Hepatocyte transplantation into diseased mouse liver. Kinetics of parenchymal repopulation and identification of the proliferative capacity of tetraploid and octaploid hepatocytes

Abstract

To examine the process of liver repopulation by transplanted hepatocytes, we developed transgenic mice carrying a mouse major urinary protein-urokinase-type plasminogen activator fusion transgene. Expression of this transgene induced diffuse hepatocellular damage beginning at 3 weeks of age, and homozygous mice supported up to 97% parenchymal repopulation by healthy donor hepatocytes transplanted into the spleen. Using this transplantation model, we determined that 1) a mean of 21% of splenically injected hepatocytes engraft in liver parenchyma; 2) a mean of 6.6% of splenically injected hepatocytes (or one-third of engrafted cells) can give rise to proliferating hepatocyte foci; 3) transplanted cells in proliferating foci display an initial cell-doubling time of 28 hours, and focus growth continues through a mean of 12 cell doublings; 4) hepatocytes isolated from young and aged adult mice display similar focus repopulation kinetics; 5) the extent of repopulated parenchyma remains stable throughout the life of the recipient mouse; and 6) tetraploid and octaploid hepatocytes can support clonal proliferation.

Figure 1.

MUP-uPA transgene construct, containing the MUP enhancer/promoter, the entire mouse uPA genomic coding sequence, and the 3′ human growth hormone-untranslated region with polyadenylation (pA) signal.

Figure 2.

Liver disease and donor cell repopulation in MUP-uPA transgenic mice. A: Serum alanine aminotransferase activity in MUP-uPA transgenic mice. All values except those at 3 and 13 weeks are significantly elevated relative to pooled controls (P < 0.03, unpaired t-test). Each point for transgenic mice represents the mean ± SD of three to seven samples. Dashed lines indicate the two standard deviation range for values from nontransgenic littermate control mice (n = 5). B: Zymographic analysis of uPA in mouse serum as a function of mouse age. The intensity of each band is proportional to the amount of uPA catalytic activity present in that sample. Non-tg: serum from 4-week-old nontransgenic control mice. Letters below each lane indicate female (F) or male (M). C: Extent of repopulation by donor hepatocytes in MUP-uPA transgenic mice as a function of recipient age. Each point represents one mouse liver. Bars indicate mean values.

Figure 3.

Rate of donor cell hepatic reconstitution. Solid triangles indicate the cross-sectional area of donor-derived foci after transplantation of hepatocytes isolated from 2- and 4-month-old hPAP transgenic donors (pooled data from two experiments). Area was measured on the liver surface, not on sectioned tissue. It therefore represents true focus cross-sectional area, and will not be biased by random cross-sectioning of foci. Each data point represents the mean ± SEM of cross-sectional area of 50 donor-derived foci from each of four to eight recipient mice. Solid squares indicate the cross-sectional area of donor-derived foci after transplantation of hepatocytes isolated from 18- and 23-month-old MT-hPAP transgenic donors (pooled data from two experiments). Each data point represents the mean ± SEM of cross-sectional area of 50 donor-derived foci from each of three to seven recipient mice. At 2, 4, and 8 weeks, data points are offset for ease of visualization.

Figure 4.

Microscopic appearance of donor-derived liver at 18 months after transplant. MT-hPAP-labeled hepatocytes were transplanted into a MUP-uPA transgenic mouse. The recipient mouse was sacrificed 18 months later, and the paraffin-embedded, sectioned liver was stained on a slide to identify hPAP-labeled donor cells. Arrow indicates portal triad (P). Arrowheads indicate central veins (CV). Notice that donor-derived parenchyma constitutes all zones of the hepatic lobule, and that the margin between donor (blue) and endogenous (unstained) parenchyma remains sharp. Original magnification, ×100.

Figure 5.

Flow cytometric analysis of a live donor hepatocyte population from a 5-month-old hPAP transgenic donor. A: Acquisition dot plot showing three relatively distinct populations of viable hepatocytes. The gated subpopulations of hepatocytes were designated as R2, R3, and R4, corresponding to diploid, tetraploid, and octaploid, respectively (see text). Note that, as expected for a mouse of this age, the diploid cell population is a minority representing 5.6% of all gated cells. In contrast, tetraploid and octaploid cells accounted for 49% and 44% of the gated populations, respectively. The population of very small particles in the lower left-hand corner of the plot is thought to represent cellular debris. The population of very large cells to the right of R4 is highly enriched for cell doublets, as determined by microscopic examination of cells isolated from this region (data not shown). B: Acquisition histogram showing three distinct peaks. The M2, M3, and M4 populations correspond to the R2, R3, and R4 populations in the acquisition dot plot. Note that fluorescent areas of M3 and M4 are two and four times as large as the M2 population fluorescent area, respectively. C: Acquisition histogram of resorted live donor hepatocyte subpopulations. Top, middle, and bottom panels represent the resort of the original M2, M3, and M4 live hepatocyte subpopulations, respectively. Hoechst dye was re-added to the cell populations before the second sort, so that Hoechst fluorescent areas of the original and resorted cell populations are not precisely equivalent. During the resort, each population was collected separately and for a variable length of time, so the number of cells in each resort population is not proportional to the fraction of that population in the original sort.