Marker tolerant, immunocompetent animals as a new tool for regenerative medicine and long-term cell tracking

<p>Abstract</p> <p>Background</p> <p>Immune-mediated rejection of labeled cells is a general problem in transplantation studies using cells labeled with any immunogenic marker, and also in gene therapy protocols. The aim of this study was to establish a syngeneic model for long-term histological cell tracking in the absence of immune-mediated rejection of labeled cells in immunocompetent animals. We used inbred transgenic Fischer 344 rats expressing human placental alkaline phosphatase (hPLAP) under the control of the ubiquitous R26 promoter for this study. hPLAP is an excellent marker enzyme, providing superb histological detection quality in paraffin and plastic sections.</p> <p>Results</p> <p>Transplantation of cells from hPLAP transgenic (hPLAP-tg) F344 rats into wild-type (WT) F344 recipients failed because of immune-mediated rejection. Here we show that this problem can be overcome by inducing tolerance to the marker gene by transplantation of bone marrow from hPLAP-tg F344 rats into WT F344 hosts after lethal irradiation, or by neonatal exposure of WT F344 rats to hPLAP-tg F344 cells. As proof-of-principle, we injected bone marrow cells (BMC) from hPLAP-tg rats into the knee joint of marker tolerant, bone marrow-transplanted WT rats, and found successful engraftment and differentiation of donor cells. In addition, hPLAP-tg BMC injected intravenously in neonatally tolerized WT F344 hosts could be traced in lymph nodes, 2 months post-injection.</p> <p>Conclusion</p> <p>In combination with the excellent marker hPLAP, marker tolerant animals may open up new perspectives for all experiments requiring long-term histological tracking of genetically labeled cells.</p

Hematopoietic bone marrow cells participate in endothelial, but not epithelial or mesenchymal cell renewal in adult rats

The extent to which bone marrow (BM) contributes to physiological cell renewal is still controversial. Using the marker human placental alkaline phosphatase (ALPP) which can readily be detected in paraffin and plastic sections by histochemistry or immunohistochemistry, and in ultrathin sections by electron microscopy after pre-embedding staining, we examined the role of endogenous BM in physiological cell renewal by analysing tissues from lethally irradiated wild-type inbred Fischer 344 (F344) rats transplanted (BMT) with unfractionated BM from ALPP-transgenic F344 rats ubiquitously expressing the marker. Histochemical, immunohistochemical and immunoelectron microscopic analysis showed that the proportion of ALPP+ capillary endothelial cells (EC) profoundly increased from 1 until 6 months after BMT in all organs except brain and adrenal medulla. In contrast, pericytes and EC in large blood vessels were ALPP–. Epithelial cells in kidney, liver, pancreas, intestine and brain were recipient-derived at all time-points. Similarly, osteoblasts, chondrocytes, striated muscle and smooth muscle cells were exclusively of recipient origin. The lack of mesenchymal BM-derived cells in peripheral tissues prompted us to examine whether BMT resulted in engraftment of mesenchymal precursors. Four weeks after BMT, all haematopoietic BM cells were of donor origin by flow cytometric analysis, whereas isolation of BM mesenchymal stem cells (MSC) failed to show engraftment of donor MSC. In conclusion, our data show that BM is an important source of physiological renewal of EC in adult rats, but raise doubt whether reconstituted irradiated rats are an apt model for BM-derived regeneration of mesenchymal cells in peripheral tissues

In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells

Background: Bone grafts are required to repair large bone defects after tumour resection or large tr

Utility of irradiated, bone marrow-transplanted, marker tolerant rats for regenerative studies and cell tracking

<p><b>Copyright information:</b></p><p>Taken from "Marker tolerant, immunocompetent animals as a new tool for regenerative medicine and long-term cell tracking"</p><p>http://www.biomedcentral.com/1472-6750/7/30</p><p>BMC Biotechnology 2007;7():30-30.</p><p>Published online 8 Jun 2007</p><p>PMCID:PMC1899491.</p><p></p> When hPLAP-tg BMC were injected into the knee joint of irradiated WT (hPLAP-BMT) rats, there is a 2 – 3 cells wide layer of donor cells (arrowheads) on the cartilage surface, 1 week post-injection (A). Inset in (A) shows lack of hPLAP-staining in the articular cartilage of an irradiated WT (hPLAP-BMT) control rat not injected with transgenic BMC. Four weeks post-injection, transgenic donor cells have adopted a fibroblast-like (arrowheads) or chondrocyte-like (arrows) appearance, and show incorporation into the surrounding chondrocyte matrix of host origin (B). Five-μm-thick undecalcified MMA sections of ethanol-fixed knee joints stained for hPLAP activity after heat pretreatment and counterstained with nuclear fast red. Bar = 50 μm

Collision of oriented NO with Ni(100) and with oriented CO on Ni(100)

Müller H, Dierks B, Hamza F, et al. Collision of oriented NO with Ni(100) and with oriented CO on Ni(100). Surface Science. 1992;269-270:207-212

Molecular adsorption on oxide surfaces: Electronic structure and orientation of NO on NiO(100)/Ni(100) and on NiO(100) as determined from electron spectroscopies and ab initio cluster calculations

We have investigated the adsorption of NO on a thin NiO(100) film of several layers thickness grown on top of a Ni(100) surface in comparison with data of an in vacuo cleaved NiO(100) single crystal. The layer exhibits a high defect density. We demonstrate via application of several surface-sensitive electron-spectroscopic techniques [i.e., x-ray photoelectron spectroscopy (XPS), angle-resolved ultraviolet photoelectron spectroscopy (ARUPS), near-edge x-ray-absorption fine structure (NEXAFS), and high-resolution electron-energy-loss spectroscopy (HREELS)] that this layer has similar occupied (ARUPS) and unoccupied (NEXAFS) states as a bulk NiO(100) sample. In spite of its limited thickness, the band structure of the film exhibits dispersions perpendicular to the surface compatible with bulk NiO(100). It is shown that the electronic structure of the oxygen sublattice can be described in a band-structure picture while for the Ni sublattice electron localization effects lead to a breakdown of the band-structure picture. NO on NiO desorbs at 220 K. This indicates weak chemisorption. The NO coverage is close to 0.2 relative to the number of Ni surface atoms as determined by XPS.HREELS reveals that there is only one species on the surface documented by the observation of only one bond-stretching frequency. NEXAFS data on the system and a comparison with previous data on the system NO/Ni(100) indicate that the molecular axis of adsorbed NO is tilted by an angle of approximately 45° relative to the surface normal. The N 1s XP spectra of the weakly chemisorbed species show giant satellites similar to the previously observed cases for weak chemisorption on metal surfaces. This is the first observation of an intense satellite structure for an adsorbate on an insulator surface, which shows that there must be sufficient screening channels even on an insulating surface. A theoretical assignment of the peaks is discussed. We compare the spectroscopic properties of the NO species on the thin-film oxide surface, which is likely to contain a certain number of defects, with NO adsorbed on a basically defect-free bulk oxide surface by thermal-desorption (TDS) and XP spectra. TDS and XP spectra of the bulk system are basically identical as compared with the oxide film, indicating that the majority of species adsorbed on the film is not adsorbed on defects but rather on regular NiO sites. Results of ab initio oxide cluster calculations are used to explain the bonding geometry of NO on regular NiO sites