins which were co-immunoprecipitated with RanBPM in each case. (<i>c</i>) HEK293T cells were co-transfected with Cend1 and Dyrk1B and immunoprecipitated with anti-FLAG antibody. Note that anti-FLAG does not immunoprecipitate either of these proteins.</p

Cend1-RanBPM interaction enhances cyclin D1 levels and cell proliferation.

<p>(<i>a</i>) Western blot analysis of Neuro 2a cells transiently transfected with Cend1 or FLAG-RanBPM or both, using the indicated antibodies. An increase in cyclin D1 levels is noted upon Cend1-RanBPM co-transfection. Non-transfected Neuro 2a cells (CTL) and mock-transfected cells with empty vector were used as controls. (<i>b</i>) Quantification and normalization of cyclin D1 protein levels relatively to α-actin in Cend1 and Cend1/RanBPM transfected cells using the Image J software. (<i>c</i>) Quantification of cyclin D1 mRNA by real-time real-time RT-qPCR (d) Co-expression of RanBPM and Cend1 enhances BrdU incorporation. Estimation of the BrdU index was made by counting BrdU⁺ cells out of all cells in control cultures (CTL); BrdU⁺/Cend1⁺ cells out of all Cend1⁺ cells in Cend1-transfected cells; BrdU⁺/RanBPM⁺ out of all RanBPM⁺ cells in RanBPM-transfected cells; BrdU⁺/Cend1⁺/RanBPM⁺ cells out of all Cend1⁺/RanBPM⁺ cells in double transfected Neuro 2a cells, following a 1-hour BrdU pulse. At least 2000 cells were counted in each case and the BrdU index was calculated as the mean value from 6 independent experiments. Error bars represent SEM; ***Student’s t-test, p<0.001. (<i>e</i>- <i>g</i>) Co-expression of Cend1 and RanBPM stabilizes cyclin D1 in the nucleus. In Neuro 2a cells transiently transfected with Cend1, cyclin D1 is either extinct (open arrowheads) or exported from the nucleus to the cytoplasm (white arrows), and only occasionally maintained in the nucleus (white arrowhead) of Cend1⁺ cells (<i>e</i>). In RanBPM-transfected cultures, cyclin D1 is found in the nucleus (white arrowheads) or is extinct (open arrowheads) in RanBPM⁺ cells similar to control untransfected cells (<i>f</i>). In contrast, in all Cend1⁺/RanBPM⁺ cells cyclin D1 exhibited entirely nuclear localization (arrowheads in <i>g</i>). Scale bar: 10 μm.</p

Schematic representation of Cend1 and RanBPM cDNA constructs and protein fragments.

<p>(<i>a</i>) Total mouse Cend1 cDNA (1241-bp) encodes for a 149 amino-acid protein, containing a C-terminal transmembrane region (TM) followed by RKK tail. A 375 bp fragment (372 bp plus a stop codon) corresponding to nucleotides 141 to 513 of Cend1 cDNA) and encoding the first 124 amino acids of the protein, was generated by high-fidelity PCR. This fragment was fused in frame with either the GAL4 binding domain or GST and served for yeast two hybrid screening or GST-pull down assays, respectively. (<i>b</i>, <i>c</i>) Diagrammatic illustration of the three RanBPM cDNA clones (<i>b</i>) and corresponding protein fragments (<i>c</i>) isolated from yeast two hybrid screening, all containing the multifunctional proline-rich SPRY-LISH-CTLH-CRA domain. Generation of GST-RanBPM chimeric molecules (corresponding to the 981 bp and 1731 bp cDNA fragments, as indicated) and the SPRY-LISH-CTLH domain (942 bp) was performed by high-fidelity PCR. </p

RanBPM facilitates Dyrk1B proteasomal degradation and stabilizes cyclin D1.

<p>(<i>a</i>) Neuro 2a cells were transiently co-transfected with Dyrk1B and increasing amounts of RanBPM (0, 1 and 5 μg of plasmid). Cells were maintained in culture for 16 h post transfection in the absence or in the presence of MG132 and cell lysates were subjected to Western blot analysis. In the absence of MG132, RanBPM drives Dyrk1B towards proteasomal degradation in a dose-dependent manner, while cyclin D1 levels are stabilized. When the proteasome is blocked, Dyrk1B accumulation is observed with simultaneous accumulation of cyclin D1, which is also degraded via the 26S-proteasome. (<i>b</i>, <i>c</i>) Double (<i>b</i>) and triple (<i>c</i>) immunofluorescence labeling for RanBPM (green) and Dyrk1B (red) or RanBPM (red), Dyrk1B (red) and cyclin D1 (blue). (<i>c</i>) Confocal microscopy shows that RanBPM promotes Dyrk1B cytoplasmic translocation in double positive RanBPM⁺/Dyrk1B⁺ Neuro 2a cells, 16 h post-transfection (arrows, <i>c</i>). Nuclei were visualized with TO-PRO3 (<i>c</i>). (<i>d</i>), In RanBPM⁺/Dyrk1B⁺ double positive cells, cyclin D1 is stabilized in the nucleus (arrow, d), while in cells expressing Dyrk1B, but not RanBPM (double arrowheads), cyclin D1 is not detectable in the nucleus. Scale bars: 8 μm. </p

Dyrk1B protein expression and turn-over in Neuro 2a cells.

<p>(<i>a</i>) Dyrk1B expression in transiently transfected Neuro 2a cells was examined at 16 h and 48 h post-transfection in the presence or in the absence of the specific proteasome inhibitor MG132. Dyrk1B is not detectable in non-transfected Neuro 2a cells (CTL) with available antibody, while transgene Dyrk1B, obvious at 16h, is decreased overtime in a proteasome-specific manner (upper panel). In the absence of MG132, cyclin D1 levels are inversely related to Dyrk1B (middle panel) while in the presence of MG132 cyclin D1, also degraded via the proteasome, is maintained despite persisting Dyrk1B expression (middle panel); β-tubulin indicates protein loading (lower panel). (<i>b</i>) Dyrk1B is localized in the nucleus of Dyrk1B⁺ Neuro 2a cells 16 h post-transfection, while at 48 h it is mainly cytoplasmic as shown by immunocytochemistry and confocal microscopy. The cells depicted by arrows at 16h and 48h, respectively, are shown at higher magnification. Different image acquisition settings were used at 16h and 24h to compensate for signal reduction at 48h. Scale bar: 10 μm. (<i>c</i>) Dyrk1B protein turn-over in Neuro 2A cells. Cells were transfected with Dyrk1B and allowed for expression 16 h after transfection. Cells were then treated with cycloheximide for different times as indicated and subjected to Western blot analysis. (<i>d</i>) Dyrk1B reduces BrdU incorporation in transiently transfected Neuro 2a cells after 16 h of expression. **Student’s t-test, p=0.00146, n=5. (<i>e</i>) In the presence of 10 μM harmine, a specific kinase inhibitor of the Dyrk1 protein family, the Dyrk1B-dependent down-regulation of cyclin D1 is inhibited in Neuro 2a cells (middle panel) without affecting Dyrk1B protein levels (upper panel). Lanes show Neuro 2a cells transiently transfected with Dyrk1B and allowed 16 h for expression, or non-transfected cells (CTL), harvested and immunoblotted with the indicated antibodies. (<i>f</i>) Quantification of Dyrk1B protein levels normalized relative to β-actin, in the presence or absence of 10 μΜ harmine. (<i>g</i>) Cyclin D1 is wiped out from the nuclei of Dyrk1B⁺ transiently transfected Neuro 2a cells (arrows, <i>i</i>-iv) but is clearly maintained in the nucleus of Dyrk1B⁺ cells in the presence of harmine (arrows, <i>v</i>-viii). Cells were double labeled for Dyrk1B (green) and cyclin D1 (red) while nuclei were visualized using TO-PRO-3 (blue). The merged pictures are shown (iv, viii). Scale bar: 8 μm.</p