Sexual Dimorphism in Hepatocyte Xenograft Models

Animals

uPA-NOG and TK-NOG mice embryos were provided by dr. Suemizu, Central Institute for Experimental Animals, Kawasaki, Japan ^9,12 and mice were bred at the Central Animal Facility of the Erasmus Medical Center (DEC nr 141-12-11). For genotyping of TK-NOG, a copy number duplex qPCR was applied using custom designed primer-probe mix F: CGATTCGCCGCGTTTACG, R: CGCCGCCCTGCAGATA and probe: [6FAM]CCGCACCGTATTGGCAA[BHQ1] targeting the TK gene. TaqMan Genotyping master mix (Life Technologies), TaqMan uPA genotyping assay (Mm00422051_cn; Life technologies, Carlsbad, CA, USA) and Tert gene references mix (Life technologies, Carlsbad, CA, USA) were used according to the manufacturer’s protocol. Assays were performed on phenol-chloroform-isoamyl alcohol (Sigma-Aldrich, St. Louis, MO, USA)-extracted genomic mouse DNA from toe snip.

Human Hepatocyte Transplantation

Homozygous uPA+/+ or TK+ mice were anesthetized and transplanted with 0.4 to 2×10⁶ viable commercially available cryopreserved PHH (Lonza, Lot:9F3003, Basel, Switzerland, and BD Gentest, Lot:342 and Lot:345, Corning, Corning, NY, USA) via the intrasplenic injection route ^9,12,17,21 . Based on our and others’ previous experience plateable cryopreserved hepatocytes from neonatal and pediatric donors were selected for liver humanization studies ^17,22,23 . Cell viability was assessed immediately after thawing using trypan blue and a cell viability of ≥ 50% was set as threshold for further transplantation. TK+ mice were challenged with an intraperitoneal ganciclovir (GCV) (stock solution is 750 µg/mL) injection at day 7 and 5 before transplantation ¹² . At indicated time points mouse blood samples (50 μL) were obtained via a tail vein bleed.

Except for the GCV preconditioning in TK-NOG mice, all housing, handling and experimental procedures were identical between TK-NOG and uPA-NOG mice. In addition, both models were mostly transplanted in parallel, but inherent variation in offspring numbers with the required uPA^+/+ and TK+ zygosity led to some differences in the age at transplatation and number of recipients per PHH donor. Hepatocyte engraftment was determined by measuring hALB in mouse serum with a sandwich ELISA method as previously described (Bethyl laboratories, Montgomery, TX, USA). Human serum and non-transplanted mouse serum samples were used as positive and negative controls, respectively. Human reference serum (RS10-110, Bethyl laboratories, Montgomery, TX, USA) dilutions were used to create a standard curve for quantification ^12,21 .

HEV Infection

Successfully engrafted mice were intravenously inoculated with at least 10⁶ geq or IU HEV gt3c isolated from a chronically infected patient or the prototype HEV gt3a Kernow strain (a kind gift from Dr Zongdi Feng, Nationswide Children Hospital, Columbus, Ohio, USA). After inoculation mice were housed individually. HEV RNA levels in weekly collected mouse fecal samples and a liver sample, collected at euthanasia, were determined using an ISO15189:2012-validated, internally controlled qRT-PCR, as described previously ^21,24 . Detection limit of the assay is 148 IU/mL and results were normalized using tissue weight and dilution factor.

High Fat Diet

Successfully engrafted mice were housed in individually ventilated cages, with a maximum of 4 mice per cage. Mice were fed al libitum with rodent chowcontaining 0.3% cholesterol (Altromin Spezialfutter GmbH & Co. KG, Lage, Germany) up to 10 weeks ¹⁶ .

Histology, Immunohistochemistry, and Oil-Red-O (ORO) Staining

Mouse livers were fixed in 4% formaldehyde (Merck Millipore, MA, USA). Standard hematoxylin and eosin (H&E) staining was performed using 4-5 µm cuts from paraffin embedded blocks. An Oil-Red-O staining method was performed to identify steatosis on 5 µm cuts from N₂ snap frozen humanized liver fragments ²⁵ . Human hepatocytes were stained using goat anti-human albumin cross-adsorbed antibody (Bethyl Laboratories Inc., TX, USA) and slides were visualized using substrate 3,3′-diaminobenzidine (DAB; Dako, Copenhagen, Denmark) detection.

RNA Isolation, cDNA Synthesis and qPCR Analysis

RNA isolation was performed using whole liver tissues stored in RNAlater RNA stabilization reagent (Qiagen, Hilden, Germany) with Qiazol (Qiagen, Hilden, Germany). Complementary DNA (cDNA) was generated by using PrimeScriptTM reverse transcriptase master mix (Takara Bio Inc., Kusatsu, Japan) according to manufacturer’s protocol. For the mRNA expression analysis following TaqMan probes were used: F: CGATTCGCCGCGTTTACG, R: CGCCGCCCTGCAGATA and probe: [6FAM]CCGCACCGTATTGGCAA[BHQ1] targeting the HSVTK mRNA and Mm00422051_cn TaqMan probe (Life technologies, Carlsbad, CA, USA) for plau mRNA. Tert gene mRNA level was used to calculate the relative expression.

ALT Measurement

Enzymatic quantification of mouse serum ALT levels was performed at the Erasmus University Medical Center Clinical Diagnostic Laboratory (TrialLab, ISO15189:2012 - M098).

Statistical Analysis

Survival analysis, correlation analysis and t-test were performed using GraphPad Prism version 5.00 for Windows, (GraphPad Software, San Diego, CA, USA) and P < 0.1 was accepted as statistically significant. *P < 0.1, **P < 0.05, ***P < 0.001.

Multivariate regression and correlation analysis were performed using Statistical Package for the Social Sciences (SPSS) for Windows, version 25.0 (SPSS Inc., Chicago, IL, USA). Multivariate regression analysis (list wise exclusion of independent variables, with both an enter and backward approach) was applied to study the effect of PHH engraftment on fecal HEV shedding upon HEV inoculation, using fecal HEV titers as dependent variable and mouse serum hALB level (µg/mL), the mouse genetic background and HEV strain as independent variables. Thereafter, linear regression was performed with the identified significant independent variable from the multivariate analysis.

To examine the relative contribution of different experimental parameters on PHH engraftment in a second multivariate regression analysis, post-transplantation (postTx) week 8 mouse serum hALB level (µg/mL) was set as dependent variable and the following parameters as independent variables: hepatocyte donor, transplanted PHH cell count, sex of the acceptor mouse, the mouse genetic background and offspring generation. As we performed transplantation with three different hepatocyte donors, we first performed a multivariate analysis without including hepatocyte donor as variable. Second, dummy data transformation was applied for each of the hepatocyte donors, using the Donor345 as our baseline, as this hepatocyte donor was applied most extensively in both xenograft models (Fig. 2B). Forward analysis was applied to see the effect of each significant parameter (SPSS, IBM SPSS Inc., Chicago, IL, USA).

UPA-NOG and TK-NOG Mice with Comparable Serum Human Albumin Levels Show Similar HEV Fecal Shedding, Irrespective of the Mouse Genetic Background.

In this large scale comparison of 2 PHH xenograft models, we initially wanted to determine the impact of the model system on HEV infection and fecal shedding, similar to our previous optimization studies for HBV and HCV infections in humanized uPA-SCID mice ¹⁷ . For this purpose, we first compared serum hALB levels, as established marker for PHH engraftment, at different time points after PHH transplantation between both model systems ^10,17,19 . Experimental design and summary of the analyzed samples and PHH recipients is illustrated in Fig. 1A and Supplemental Table S1.

To examine the differences in serum hALB kinetics, PHH transplanted animals were divided into three categories according to serum hALB levels at week 5 postTx: <1 log₁₀ µg/mL, >1 log₁₀ µg/mL, and >2 log₁₀ µg/mL. Serum hALB levels were monitored in unchallenged animals up to 24 weeks postTx. As shown in Fig. 1B, increases in serum hALB levels of PHH transplanted uPA-NOG mice are small over the course of 16 weeks. Compared to animals with early hALB levels >1 log₁₀ µg/mL, those with >2 log₁₀ µg/mL show less pronounced increases in serum hALB levels (mean 9.8-fold vs 2.8-fold, respectively), but at week 16 postTx the latter (initial levels >2 log₁₀ µg/mL) still remain significantly higher than the former (initial levels >1 log₁₀ µg/mL) (P-value < 0.05). PHH transplanted TK-NOG mice show a different serum hALB kinetics pattern (Fig. 1C); overall week 5 post Tx serum hALB levels were lower compared to uPA-NOG animals. However, serum hALB levels in animals with lowest week 5 post Tx levels (<1 log₁₀ µg/mL hALB) progressively increased and reached levels that were not significantly different at week 20–24 post Tx from animals that showed highest week 5 post Tx serum hALB levels (>2 log₁₀ µg/mL hALB). Serum hALB increments of up to 3 log₁₀ µg/mL were observed in these animals, which is significantly higher than the increments observed for uPA-NOG (mean ± SEM hALB change is 175.1 ± 66.35 µg/mL and 708.6 ± 148.6; for uPA-NOG and TK-NOG resp; P < 0.05). These serum hALB increments paralleled true PHH repopulation as shown by representative H&E and hALB stains of liver fragments (Fig. 1B, C). Combined these data indicate that week 5 post Tx serum hALB levels in uPA-NOG mice is higher, but serum hALB increase until week 20–24 post Tx is significantly lower compared to TK-NOG mice.

We next compared serum hALB kinetics under external stress factors, such as a HEV infection or high fat diet (HFD) induced steatosis. Under these conditions, both TK-NOG and uPA-NOG models were shown to keep their characteristic serum hALB kinetics (Fig. 1D, E): Average serum hALB increments upon HEV infections were +114.8 and +161.7 µg/mL in uPA-NOG and TK-NOG respectively. Similarly, a HFD resulted in comparable serum hALB changes of +154.4 and +2272.3 µg/mL (10 weeks of HFD) in uPA-NOG and TK-NOG, respectively, despite histologically confirmed steatosis in the xenograft (Fig. 1D, E).

We previously found that HEV RNA titers in liver humanized uPA-NOG correlated with the degree of liver chimerism ²¹ . In addition, we and others identified mouse feces as a straight-forward non-invasive compartment to monitor HEV infections in vivo ^{26 –28} . To determine the HEV infection efficiency in humanized uPA-NOG and TK-NOG mice, we next examined the association between serum hALB levels and HEV fecal shedding in both xenograft models. As shown in Fig. 1F, week 4 fecal HEV titers positively correlated with serum hALB levels (P < 0.01; Supplemental Table S2A). In a multivariate regression analysis, neither the model background nor the infecting HEV strain were independent predictors for the observed fecal HEV RNA loads (Supplemental Table S2B). Therefore, uPA-NOG and TK-NOG models behave similar in HEV infection studies, once a minimal level of serum human albumin is reached. Only 2 animals out of 50 were found to have HEV fecal shedding with serum hALB levels < 2 log₁₀ µg/mL, which therefore qualifies as an optimal engraftment criterion for HEV infection studies.

Multivariate Analysis Identifies PHH Donor Type and Male Sex to Be the Most Important Parameters for PHH Engraftment

Next, to examine the relative contribution of donor and host characteristics to serum human albumin levels, as established marker of PHH engraftment, we performed a comprehensive analysis of 399 uPA-NOG and 198 TK-NOG PHH transplanted mice. Offspring were backcrossed onto the previous generation or crossed within the same generation to maintain genomic stability ^29,30 . High human serum albumin levels were observed up to generation 22 and 15 in uPA- and TK-NOG respectively, indicating no loss of phenotype with further inbreeding (Fig. 2A; P = 0.36).

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Figure 1.

Serum hALB kinetics after PHH transplantation and the correlation with HEV RNA feces shedding. (A) Experimental design and summary of the analyzed samples. (B) Serum hALb levels of non-challenged uPA-NOG mice starting with >1 log₁₀ µg/mL (n = 8) and >2 log₁₀ µg/mL (n = 3) at week 5 postTx resp. Liver H&E and hALB staining of a representative uPA-NOG mouse which had 216 µg/mL and 251 µg/mL serum hALB at week 8 and 18 postTx resp. (C) Serum hALb levels of non-challenged TK-NOG mice starting with <1 log₁₀ µg/mL (n = 11), >1 log₁₀ µg/mL (n = 7) and >2 log₁₀ µg/mL (n = 3) at week 5 postTx resp. Liver H&E and hALB staining of a representative TK-NOG mouse which had >1 log₁₀ µg/mL and 2836.5 µg/mL hALB by week 8 and 16 postTx resp. (D) hALB levels of uPA-NOG mice following HEV infection (n = 9) and high fat diet (HFD) (0.3% cholesterol, n = 2). Liver ORO staining following 10 weeks of HFD. (E) Serum hALB level change of TK-NOG mice following HEV infection (n = 10) and HFD (0.3% cholesterol, n = 1). Liver ORO staining following 10 weeks of HFD. Data shown in B, C, D, and E were obtained from mice transplanted with PHH D345. (F) Correlation analysis between week 4 feces HEV RNA titers and serum human albumin levels of xenografted mice infected with a clinical HEV gt3c strain (HEV1, n = 68) and the prototype HEV gt3a Kernow strain (HEV2, n = 7). Mice transplanted with D342, D345, or D3F3003. Statistical analysis was performed using GraphPad Prism and *P < 0.1.

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Figure 2.

The effect of generation, PHH donor, transplanted PHH counts and age at transplantation on PHH xenografting success in uPA-NOG and TK-NOG models. (A) The effect of generation on PHH engraftment. Left Panel: week 8 postTx serum hALB levels (µg/mL) of uPA-NOG mice transplanted with 342, D345 and D9F3003. (Right panel) TK-NOG mice transplanted with D342 and D345. (B) The effect of PHH donor on transplantation success. Week 8 postTx serum hALB levels (µg/mL) of uPA-NOG mice (n = 328, left) and TK-NOG mice (n = 144, right) transplanted with PHH donors 342, 345, and 9F3003. (C) The effect of the number of transplanted PHH cells on serum hAlb levels. Week 8 postTx serum hALB levels (µg/mL) of uPA-NOG (n = 263, left) and TK-NOG (n = 170, right) mice transplanted with PHH donors 342, 345, and 9F3003. Cell counts are also split into two categories irrespective of the recipient background and donors: <0.5 × 10⁶/mouse and >1.5 × 10⁶/mouse. Groups are compared using a t-test. (D) The correlation between week 8 serum hALB level (µg/mL) and age at transplantation for 373 uPA-NOG (left) and 109 TK-NOG (right) mice. GraphPad Prism was used for statistical analysis and *P < 0.1, **P < 0.05, ***P < 0.001.

Previously, the contribution of donor type to engraftment success has been reported by us and others ^17,20 . Details of the PHH donors are provided in Table 1. Analysis of week 8 postTx serum hALB levels of uPA-NOG and TK-NOG mice transplanted with three different PHH donors (Fig. 2B, Table 2), demonstrated significant differences between the donors but also a large variation within different acceptor mice, suggesting that factors other than donor type add to the serum human albumin levels and engraftment success in these mice. As a high number of transplanted cells was identified to contribute to humanization ratios ^19,31 , we next analyzed the effect of the transplanted PHH counts for each PHH donor. Except for higher serum human albumin levels in uPA-NOG animals receiving > 10⁶ donor345 cells, this effect was not observed in TK-NOG mice; nor with donor9F3003 (Supplemental Fig. S1A). Therefore, we regard 0.5–1 × 10⁶ PHH/mouse to be the most optimal transplanted cell dose (Fig. 2C). We additionally examined the mouse age at PHH transplantation. As shown in Fig. 2D, we identified the optimal age at transplantation to be <80 days for uPA-NOG (P < 0.0001). For TK-NOG mice age was not significantly associated with serum human albumin levels (P = 0.19). Overall, in this univariate analysis PHH donor, transplanted cell number and recipient age at transplantation are associated with serum human albumin levels, as marker for PHH engraftment success.

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Table 1.

Demographic Data of Cryopreserved Primary Human Hepatocyte (PHH) Donors.

Lot number	Sex	Age	Race
342	Female	2 years	Caucasian
345	Female	7 months	Caucasian
9F3003	Male	2 years	Caucasian

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Table 2.

The Distribution of Male and Female Sex in Recipient uPANOG and TKNOG Mice Transplanted with PHH D342, D342, or D9F3003.

		Genetic Background of Mice					Total
		uPANOG			TKNOG
PHH Donor		D342	D345	D9F3003	D342	D345
Sex	Female	4	24	36	9	21	94
	Male	75	126	63	20	94	378
Total		328			144		472

To further quantify the relative contributions of donor and host characteristics, we performed a multivariate regression analysis (Supplemental Table S3A, S3B). First, we performed a multivariate analysis to study the contributions of recipient sex, age at transplantation, transplanted PHH count, generation and genetic background to predict week 8 postTx serum hALb levels (µg/mL). Interestingly, and confirming our previous data, neither the background of the mouse strain, nor the mouse generation determined serum hALB levels. Recipient sex was found to be the first most significant independent variable (adjusted R ² 0.020, P = 0.003) and age at transplantation the second (adjusted R ² addition 0.016, P = 0.011) (Supplemental Table S3A, 3B). To be able to include the PHH donor as a variable, we performed dummy data transformation. Donor345 was set as baseline for comparison and we re-performed the multivariate regression analysis (Supplemental Table S3C). Again recipient sex was one of the most significant variables (P < 0.0001), with female recipient sex resulting in an average 442.1 fewer units serum hAlb (expressed in μg/mL) compared to male mice. Donor9F3003 was shown to increase the serum hALB level on average with 683.9 units μg/mL relative to PHH donor345 (P < 0.0001). Other significant independent variables were age at transplantation (P < 0.05, –4.5 units serum hAlb per day). The transplanted PHH count was found to significantly contribute to engraftment, but its effect size was low, suggesting that 1 unit μg/mL serum hAlb increase would require 9.6 × 10⁵ more initially transplanted PHH (P < 0.05). Our data show that male recipient sex and a good PHH donor (in our case PHH Donor9F3003) can result in serum hALB levels that are up to 1000 µg/mL higher.

In summary, both PHH donor type and recipient sex are the most important factors contributing to serum human albumin levels, as marker for PHH engraftment in uPANOG and TKNOG mice.

Sexual Imbalance in PHH Xenografting is Related to Higher Baseline ALT Levels and Sensitivity for Ganciclovir in Males

To further examine the effect of recipient sex in xenografting success, we listed serum hALB levels per recipient sex and xenograft model (Fig. 3A, B). Except for 1 young TKNOG female transplanted at 40 days of age (Fig. 3B), maximum serum human albumin levels remained well below 500 μg/mL, irrespective of the applied model (Supplemental Fig. S1B). We recorded no differences in serum human albumin levels in male UPANOG recipients transplanted with either male or female PHH donors, indicating that the observed difference stems from a host gender imbalance (Supplemental Fig. S1C). We also analyzed GCV induced morbidity (Supplemental Fig. S2) and mortality for both male and female TK-NOG mice separately. Again, recipient sex was the major determinant of survival (Fig. 3C). Mortality after GCV reached more than 30% at doses of 6 mg/kg in males, while TK-NOG females tolerated doses up to 12 mg/kg. Increasing the GCV posology in female TK-NOG mice to 14 mg/kg resulted in significantly lower survival rates of 75% and serum hAlb levels <500 μg/mL that remained well below those of males challenged with 4 mg/kg GCV (Fig. 3B, C). Substantial increases in female mortality prohibited the use of even higher GCV doses (Fig. 3C). On the other hand, excellent survival rates of > 95.5% and serum hAlb upto 2650 μg/mL levels were seen in male TK-NOG animals after applying GCV at 3 or 4 mg/kg. (Fig. 3B, C).

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Figure 3.

Sexual dimorphism in serum hAlb levels after PHH transplantation, GCV tolerance and liver damage parameters in uPA-NOG and TK-NOG models. (A) The correlation between week 8 serum hALb levels (µg/mL) and age at transplantation (days) for male (left, n = 309) and female (right, n = 59, respectively) uPA-NOG mice. (B) Age (days) and week 8 postTx serum hALB level (µg/mL) correlation of male TK-NOG mice which received 3mg/kg (n = 8) and 4mg/kg GCV (n = 64) 7 and 5 days prior to transplantation (left). Age (days) and week 8 post Tx serum hALB level (µg/mL) correlation of TK-NOG female mice which received 6, 9 or 12mg/kg (n = 13) and 14mg/kg (n = 12) GCV at 7 and 5 days prior to transplantation (right). (C) Survival plots of male (left.) and female (right) TK-NOG mice. Both sexes received different GCV doses i.p. 7 and 5 days prior to transplantation, as indicated in the graph legend. Survival plots show the response of animals until week 8 postTx. Time point of first GCV dose is set at 0. (D) Liver plau relative mRNA expression 40–60 days old non-transplanted uPA-NOG male (n = 6) and female mice (n = 6). (E) Liver HSVTK relative mRNA expression of 130–160 days old male (n = 3) and female (n = 3) non-transplanted TK-NOG mice. Tert gene mRNA level was used to calculate the relative expression and relative expression level was calculated using 2^-deltaCt transformation. (F) Serum ALT analysis of 40-60 days old non-transplanted uPA-NOG male (n = 6) and female mice (n = 6) (left) and 130–160 days old male and female non-transplanted TK+ and TK- mice (right). Baseline serum samples of TK-NOG mice were obtained before treatment and then TK+ mice were challenged with GCV at day 0 and 2 and post-challenge serum samples were obtained at day 7 (n = 4 mice/group). (G) The comparison of week 8 postTx serum hALB levels (µg/mL) of uPA-NOG male mice (n = 150) and female mice (n = 25), at age 40–60 days at transplantation (left) and 90–130 days old female (n = 12) and male (n = 48) TK-NOG mice (right). Female TK-NOG mice received 14 mg/GCV and TK-NOG male received 4 mg/kg GCV prior to Tx. Correlation analysis and t test were performed using GraphPad Prism. *P < 0.1, **P < 0.05 and ***P < 0.001.

To understand the sexual imbalance in both models, we examined the relative transgene expression and baseline serum ALT levels of non-transplanted uPA-NOG and TK-NOG mice. While liver Plasminogen activator (Plau) mRNA levels were not significantly different between both uPA-NOG sexes, HSVTK mRNA levels were higher in male compared to female TK-NOG mice (Fig. 3D, E). Nevertheless, serum ALT levels at baseline were significantly higher in males than their female counterparts, irrespective of the examined model. Furthermore, the optimal GCV dose in TKNOG mice (cfr supra) led to higher ALT peaks in males compared to female mice (Fig. 3F). Parallel to higher ALT levels, uPA-NOG and TK-NOG males had significantly higher levels of serum hALB at week 8 postTx (Fig. 3G). In conclusion, despite titrating GCV to highest tolerated doses in females, TK-NOG males remain better PHH acceptors. Therefore, irrespective of the applied xenograft model, recipient males present higher ALT levels, which parallel serum hAlb levels after PHH transplantation.