Progenitor Cell Characteristics

ORIGINAL RESEARCH

59 60 61 62 63 64 65 Takeshi Katsuda,1,* Kazunori Hosaka,1,2,* Juntaro Matsuzaki,1 Wataru Usuba,1 66 67 Marta Prieto-Vila,1 Tomoko Yamaguchi,1,3 Atsunori Tsuchiya,2 Shuji Terai,2 and 1 68 Takahiro Ochiya 69 70 1 Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan; 2Division of 71 Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, Aasahimachi-Dori, 3 72 Chuo-Ku, Niigata, Japan; Institute of Medical Science, Tokyo Medical University, Shinjuku, Tokyo, Japan 73 74 75 Bulk transcriptome Abundance of Glul+ 76 (2c vs 4c vs 8c) 2c hepatocytes Cyp1a2hi 77 Ahrhi 78 79 Progenitor ? 80 4c 8c 2c 81 Axin2+ 82 scPCR (2c vs 4c) Prom1+ …. 83 + Lgr5 …. 84 Adult rat …. 85 Isolation of hepatocytes …. Cell 86 with different ploidy 87 statuses …. 88 89 Gene 90 91 92 RESULTS: Microarray analysis showed that cell cycle–related 93 SUMMARY genes were enriched in 8c hepatocytes, which is in line with 94 the established notion that polyploidy is formed via cell diviBy using rat hepatocytes with different ploidy statuses, a sion failure. Surprisingly, in contrast to the general consensus 95 bulk transcriptome and single-cell quantitative reversethat 2c hepatocytes reside in the periportal region, in our bulk 96 transcription polymerase chain reaction show that diploid transcriptome and scPCR analyses, the 2c hepatocytes consis- 97 hepatocytes are preferentially located in the pericentral retently showed pericentral hepatocyte-enriched characteristics. 98 gion. Single-cell analysis further identifies a subpopulation In addition, scPCR analysis identified a subpopulation within 99 within the 2c hepatocytes that co-express the mature hethe 2c hepatocytes that co-express the liver progenitor cell 100 patocyte markers and liver progenitor cell markers. markers Axin2, Prom1, and Lgr5, implying the potential bio- 101 102 logical relevance of this subpopulation. 103 BACKGROUND & AIMS: There is a long-standing debate CONCLUSIONS: This study provides new insights into hepato- 104 regarding the biological significance of polyploidy in hepato- cyte heterogeneity, namely 2c hepatocytes are preferentially 105 cytes. Recent studies have provided increasing evidence that localized to the pericentral region, and a subpopulation of 2c 106 hepatocytes with different ploidy statuses behave differently in hepatocytes show liver progenitor cell–like features in terms of 107 a context-dependent manner (eg, susceptibility to oncogenesis, liver progenitor cell marker expression (Axin2, Prom1, and 108 regenerative ability after injury, and in vitro proliferative ca- Lgr5). (Cell Mol Gastroenterol Hepatol 2019;-:-–-; https:// 109 pacity). However, their overall transcriptomic differences in a doi.org/10.1016/j.jcmgh.2019.08.011) 110 physiological context is not known. 111 Keywords: Hepatocyte; Ploidy; Zonation; Single-Cell PCR; 112 METHODS: By using microarray transcriptome analysis, we Transcriptome. 113 investigated the heterogeneity of hepatocyte populations with 114 different ploidy statuses. Moreover, by using single-cell quanolyploidy is a characteristic feature of hepatocytes, Q6115 titative reverse-transcription polymerase chain reaction but its biological significance is largely unknown.1–4 Q7116 (scPCR) analysis, we investigated the intrapopulational tranHepatocytes are diploid (2c) at birth, but, after weaning, Q8 scriptome heterogeneity of 2c and 4c hepatocytes.

Transcriptomic Dissection of Hepatocyte Heterogeneity: Linking Ploidy, Zonation, and Stem/Progenitor Cell Characteristics

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their DNA content increases. In the adult rodent liver, up to 90% of hepatocytes are polyploid under physiological conditions.5 The majority of polyploid hepatocytes are tetraploid (4c), but some are octoploid (8c) or even greater (
16c). Although awareness of this heterogeneity is not new, whether hepatocytes with different ploidy statuses have different characteristics is still under debate. Recent studies have provided evidence that hepatocytes with distinct ploidy statuses have different phenotypes. For example, Wang et al6 showed that pericentral Axin2þ hepatocytes are enriched with a 2c population and Axin2þ 2c hepatocytes contribute to hepatocyte turnover and the maintenance of liver homeostasis. Our group found that 2c hepatocytes, in response to growth stimuli (ie, a small molecule cocktail), acquired a higher in vitro proliferative capacity than that of their 4c and 8c counterparts.7 Similarly, Wilkinson et al8 reported that the polyploid state restricts hepatocyte proliferation and liver regeneration. On the other hand, the hepatocyte polyploidization prevents tumorigenesis by decreasing their susceptibility to genomic aberrations.8,9 These reports suggested that ploidy status affects the phenotype of hepatocytes; however, the studies focused on specific processes. More comprehensive analyses are required to gain broader, more holistic insights into this phenomenon. In this study, we performed a microarray analysis to compare the transcriptomes of 2c, 4c, and 8c rat hepatocytes. In addition, to address any potential transcriptomic heterogeneity in hepatocytes with different ploidy statuses, we performed single-cell quantitative reverse-transcription polymerase chain reaction (scPCR) using a set of hepatocyte and liver progenitor cell (LPC) marker genes. Contrary to the widely accepted notion that 2c hepatocytes reside in the periportal region,5,10–13 both our bulk transcriptome and scPCR results showed that 2c hepatocytes are preferentially located in the pericentral region. In addition, scPCR analysis showed the existence of a progenitor-like population of 2c hepatocytes.

Results Microarray Transcriptome Analysis Identified 8c Hepatocytes as Cells With Typical Polyploid Characteristics By using freshly isolated rat adult hepatocytes, we obtained 2c, 4c, and 8c hepatocytes using fluorescenceactivated cell sorting (FACS) based on the fluorescence intensity of the DNA stain Hoechst 33342 (Figure 1A). We validated the accuracy of this sorting method by performing microscope observations, which confirmed that none of the cells sorted into the 2c population were binucleated, whereas 42.0% ± 4.44% and 92.0% ± 7.10% of 4c- and 8csorted cells were binucleated, respectively (means ± SEM) (Figure 1B and C). Importantly, by phase-contrast imaging, we confirmed that the nucleus size of the mononucleated hepatocytes in the 4c fraction was larger than that in the 2c fraction (Figure 1B). Likewise, the nucleus size of the binucleated hepatocytes in the 8c fraction was larger than that in the 4c fraction (Figure 1B).

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We next performed a microarray transcriptome analysis on the sorted populations of hepatocytes. After excluding probes without gene annotations and those with low expression levels (see the Materials and Methods section for details), we performed hierarchical clustering analysis at the whole-transcriptome level. As shown in Figure 2A, we did not observe a clear difference between 2c, 4c, and 8c hepatocytes. As expected, the expression levels of typical hepatocyte marker genes, including Serpina1, Tf, and Ttr, were almost the same in these 3 populations (Figure 2B), with an exception of the expression level of Hnf4a in 2c hepatocytes, which was slightly but significantly lower than the 8c counterpart. Under physiological conditions, the polyploidization of hepatocytes is caused by cytokinesis failure during the cell cycle.3,4 We found that most of the gene ontology (GO) bioprocess pathways enriched in 8c hepatocytes were associated with the cell cycle (Figure 2C). This observation was supported further by gene signature enrichment analysis (Figure 2D). Furthermore, we confirmed that the representative cell cycle– and cell division–associated genes were expressed at higher levels exclusively in 8c hepatocytes (Figure 2E). These results collectively show that the transcriptomes were overall similar among hepatocytes with different ploidy statuses, with the exception of the transcriptome of 8c hepatocytes. Thus, the transcriptome data are consistent with known differences between hepatocytes with different polyploidy statuses, indicating that the analysis was valid and biologically relevant.

2c Hepatocytes Show a Zone 3–Enriched Gene Signature We reported previously that 2c hepatocytes and, to a lesser extent, 4c hepatocytes have in vitro colony forming ability in the presence of proliferative signals, whereas 8c hepatocytes do not7 (reproduced in this study, as shown in Figure 3A and B). This finding emphasizes that, although the overall transcriptome is similar between 2c, 4c, and 8c hepatocytes, there must still be undetected differences among these populations. Indeed, unlike the hierarchical clustering analysis, in a principal component analysis (PCA), although the first principal component (PC1) did not separate the hepatocytes with different ploidy statuses, PC2 separated 2c hepatocytes from 4c and 8c hepatocytes (Figure 4A). As indicated by the plot labels in Figure 4A, PC1 reflects the variance derived from the batch difference of microarray experiments. Given this technical issue, PC2-based *Authors share co-first authorship. Abbreviations used in this paper: ANOVA, analysis of variance; BEC, biliary epithelial cell; FACS, fluorescence-activated cell sorting; GO, gene ontology; LPC, liver progenitor cell; NPC, nonparenchymal cell; PCA, principal component analysis; RM, repeated-measures; scPCR, single-cell quantitative reverse-transcription polymerase chain reaction; TP, triple positive; Wnt, _____________.

© 2019 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2352-345X https://doi.org/10.1016/j.jcmgh.2019.08.011

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294 295 296 297 298 299 2c 4c 8c 300 301 302 303 304 305 FSC-A PI-A FSC-H Hoechst-A 306 307 308 309 2c 4c 8c 310 311 312 313 314 50 m 315 316 317 318 319 320 321 322 323 Figure 1. Validation of FACS sorting of hepatocytes with different ploidy statuses. (A) Representative gating for rat hepatocytes with 2c, 4c, and 8c DNA content as assessed by Hoechst 33342 fluorescent intensity. (B) Representative images of 324 single-sorted 2c, 4c, and 8c hepatocytes. Ploidy statuses of the sorted hepatocytes were validated via microscopy on day 1 325 after plating. Images were taken using the BZX-710 microscope (Keyence). (C) Microscopic validation of nuclear numbers of 326 hepatocytes. The data indicate the means ± SEM of 4 independent experiments. FSC-A, _________; FSC-H, _______; PI-A, Q36327 ________; SSC-A, __________. 328 329 information suggests that 2c hepatocytes have distinct well-characterized factor affecting the heterogeneity of he- 330 patocytes in terms of phenotype, such as those related to 331 phenotypes from 4c and 8c hepatocytes. We further investigated for differentially expressed metabolic and secretory functions.14–17 We examined for Q10332 genes among these 3 populations. After filtering the probes sets of genes that have been reported to be enriched in zone 333 with P values less than .05, determined by repeated- 1, zone 2, and zone 3 hepatocytes.18 We found that multiple 334 measures (RM) 1-way analysis of variance (ANOVA), we zone 3–enriched genes were expressed at significantly 335 identified 3080 probes that were differentially expressed higher levels in 2c hepatocytes (Figure 4C), including Glul 336 among 2c, 4c, and 8c hepatocytes. By using these probes, we (P ¼ 2.1  10-5), Cyp7a1 (P ¼ 3.4  10-3), and Slc1a2 (P ¼ 337 performed hierarchical clustering, which confirmed that the 2.7  10-5). We then manually prepared a “zone 3 signature 338 2c hepatocytes were distinct from the other 2 fractions gene set” by assembling 67 genes that were enriched Q11339 (Figure 4B). A GO analysis identified multiple bioprocess significantly in zone 3 hepatocytes as reported by Halpern 340 pathways that were enriched in 2c hepatocytes. However, et al.18 Gene signature enrichment analysis showed that this 341 despite careful analysis, we could not identify meaningful zone 3 signature gene set was enriched significantly in 2c 342 patterns within these GO terms. We did not find any GO hepatocytes (nominal P ¼ .0082) (Figure 4D and E). 343 bioprocess pathways enriched in 4c hepatocytes, suggesting Consistently, 2c hepatocytes had higher Wnt signaling ac- Q12344 that these cells share most of the features of 2c and 8c tivity, a characteristic of the pericentral region 345 (Figure 4F)19,20: nominal P ¼ .079 for Wnt Signaling 346 hepatocytes. We next asked whether ploidy status is associated with (contributed by SuperArray); nominal P ¼ .016 for Q13347 hepatic zone. The structure of the liver lobule generally is KEGG_Wnt_signaling_pathway; P ¼ .055 for WIL- Q14348 divided into 3 regions: zone 1 (periportal region), zone 3 LERT_Wnt_signaling (by the comparison of 2c vs 4c and 8c). 349 (pericentral region), and zone 2 (intermediate region be- Expression analysis of representative zone 1–enriched 350 tween zone 1 and zone 3). This hepatic zonation is the most genes showed that several genes were significantly 351 352

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412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 Figure 2. Microarray transcriptome analysis of rat hepatocytes with different ploidy statuses. (A) Heatmap with hierar- 456 chical clustering of whole-transcriptome analysis of 18,601 probes. Experiments were performed with 5 rats. (B) Expression of 457 general hepatocyte marker genes. Data are means ± SEM (n ¼ 5). (C) 8c hepatocyte-enriched pathways with GO terms 458 (biological process) are shown. Dots represent term enrichment with color coding. The sizes of the dots represent the percentage of each GO term. (D) Gene signature enrichment analysis of 8c hepatocytes and 2c and 4c hepatocytes using the 459 KEGG cell-cycle gene set. (E) Expression of cell cycle–related genes. Data are means ± SEM (n ¼ 5). (B and E) Bottom: P 460 values were calculated by RM 1-way ANOVA, followed by the Holm multiple comparisons test. P values for post hoc tests are 461 presented as follows: *P < .05, **P < .01, ***P < .001. Significance symbols on the left, middle, and right in each panel indicate 462 the comparison between 2c and 4c hepatocytes, 2c and 8c hepatocytes, and 4c and 8c hepatocytes, respectively. BP, ____; Q37463 KEGG, _________; Nom, nominal. 464 465 differentially expressed between the 3 fractions of hepato- genes (Figure 4C). As expected, nonmonotonic genes were 466 cytes, including Asl (P ¼ .0495), Ass1 (P ¼ .027), and G6pc expressed almost evenly throughout the 3 fractions 467 (P ¼ 1.1  10-4) (Figure 5, top), but these differences were (Figure 5, middle). By contrast, the expression levels of zone 468 much smaller than those observed for zone 3–enriched 2–enriched genes Hamp (P ¼ 2.1  10-4) and Igfbp2 (P ¼ 469 470

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Figure 3. In vitro phenotypic validation of FACS-sorted hepatocytes. (A) Colony formation assay with single-sorted 2c, 4c, and 8c hepatocytes. Images were taken using the BZX-710 microscope (Keyence). (B) Colony-forming capacity was evaluated on day 10. Colonies with 20 or more cells were counted for each fraction. The data are shown as means ± SEM (n ¼ 4). RM 1-way ANOVA was used to determine significant differences among the 3 groups (P ¼ 7.4  10-4), followed by the Tukey–Kramer post hoc test as indicated in the panel. *P < .05. D, day.

3.6  10-4) were lower in 2c hepatocytes than in the other 2 fractions (Figure 5, middle). These results strongly suggest that 2c hepatocytes are enriched in zone 3.

Some LPC Marker Genes Are Expressed Differentially According to Ploidy Status One question yet to be fully addressed is whether any specific subpopulation(s) of hepatocytes have stem/progenitor cell–like characteristics. Previous work has identified putative resident LPCs that are characterized by specific marker genes (eg, Afp, Epcam, and Prom1/Cd133) (reviewed by Miyajima et al21). Furthermore, several groups recently identified subsets of hepatocytes that phenotypically show LPC-like features,6,22–24 while other groups have reported that mature hepatocytes have the capacity to be reprogrammed into LPCs.25–30 In particular, Wang et al6 recently reported that Axin2-positive pericentral hepatocytes, which contribute to hepatocyte turnover under physiological conditions, are enriched in 2c hepatocytes. Our analysis of zone 3–enriched genes showed that 2c hepatocytes showed a slight but significant increase in Axin2 expression (P ¼ .024) compared with other hepatocyte populations (Figure 4C). In addition, other genes, including Epcam (P ¼ 9.7  10-4), Notch2 (P ¼ 2.5  10-3), Prom1 (P ¼ 1.0  10-3), and Tbx3 (P ¼ .012), were expressed differentially with statistical significance (Figure 5, bottom).

Dissecting 2c and 4c Hepatocyte Heterogeneity at the Single-Cell Level Microarray transcriptome analysis provided insight into the association between ploidy status and hepatic phenotype, but we were unable to determine whether the differences observed in the bulk analysis were attributable to global (averaged) differences between these fractions or to subpopulations that behave in a very different manner relative to the majority of the population. This question is particularly important when considering the existence of LPCs, which are supposed to be a rare population. To examine the heterogeneity of hepatocytes from the point of view of different ploidy status, we performed scPCR analysis on FACS-sorted primary hepatocytes. To avoid contamination with doublet cells, we used the Fluidigm C1

system, which is designed to capture cells with diameters ranging between 17 and 25 mm. Because the majority of 8c hepatocytes were larger than 25 mm (Figure 6A), the maximum diameter valid in the C1 platform, we excluded 8c hepatocytes from the scPCR analysis and focused on comparing 2c and 4c hepatocytes. We prepared a TaqMan probe set targeting 47 genes and 1 negative control (no probe added): these probes included 2 housekeeping genes, general hepatic marker genes, zone-related genes, biliary epithelial cell (BEC) marker genes, and LPC marker genes (Table 1). Because some of the LPC marker genes also are expressed by BECs, we first compared hepatocytes with the nonparenchymal cell (NPC) fraction, which contains BECs. We confirmed that 10 of 96 NPCs analyzed expressed Krt19, a definitive BEC marker, whereas only 2 of 337 hepatocytes, both of which were 2c, showed Krt19 expression, which was lower than that in Krt19þ NPCs (Figure 6B). In particular, 5 of 10 Krt19þ NPCs expressed Gstp1 and Sox9, which also are BEC markers, and thus we regarded these 5 NPCs as BECs. PCA of these 5 BECs and the 337 hepatocytes clearly discriminated the BECs from the hepatocytes (Figure 6C). Indeed, except for Krt7, which was not detected in our experiment and thus was excluded from the analysis, almost all of the BEC marker genes were expressed exclusively in BECs (Figure 6B). Accordingly, BECs showed no or much lower levels of expression of hepatocyte marker genes (Figure 6B). Importantly, the earlier-mentioned 2 Krt19positive 2c hepatocytes were confirmed to be authentic hepatocytes because they showed robust expression of hepatic marker genes (eg, Alb, Ttr, and G6pc). A correlation heatmap for whole genes (43 genes, except Krt7, Afp, and Ncam1, which were expressed in <1% of analyzed cells) showed that the expression profiles of BEC/LPC markers and hepatocyte markers were clearly different (Figure 6D). Thus, we conclude that both the 2c and 4c hepatocytes investigated in this study were not contaminated with BECs. By using 92 2c hepatocytes and 245 4c hepatocytes, we compared the expression levels of zone-related genes. A correlation heatmap of BEC/LPC makers (except Krt19, Gstp1, and Spp1, which were expressed in <1% of hepatocytes) and hepatic marker genes with zone information showed that hepatic genes were clustered in a zonedependent manner (Figure 6E). Consistent with the

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766 767 Zone 1-enriched genes 768 769 770 ns ns ns *** 771 ns ns ns ns ns ns ** * 772 773 774 775 776 p = 0.83 p = 0.0495 p = 0.027 p = 0.056 p = 0.030 p = 1.1 x 10-4 p = 0.71 p = 0.096 p = 0.059 p = 0.38 777 778 Non-monotonically expressed genes / Relatively zone 2-enriched genes 779 780 781 ** ns * ns 782 ** ** 783 784 785 786 787 788 p = 0.13 p = 0.52 p = 0.81 p = 0.72 p = 0.0030 p = 0.056 p = 0.73 p = 2.1 x 10-4 p = 3.6 x 10-4 789 Non-monotonically expressed genes Relatively zone 2-enriched 790 791 792 LPC marker genes 793 794 795 ns ** * * * ns ns ns ns * ns * ns * ns 796 797 798 799 800 801 p = 0.011 p = 9.7 x 10-4 p = 0.79 p = 0.83 p = 0.21 p = 0.0025 p = 0.0010 p = 0.88 p = 0.012 p = 0.13 802 803 *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant 804 805 Figure 5. Further characterization of FACS-sorted hepatocytes. Expression of zone 1–enriched genes (top), non806 monotonically expressed or relatively zone 2–enriched genes (middle), and LPC marker genes (bottom). Data are represented as means ± SEM (n ¼ 5). Bottom: P values were calculated by RM 1-way ANOVA, followed by the Holm multiple comparisons 807 test. P values for post hoc tests are presented as follows: *P < .05, **P < .01, ***P < .001. Significance symbols on the left, 808 middle, and right in each panel indicate the comparison between 2c and 4c hepatocytes, 2c and 8c hepatocytes, and 4c and 809 8c hepatocytes, respectively. 810 811 812 813 814 Figure 4. (See previous page). Characterization of differentially expressed genes in 2c, 4c, and 8c hepatocytes. (A) PCA for whole-transcriptome analysis of 18,601 probes. Labels for each plot provide the ploidy information (2c, 4c, and 8c) with 815 batch information of 5 experiments (_1, _2, _3, _4, and _5). (B) Heatmap with hierarchical clustering of the 3080 differentially 816 expressed probes (defined as probes with P < .05 by RM 1-way ANOVA). (C) Expression of zone 3–enriched genes. Data are 817 represented as means ± SEM (n ¼ 5). P values at the bottom of each panel were calculated by RM 1-way ANOVA, followed by 818 the Holm multiple comparisons test. P values for post hoc tests are presented as follows: *P < .05, **P < 0.01. Significance 819 symbols on the left, middle, and right in each panel indicate the comparison between 2c and 4c hepatocytes, 2c and 8c 820 hepatocytes, and 4c and 8c hepatocytes, respectively. (D) Gene signature enrichment analysis of 2c hepatocytes and 4c and 821 8c hepatocytes using a zone 3 signature gene set that was generated manually (the gene list is shown in panel E). (E) Heatmap of the zone 3–enriched gene set. (F) Gene signature enrichment analysis of 2c hepatocytes compared with 4c and 8c he- 822 Q38 823 patocytes using Wnt signaling gene sets. KEGG, ________; NES, ______; Nom, nominal; WILLERT, _______. 824 Normalized expression (log2)

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almost restricted to the 2c cells (Figure 8B, solid ellipses). 1002 Indeed, the top 10% of cells ranked according to Glul 1003 expression level (34 of 337 cells) were 2c hepatocytes. 1004 Convincingly, cells with the highest expression of Glul 1005 showed lower expression levels of zone 1–enriched genes 1006 such as Alb, Ass1, G6pc, Pck1, and Tat (Figure 8C, solid el- 1007 lipses). On the other hand, the subpopulation of 4c hepa- 1008 tocytes with partial expression of zone 3 marker genes also 1009 had high expression levels of zone 1–enriched genes 1010 (Figure 8C, dotted ellipses). Compared with zone 1– and 1011 zone 3–enriched genes, we confirmed that 2c-enriched/ 1012 nonmonotonic genes showed similar levels of expression 1013 among the analyzed cells (Figure 8D). 1014 As described earlier, hierarchical clustering showed a 2c 1015 hepatocyte-rich subpopulation that was enriched with cells 1016 expressing Axin2, Prom1, and Lgr5 (Figure 7B). A Venn di- 1017 agram of the whole hepatocyte population indicated that 1018 100% (20 of 20) and 70.0% (14 of 20) of Lgr5þ hepato- 1019 cytes expressed Axin2 and Prom1, respectively, and that 1020 82.9% (68 of 82) of Prom1-positive hepatocytes expressed 1021 Axin2 (Figure 9A). These results were suggestive of a pop- 1022 ulation hierarchy among LPC marker–expressing hepato- 1023 cytes in which a population positive for all 3 LPC markers 1024 (hereafter designated as triple positive [TP] population) 1025 resided at the top. The TP population comprised 4.15% of 1026 the entire hepatocyte pool (14 of 337 hepatocytes). The 1027 ratio of TP cells was higher in 2c hepatocytes (12.0%; 11 of 1028 92 cells) than in 4c hepatocytes (1.2%; 3 of 245 cells) 1029 (Figure 9B, see also Figure 9C for detailed co-expression 1030 profiles), confirming that the TP population is highly 1031 enriched among 2c hepatocytes. 1032 We further explored the expression profiles of all of the 1033 LPC markers, including Lgr5, Axin2, Prom1, Epcam, Icam1, 1034 Sox9, and Notch2 in the TP population. We found that the TP 1035 PCA of Single Cells Using Zonation and LPC population also was enriched for Epcam, Icam1, and Tbx3 1036 Signature (Figure 9B). By contrast, these cells did not express Sox9 1037 We next visualized each of the analyzed cells by PCA and (Figure 9B). Sox9 expression is reported to be limited to a 1038 confirmed that the 2c and 4c hepatocytes form distinct subpopulation of zone 1 hepatocytes called “hybrid hepa- 1039 populations (Figure 8A). Loading these PCA plots with the tocytes.”23,32 Thus, the TP population can be regarded as a Q161040 expression levels of zone-associated genes indicated that 2c population distinct from hybrid hepatocytes. We also 1041 hepatocytes have a pericentral expression signature confirmed that all of the TP population cells (14 of 14) 1042 (Figure 8B–D). Some of the 4c hepatocytes expressed zone 3 expressed Glul, strongly suggesting that these cells are 1043 marker genes, including Axin2, Cyp7a1, and Nr1i3 localized to the pericentral region. Indeed, Planas et al33 Q171044 (Figure 8B, dotted ellipses), showing that not only 2c but recently reported that, in mice, Lgr5 messenger RNA 1045 also 4c hepatocytes also distribute to zone 3. However, the expression was observed preferentially in pericentral 1046 highest expression levels of Glul, Cyp1a2, and Cyp2e1 were hepatocytes. 1047 1048 1049 1050 Figure 6. (See previous page). Validation of the results of scPCR. (A) Cell size of FACS-sorted 2c, 4c, and 8c hepatocytes. A 1051 total of 65, 88, and 21 cells were analyzed for 2c, 4c, and 8c cells, respectively. One-way ANOVA was used to determine 1052 significant differences among the 3 groups (P ¼ 3.8  10-30), followed by the Tukey multiple comparisons test. (B) Violin plots 1053 of BEC marker genes (left) and hepatocyte marker genes (right). Bottom: P values were calculated by 1-way ANOVA, followed 1054 by the Tukey multiple comparisons test. P values for post hoc tests are presented as follows: *P < .05, **P < .01, ***P < .001. Q391055 Significance symbols on the left, middle, and right in each panel indicate the comparison between BEC and 2c hepatocytes, 1056 BEC and 4c hepatocytes, and 2c and 4c hepatocytes, respectively. (C) PCA of 2c and 4c hepatocytes with 5 þ þ þ Krt19 Sox9 Gstp1 BECs using 43 genes. (D) Correlation heatmap for whole genes (43 genes, except Krt7, Afp, and Ncam1, 1057 which were expressed in <1% of analyzed cells) using 5 BECs and 337 hepatocytes. (E) Correlation heatmap of BEC/LPC 1058 makers (except Krt19, Gstp1, and Spp1, which were expressed in <1% of hepatocytes) and hepatic marker genes with zone 1059 information. 1060

observations from the bulk transcriptome, we confirmed that 2c hepatocytes showed higher expression levels for most of the zone 3–enriched genes (Figure 7A, middle).18 Specifically, 94.6% of 2c hepatocytes expressed Glul, whose expression is sharply localized in proximity to the central vein, whereas only 58.8% of 4c cells expressed this gene. Accordingly, the zone 1–enriched genes Alb (P ¼ 4.2  10-9), Ass1 (P ¼ 3.2  10-21), G6pc (P ¼ 1.6  10-27), Pck1 (P ¼ 1.3  10-15), and Tat (P ¼ 2.5  10-29) were expressed consistently at lower levels in 2c hepatocytes (Figure 7A, bottom left). We also confirmed that nonmonotonic genes were expressed at relatively similar levels in 2c and 4c hepatocytes (Figure 7A, bottom left). We also investigated the expression of LPC marker genes, including Epcam, Icam1, Lgr5, Notch2, Prom1, and Tbx3. Consistent with the bulk transcriptome results, Epcam (P ¼ .029), Prom1 (P ¼ .013), Tbx3 (P ¼ 1.0  10-5), and Axin2 (P ¼ 2.1  10-6) were expressed at higher levels in 2c than in 4c hepatocytes (Figure 7A, bottom right, see middle for Axin2). In addition, we found that a small number of cells expressed Lgr5, whose expression levels were very low in the bulk microarray (Figure 5, bottom), presumably because the majority of hepatocytes, irrespective of their ploidy status, do not express this gene.31 Hierarchical clustering of the 40 genes expressed in at least 1% of the total cells analyzed (
4 cells) clustered the 2c and 4c hepatocytes into distinct groups (Figure 7B, lower column side bar). As highlighted in the upper column side bar (see the purple-colored cluster on the far left), the 2c hepatocyte-rich subpopulation showed higher expression of multiple LPC markers including Axin2, Prom1, and Lgr5 (Figure 7B, upper column side bar).

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1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 10

Table 1.Gene List for scPCR Analysis Zone 1 marker

Zone 2/ nonmonotonic marker

Zone 3 marker

LPC marker

BEC marker

Description/note

Reference

B

Reported to have no zonation Used for normalizing gene expression

18

Gapdh Rn01775763_g1

B

Reported to have no zonation, but relatively more deviated than Actb18

18

Alb Rn00592480_m1

B

Hepatocyte-secreted serum protein

15,18

Tat Rn00562011_m1

B

Hepatocyte-secreted serum protein

18

Ass1 Rn00565808_g1

B

Involved in the synthesis of creatine, polyamines, arginine, urea, and nitric oxide

18

G6pc Rn00689876_m1

B

Functions in gluconeogenesis and glycogenolysis

Pck1 Rn01529014_m1

B

Main regulator in gluconeogenesis

18,52

15,18,53

Abcb11 Rn01515444_m1

B

ABC transporter expressed along bile canaliculi

Cyp2c6v1 Rn03417171_gH

B

Phase I modification enzyme

Cyp2d4 Rn01504629_m1

B

Phase I modification enzyme A rat orthologue of human CYP2D6 and mouse Cyp2d22

18

Gsta1 Rn04223027_m1

B

Phase II conjugation enzyme

18

Baat Rn00568867_m1

B

Catalyzes the transfer of C24 bile acids from the acyl-CoA thioester to either glycine or taurine

18

Hnf4a Rn04339144_m1

B

Catalyzes the rate-limiting step in the synthesis of glycogen

18

Gys2 Rn00565296_m1

B

Bile acid transporter located at the basal side of hepatocytes

18

Slc10a1 Rn00566894_m1

B

Detected in no more than 1% of analyzed cells, thus not included in analysis

18

Nr1i2 Rn00583887_m1

B

Phase II conjugation enzyme

Glul Rn01483107_m1

B

Catalyzes the synthesis of glutamine from glutamate and ammonia

Abcc2 Rn00563231_m1

B

ABC transporter expressed along bile canaliculi

18

7

Human protein atlas: www.proteinatlas.org 15,18,53–55

18

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Actb Rn00667869_m1

18

Katsuda et al

Gene

House keeping marker

-,

No. -

1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178

1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 2019

Table 1. Continued

Gene

House keeping marker

Zone 1 marker

Zone 2/ nonmonotonic marker

Zone 3 marker

LPC marker

BEC marker

Description/note

Reference 18

B

The next enzyme after alcohol dehydrogenase in the alcohol metabolism pathway

Ahr Rn00565750_m1

B

Transcription factor that regulates CYP enzyme expression

Nr1i3 Rn04339043_m1

B

Nuclear receptor, also known as constitutive androstane receptor, which regulates xenobiotic and endobiotic metabolism

Cyp1a2 Rn00561082_m1

B

Phase I modification enzyme

15,16,18,54,55

Cyp2e1 Rn00580624_m1

B

Phase I modification enzyme

15,16,18,53–55

Cyp2f4 Rn00570779_m1

B

Phase I modification enzyme A rat orthologue of mouse Cyp2f2

Cyp3a23 /3a1 Rn01412959_g1

B

Phase I modification enzyme

16

Cyp7a1 Rn00564065_m1

B

Phase I modification enzyme

7,18,54

Cyp27a1 Rn00710297_m1

B

Phase I modification enzyme

16,18,54

Ugt1a1 Rn00754947_m1

B

Transcription factor that plays an important role in liver development and liver functions

Ttr Rn00562124_m1

B

Phase II conjugation enzyme

Cyp2b3 Rn01476084_m1

B

Hepatocyte-secreted serum protein

Sox9 Rn01751070_m1

Phase I modification enzyme No zonation-related information available

Axin2 Rn00577441_m1

B

B

Plays an important role in the regulation of the stability of b-catenin

Tbx3 Rn00710902_m1

B

B

Transcription factor involved in the regulation of various developmental processes

Afp Rn00560661_m1

B

B

Ncam1 Rn01418541_m1

B

Epcam Rn01473202_m1

B

B

Transcription factor regulating the bile duct development of BECs Also reported to be expressed by periportal adult LPCs A major plasma protein produced by fetal liver cells Also expressed by injury-induced rat LPCs

B

A marker expressed in developing bile ducts and LPCs

15,16,18,54,55

Q42

15,18,55

18,54

18

16,18

18

N/A 6,15,18

18

23,32,56

21

57,58

Ploidal Heterogeneity of Mature Hepatocytes

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Aldh1a1 Rn00755484_m1

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1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296

1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 12

Table 1. Continued Zone 3 marker

Prom1 Rn00572720_m1

B

Notch2 Rn01534371_m1

B

Icam1 Rn00564227_m1

B

Itga6 Rn01512708_m1

B

Lgr5 Rn01509662_m1 Krt19 Rn01496867_m1

Description/note

Reference

B

A marker expressed in fetal hepatoblasts, BECs, and injuryinduced adult LPCs

21

B

A marker expressed in fetal hepatoblasts, BECs, and injuryinduced adult LPCs

21

A marker expressed in fetal hepatoblasts

59

B

A marker expressed in fetal hepatoblasts, BECs, and injuryinduced adult LPCs

60

B

B

A marker expressed in fetal hepatoblasts, BECs, and injuryinduced adult LPCs

21

B

B

A marker expressed in BECs/LPCs upon injury

31

Krt7 Rn04224249_u1

B

A marker of BECs

61

Gstp1 Rn00561378_gH

B

A marker of BECs Detected in <1% of analyzed cells, thus excluded from study

61

B

A marker of BECs and LPCs

Hnf1b Rn00447453_m1

B

62–64

Human protein atlas: www.proteinatlas.org

Sult2a1 Rn04223057_mH

B

A marker of BECs

61

Spp1 Rn00681031_m1

B

A marker of BECs

65

None

Negative control

N/A

NOTE. For zonation information obtained from Halpern et al,18 we referred to Supplementary Table 3 in their article. We defined genes with zonation as those with a P value (Kruskal–Wallis) < .01. Genes with their expression peak in layers 1–3, 4–6, and 7–9 were defined as zone 1, zone 2, and zone 3 markers, respectively.

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Zone 1 marker

Zone 2/ nonmonotonic marker

LPC marker

Gene

House keeping marker

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No. -

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Ploidal Heterogeneity of Mature Hepatocytes

Mononucleated 4c Hepatocytes Are More Zone 3–Enriched Than Binucleated 4c Hepatocytes Finally, we investigated whether nuclearity affects the gene expression profiles of 4c hepatocytes. A recent study examined the difference in metabolic capacity between mononucleated and binucleated hepatocytes.34 In the present study, we asked whether zonation varies between mononucleated and binucleated 4c hepatocytes. Taking advantage of the single-cell–capturing ability of the C1 platform, we annotated 175 4c hepatocytes with mononuclear or binuclear states (Figure 10A). PCA mapping of 106 mononucleated and 69 binucleated hepatocytes did not show a clear separation (Figure 10B). However, individual comparison of the expression levels of zonation-related genes showed that mononucleated 4c hepatocytes expressed zone 3–enriched (Figure 10C, middle) and zone 1–enriched (Figure 5C, bottom left) genes at higher and lower levels, respectively, than binucleated hepatocytes, while nonmonotonic genes were mostly expressed evenly (Figure 10C, top row). Except for Tbx3 (P ¼ 7.9  10-5), most LPC marker genes were not expressed differentially between mononucleated and binucleated 4c hepatocytes (Figure 10C, bottom right). Tbx3 is a downstream gene in the Wnt signaling pathway similar to Axin2, and therefore its expression supports the idea that mononucleated 4c hepatocytes are enriched in zone 3.35 Despite this finding, the zonal signature is much more clearly distinguishable between 2c and 4c hepatocytes (Figure 11).

Discussion This study provides new insights into hepatocyte heterogeneity, namely 2c hepatocytes are preferentially localized to the pericentral region; and a subpopulation of 2c hepatocytes show LPC-like features in terms of LPC marker expression (Axin2, Prom1, and Lgr5). In a previous study, Lu et al36 reported no major differences in the gene expression patterns between mouse hepatocytes of different ploidy. Inconsistencies between their observations and ours might derive from differences in, first, the analyzed probe numbers: approximately 12,000 probes (designed based on the information available in 2002) were included in the Lu et al36 study, whereas approximately 45,000 probes were used in our study; and, second, the microarray platforms: Lu et al36 used the Affymetrix platform, whereas we used the Agilent platform. However, we confirmed that the differentially expressed genes reported by Lu et al36 also were expressed differentially in our study. Eleven of the 75 probes that correspond to the 56 genes identified as being expressed differentially by Lu et al36 also were identified as expressed differentially in the present study (P < .05, RM 1-way ANOVA). The P values for these 75 probes were significantly lower than those of the whole 35,852 probes (P ¼ 1.2  10-4 by the Wilcoxon test) (Figure 12), confirming, at least partly, consistency between the observations of Lu et al36 and those described here. In addition, we confirmed the enrichment of cell cycle–related genes in 8c hepatocytes, which is consistent with the established notion that polyploidy is formed

13

via cell division failure. Thus, we believe that our analysis 1474 successfully identified characteristic features of hepatocytes 1475 with different ploidy statuses. 1476 Zonal localization of hepatocytes with different ploidy 1477 statuses is an unresolved issue in the field. Our bulk tran- 1478 scriptome and scPCR analysis consistently showed that 2c 1479 hepatocytes are localized preferentially to the pericentral 1480 region. Specifically, scPCR showed that the hepatocytes with 1481 the highest expression levels of Glul, one of the most strin- 1482 gent pericentral marker genes, were almost exclusively 2c. 1483 The proposition of Wang et al6 that pericentral hepatocytes 1484 are enriched among 2c hepatocytes is consistent with ours. 1485 However, their finding does not necessarily indicate that 2c 1486 hepatocytes are located in the pericentral region, whereas 1487 the present study directly showed that 2c hepatocytes are 1488 localized preferentially to the pericentral region. At the 1489 same time, the finding that 2c hepatocytes show zone 1490 3–enriched features was surprising because it is widely 1491 accepted that diploid hepatocytes are enriched in the peri- 1492 portal region and polyploid hepatocytes are enriched in the 1493 pericentral region.5,10–13 At present, we cannot resolve this 1494 discrepancy, but one possible explanation is the 1495 methodology-dependent bias of isolated hepatocytes. In the 1496 present study, to avoid contamination with BECs, we used a 1497 stringent hepatocyte enrichment protocol in which digested 1498 whole-liver cells were subjected to sequential centrifugation 1499 steps (57  g for 1 min, 2 cycles) and then separated further 1500 using Percoll density gradient centrifugation (see the Ma- 1501 terials and Methods section). Our method highly enriches 1502 mature hepatocytes but, in turn, possibly loses a minor 1503 fraction of smaller hepatocytes, the so-called “small hepa- 1504 tocytes,” which are found in both parenchymal and non- Q201505 parenchymal fractions.37 Indeed, in this study, the 2c 1506 fraction accounted for approximately 5.4% ± 2.2% (means 1507 ± SD, n ¼ 16 rats aged between 5 and 14 weeks) of total 1508 hepatocytes. This is lower than the widely accepted per- 1509 centage of 2c hepatocytes: it generally is accepted that 1510 approximately 10%–15% of hepatocytes are diploid in rats 1511 and mice.3,5 We cannot rule out the possibility that, in our 1512 experiments, some of the 2c hepatocytes were lost. None- 1513 theless, it still is noteworthy that a substantial number of 2c 1514 hepatocytes showed zone 3–enriched characteristics. 1515 Moreover, unlike previous studies in which conclusions 1516 were drawn based on histologic analyses, this study pro- 1517 vides novel insights from the transcriptomic perspective. 1518 This study also provides novel insights into LPC biology. 1519 It remains controversial whether the liver has resident 1520 stem/progenitor cells. Our scPCR study failed to provide 1521 evidence for their existence but shows instead that 2c he- 1522 patocytes are more enriched in well-established LPC 1523 markers than 4c hepatocytes. In particular, our data suggest 1524 that these progenitor-like cells have a hierarchy in which 1525 Lgr5þProm1þAxin2þ TP cells reside at the top (at least ac- 1526 cording to the gene set used here). On the other hand, 1527 because the sample size in this study was small, in partic- 1528 ular for the TP population (11 of 92 and 3 of 245 cells for 2c 1529 and 4c populations, respectively), we have to be careful 1530 about the conclusion. In addition, our single-cell analysis 1531 was based on a limited panel of genes, thereby leaving the 1532

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A

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1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591

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1651 1652 1653 1654 1655 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675 1676 1677 1678 1679 1680 1681 1682 1683 1684 1685 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704 1705 1706 1707 1708 1709

Ploidal Heterogeneity of Mature Hepatocytes

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1710 1711 1712 1713 1714 1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725 1726 1727 1728 1729 1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743 1744 1745 1746 1747 1748 1749 1750 1751 1752 1753 Figure 8. Characterization of 2c and 4c hepatocytes by PCA mapping. (A) PCA mapping of 92 2c (green) and 245 4c (blue) 1754 hepatocytes. (B–D) Each cell is colored according to the designated gene expression level as scaled with the color key. (B) 1755 Solid ellipses and dotted ellipses indicate a Glul-high 2c cell-rich population and zone 3–oriented 4c cell-rich population, Q411756 respectively. In panels with asterisks, some cells were excluded from the analysis because their inclusion reduced the res- 1757 olution of the expression profiles for the designated genes. Complete mapping of these genes is shown in panel E. (E) 1758 Complete PCA mapping of 337 hepatocytes for Axin2, Cyp1a2, Cyp2c6v1, Gys2, Icam1, and Notch2. 1759 1760 1761 1762 Figure 7. (See previous page). scPCR-based expression profile of hepatocyte and LPC markers in 2c and 4c hepa- 1763 tocytes. (A) Violin plots of nonmonotonically expressed hepatic genes (top), zone 3–enriched genes (middle), zone 1–enriched 1764 genes (bottom left), and LPC marker genes (bottom right). Expression level for each gene was normalized with that of Actb. 1765 White diamonds indicate mean values. P values were calculated using the Welch t test (n ¼ 92 and 245 for 2c and 4c he- 1766 patocytes, respectively). (B) Heatmap with hierarchical clustering of 92 2c and 245 4c hepatocytes using 40 genes. The upper 1767 Q40 and lower column side color bars indicate 12 cell clusters and hepatocyte ploidy, respectively. 1768

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Katsuda et al

Figure 9. Characterization of LPC-like subpopulation in 2c hepatocytes. (A) Venn diagram describing the co-expression profile of 3 LPC marker genes: Axin2, Prom1, and Lgr5. (B) Cells of Axin2þProm1þLgr5þ TP population are highlighted in magenta (upper left). The ploidy status of TP population cells is indicated in green (2c) and blue (4c) (lower left). Expression levels for all of the analyzed LPC marker genes are shown (right). In panels with asterisks, some cells were excluded from the analysis because their inclusion reduced the resolution of the expression profiles for the designated genes. Complete mapping of these genes is shown in Figure 8E. (C) Scatter plot with density estimations for Axin2 vs Prom1 and Prom1 vs Lgr5. Expression levels for each gene are normalized to Actb. (D) Scatter plot with density estimations for Axin2 vs Prom1 and Prom1 vs Lgr5. Expression levels for each gene are normalized to Actb.

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Ploidal Heterogeneity of Mature Hepatocytes

Figure 10. Nuclearity-based dissection of 4c hepatocytes. (A) Microscopic identification of the nuclearity of 4c hepatocytes using the C1 microfluidics platform. A total of 106 mononucleated and 69 binucleated hepatocytes were included in this analysis. Images were taken using the BZX-710 microscope (Keyence). (B) PCA for mononucleated and binucleated 4c hepatocytes using 40 genes. (C) Violin plots of nonmonotonically expressed hepatic genes (top), zone 3–enriched genes (middle), zone 1–enriched genes (bottom left), and LPC marker genes (bottom right). P values were calculated using the Welch t test.

possibility that we have missed other LPC markers that would further dissect the hepatocyte population. Further exploration will require single-cell RNA sequencing with a larger sample scale. Nonetheless, we propose that this study

provides a wealth of transcriptomic data and provides some novel insights about localization and function of the heterogeneous population of hepatocytes, which can be tested in the future.

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1946 1947 1948 1949 1950 1951 1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

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2064 2065 2066 2067 2068 2069 2070 2071 2072 2073 2074 2075 2076 2077 2078 2079 2080 2081 2082 2083 2084 2085 2086 2087 2088 2089 2090 2091 2092 2093 Figure 11. Comparison between mononucleated and binucleated 4c hepatocytes and 2c hepatocytes. Violin plots of 2094 nonmonotonically expressed hepatic genes (top), zone 3–enriched genes (middle), zone 1–enriched genes (bottom left), and 2095 LPC marker genes (bottom right). A total of 92 2c hepatocytes, 106 mononucleated 4c hepatocytes, and 69 binucleated 4c 2096 hepatocytes were used. P values at the bottom of each panel were calculated by 1-way ANOVA, followed by the Tukey 2097 multiple comparisons test. P values for post hoc tests are presented as follows: *P < .05, **P < .01, ***P < .001. Significance 2098 symbols on the left, middle, and right in each panel indicate the comparison between 2c and mononucleated 4c hepatocytes, 2099 2c and binonucleated 4c hepatocytes, and mononucleated and binucleated 4c hepatocytes, respectively. 2100 2101 Finally, we clarify the limitation of the present study. Laboratory Animal Research of the National Cancer Center 2102 First, given the interspecies difference in ploidy profile of Research Institute. The protocol was approved by the 2103 hepatocytes, the relevance of our findings here is not Committee on the Ethics of Animal Experiments of the Na- 2104 applicable simply to human liver biology. The degree of liver tional Cancer Center Research Institute. Rats were housed in 2105 polyploidization varies between mammals: although 80%– specific pathogen-free facilities on a 12-hour light/dark cy- 2106 97%9,34,38–40 and 70%–95%41–44 of adult mouse and rat cle and were given food and water ad libitum. All animals 2107 hepatocytes are polyploid, respectively, only 10%–40% of used in this study were female Wistar rats aged between 5 2108 adult human hepatocytes are polyploid.45–47 Strikingly, and 14 weeks (CLEA Japan, Shizuoka, Japan). Details Q222109 although polyploid hepatocytes become the majority within regarding the animals used in the study are described for 2110 a week after weaning in rodents, polyploidy cells generally each part of the experiment in the Materials and Methods 2111 2112 do not exceed 15% in 20-year-old human beings. These section. 2113 reports collectively suggest that the role division that might 2114 be provided by the ploidy heterogeneity is different beIsolation of Rat Adult Hepatocytes 2115 tween rodents and human beings. Further study with the 2116 Adult rat hepatocytes were isolated from adult rats using use of freshly isolated human hepatocytes will be required. the procedure described by Seglen48 with minor modifica- 2117 tions (for details, see Katsuda et al33). Briefly, after 2118 Materials and Methods preperfusion with Ca2þ-free Hank’s/ethylene glycol-bis(b- 2119 Rats aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid solution 2120 Animal experiments in this study were performed in through the portal vein, the liver was perfused with 2121 compliance with the guidelines of the Institute for approximately 400 mL Hank’s solution containing 0.05% 2122

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2182 2183 2184 2185 2186 2187 2188 2189 Isolation of BEC-Enriched NPCs 2190 NPCs were isolated from 7-week-old rats as described 2191 previously.49,50 Briefly, we harvested bile duct containing 2192 remnant tissue after 2-step collagenase perfusion of an 2193 adult rat liver as described earlier. Then, the remnant tissue 2194  was digested by shaking at 37 C for 30 minutes in Leibovitz 2195 L-15 medium (Invitrogen) supplemented with 400 U/mL Q25 2196 collagenase (Wako), 700 U/mL hyaluronidase (Sigma2197 Aldrich), 10-7 mol/L insulin, and 10-7 mol/L dexamethasone. 2198 After filtration with a 40-mm cell strainer (BD Biosciences), 2199 the cells were collected by centrifugation at 800  g for 10 2200 minutes. Then the pellet was resuspended in Dulbecco’s 2201 modified Eagle medium (Invitrogen) supplemented with 2202 10% fetal bovine serum (Invitrogen) and centrifuged twice 2203 at 800  g for 5 minutes. Purified NPCs were captured by 2204 Q26 the C1 system and analyzed by scPCR. 2205 2206 2207 FACS of Hepatocytes With Different Ploidy 2208 Statuses To fractionate the 2c, 4c, and 8c hepatocytes, FACS-based 2209 single-cell isolation was performed according to a previ- 2210 ously described protocol.39 Briefly, rat hepatocytes har- 2211 vested from 5- to 14-week old rats were suspended in 10% 2212 fetal bovine serum–Dulbecco’s modified Eagle medium 2213 supplemented with 15 mg/mL Hoechst 33342 and 5 mmol/ 2214 L reserpine and incubated at 37 C for 10 minutes. After the 2215 addition of 5 mg/mL propidium iodide (BD, Franklin Lakes, Q272216 NJ), the cells were sorted using a FACSAria II or FACSAria III 2217 2218 (BD). 2219 2220 Colony Formation Assay of Single 2c, 4c, and 8c 2221 Rat Hepatocytes 2222 Hepatocytes harvested from 5- to 8-week-old rats were 2223 sorted into 96-well collagen-coated plates filled with 100 2224 mL/well of Dulbecco’s modified Eagle medium/F12 (Life 2225 Technologies) containing 2.4 g/L NaHCO3 and L-glutamine, 2226 which was supplemented with 5 mmol/L HEPES (Sigma), 30 2227 mg/L L-proline (Sigma), 0.05% bovine serum albumin 2228 (Sigma), 10 ng/mL epidermal growth factor (Sigma), 2229 insulin-transferrin-serine-X (Life Technologies), 10-7 mol/L 2230 dexamethasone (Sigma), 10 mmol/L nicotinamide (Sigma), 2231 1 mmol/L ascorbic acid-2 phosphate (Wako), an antibiotic/ 2232 antimycotic solution (Life Technologies), and 3 small mol- 2233 ecules: 10 mmol/L Y-27632 (Wako), 0.5 mmol/L A-83-01 2234 (Wako), and 3 mmol/L CHIR99021 (Axon Medchem). On Q282235 day 1, the cell and nuclei number for each well was deter- 2236 mined, and wells that contained dead cells and, in the 8c 2237 plates, cells greater than doublets were excluded from 2238 further analysis. Ten days after seeding, cells from each of 2239 the formed colonies were counted manually. 2240

mL 10 Hank’s balanced salt solution(-) [Life Technologies], and 21.6 mL Percoll [GE Healthcare]), and dead cells were removed via centrifugation at 57  g for 10 minutes. Finally, the cells were washed in 50 mL/tube E-MEM twice via centrifugation at 57  g for 2 minutes. The purified hepatocytes were used for the downstream experiments.

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Figure 12. Analysis of genes that were reported to be expressed differentially among hepatocytes with different ploidy status. P values were calculated by RM 1-way ANOVA using 2c, 4c, and 8c hepatocytes (n ¼ 5 experiments) for 75 probes, which correspond to the 56 differentially expressed genes reported by Lu et al36 and compared with the P values for whole probes (n ¼ 75 probes for Lu et al36; n ¼ 18,021 probes for whole probes).

collagenase at 25–30 mL/min. The extracted liver was mechanically digested with a surgical knife and then enzymatically digested in a mixture of 0.05% collagenase solution and 20 mL E-MEM (Sigma) at 37 C for 15 minutes. The digested liver then was filtered twice through a sterilized cotton mesh (single-folded and then double-folded). The cell suspension was aliquoted into two 50-mL tubes, which then were filled with E-MEM, and the cells were collected via centrifugation at 57  g for 1 minute. After resuspension in a 50 mL/tube E-MEM, large-cell aggregates were eliminated by filtering the cell suspension through a 60-mm stainless double-mesh cell strainer (Ikemoto Scientific Technology Co, Ltd, Tokyo, Japan) and the cells were collected by centrifuging the filtrate at 57  g for 1 minute. Next, the cells were resuspended in 24.5 mL/tube complete Percoll medium (25 mL L-15 medium [Life Technologies] supplemented with 0.429 g/L HEPES [Sigma], 2 g/L bovine serum albumin [Sigma], 1  10-7 mol/L insulin [Sigma], 2.4

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Microarray Analysis Hepatocytes isolated from 5 rats aged between 5 and 14 weeks were used. A 1-color microarray-based gene expression analysis system (Agilent Technologies) using the SurePrint G3 Rat GE 8  60 K Kit (G4853A) was used following the manufacturer’s instructions. Normalization was performed using Agilent GeneSpring version 12.6.1 (per chip: normalization to 75th percentile shift; per gene: normalization to the median of all samples). Transcriptome data were obtained from 5 rats. Probes with signal values lower than 5 in average (75 of total signal value for the 15 samples) were excluded from further analysis. The intensity values were log2-transformed, and RM 1-way ANOVA was performed using the lmne R package to identify differentially expressed genes. Probes with P values less than .05 were regarded as differentially expressed. Unsupervised clustering was performed using the heatmap.2 or heatmap.3 R package. PCA was performed using the prcomp R package, and mapped with the ggplot2 R package. Pathway analysis was performed using the clusterProfiler R package.51

Measurement of Cell Diameter of 2c, 4c, and 8c Hepatocytes FACS-sorted hepatocytes isolated from a 9-week-old rat were sparsely loaded into a hemocytometer and imaged with a Keyence BZX-710 camera (Keyence, Osaka, Japan). Cell area was calculated using the Hybrid Cell Count program (Keyence). Cellp diameter ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiwas determined using the following equation: 2 ðarea=pÞ.

Capturing Single Cells Single-cell capture, lysis, and RT pre-amplification experiments were performed using the C1 Auto Prep System (Fluidigm, SF) according to the manufacturer’s instructions. Isolated hepatocytes and NPCs were loaded into the C1 integrated fluidic circuit at a concentration of 300 cells/mL in phosphate-buffered saline containing 5% fetal bovine serum and 5 mmol/L of EDTA (Nacalai Tesque, San Diego, CA). Once cell capture was archived, cells were assessed for viability stain using Calcein-AM and ethidium homodimer 1 (Live/Dead Kit; Life Technologies). Chips were imaged using a BZ-X700 (Keyence) and chambers containing 2 or more, none, or death cells were removed from further analysis. After lysis, reverse transcription (25 C for 10 min, 42 C for 60 min, 85 C for 5 min) and pre-amplification for 18 cycles (each cycle: 95 C for 15 sec, 60 C for 4 min), single-cell complementary DNA was harvested, transferred to a 96well plate, and diluted 6 times with complementary DNA dilution buffer.

Single-Cell scPCR For this study, hepatocytes isolated from 5- to 7-weekold rats were used. Single-cell gene-expression experiments were performed using Fluidigm’s 96.96 or 48.48 quantitative PCR DynamicArray microfluidic chips (Fluidigm, SF) according to the manufacturer’s instructions. Individual probe assay mixes were generated by loading 2.5 mL of 2

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Assay Loading Reagent (Fluidigm, SF) and 2.5 mL 20 2300 TaqMan gene expression assay (Applied Biosystems, Bev- 2301 erly, MA). Sample mixes were generated by 2.5 mL 2 2302 TaqMan Universal PCR Master Mix (Applied Biosystems), 2303 0.25 mL of 20 GE Sample Loading Reagent (Fluidigm, SF), 2304 and 2.25 mL of diluted complementary DNA. Plates were 2305 vortexed and centrifuged to homogenize the solutions. 2306 Before loading probe assays and sample mixes into 96.96 or 2307 48.48 DynamicArray microfluidic chips, chips were primed 2308 in a HX integrated fluidic circuit controller (Fluidigm, SF) Q322309 machine. After priming, 5 mL of both sample and probe mix 2310 were loaded individually in the same machine. Then, 2311 chips were transferred into the BioMarkHD real-time 2312 quantitative PCR (Fluidigm, SF) and run according to the 2313 manufacturer’s instructions. Expression levels were 2314 normalized with Actb. 2315 2316 2317 Statistics When comparing 2 means, P values were calculated by 2318 the Welch t test unless otherwise mentioned. Paired com- 2319 parisons for more than 2 means was conducted using RM 1- 2320 way ANOVA, followed by the Holm multiple comparisons 2321 test. Unpaired comparisons for more than 2 means were 2322 conducted using 1-way ANOVA, followed by the Tukey 2323 2324 multiple comparisons test. 2325 2326 Accession Numbers 2327 The microarray data are deposited in GEO under acces2328 sion number GSE132409. The raw data of scPCR (Ct values) Q33 2329 are deposited in GEO under accession number GSE132459. 2330 2331 2332 References 1. Gentric G, Celton-Morizur S, Desdouets C. Polyploidy 2333 and liver proliferation. Clin Res Hepatol Gastroenterol 2334 2335 2012;36:29–34. 2. Wang MJ, Chen F, Lau JTY, Hu YP. Hepatocyte poly- 2336 ploidization and its association with pathophysiological 2337 2338 processes. Cell Death Dis 2017;8:e2805. 3. Gentric G, Desdouets C. Polyploidization in liver tissue. 2339 2340 Am J Pathol 2014;184:322–331. 4. Pandit SK, Westendorp B, De Bruin A. Physiological 2341 significance of polyploidization in mammalian cells. 2342 2343 Trends Cell Biol 2013;23:556–566. 5. Duncan AW. Aneuploidy, polyploidy and ploidy reversal 2344 2345 in the liver. Semin Cell Dev Biol 2013;24:347–356. 6. Wang B, Zhao L, Fish M, Logan CY, Nusse R. Self- 2346 renewing diploid Axin2þ cells fuel homeostatic renewal 2347 of the liver. Nature 2015;524:180–185. 2348 7. Katsuda T, Kawamata M, Hagiwara K, et al. Conversion 2349 of terminally committed hepatocytes to culturable bipo- 2350 tent progenitor cells with regenerative capacity. Cell 2351 Q34 Stem Cell 2017;20:41–55. 2352 8. Wilkinson PD, Delgado ER, Alencastro F, et al. The 2353 polyploid state restricts hepatocyte proliferation and liver 2354 regeneration. Hepatology 2019;69:1242–1258. 2355 9. Zhang S, Zhou K, Luo X, et al. The polyploid state plays a 2356 tumor-suppressive role in the liver. Dev Cell 2018; 2357 44:447–459.e5. 2358

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2536 2537 61. 2538 2539 2540 62. 2541 2542 2543 2544 63. 2545 2546 2547 2548 64. 2549 2550 2551 2552 65. 2553 2554 2555 2556 2557 2558 Received March 31, 2019. Accepted August 22, 2019. 2559 Correspondence 2560 Address correspondence to: Takahiro Ochiya, MD, Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, 5-1-1, Tsukiji, Q12561 Chuo-ku, Tokyo 104-0045, Japan. e-mail: [email protected]; fax: (81) Q22562 3-3541-2685. 2563 Acknowledgments 2564 The authors thank Ryou-u Takahashi and Hirokazu Ohata for assistance with 2565 FACS experiments, Ayako Inoue for technical assistance, and Luc Gailhouste for constructive discussion. The authors also thank Hideki 2566 Nishikawa (Keyence Corporation) for assistance with the microscopic 2567 analyses. 2568 Author contributions 2569 Takahiro Ochiya supervised the project; Takeshi Katsuda and Kazunori Hosaka designed the experiments; Kazunori Hosaka performed the majority of the 2570 experiments with support from Takeshi Katsuda, Juntaro Matsuzaki, Wataru 2571 Usuba, Marta Prieto-Vila, and Tomoko Yamaguchi; Takeshi Katsuda 2572 analyzed the data with assistance from Kazunori Hosaka; Atsunori Tsuchiya and Shuji Terai assisted with data interpretation and provided helpful 2573 discussions; Takeshi Katsuda wrote the manuscript with support from 2574 Kazunori Hosaka, Juntaro Matsuzaki, and Takahiro Ochiya; and Takahiro Ochiya and Takeshi Katsuda obtained funding for the study. 2575 Q32576 Conflicts of interest The authors disclose no conflicts. 2577 2578 Funding Supported by Grants-in-Aid from the Research Program on Hepatitis from the Q42579 Japan Agency for Medical Research and Development (AMED: Q432580 16fk0310512h0005 and 17fk0310101h0001 to T.O.) and a Grant-in-Aid for Q5 2581 Young Scientists B (16K16643 to T.K.). 2582 2583 2584 2585 2586 2587 2588 2589 2590 2591 2592 2593 2594 differentiate to functional hepatocytes in vitro and repopulate liver tissue. Stem Cells 2016;34:2889–2901. Tanimizu N, Miyajima A. Molecular mechanism of liver development and regeneration. Int Rev Cytol 2007; 259:1–48. Mitaka T, Sato F, Mizuguchi T, Yokono T, Mochizuki Y. Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells. Hepatology 1999;29:111–125. Lowes KN, Brennan BA, Yeoh GC, Olynyk JK. Oval cell numbers in human chronic liver diseases are directly related to disease severity. Am J Pathol 1999; 154:537–541. Sell S. Comparison of liver progenitor cells in human atypical ductular reactions with those seen in experimental models of liver injury. Hepatology 1998; 27:317–331. Español-Suñer R, Carpentier R, Van Hul N, et al. Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice. Gastroenterology 2012; 143:1564–1575.e7.

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