Citation

A reporter system for cell fusion in the injured mouse liver

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Abstract:

Background and Aims: In the fumaryl acetoacetate hydrolase (FAH) knockout mouse model of hereditary tyrosinemia, stable fusion between diseased hepatocytes and transplanted normal macrophages restores FAH enzyme function through poorly understood nuclear reprogramming events. Expression of FAH protein in fusion-derived hepatocytes is positively selected in vivo, leading to therapeutic liver repopulation. Whether cell fusion occurs during normal liver development, or in response to other liver injuries, is not known. Our aim was to develop a reporter system which would allow us to monitor cell fusion events in vivo and to facilitate analysis of changes in gene expression resulting from cell fusion.

Methods: Embryonic stem (ES) cell chimeric mice were prepared by blastocyst microinjection and embryo transfer. The ES cells carried a transgene encoding a Stop-Lox-Yellow Fluorescent Protein (StopLox-YFP) reporter, in which YFP expression is completely blocked until a regulatory sequence is removed by Cre recombinase. The embryonic day 4.5 blastocysts carried a transgene encoding Cre recombinase under the control of the albumin promoter. Fusion of a Cre-expressing blastocyst-derived hepatocyte with an ES-derived StopLox-YFP cell was therefore expected to activate YFP expression in an irreversible manner. Adult fusion reporter chimeric mice were treated with intraperitoneal injections of CCl4 (0.4 ml/kg, n=4) or mineral oil vehicle (n=3) once per week for six weeks, and were sacrificed one week after the final dose. Additional chimeric mice (n=4) served as untreated controls. Expression of YFP in the liver was detected by immunofluorescence in formalin-fixed paraffin sections.

Results: The overall ES contribution was estimated at 25-50% based on coat color chimerism. The livers of mice treated with CCl4 recovered with minimal fibrosis and inflammation one week after the final dose. Mineral oil vehicle induced an unexpected degree of steatosis and inflammation. Numerous YFP-positive hepatocytes were seen as single cells and small clusters in both CCl4-treated and vehicle-treated mice. Some YFP-positive fused cells contained as many as 3 nuclei. No YFP-expressing hepatocytes were found in untreated chimeric controls.

Conclusions: We found no evidence of cell fusion during normal liver development. However,hepatocyte fusion did occur in response to liver injury with CCl4 or mineral oil. This reporter system will allow us to evaluate hepatocyte fusion in response to other types of injury and will facilitate flow cytometric sorting of fused cells for analysis of changes in gene expression by microarray.
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Association:
Name: Connecticut's Stem Cell Research International Symposium
URL:
http://stemconn.org


Citation:
URL: http://citation.allacademic.com/meta/p318639_index.html
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MLA Citation:

Swenson, Scott., Przybylo, Jennifer., Mohamadnejad, Mehdi., Theise, Neil. and Krause, Diane. "A reporter system for cell fusion in the injured mouse liver" Paper presented at the annual meeting of the Connecticut's Stem Cell Research International Symposium, Omni Hotel, New Haven, CT, Mar 23, 2009 <Not Available>. 2014-11-29 <http://citation.allacademic.com/meta/p318639_index.html>

APA Citation:

Swenson, S. , Przybylo, J. , Mohamadnejad, M. , Theise, N. and Krause, D. , 2009-03-23 "A reporter system for cell fusion in the injured mouse liver" Paper presented at the annual meeting of the Connecticut's Stem Cell Research International Symposium, Omni Hotel, New Haven, CT <Not Available>. 2014-11-29 from http://citation.allacademic.com/meta/p318639_index.html

Publication Type: Poster
Review Method: Peer Reviewed
Abstract: Background and Aims: In the fumaryl acetoacetate hydrolase (FAH) knockout mouse model of hereditary tyrosinemia, stable fusion between diseased hepatocytes and transplanted normal macrophages restores FAH enzyme function through poorly understood nuclear reprogramming events. Expression of FAH protein in fusion-derived hepatocytes is positively selected in vivo, leading to therapeutic liver repopulation. Whether cell fusion occurs during normal liver development, or in response to other liver injuries, is not known. Our aim was to develop a reporter system which would allow us to monitor cell fusion events in vivo and to facilitate analysis of changes in gene expression resulting from cell fusion.

Methods: Embryonic stem (ES) cell chimeric mice were prepared by blastocyst microinjection and embryo transfer. The ES cells carried a transgene encoding a Stop-Lox-Yellow Fluorescent Protein (StopLox-YFP) reporter, in which YFP expression is completely blocked until a regulatory sequence is removed by Cre recombinase. The embryonic day 4.5 blastocysts carried a transgene encoding Cre recombinase under the control of the albumin promoter. Fusion of a Cre-expressing blastocyst-derived hepatocyte with an ES-derived StopLox-YFP cell was therefore expected to activate YFP expression in an irreversible manner. Adult fusion reporter chimeric mice were treated with intraperitoneal injections of CCl4 (0.4 ml/kg, n=4) or mineral oil vehicle (n=3) once per week for six weeks, and were sacrificed one week after the final dose. Additional chimeric mice (n=4) served as untreated controls. Expression of YFP in the liver was detected by immunofluorescence in formalin-fixed paraffin sections.

Results: The overall ES contribution was estimated at 25-50% based on coat color chimerism. The livers of mice treated with CCl4 recovered with minimal fibrosis and inflammation one week after the final dose. Mineral oil vehicle induced an unexpected degree of steatosis and inflammation. Numerous YFP-positive hepatocytes were seen as single cells and small clusters in both CCl4-treated and vehicle-treated mice. Some YFP-positive fused cells contained as many as 3 nuclei. No YFP-expressing hepatocytes were found in untreated chimeric controls.

Conclusions: We found no evidence of cell fusion during normal liver development. However,hepatocyte fusion did occur in response to liver injury with CCl4 or mineral oil. This reporter system will allow us to evaluate hepatocyte fusion in response to other types of injury and will facilitate flow cytometric sorting of fused cells for analysis of changes in gene expression by microarray.


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