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• W2568971054 abstract "•Bone marrow cell migration after liver hepatectomy is key for liver regeneration•Migrated bone marrow cells fuse with hepatocytes•Hybrids are essential for liver regeneration•Mathematical modeling unveils the hybrid function for liver regeneration In rodents and humans, the liver can efficiently restore its mass after hepatectomy. This is largely attributed to the proliferation and cell cycle re-entry of hepatocytes. On the other hand, bone marrow cells (BMCs) migrate into the liver after resection. Here, we find that a block of BMC recruitment into the liver severely impairs its regeneration after the surgery. Mobilized hematopoietic stem and progenitor cells (HSPCs) in the resected liver can fuse with hepatocytes, and the hybrids proliferate earlier than the hepatocytes. Genetic ablation of the hybrids severely impairs hepatocyte proliferation and liver mass regeneration. Mathematical modeling reveals a key role of bone marrow (BM)-derived hybrids to drive proliferation in the regeneration process, and predicts regeneration efficiency in experimentally non-testable conditions. In conclusion, BM-derived hybrids are essential to trigger efficient liver regeneration after hepatectomy. In rodents and humans, the liver can efficiently restore its mass after hepatectomy. This is largely attributed to the proliferation and cell cycle re-entry of hepatocytes. On the other hand, bone marrow cells (BMCs) migrate into the liver after resection. Here, we find that a block of BMC recruitment into the liver severely impairs its regeneration after the surgery. Mobilized hematopoietic stem and progenitor cells (HSPCs) in the resected liver can fuse with hepatocytes, and the hybrids proliferate earlier than the hepatocytes. Genetic ablation of the hybrids severely impairs hepatocyte proliferation and liver mass regeneration. Mathematical modeling reveals a key role of bone marrow (BM)-derived hybrids to drive proliferation in the regeneration process, and predicts regeneration efficiency in experimentally non-testable conditions. In conclusion, BM-derived hybrids are essential to trigger efficient liver regeneration after hepatectomy. Mathematical modeling is a powerful tool to describe complex biological processes, formalize interactions between components, analyze temporal dynamics, and predict the effects of perturbations (Kitano, 2002Kitano H. Computational systems biology.Nature. 2002; 420: 206-210Crossref PubMed Scopus (1583) Google Scholar). Modeling has been used to describe liver functions and dynamics in mammals under normal and pathological conditions (Cook et al., 2015Cook D. Ogunnaike B.A. Vadigepalli R. Systems analysis of non-parenchymal cell modulation of liver repair across multiple regeneration modes.BMC Syst. Biol. 2015; 9: 71Crossref PubMed Scopus (19) Google Scholar, Furchtgott et al., 2009Furchtgott L.A. Chow C.C. Periwal V. A model of liver regeneration.Biophys. J. 2009; 96: 3926-3935Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, Holzhütter et al., 2012Holzhütter H.G. Drasdo D. Preusser T. Lippert J. Henney A.M. The virtual liver: a multidisciplinary, multilevel challenge for systems biology.Wiley Interdiscip. Rev. Syst. Biol. Med. 2012; 4: 221-235Crossref PubMed Scopus (88) Google Scholar, Periwal et al., 2014Periwal V. Gaillard J.R. Needleman L. Doria C. Mathematical model of liver regeneration in human live donors.J. Cell. Physiol. 2014; 229: 599-606Crossref PubMed Scopus (13) Google Scholar). The liver is the main detoxifying organ of the body, which can be injured by ingested toxins and infections. In response to these insults, hepatocytes can proliferate (Michalopoulos and DeFrances, 1997Michalopoulos G.K. DeFrances M.C. Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2900) Google Scholar), and regeneration of the liver has evolved as a protective mechanism (Taub, 2004Taub R. Liver regeneration: from myth to mechanism.Nat. Rev. Mol. Cell Biol. 2004; 5: 836-847Crossref PubMed Scopus (1268) Google Scholar). Indeed, the mammalian liver displays a high regeneration potential (Fausto et al., 2006Fausto N. Campbell J.S. Riehle K.J. Liver regeneration.Hepatology. 2006; 43: S45-S53Crossref PubMed Scopus (1265) Google Scholar, Michalopoulos and DeFrances, 1997Michalopoulos G.K. DeFrances M.C. Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2900) Google Scholar, Taub, 2004Taub R. Liver regeneration: from myth to mechanism.Nat. Rev. Mol. Cell Biol. 2004; 5: 836-847Crossref PubMed Scopus (1268) Google Scholar), and this phenomenon was described in rats a long time ago through the two-thirds partial hepatectomy model (Higgins and Anderson, 1931Higgins G.M. Anderson R.M. Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal.Arch. Pathol. (Chic). 1931; 12: 186-202Google Scholar). After partial hepatectomy, the remaining lobes grow and liver mass is restored in approximately 1 week in rodents (Duncan et al., 2009Duncan A.W. Dorrell C. Grompe M. Stem cells and liver regeneration.Gastroenterology. 2009; 137: 466-481Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). The regeneration mechanism is largely attributed to the re-entry of the hepatocytes into the cell cycle and their proliferation (Fausto et al., 2006Fausto N. Campbell J.S. Riehle K.J. Liver regeneration.Hepatology. 2006; 43: S45-S53Crossref PubMed Scopus (1265) Google Scholar, Michalopoulos, 2007Michalopoulos G.K. Liver regeneration.J. Cell. Physiol. 2007; 213: 286-300Crossref PubMed Scopus (1154) Google Scholar), which peaks 48 hr after resection in mice (Miyaoka et al., 2012Miyaoka Y. Ebato K. Kato H. Arakawa S. Shimizu S. Miyajima A. Hypertrophy and unconventional cell division of hepatocytes underlie liver regeneration.Curr. Biol. 2012; 22: 1166-1175Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Cooperative signals induced by growth factors (such as hepatocyte, transforming, and epidermal growth factors, insulin, and glucagons) and cytokines (such as tumor necrosis factor and interleukin 6) are thought to be responsible for hepatocyte re-entry into the cell cycle, DNA replication, proliferation, and consequent liver mass regeneration (Costa et al., 2003Costa R.H. Kalinichenko V.V. Holterman A.X. Wang X. Transcription factors in liver development, differentiation, and regeneration.Hepatology. 2003; 38: 1331-1347Crossref PubMed Scopus (348) Google Scholar). However, there are still many unresolved key aspects in this process. The cell volume of hepatocytes enlarges (Gentric et al., 2012Gentric G. Celton-Morizur S. Desdouets C. Polyploidy and liver proliferation.Clin. Res. Hepatol. Gastroenterol. 2012; 36: 29-34Crossref PubMed Scopus (55) Google Scholar, Miyaoka et al., 2012Miyaoka Y. Ebato K. Kato H. Arakawa S. Shimizu S. Miyajima A. Hypertrophy and unconventional cell division of hepatocytes underlie liver regeneration.Curr. Biol. 2012; 22: 1166-1175Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar), and there is a massive increase of hematopoietic stem cells (HSCs) in the peripheral blood and in the liver itself (De Silvestro et al., 2004De Silvestro G. Vicarioto M. Donadel C. Menegazzo M. Marson P. Corsini A. Mobilization of peripheral blood hematopoietic stem cells following liver resection surgery.Hepatogastroenterology. 2004; 51: 805-810PubMed Google Scholar, Fujii et al., 2002Fujii H. Hirose T. Oe S. Yasuchika K. Azuma H. Fujikawa T. Nagao M. Yamaoka Y. Contribution of bone marrow cells to liver regeneration after partial hepatectomy in mice.J. Hepatol. 2002; 36: 653-659Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, Lemoli et al., 2006Lemoli R.M. Catani L. Talarico S. Loggi E. Gramenzi A. Baccarani U. Fogli M. Grazi G.L. Aluigi M. Marzocchi G. et al.Mobilization of bone marrow-derived hematopoietic and endothelial stem cells after orthotopic liver transplantation and liver resection.Stem Cells. 2006; 24: 2817-2825Crossref PubMed Scopus (79) Google Scholar), whose role is not clear. In addition, there is some diverging evidence indicating that bone marrow (BM)-derived cells can either transdifferentiate in vivo in the mouse liver (Alison et al., 2000Alison M.R. Poulsom R. Jeffery R. Dhillon A.P. Quaglia A. Jacob J. Novelli M. Prentice G. Williamson J. Wright N.A. Hepatocytes from non-hepatic adult stem cells.Nature. 2000; 406: 257Crossref PubMed Scopus (959) Google Scholar, Lagasse et al., 2000Lagasse E. Connors H. Al-Dhalimy M. Reitsma M. Dohse M. Osborne L. Wang X. Finegold M. Weissman I.L. Grompe M. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo.Nat. Med. 2000; 6: 1229-1234Crossref PubMed Scopus (2148) Google Scholar) or can fuse with hepatocytes in fumarylacetoacetate hydrolase (Fah)-deficient mice (Vassilopoulos et al., 2003Vassilopoulos G. Wang P.R. Russell D.W. Transplanted bone marrow regenerates liver by cell fusion.Nature. 2003; 422: 901-904Crossref PubMed Scopus (1185) Google Scholar, Wang et al., 2003Wang X. Willenbring H. Akkari Y. Torimaru Y. Foster M. Al-Dhalimy M. Lagasse E. Finegold M. Olson S. Grompe M. Cell fusion is the principal source of bone-marrow-derived hepatocytes.Nature. 2003; 422: 897-901Crossref PubMed Scopus (1460) Google Scholar). Here, using modeling and experimental approaches, we prove a crucial role of bone marrow cells (BMCs) and of BM-hepatocyte hybrids in the dynamics and efficiency of mouse liver regeneration upon 30% and 70% partial hepatectomy. A mathematical model, fitted on experimental data, unveils the critical role of BMC recruitment and hybrid formation in enhancing proliferation and, ultimately, liver regeneration. In earlier work, a mathematical model for white rat liver regeneration upon partial hepatectomy was proposed (Furchtgott et al., 2009Furchtgott L.A. Chow C.C. Periwal V. A model of liver regeneration.Biophys. J. 2009; 96: 3926-3935Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), which incorporates the main phenomenology and underlying signaling. Similarly, the mathematical formalism of our delay differential equations (DDEs) captures the rate of change in cell numbers, considering the three populations previously suggested to contribute to liver regeneration (Fausto et al., 2006Fausto N. Campbell J.S. Riehle K.J. Liver regeneration.Hepatology. 2006; 43: S45-S53Crossref PubMed Scopus (1265) Google Scholar): quiescent (Q), primed to replicate (P), and replicating (R) cells (Figure 1A). Coupled to cellular equations (Figure S1A), molecular equations describe immediate-early genes, cytokines, and growth factors that, activated upon liver resection, determine the transition among cell states (Figure S1B; Supplemental Experimental Procedures). The premise of our model is the focus on regeneration dynamics rather than on cellular species. Thus, we adapted the phenomenological parameters in the cellular equations, whereas the molecular equations and the related parameters were kept as intact as possible (Table S1). Notably, the same approach has been successfully used in adapting the rat model (Furchtgott et al., 2009Furchtgott L.A. Chow C.C. Periwal V. A model of liver regeneration.Biophys. J. 2009; 96: 3926-3935Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) to reproduce data from humans (Periwal et al., 2014Periwal V. Gaillard J.R. Needleman L. Doria C. Mathematical model of liver regeneration in human live donors.J. Cell. Physiol. 2014; 229: 599-606Crossref PubMed Scopus (13) Google Scholar) because the biochemistry of liver regeneration is probably similar in different mammals. To adapt the rat model, we noticed that, while in the rat, hepatocyte proliferation starts soon after hepatectomy (Furchtgott et al., 2009Furchtgott L.A. Chow C.C. Periwal V. A model of liver regeneration.Biophys. J. 2009; 96: 3926-3935Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), in mice, the proliferation is delayed and peaks at 48 hr (Miyaoka et al., 2012Miyaoka Y. Ebato K. Kato H. Arakawa S. Shimizu S. Miyajima A. Hypertrophy and unconventional cell division of hepatocytes underlie liver regeneration.Curr. Biol. 2012; 22: 1166-1175Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, Weglarz and Sandgren, 2000Weglarz T.C. Sandgren E.P. Timing of hepatocyte entry into DNA synthesis after partial hepatectomy is cell autonomous.Proc. Natl. Acad. Sci. USA. 2000; 97: 12595-12600Crossref PubMed Scopus (78) Google Scholar). As expected, 24 hr upon 70% resection in wild-type mice, liver cells did not proliferate (Figure S1C) (Shu et al., 2009Shu R.Z. Zhang F. Wang F. Feng D.C. Li X.H. Ren W.H. Wu X.L. Yang X. Liao X.D. Huang L. et al.Adiponectin deficiency impairs liver regeneration through attenuating STAT3 phosphorylation in mice.Lab. Invest. 2009; 89: 1043-1052Crossref PubMed Scopus (32) Google Scholar). We evaluated liver mass regeneration 7 days after resection (Figure 1B, no AMD3100) because this is a standard time range to analyze regeneration (Zhang et al., 2015Zhang Y. Desai A. Yang S.Y. Bae K.B. Antczak M.I. Fink S.P. Tiwari S. Willis J.E. Williams N.S. Dawson D.M. et al.Tissue regeneration. Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration.Science. 2015; 348: aaa2340Crossref PubMed Scopus (173) Google Scholar). Interestingly, we observed a small but significant (p < 0.0001 between post-hepatectomy and day 1) increase in liver mass at day 1 (Figure 1B, day 1 no AMD3100) before cycling cells appeared (Figure S1C). This was likely due to the recruitment of hematopoietic cells in early stages of regeneration (De Silvestro et al., 2004De Silvestro G. Vicarioto M. Donadel C. Menegazzo M. Marson P. Corsini A. Mobilization of peripheral blood hematopoietic stem cells following liver resection surgery.Hepatogastroenterology. 2004; 51: 805-810PubMed Google Scholar, Lemoli et al., 2006Lemoli R.M. Catani L. Talarico S. Loggi E. Gramenzi A. Baccarani U. Fogli M. Grazi G.L. Aluigi M. Marzocchi G. et al.Mobilization of bone marrow-derived hematopoietic and endothelial stem cells after orthotopic liver transplantation and liver resection.Stem Cells. 2006; 24: 2817-2825Crossref PubMed Scopus (79) Google Scholar). To confirm this hypothesis, we applied 70% liver resection to a group of transgenic mice expressing the yellow fluorescent protein (YFP) from the Rosa26-LoxP-stop-LoxP-YFP allele in the hematopoietic cells (BMYFP) (Figure 1C). We found up to 30% of YFP+ cells in the liver, indicating a massive recruitment of hematopoietic cells within 24 hr from surgery (Figures 1D and S1D). Next, to determine the identity of the recruited YFP+ cells, we examined the expression of markers of mature circulating blood cells or bone-marrow-derived progenitors. YFP+ cells expressed HSPC (c-kit+/sca1+) and granulocyte monocyte progenitor (GMP) (c-Kit+/Sca1−/Cd34+/Cd16.32+) markers (Figure S1E). In contrast, we excluded recruitment of cells from the peripheral blood because lineage-positive cells, such as B (B220+), T (Cd3+), and NK (CD49b+/CD3− and CD49b+/CD3+) cells, and macrophages (CD11b+ and CD11b+/F4-80+) did not increase into the resected liver after hepatectomy (Figure S1F), suggesting that recruited YFP+ cells include mostly BMCs. Therefore, we changed the rat model to account for both the role that BMC mobilization can play in liver regeneration in a mouse and the different timing of hepatocyte proliferation and regeneration. We included an explicit term for BMC recruitment, and added two time delays (τ and θ) between the Q and P states and the P and R states (Figures 1A and S1A). We fitted the mathematical model to time-courses (7 day experiments) of wild-type mice that underwent hepatectomy; the dynamics of transition among the Q, P, and R states depend on BMC recruitment. The fitting accurately matches experimental proliferation and regeneration dynamics (Figures 1E and 1F). C-X-C motif chemokine receptor type 4 (CXCR4) and its ligand, SDF-1/CXCL12 (stromal cell-derived factor 1/C-X-C motif chemokine 12), are essential for the mobilization and migration of BMCs from the niche (Dalakas et al., 2005Dalakas E. Newsome P.N. Harrison D.J. Plevris J.N. Hematopoietic stem cell trafficking in liver injury.FASEB J. 2005; 19: 1225-1231Crossref PubMed Scopus (105) Google Scholar, Hatch et al., 2002Hatch H.M. Zheng D. Jorgensen M.L. Petersen B.E. SDF-1alpha/CXCR4: a mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats.Cloning Stem Cells. 2002; 4: 339-351Crossref PubMed Scopus (221) Google Scholar, Kollet et al., 2003Kollet O. Shivtiel S. Chen Y.Q. Suriawinata J. Thung S.N. Dabeva M.D. Kahn J. Spiegel A. Dar A. Samira S. et al.HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver.J. Clin. Invest. 2003; 112: 160-169Crossref PubMed Scopus (557) Google Scholar). Thus, to investigate if the recruitment of BMCs into the liver was critical for its regeneration, we analyzed BMC recruitment after 70% resection in the CXCR4fl/fl/Vav-CRE/R26Y model, which carries BMCs deleted for CXCR4 and expressing YFP (BMYFP/CXCR4−/−) (Figure 2A). Of note, CXCR4fl/fl-VavCRE mice are normal and fertile and do not show apparent phenotypic defects, which could be ascribed to a bone marrow dysfunction. Indeed, it has been shown that Flt3-LSK cells in CXCR4−/− mice are in a normal number as compared to wild-type mice and sustained long-term hematopoiesis (Nie et al., 2008Nie Y. Han Y.C. Zou Y.R. CXCR4 is required for the quiescence of primitive hematopoietic cells.J. Exp. Med. 2008; 205: 777-783Crossref PubMed Scopus (298) Google Scholar). Moreover, no major differences were found in the number of HSPCs in the fetal liver of CXCR4−/− E14.5 embryos as compared to wild-type mice (Foudi et al., 2006Foudi A. Jarrier P. Zhang Y. Wittner M. Geay J.F. Lecluse Y. Nagasawa T. Vainchenker W. Louache F. Reduced retention of radioprotective hematopoietic cells within the bone marrow microenvironment in CXCR4-/- chimeric mice.Blood. 2006; 107: 2243-2251Crossref PubMed Scopus (97) Google Scholar). As opposed to the BMYFP wild-type mice, we observed a massive impairment of YFP+ BMC recruitment in BMYFP/CXCR4−/− (Figures 2B and S2A). Importantly, liver regeneration in BMCXCR4−/− animals was severely compromised and, up to 30 days after resection, BMCXCR4−/− mice could not entirely restore their liver mass (Figure 2C). Moreover, the block of liver mass regeneration was associated with an impairment of liver cell proliferation; the mitotic index and Ki67+ cells measured in liver sections were drastically reduced 3 days after hepatectomy in BMCXCR4−/− mice (Figures 2D, 2E, S2B, and S2C). Reduction of proliferation was confirmed by fluorescence-activated cell sorting (FACS) analysis, although it was recovered at late time points after the surgery, likely representing a compensatory effect through late liver parenchymal cell replication (Figures S2D and S2E). Model fitting confirmed the crucial role for BMCs in triggering the proliferation and, consequently, the regeneration processes. When reproducing regeneration and proliferation dynamics in BMCXCR4−/− mice (Figures S2F and S2G), kQ (the parameter governing the propensity of cells to become primed to proliferate) was decreased, whereas kreq and kR (the parameters describing the return to the quiescent state), as well as the two delays (τ and θ), were increased as compared to their values in the control conditions (Figure 2F; Table S1). Hence, by removing BMCs from the system, the transition of cells into a proliferative state is delayed and less effective, the transition from the primed to the replicating state is also delayed, and the sensitivity to requiescence signals is increased. Importantly, after simulating the model for a longer time (30 days), incomplete regeneration was observed in BMCXCR4−/− mice (Figure 2G). Given that a small fraction of BMC population persists in BMCXCR4−/− mice (Figure 2B), the mathematical model was used to predict regeneration dynamics in the case of more severe reduction of BMCs. We found that the strength and timing of regeneration were further impaired compared to the actual experimental observations (Figure 2H). Finally, we used the model to predict liver regeneration dynamics upon perturbation of BMC migration. Experimentally, it was not possible to assess for how long BMCs were recruited during the whole regeneration process. Thus, we ran simulations, stopping BMC recruitment 12 hr after resection. This resulted in a considerably impaired regeneration profile (Figure 2I), thereby suggesting that BMC recruitment should take place for at least 12 hr after surgery. Cell fusion is a well-known developmental process and an essential mechanism of regeneration after an injury (Johansson et al., 2008Johansson C.B. Youssef S. Koleckar K. Holbrook C. Doyonnas R. Corbel S.Y. Steinman L. Rossi F.M. Blau H.M. Extensive fusion of haematopoietic cells with Purkinje neurons in response to chronic inflammation.Nat. Cell Biol. 2008; 10: 575-583Crossref PubMed Scopus (189) Google Scholar, Lluis and Cosma, 2010Lluis F. Cosma M.P. Cell-fusion-mediated somatic-cell reprogramming: a mechanism for tissue regeneration.J. Cell. Physiol. 2010; 223: 6-13PubMed Google Scholar, Sanges et al., 2013Sanges D. Romo N. Simonte G. Di Vicino U. Tahoces A.D. Fernández E. Cosma M.P. Wnt/β-catenin signaling triggers neuron reprogramming and regeneration in the mouse retina.Cell Rep. 2013; 4: 271-286Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, Sanges et al., 2016Sanges D. Simonte G. Di Vicino U. Romo N. Pinilla I. Nicolás M. Cosma M.P. Reprogramming Müller glia via in vivo cell fusion regenerates murine photoreceptors.J. Clin. Invest. 2016; 126: 3104-3116Crossref PubMed Scopus (48) Google Scholar, Sullivan and Eggan, 2006Sullivan S. Eggan K. The potential of cell fusion for human therapy.Stem Cell Rev. 2006; 2: 341-349Crossref PubMed Scopus (26) Google Scholar, Altarche-Xifro et al., 2016Altarche-Xifro W. di Vicino U. Muñoz-Martin M.I. Bortolozzi A. Bové J. Vila M. Cosma M.P. Functional rescue of dopaminergic neuron loss in Parkinson’s disease mice after transplantation of hematopoietic stem and progenitor cells.EBioMedicine. 2016; 8: 83-95Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). We therefore aimed to investigate whether mobilized BMCs could fuse with liver cells and promote regeneration after hepatectomy. We subjected 70% liver resection to a group of chimeric mice carrying the R26Y transgene, in which the BM was replaced with a double transgenic CAG-RFP/VAV-CRE BM from donor mice (R26Y-BMRFP/CRE) (Figure 3A). Vav-Cre is expressed only in the BM of transgenic mice (Stadtfeld and Graf, 2005Stadtfeld M. Graf T. Assessing the role of hematopoietic plasticity for endothelial and hepatocyte development by non-invasive lineage tracing.Development. 2005; 132: 203-213Crossref PubMed Scopus (184) Google Scholar); furthermore, we excluded its expression in liver cells. We found a limited number of positive cells in sections, which likely corresponded to liver resident hematopoietic cells (Figure S3A). Hepatectomy was performed 6 weeks after BM repopulation when peripheral blood and bone marrow chimerisms were around 30% and 42%, respectively (Figure S3B). Up to 3 days after resection, we found that 10%–15% of recruited RFP+ cells in the liver were also YFP+, indicating fusion events. This percentage increased to ∼50% from 7 days up to 3 weeks after surgery, whereas recruited RFP+ cells decreased (Figures 3B, left plot, 3C, and S3C), suggesting an increase of hybrids and a decrease of BMCs in time in the resected liver. Importantly, we excluded major cell fusion events between BMCs and non-parenchymal liver cells (Figures 3B, right plot, and S3D). As the control experiment, to exclude a possible leakiness of the R26Y transgene and therefore expression of YFP independently of Cre-mediated STOP codon excision, we transplanted R26Y mice with a BMRFP (not expressing Cre). After hepatectomy, we observed neither YFP+/RFP+ nor YFP+/RFP− cells in R26Y-BMRFP chimeric mice (Figure S3C). Furthermore, we also excluded formation of hybrids in the BM and peripheral blood of R26Y-BMRFP/CRE (Figure S3B). The increase of the hybrids at different days after surgery was also evident by counting the number of YFP+ cells for each section at different days after resection (Figure 3D). These results were also confirmed by immunohistochemistry on sections (Figure 3E). Of note, we found several YFP+ binucleated cells (arrows in Figure 3D). The majority of the hybrids (YFP+/RFP+ population) were positive for markers of HSPCs (c-kit+/sca-1+) 1 day after hepatectomy (Figures 3F and S3E) and for the hepatocyte markers Albumin, hepatocyte nuclear factor 1 (Hnf1), and hepatocyte nuclear factor 4 alpha (HNF4α) from 1 day up to 21 days after surgery, indicating fusion of HSPCs with hepatocytes (Figures 3G and S3F). We confirmed these results by performing HNF4α and c-kit immunostaining on YFP+/RFP+ hybrids sorted from the livers of R26Y-BMRFP/CRE 24 hrs after surgery. We found cells that were positive for both HNF4α and c-Kit expression (Figure 3H). Furthermore, 24 hr after surgery, the hybrids expressed the cycling cell marker Ki67 and were polyploid (Figures 3I, S3G, and S3H). In contrast, the unfused BMCs (RFP+/YFP−) and parenchymal liver cells (PCs) did not express Ki67 at this time point (Figures 3I, S1C, and S3G). In order to further prove whether the hepatocytes were BMC fusion partners, we used the hepatocyte-specific Albumin-CRE chimeric mice (Postic et al., 1999Postic C. Shiota M. Niswender K.D. Jetton T.L. Chen Y. Moates J.M. Shelton K.D. Lindner J. Cherrington A.D. Magnuson M.A. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase.J. Biol. Chem. 1999; 274: 305-315Crossref PubMed Scopus (1024) Google Scholar) carrying the R26Y bone marrow (AlbCRE-BMR26Y) (Figure 4A). The hybrids largely increased at different days after surgery (Figures 4B–4D), indicating fusion of BMCs with hepatocytes. Finally, we observed that lineage-depleted bone marrow cells that are enriched for HSPCs could also fuse in vitro with PCs purified after liver hepatectomy, resulting in hybrids, which expressed HNF4α (Figures S4A and S4B). In contrast, lineage-positive cells did not fuse efficiently in vitro and neither did their fusion capability increase after hepatectomy (Figure S4A). Overall, these results show that HSPCs can fuse with hepatocytes after liver resection and the hybrids have already entered the cell cycle 24 hr after the surgery, at a time when hepatocytes are still in the G0 resting phase of the cell cycle. Because we showed that BMC migration in the liver of BMCXCR4−/− animals after hepatectomy is impaired, we then aimed to investigate whether this block of BMC recruitment affects hybrid formation. Thus, we injected the CXCR4 antagonist AMD3100 (De Clercq, 2009De Clercq E. The AMD3100 story: the path to the discovery of a stem cell mobilizer (Mozobil).Biochem. Pharmacol. 2009; 77: 1655-1664Crossref PubMed Scopus (240) Google Scholar) into a group of chimeric R26Y-BMRFP/CRE mice, which received 70% liver resection and were analyzed from 24 hr to 7 days after the surgery (Figure 4E). In the group of AMD3100-treated mice, migration of RFP+ BMCs (Figure 4F) and fusion of the recruited BMCs with the hepatocytes (YFP+ over RFP+ cells) were largely reduced after the hepatectomy (Figures 4G and S4C). Interestingly, regeneration was largely impaired because liver mass corrected on body weight did not reach the level observed in the untreated mice (Figure 1B). Fitting the mathematical model on AMD3100-treated versus control mice, regeneration data confirmed impaired transition into the proliferative and replicating states, as in the comparison between BMCXCR4−/− and BMCXCR4fl/fl mice discussed above (Figure S4D; Table S1). Of note, due to the lack of BMC recruitment in BMYFP/CXCR4−/− mice and the reduced fusion after CXCR4 inhibition by AMD3100, polyploidy was accordingly impaired in BMCXCR4−/− mice (Figure S4E), indicating the major contribution of BMC recruitment and bone-marrow-derived hybrids to liver regeneration after hepatectomy. To definitively prove that the hybrids play an essential role to induce liver regeneration, we used a mouse model that allows ablation of the hybrids in the liver after surgery. We obtained chimeric mice carrying the Rosa26-LoxP-STOP-LoxP-DTR (R26-diphtheria toxin receptor) transgene, in whom the BM was replaced with the CAG-RFP/VAV-CRE BM from donor mice (R26DTR-BMRFP/CRE) (Buch et al., 2005Buch T. Heppner F.L. Tertilt C. Heinen T.J. Kremer M. Wunderlich F.T. Jung S. Waisman A. A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration.Nat. Methods. 2005; 2: 419-426Crossref PubMed Scopus (603) Google Scholar). Upon 70% liver resection, BM-derived hybrids expressed both RFP and DTR, making them sensitive to diphtheria toxin injection; thus, they were selectively ablated (Figure 5A). We found a substantial, although not complete, ablation of the hybrids (DTR+ over RFP+ cells) in the liver at the different days after surgery, which, as expected, paralleled the reduction of RFP+ liver cells (Figures 4B and 4C). Leakiness of the R26DTR promoter was excluded because neither DTR+/RFP+ nor DTR+/RFP− cells were found in R26DTR-BMRFP mice (not expressing Cre) (Figure S5A). Importantly, we noticed a significant impairment of liver regeneration in the group of toxin-treated mice (Figure 5D). Accordingly, we found a severe reduction of the cell mitotic index, Ki67+ cell number, and polyploidy in toxin-treated mic" @default.
• W2568971054 created "2017-01-13" @default.
• W2568971054 creator A5010265948 @default.
• W2568971054 creator A5013604041 @default.
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• W2568971054 date "2017-01-01" @default.
• W2568971054 modified "2023-10-17" @default.
• W2568971054 title "Modeling Dynamics and Function of Bone Marrow Cells in Mouse Liver Regeneration" @default.
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