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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 4, 2003 as doi:10.1096/fj.03-0214fje. |
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,2
* Department of Neurology, Yale University School of Medicine, New Haven, an Neuroscience Research Center, VA Medical Center, West Haven, Connecticut, USA;
* Alexion Pharmaceuticals Inc., Cheshire, Connecticut, USA; and
CTR. Anatomy, Hannover Medical School, Hannover, Germany
2 Correspondence: J.D.K., Yale University School of Medicine, Neuroscience Research Center (127A), VAMC, West Haven, CT 06516, USA; E-mail: jeffery.kocsis{at}yale.edu or W.L.F., Department of Molecular and Cellular Biology, CT Center for Regenerative Biology, University of Connecticut, Storrs, CT 06269-4243, USA; E-mail: william.fodor{at}uconn.edu
SPECIFIC AIM
Olfactory ensheathing cells (OECs) can remyelinate central demyelinated lesions. The primary objective of this study was to determine whether xenotransplantation of characterized OECs derived from transgenic pigs expressing the 
1,2 fucosyltransferase gene (H-transferase; HT) could remyelinate the spinal cord of nonhuman primates.
PRINCIPAL FINDINGS
1. Transplantation of HT transgenic pig OECs into monkey spinal cord
We transplanted HT OECs into demyelinated lesions by microinjection 3 days after lesion induction. All monkey recipients were immunosuppressed with cyclosporine A (CsA, 15 mg·kg–1·day–1, i.m.) beginning 1 day before transplantation and continuing throughout the course of the experiment. The transplanted spinal cords were prepared for histological analysis 3–5 wk post-injection. Evidence of remyelination was detected in 10 of 16 (62.5%) transplant recipient sites as determined by histological analysis of serial sections through the lesion area. A representative spinal cord from an HT OEC recipient 4 wk post-transplantation illustrated the area of the lesion and the transplantation site within the dorsal funiculus (Fig. 1
A). Area of the dorsal funiculus inside the white dashed lines was densely remyelinated (Fig. 1B
). Some remyelinated axons are associated with ensheathing cells that contain large cytoplasmic compartments and large nuclei (arrows, Fig. 1C
). We observed that remyelination did not extend across the entire cross-sectional area of the lesion. Analysis of the border between the transplant zone and the demyelinated region, revealed demyelinated axons adjacent to remyelinated axons (Fig. 1D
).
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2. Morphology and purity of transgenic pig OECs in culture
OECs were prepared from pig olfactory bulbs and maintained in culture for 5–7 days. The prevalence of p75 nerve growth factor receptor (NGFR) -labeled cells confirmed the identity and purity of the OECs, as they are the only cell type in the olfactory bulb that expresses p75 NGFR. The cultures were counterstained with nuclear yellow (Hoechst 33250) to identify individual cells, and the vast majority (
98%) of the cells were p75 NGFR positive. In addition, OECs are known to express both S100 and GFAP. There was a high degree of coexpression of S100 or GFAP immunoreactivity with p75 NGFR immunoreactivity. These data indicate that, similar to rodent OECs, cultured HT transgenic pig OECs express p75 NGFR, S100, and GFAP. However, unlike rodent OECs, the porcine OECS could be harvested at high purity without additional purification strategies such as immunopanning.
3. Carbohydrate expression analyses and complement activation on nontransgenic and transgenic pig OECs
HT leads to expression of the universally accepted human type O blood group phenotype (H epitope), which in turn reduces Gal1,3Gal expression, resulting in reduced human antibody reactivity and complement-mediated cell lysis. OECs isolated from nontransgenic and HT transgenic pigs were analyzed for H epitope and Gal1,3Gal epitope expression by flow cytometry (Fig. 2
). A reciprocal expression pattern was seen in the flow cytometric profiles, where an increase in H epitope expression resulted in a decrease in Gal1,3Gal (Fig. 2A, B)
and correlated with the immunocytochemical analyses. To further illustrate the reciprocal relationship between Gal1,3Gal antigen and H antigen expression of nontransgenic and HT transgenic pig OECs, we performed semiquantitative analysis by calculating the mean fluorescence intensity (FL-1) from several flow cytometry experiments (Fig. 2C
). These data demonstrate that a 100-fold increase in HT expression leads to 7-fold reduction in Gal1,3Gal expression (Fig. 2C
). We monitored the expression level of two control antigens, SLA class I and CD44, to ensure comparability in the flow cytometry analysis (Fig. 2C
). There was no difference in the expression levels of the porcine SLA class I or CD44 between the nontransgenic and the transgenic OECs and indicated that changes in the carbohydrate phenotype did not alter the expression of other cell surface molecules in the transgenic OECs. To determine the effect of H-transferase expression on complement activation in the transgenic OECs, we analyzed HT and nontransgenic OECs in a complement activation assay. This assay activates complement on the target cell surface without lysing the cell and allows for detection of the activated human complement component C3d on the cell surface using an anti-human C3d monoclonal antibody and flow cytometry. Complement activation was reduced >twofold in the HT OECs compared with the nontransgenic cells (Fig. 2D
), indicating that the expression of HT in the transgenic OECs leads to protection from antibody reactivity and complement activation.
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CONCLUSIONS AND SIGNIFICANCE
We determined that the HT transgenic OECs indeed expressed the transgene and displayed antigenic properties similar to the well-characterized rodent OECs, including S100, glial fibrillary acidic protein (GFAP), and p75 NGFR expression. The OECs were characterized in culture and could be consistently prepared in large number, thus allowing transplantation of cells that displayed consistent and identified phenotypic properties. Moreover, an in vitro functional assay indicated that the porcine HT OECs have reduced complement deposition on the cell surface when exposed to human serum compared with nontransgenic control OECs. The repair potential of OECs transplanted into the injured rodent spinal cord has been demonstrated, but the ability of OECs to repair the nonhuman primate spinal cord has not been tested. We demonstrate that transplanting phenotypically characterized pig OECs expressing the H antigen into the demyelinated nonhuman primate spinal cord results in remyelination with a morphological architecture characteristic of peripheral myelin (Fig. 3
). Thus, the repair potential observed in rodents with transplantation of transgenic pig OECs can be extrapolated to the primate, and the expression of the HT transgene does not appear to affect the repair potential of the OECs.
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The development of porcine cell- and tissue-based therapies to treat human diseases has gained much attention and currently there are several potential clinical applications for the use of porcine tissues. Examples include the treatment of Parkinson’s disease patients with embryonic porcine mesencephalic neurons and the use of porcine hepatocytes for liver support systems in cases of acute liver failure. Many porcine cell types potentially useful for transplantation, such as cardiomyocytes for damaged heart muscle or OECs for axonal repair, would be expected to trigger a humoral and cellular immune response. Therefore, the demonstration that genetically engineered pig OECs, modified to resist humoral immunity, exhibit characteristic OEC phenotypic properties and can repair demyelinated primate spinal cord lesions reinforces the potential utility of transgenic porcine tissues for human cell therapy.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0214fje 
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0.05. D) Cell surface complement activation on HT transgenic (n=3) and NT control (n=3) olfactory ensheathing cells was determined by flow cytometry using an antibody to the activated complement component, human C3d. Mean fluorescence intensity (FL-1) was calculated from 3 independent cell isolates (P=0.0036).