Organ Size Control Figure 2. Angiogenesis pattern for the E42 spleen implants growth. (A). Morphometric analysis of endothelial density based on CD31 positive cells following implantation of E-42 spleen grafts into NOD-SCID or Factor VIII KO SCID recipients. (B) Quantitative evaluation of the numbers of proliferating endothelial cells per total number of nucleated proliferating cells in a square mm of the growing tissues. Analysis was performed at 2 weeks post transplantation. (C) Representative double staining of CD31+ endothelial cells (red) and Ki67+ proliferating cells (green). Nuclei are in stained in blue. Arrows outline proliferating endothelial cells (yellow) in the grafts. doi:10.1371/journal.pone.0008362.g002 events associated with graft accommodation and formation of vasculature, which may potentially differ in Factor VIII KO versus non-hemophilic SCID recipients, in a manner that is independent of the origin of the donor tissue. To address this possibility, similar implantation experiments were repeated using embryonic tissues of mouse origin. In contrast to the pig implants, no differences in organ size were found between SCID Factor VIII KO and non- hemophilic SCID recipients following implantation of mouse E15 gestational age tissues. Spleen grafts of mouse origin exhibited an similar size, 3 months post transplantation of 8.9862.55 in SCID and 9.1261.14 SCID Factor VIII KO recipients. Similar results were obtained for mouse E16 pancreas and liver transplants (data not shown). Furthermore, no differences in size were found following transplantation of embryonic mouse tissues obtained from hemophilic donors into Factor VIII KO-SCID versus non- hemophilic SCID mice. Thus, our results suggest that while the final size of heterologous embryonic pig implants is affected by the presence or absence of mouse Factor VIII, embryonic mouse implants attain their final size regardless of the host environment. The lack of any effect on mouse embryonic transplants, regardless of the expression of Factor VIII by the host or the donor, suggests that the role of Factor VIII is likely limited to a checkpoint of excessive growth, which operates upon implantation of tissue from a larger animal, such as the pig. More specifically, it is possible that Factor VIII or one of its derivatives might interfere with the activity of a putative survival factor, and thereby may have an important role in defining the maximum tolerable tissue mass. Thus, if we assume that mouse and pig embryonic implants are endowed with stem cell pools of different sizes prior to transplantation, they are likely to attain different organ size upon completion of growth and differentiation in the mouse recipients. Consequently, in hemophilic mice lacking the putative inhibitory activity (i.e. overgrowth checkpoint) mediated by Factor VIII, the size of the pig implants is likely to be larger, while mouse implants growing to their expected [mouse] size will not be subject to Factor VIII control. The Role of Downstream Members of the Coagulation Pathway in Organ Size Control Our present findings indicate a role for Factor VIII in organ size control, through an unknown mechanism. This intriguing role could be mediated directly by this molecule or through one of the other coagulation factors activated along the cascade triggered by Factor VIII. Factor Xa is activated by Factor VIII and, in turn, PLoS ONE | www.plosone.org 4 December 2009 | Volume 4 | Issue 12 | e8362