Species specific analysis of HGF and MET gene expression in wild-type C57BL/6 mice and homozygous humanized B-hHGF/hMET mice by RT-PCR. The RNAs of the liver, lung and kidney were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hHGF/hMET mice(H/H; H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human HGF and MET primers. Human HGF and MET mRNA were detectable only in homozygous B-hHGF/hMET mice but not in wild-type mice. Mouse Met mRNA was detectable in wild-type C57BL/6 mice.

Strain specific HGF expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hHGF/hMET mice by ELISA. The serum was collected from wild-type C57BL/6 mice (+/+) and homozygous B-hHGF/hMET mice(H/H; H/H) (female, n=3, 9-week-old). Expression level of mouse and human HGF was analyzed by ELISA(anti-mouse HGF ELISA kit: R&D, MHG00; anti-human HGF ELISA kit: Abcam, ab275901). Mouse HGF was only detectable in wild-type C57BL/6 mice. Human HGF was exclusively detectable in homozygous B-hHGF/hMET mice. Values are expressed as mean ± SEM. ND: not detectable.

Western blot analysis of MET protein expression in homozygous B-hHGF/hMET mice. Liver, lung and kidney lysates were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hHGF/hMET mice (H/H, H/H), and then analyzed by western blot with anti-m/hMET antibody (CST, 3127S). 60 μg total proteins were loaded for western blotting analysis. MET was detected in the liver, lung, and kidney of wild-type mice and homozygous B-hHGF/hMET mice, as the antibody cross-reacted with both mouse and human MET.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice and homozygous B-hHGF/hMET mice (female, 9-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hHGF/hMET mice were similar to those in C57BL/6 mice, demonstrating that humanization of HGF and MET do not change the frequency or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6 mice and homozygous B-hHGF/hMET mice (female, 6-week-old, n=3). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hHGF/hMET mice were similar to those in C57BL/6 mice, demonstrating that humanization of HGF and MET do not change the frequency or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in lymph node by flow cytometry. Leukocytes were isolated from wild-type C57BL/6 mice and homozygous B-hHGF/hMET mice (female, 6-week-old, n=3). A. Flow cytometry analysis of the leukocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hHGF/hMET mice were similar to those in C57BL/6 mice, demonstrating that humanization of HGF and MET do not change the frequency or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.

Complete blood count (CBC) of C57BL/6JNifdc mice. Values are expressed as mean ± SD.

Complete blood count (CBC) of B-hHGF/hMET mice. Values are expressed as mean ± SD.

Biochemical test of C57BL/6JNifdc mice. Values are expressed as mean ± SD.

Biochemical test of B-hHGF/hMET mice. Values are expressed as mean ± SD.

The organs of female C57BL/6JNifdc mice and B-hHGF/hMET mice  (7-week-old, n=5).

The organs of male C57BL/6JNifdc mice and B-hHGF/hMET mice  (7-week-old, n=5).

Average weight of the main organs of C57BL/6JNifdc mice. Values are expressed as mean ± SD.

Average weight of the main organs of B-hHGF/hMET mice. Values are expressed as mean ± SD.

Histopathological analysis of organs in B-hHGF/hMET mice. The main organs of B-hHGF/hMET mice were isolated at 8 weeks of age and analyzed with H&E staining (female, n=5). The results showed that no significant abnormalities were found in any organs except the uterus, including the heart, liver, spleen, lung, kidney, brain, stomach, small intestine, large intestine and ovary. The uterus exhibited significant dilation, with morphological alterations in the stromal cells of the endometrial layer—specifically, the cells had transformed into polygonal or rounded shapes, with enlarged nuclei and increased eosinophils. Scale bar: 200 μm.

Histopathological analysis of organs in B-hHGF/hMET mice. The main organs of B-hHGF/hMET mice were isolated at 8 weeks of age and analyzed with H&E staining (male, n=5). Results showed that no obvious abnormalities were found in all of the organs (heart, liver, spleen, lung, kidney, brain, stomach, small intestine, large intestine and testis). Scale bar: 200 μm.