Engraftment of human hepatocytes clusters expressing mCD47/eGFP in comparison to

Human hepatocytes were either transduced with LV-mCD47-eGFP or LV-eGFP as control prior to transplantation into BALB Rag/γc uPA mice. Transduction of human hepatocytes generated a mixture of mCD47⁺/eGFP⁺ (control BD-eGFP: mCD47^-/eGFP⁺) and non-transduced mCD47^-/eGFP^- cells. The aim was to show whether the proportion of engrafted eGFP⁺ hepatocytes changed in respect to the proportion of eGFP⁺ hepatocytes transplanted into the mice. Latter was determined by FACS analysis of an aliquot of transplanted cell that had been cultured 5 days after transplantation.

8 weeks after transplantation, mice showing detectable human serum albumin levels were analysed for engraftment of human hepatocyte clusters. The clusters from various lobes of the mouse liver were analysed for expression of human serum albumin, mCD47 and eGFP by the means of immunofluorescence staining (fig. 45).

Figure 45: Judgement of human hepatocyte clusters by immunofluorescence staining. Liver sections from all transplanted mice were prepared for immunofluorescence staining using antibodies against human serum albumin (orange), GFP (green) and Hoechst 33342 for cell nucleus staining (blue). The clusters were analysed for GFP (yes/no) and human albumin expression and this judgement represented the basis for the statistical analysis of improved, mCD47-related engraftment in transplanted mice. The total number of eGFP expressing clusters in one mouse was calculated and related to the number of human albumin expressing clusters to determine the percentage of eGFP expressing clusters. Bars represent 250 µm.

The percentage of mCD47-eGFP-expressing clusters of total human albumin expressing humane hepatocyte clusters was determined. This value was divided with the percentage of transplanted eGFP⁺ human hepatocytes determined by FACS analysis 5 days to acquire a ratio. This ratio expressed whether mCD47⁺/eGFP⁺ clusters showed favoured long-term

engraftment (>1.0) or not (<1.0). Fig. 46 to 48 show the evaluation of mouse no 37501 with relatively moderate engraftment of human hepatocyte clusters. Immunofluorescence staining of mouse 37501 demonstrated engraftment of 7 clusters expressing mCD47/eGFP while a total 19 of clusters expressing human albumin were identified. The percentage of eGFP expressing clusters was calculated to 36.84 %. In comparison, FACS analysis showed that transplanted hepatocytes were 21.2 % eGFP positive 5 days after transplantation. This represents a ratio of 36.84 % / 21.2 % = 1.74 and long-term engraftment of mCD47/eGFP is favoured in comparison to human hepatocytes not expressing mCD47/eGFP.

Figure 46: Judgment of human hepatocyte clusters in mouse no 37501, clusters 1-7. Mouse no 37501 was transplanted with BD-mCD47 and sacrificed 8 weeks after transplantation. Liver sections from all transplanted mice were prepared for immunofluorescence staining using antibodies against human serum albumin (orange),0 GFP (green) and Hoechst 33342 (cell nucleus staining, blue) to identify isolated clusters. In mouse 37501, 7 out of 19 human albumin expressing clusters were eGFP positive (36.84 %) compared to 21.2 % eGFP⁺ cells upon transplantation. Ratio was calculated to 36.84 % / 21.2 % = 1.74.

Figures 46-48 show the judgement of all human hepatocytes clusters identified in mouse no. 37501 that are randomly and sparesly distributed throughout the liver. This analytical approach was used to determine engraftment statistics in mice 20908, 26570, 26571, 37497, 37503, 37505, 44701, 44705, and 44708-44710.

Bars represent 250 µm.

Figure 47: Judgment of human hepatocyte clusters in mouse no 37501, clusters 8-13. Mouse no 37501 was transplanted with BD-mCD47 and sacrificed 8 weeks after transplantation. Liver sections from all transplanted mice were prepared for immunofluorescence staining using antibodies against human serum albumin (orange),0 GFP (green) and Hoechst 33342 (cell nucleus staining, blue). Clusters 10/11 and 12/13 are in close proximity to each other and are marked with an arrow with respective judgement. Bars represent 250 µm.

Figure 48: Judgment of human hepatocyte clusters in mouse no 37501, clusters 14-19. Mouse no 37501 was transplanted with BD-mCD47 and sacrificed 8 weeks after transplantation. Liver sections from all transplanted mice were prepared for immunofluorescence staining using antibodies against human serum albumin (orange),0 GFP (green) and Hoechst 33342 (cell nucleus staining, blue).

Some mice transplanted with BD-mCD47 and BD-eGFP demonstrated substantial engraftment of human hepatocytes with repopulations of more than 30 % of the mouse liver.

To evaluate the characteristics of the engrafted human hepatocytes efficiently, it was necessary to perform a sequential fluorescence image scan of the entire liver making it possible to create an overview of the entire liver. 4 out of 12 mice transplanted with BD- mCD47-eGFP demonstrated high engraftment, which made it necessary to perform sequential scanning of the entire liver to acquire an overview picture. Figure 49 shows one liver lobe of mouse no 37505 that engrafted 44 human albumin expressing human hepatocyte clusters. 14 clusters are mCD47/eGFP positive (31.8 %). Transplanted cells were 21.2 % eGFP positive.

Considering just this liver lobe, the ratio was calculated to 1.5 for mouse 37505. Later during this study, further lobes from mouse 37505 were analysed for eGFP and human albumin clusters. A total of 98 cluster were found of which 31 were eGFP positive. This represents a GFP percentage of 31.63 and the total ratio was calculated to 1.49.

Figure 49: Immunofluorescence staining of mouse no 37505. Mouse no 37505 was transplanted with BD-mCD47 and sacrificed 8 weeks after transplantation. Liver sections from mouse no 37505 were prepared for immunofluorescence staining using antibodies against human serum albumin (orange) and GFP (green). Hoechst 33342 was used for cell nucleus staining (blue). This picture demonstrates one liver lobe of mouse 37505 that has been ripped apart. The right edge of the big part matches to the left edge off the small part. 14 out of 44 human albumin expressing clusters were eGFP positive (31.8 %). B Transplanted hepatocytes demonstrated 21.2 % eGFP positive cells 5 days after transplantation. Ratio was calculated to 31.8 % / 21.2 % = 1.5. This overlay image has been assembled from approximately 400 single one-colour images. Assembled images were used to analyse the engraftment ratios in transplanted mice 37505, 42274, 44707, and 44711. The bar in the right bottom corner represents 1 mm.

In total, 9 mice were transplanted with BD-mCD47 and survived. For all mice, repopulation advantage of CD47⁺/eGFP⁺ human clusters was determined by comparing the percentage of eGFP⁺ human hepatocyte clusters with respective transduction efficiencies at 5 days after transplantation. The overall result was that CD47⁺/eGFP⁺ clusters demonstrate a repopulation rate that is 101.6 % (ratio 2.016) higher than the transplanted population of CD47⁺/eGFP⁺ hepatocytes at the time of transplantation. In detail, improvement of engraftment ranged from 21.2 % (ratio 1.212) for mouse no 26571 to 212.1 % (ratio 3.121) for mouse no 44701. For control mice transplanted with BD-eGFP, fewer hepatocytes expressing eGFP showed

engraftment compared with the percentage of eGFP⁺ transplanted hepatocytes. Only 39.2 % (ratio 0.392) of transduced eGFP⁺ expressing hepatocytes had engrafted into the murine livers (table 16, figure 50).

Despite the fact that much effort was put into scanning the complete liver, no eGFP clusters were found in control mice 44708 – 44710. In general, these mice demonstrated less repopulation of the liver by human hepatocytes.

Table 15: Repopulation of uPA mice by mCD47⁺ human hepatocytes. BALB Rag/γc uPA mice were transplanted with BD-mCD47 or BD-eGFP. Mice were sacrificed 8 weeks after transplantation and liver sections were analysed for human hepatocyte clusters expressing eGFP (GFP+) or human albumin (ALB+) only. The percentage of eGFP-expressing clusters in liver sections was divided by the transduction efficiency to calculate the repopulation ratio (ratio = GFP [%] / transduction efficiency [%]). Engraftment of mCD47-eGFP expressing clusters had doubled (ratio 2.016) in comparison to the transplanted mCD47/eGFP-expressing hepatocyte population. Control mice demonstrated less engraftment of transplanted eGFP expressing control hepatocytes (ratio 0.392)

BD-mCD47

mouse no transduction efficiency [%] GFP+ ALB+ total GFP [%] Repopulation ratio

20908 5.18 1 7 8 12.50 2.413

mouse no transduction efficiency [%] GFP+ ALB+ total GFP [%] Repopulation ratio

42274 4.3 9 217 226 3.98 0.926