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Supporting Information Figure 1: Sox9-CreERT2 marks hepatocytes with high tamoxifen doses

Recombination was induced in Sox9-CreERT2 R26R-lacZ mice with 250mg/kg tamoxifen. Mice were examined (a) after 24 hours normal chow or (b) after prototypical steatosis injury that included a 1-week rest period, 3 weeks choline-deficient, ethionine supplemented diet, and a 2 week recovery period. (c) Quantification of Sox9-CreERT2 marked cells by epithelial cell type after normal chow or CDE injury recreates the increase in marked hepatocytes after injury previously reported in the literature. (d) 250mg/kg tamoxifen induced recombination Sox9-CreERT2 R26R-confetti recombination in both hepatocytes and ducts. Hepatocytes and ducts were identified in each of three Confetti colors (mCerulean, RFP, and YFP). (e) High dose tamoxifen tracing in Sox9-CreERT2 R26R-Confetti mice injured with CDE for 3 weeks and a 1-week recovery results in marked hepatocytes (arrow head) and Opn+ ductal cells (arrow). (f) High dose tamoxifen tracing in Sox9-CreERT2 R26R-Confetti in mice injured with repeated CCl4 administration results in marked HNF4a+ hepatocytes (arrowhead) and ductal cells (arrow).

Supporting Information Figure 2: Sox9-CreERT2 marked ducts and periportal hepatocytes do not continuously “stream” in homeostasis

Sox9-CreERT2 R26R-lacZ mice were treated with (a) 32mg/kg tamoxifen or (b) 125mg/kg tamoxifen and maintained on normal chow for 6 months. Marked ducts (arrows) or periportal hepatocytes (arrowheads) did not progressively replace the bulk of the hepatocyte mass (bar = 200 μm, inset bar = 50μm). (c) Recombination in Sox9-CreERT2 R26R-Confetti mice treated with 125mg/kg tamoxifen showed recombination primarily in Sox9+ cells that did not give rise to hepatocytes after 3 months homeostasis. (d) Rare perioportal hepatocyte (arrowhead) maintained a periportal position and did not proliferate or replace the bulk of hepatocytes after 3 months homeostasis.

Supporting Information Figure 3: FACS-based analysis of Cre-marked Confetti cells

(a) Single-cell suspensions of liver nonparenchymal cells were FACS sorted with the following gating strategy to identify biliary progenitors. Gates were FSC/SSC, low trigger pulse width (not shown), propridium iodide negative (live cells, not shown), followed by MIC1-1C3+ Cd31- Cd45- Cd11b-. (b) MIC1-1C3 cells were then scored based on eYFP, mCerulean, and tdimer RFP status in a mouse treated with CDE and 125mg/kg tamoxifen (c) or CDE and 16mg/kg tamoxifen. (d) MIC1-1C3 ductal cells were unmarked in AAV-Ttr-Cre treated Confetti mice (and sesame oil treated Sox9-CreERT2 R26R-Confetti mice). (e) Gravity enriched hepatocytes were identified based on FSC/SSC and were PI-, OC2-2F8+, Cd31-, Cd45-. (f) Hepatocytes were interrogated for eYFP and tdimer RFP in injured Sox9-CreERT2 R26R-Confetti animals and (g) virally marked AAV8-Ttr-Cre marked hepatocytes (1-3% marked). (h) FACS-based quantification of Sox9-CreERT2 R26R-Confetti marked hepatocytes confirmed they were rare. (i) FACS-based analysis was used to confirm image based scoring of chimeric Fah-/- mice mTomato hepatocyte chimeras. The percentage of host mT-negative hepatocytes were plotted for each of three groups (mean± SEM, n=3 per group).

Supporting Information Figure 4: Sox9-CreERT2 marks phenotypically defined MIC1-1C3+ cells that form liver organoids

Non-parenchymal liver cells from Sox9-CreERT2 R26R-Confetti FACS were FACS sorted (YFP+ MIC1-1C3+ CD31- CD45- CD11b-) and seeded into organoid culture conditions. Organoids formed from YFP+ cells at equivalent rates in mice treated with (a) 32mg/kg or (b) 250mg/kg tamoxifen after 12 days culture. (c) Albumin mRNA expression in confetti+ MIC1-1C3+ CD31- CD45- CD11b- marked with high (250mg/kg) or low tamoxifen (32mg/kg) and differentiated towards a hepatic fate. (d) Clonally labeled ducts formed organoids retained the ability to form organoids in vitro after injury with CDE diet or (e) chronic CCl4 injury.

Supporting information 5: Sox9+ ducts rarely give rise to hepatocytes in acute CCl4 injury

(a) Experimental scheme for acute CCl4 tracing: Sox9-CreERT2 R26R-Confetti+/- mice were given a single acute toxic injury (1ul/kg CCl4) (b) 21 days recovery after injury, most Sox9-CreERT2 marked cells co-localized with ductal marker Opn (arrow = unique clone). (c) A single RFP+ Hnf4a+ cluster of Sox9-CreERT2 marked hepatocytes (arrow head) adjacent to a cell with a ductal Hnf4a- ductal cell is suggestive of a clonal relationship. (d) FACS quantification of Sox9-CreERT2 marked cells after acute CCl4 regeneration in phenotypically defined biliary cells (MIC1-1C3+) where approximately 4% of ducts are marked RFP or YFP+ with 32mg/kg tamoxifen. (e) OC2-2F8+ hepatocyte fractions show regeneration following CCl4 injury is associated with a 2.5-fold increase marked hepatocytes compared with in corn oil only. Less than 0.01% of hepatocytes were Confetti marked.

Supporting Information Figure 6: Hepatocyte transplantation into Fah-/- mice specifically replaced hepatocytes but not other cell types.

(a) 6 weeks after transplant with mTomato (red) marked mature donor hepatocytes, donor cells express hepatocyte marker Fah (green), but not duct marker A6 (white), demonstrating the detection of Fah-negative mTomato-negative host hepatocytes near the capsule (arrowhead), (bar = 50 μm). (b) mTomato donor marker (blue) surrounds Hnf4a+ hepatocytes (green) but not nonparenchymal cells nuclei (Hoescht only = red) c) CD31+ endothelium large vessels and sinusoids (d) CD11b+ mononuclear cells, and (e) GFAP+ stellate cells, and do not localize with mTomato, indicating these nonparenchymal cells are of host origin. (f) Fah immunostaining (green) shows donor hepatocyte repopulation was near complete but some Fah-/- hepatocytes (arrow) were retained in the periportal region adjacent to Opn+ cells (g) FACS-based analysis mTomato repopulated chimeric animals indicated no ductal cells were donor derived (0/4308 MIC1-1C3+).

Supporting Information Figure 7: Chimeric Fah mice treated with DDC and CDE do not require NTBC

(a) Schematic for testing the function of new progenitor derived hepatocytes in chimeric mice. Biliary and nonparenchymal cells are Fah-deficient. Fah-mutant hepatocyte require treatment with compound NTBC for normal survival. The liver stem cell model predicted NTBC rescue of progenitor-derived hepatocytes would be necessary for regeneration in when hepatocyte replication was disabled in oval cell/chronic injury. (b) Liver weights as a percentage of body weight were measured in chimeric animals treated with 10 weeks CCl4 injury with 1 week recovery or 3 weeks CDE. NTBC rescue of putative progenitor-derived hepatocytes was not required for chimeric animal survival or to maintain global measures of liver function such as body weight. (c) Body weight changes in chimeras treated with CDE injury (±NTBC) or NTBC only (d) chronic CCl4 injury and (e) DDC injury. (f) Quantification of body weight changes in cohorts of chimeric animals at the peak of oval cell injury. (g) FACS-based quantification of mTomato-negative hepatocytes in chimeras after DDC injury (n=3 per group). (h) Chimeric mice recovering from chronic CCl4 injury (2 hour BrdU pulse) showed BrdU incorporation in both host portal non-parenchymal cells (arrow) and hepatocytes (arrowhead). (i) Fah-immunohistochemistry in serial sections showed replicating hepatocytes (arrowhead) to be donor derived mature hepatocytes. (j) BrdU+ hepatocytes marked after 1 week BrdU administration in the recovery period following 10 weeks CCl4 . (k) Hepatocyte BrdU incorporation (2 hour pulse) in uninjured chimeras was observed in ∼1 in 25 high-powered fields.

Supporting Information Figure 8: AAV8-Ttr-Cre specificity and hepatocyte clonal dynamics

AAV8-Ttr-Cre induced recombination in R26R-Confetti+/- mice specifically in (a) Hnf4a+ hepatocytes but not (b) Osteopontin+ ducts. (c) Confetti marked clones after acute CCl4 (1ul/kg) mediated pericentral hepatocyte injury and 21 days regeneration (d) The cumulative percentage of clonally marked hepatocytes that are part of a large cluster increases following injury, demonstrating that mature hepatocytes are a common source of new hepatocytes. (e) Histogram representation of hepatocyte colony size following regeneration. (f) Distance of each Confetti marked clone to the nearest portal vein was measured at baseline (n=4) and after 3 or more months of homeostasis (n=2).

Supplementary Information Figure 9: NTBC rescues Fah-/- hepatocyte replication in regeneration from partial hepatectomy

(a) Ten days prior to 2/3 partial hepatectomy Fah-/- animals were assigned to continue receiving NTBC (8mg/L water) or taken off the drug. Partially repopulated Fah-/- hepatocyte chimeric mice were put back on NTBC at this time. Upon 2/3 partial heptectomy, BrdU was continously administered in the drinking water. Replication was assessed after 4 days partial hepatectomy. (b) Fah-/- animals on NTBC showed robust BrdU incorporation in parenchymal and non-parenchymal cells following regeneration from partial hepatectomy. (c) Fah-/- off NTBC showed BrdU incorporation primarily in non-prenchymal cells following partial hepatectomy due to hepatocyte replication blockade. (d) Chimeric Fah-/- animal put back on NTBC showed BrdU incorporation in parenchymal cells that (e) were both Fah-/- and Fah+/+ based on Fah-immunostaining in a serial section.

Supporting Information Figure 10: Extended recovery does not induce hepatic differentiation in Sox9-derived oval cells

(a) 32mg/kg tamoxifen was administered i.p. to Sox9CreERT2 R26R-Confetti mice at least two weeks prior to initiate of oval cell activation injury, as previously described (Fig. 2).

CDE injury was given for 3 weeks, chronic CCl4 and DDC injury were given for 5 weeks. For recovery, animals were given normal chow (5LOD diet) for 4-15 weeks, as indicated.

(b) Following CDE injury and 4 weeks recovery A6+ ductal proliferations persist. Confetti+ cells continue to colocalize with A6+ cells (200/200 clones, n=1 animal). (c) Following CCl4 injury and 4 weeks recovery limited ductal proliferation is observed. Confetti+ cells continue to colocalize with A6+ cells (75/75 clones, n=1 animal). (d) Following DDC injury and 4 or 15 weeks recovery, mild ductal proliferation still persists. Confetti+ cells were arranged in distinctly ductal morphology (200/200 clones, n=1 animal).

Supporting Information Figure 11: CreERT2 recombination continues a week after tamoxifen administration

(a) ROSACreERT2 were crossed with ROSAmT/mG mice to generate a mouse line that irreversibly switches from red to green fluorescence upon tamoxifen-mediated recombination. (b) In vitro tamoxifen administration to ROSACreERT2/mTmG primary hepatocytes switched cells from red to green fluorescence (bar =20 um). Fluorescence was monitored 2 and 6 days after tamoxifen treatment. (c) Transplantation experiment schematic: Fah-/- mice were given 100mg/kg i.p. tamoxifen (or vehicle) 1 or 7 days before intrasplenic transplant with ROSACreERT2/mTmG hepatocytes. NTBC water was withdrawn 1 day before transplant and 7 days were allowed for graft selection and fluorescent protein turnover. (d)The recombination rate in donor ROSACreERT2/mTmG hepatocytes was assessed by immunofluorescence in the livers of transplanted mice and (e) was quantified as the percentage of donor cells that were mGFP+.

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