Results The Role of Factor VIII in Defining Organ Size, Following Implantation of Pig Embryonic Tissues We have previously shown that implantation of pig embryonic spleen precursor tissue can correct hemophilia in factor VIII KO NOD-SCID mice by providing the necessary Factor VIII[11]. While performing this study, we observed that the size of the implanted spleen of these hemophilic mice was significantly larger compared to that growing in non-hemophilic control mice. Fig. 1A shows a typical oversized pig E42 spleen implant grown for 12 weeks in Factor VIII KO SCID recipient, versus the size of a similar implant grown in a Factor VIII expressing SCID recipient. The total average weight of a series of implants in Factor VIII KO SCID mice was 6.7862.16gr versus 1.4660.82gr in the normal SCID (Fig. 1C,), suggesting that the absence of Factor VIII enhanced spleen weight by a factor of 4 (p,0.05). Histological examination of the growing spleen implants (Fig. 1A, lower panel) revealed normal growth, development and vascularization pat- terns, comparable to those found in the corresponding Factor VIII wild type mice, ruling out potential induction of a malignant process. Furthermore, as shown by immuno-staining with pig specific antibodies for vimentin and ki67 (Fig. 1B), the bulk of the growing implant in factor VIII KO recipients was of pig origin. Weekly follow up of E42 spleen transplants revealed that disparity in transplant size between NOD-SCID and Factor VIII KO-SCID mice occurs as early as 2 weeks post transplantation (Fig. 1D, left). Further tracking of E42 spleen transplants showed exponential growth until 12 weeks post transplantation, whereby the final implant size has been reached without further growth during an additional 2 months follow-up period (Fig. 1D, right). This conclusion was also supported by morphmetric quantitative examination of pig dividing cells, stained by pig specific anti-ki67 antibody. As can be seen in Fig. 1E, 2 weeks post transplantation a significant increase in the frequency of dividing cells was found in Factor VIII KO-SCID compared to NOD-SCID mice. However, beginning on the 3rd week a gradual decrease in dividing cells is observed, reaching a low level at 12 weeks post transplantation in Factor VIII KO recipients (2.761.3) similarly to that found in the non-hemophilic recipients (2.161.2). Thus, early post transplant events during the initial two weeks are critically affected by the lack of factor VIII in the recipient mice. Considering that angiogenesis is an absolute requirement for the implant growth we also performed morphometric analysis of CD31 positive cells in the implant. As can be seen in Fig. 2A, similar density of endothelial cells was found in both types of recipients, with no significant enhancement in the hemophilic recipients at any time point post transplantation. This observation implies that the enhanced growth of transplants in Factor VIII KO-SCID mice is not due to angiogenesis advantage. Further- more, quantitative evaluation of the numbers of proliferating endothelial cells per total number of nucleated proliferating cells did not reveal a significant difference between hemophilic 21.961.9% and non-hemophilic 22.462.3% recipients (Fig. 2B). To define whether our observation on the growth of embryonic spleen is also applicable to other tissues, we used the same experimental system and evaluated growth of pig embryonic liver and pancreas precursor tissue harvested at previously defined optimal ‘windows’, namely E28 and E42, respectively[8]. The growth of pancreatic tissues was determined in the SCID recipient mice by monitoring blood levels of pig insulin using a specific ELISA, previously shown to correlate with implant size[8]. As can be seen in Fig. 3A, pig insulin blood levels were markedly enhanced in Factor VIII KO SCID mice compared to Factor VIII Organ Size Control positive SCID mice, reflecting a growth advantage in the Factor VIII KO recipients. Since these differences in insulin levels might be attributed to differences in functionality of b-cells rather than to the total b -cell number, we determined the total volume of the implants as well as the fraction of b-insulin positive cells out of the total volume (Fig. 3B), using morphometric analysis. Enhancement of pig pancreas size measured by this parameter was found to closely parallel the difference in insulin blood levels. Thus, the total volume of insulin positive cells was 0.7560.003 mm3 in Factor VIII SCID, versus 0.2360.003 mm3 in non-hemophilic SCID recipients, respectively, suggesting an overall enhancement of implant size by at least a factor of three (p,0.05). As previously reported, E42 pancreatic implants into NOD- SCID mice are predominantly composed of endocrine tissue with minimal exocrine activity[11]. Only a minimal number of exocrine cells were detected in the E42 graft 3 months after transplantation, while most of the cells were of the endocrine lineage. Similar architecture of the endocrine compartment of the growing pig pancreas was noted in Factor VIII KO NOD-SCID recipients (Fig. 3C). Thus, the significant size difference was not associated with a difference in the architecture of the growing implant. A significant enhancement, by at least a factor of two, of implant size was also found upon implantation of embryonic pig liver in Factor VIII KO SCID mice, as evaluated by ELISA for pig blood albumin levels (Fig. 3D). As in the case of spleen and pancreas, despite its enhanced growth in Factor VIII KO hosts, the growing pig liver exhibited similar architecture in both types of recipients (Fig. 3E–G). RAG2/2Factor VIII KO Mice Also Exhibit Enhanced Implant Size Following Transplantation of Pig Embryonic Tissues It is possible that the observed enhancement of growth in Factor VIII KO NOD- SCID mice is due to a potential genetic abnormality affecting some other aspect of their metabolism. Prkdcscid (commonly referred to as SCID) mice carry a spontaneous mutation in chromosome 16. In NOD-SCID mice, this mutation was backcrossed onto the NOD/ShiLt background. Hemophilic (Factor VIII KO) mice are homozygous for a targeted, X chromosome-linked mutant allele, inserted using a neo cassette, which was used to disrupt exon 16 of the Factor VIII gene. The Factor VIII KO mice on a NOD-SCID background that we used were derived by crossing these two strains[11]. However, in order to rule out any potential artifacts specific to this strain combination, we introduced the Factor VIII KO mutation into a different SCID mouse, namely, SCID induced by knockout of the RAG gene. RAG2/2 mice were produced by inserting a targeted mutation on chromosome 2, resulting in a ‘‘non-leaky’’ severe combined immune deficiency[12]. A new RAG2/2 FVIII KO colony was therefore established to confirm our findings on the effect of FVIII KO on organ size. As can be seen in Fig. 4, RAG2/2 FVIII KO recipients of an E42 pig spleen implant develop an oversized spleen implant, compared to their non-hemophilic RAG2/2 counterparts. Thus, the Factor VIII KO mutation was associated with enhanced growth in this strain combination, as well, ruling out potential artifacts due to confounding factors other than Factor VIII deficiency. Embryonic Mouse Spleen, Pancreas or Liver Implanted in Factor VIII KO SCID Mice Exhibit No Size Enhancement The enhancement of organ size following implantation of pig embryonic spleen, pancreas and liver could be related to early PLoS ONE | www.plosone.org 2 December 2009 | Volume 4 | Issue 12 | e8362