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Human fetal liver cells for regulated ex vivo erythropoietin gene therapy.

El Filali E, Duijst S, Hiralall JK, Legrand N, van Gulik T, Hoekstra R, Seppen J - Mol Ther Methods Clin Dev (2014)

Bottom Line: Liver cell transplantation is, at this moment, not a feasible alternative because engraftment in the liver is poor.Human liver endothelial cells were subsequently transduced with a lentiviral autoregulatory erythropoietin expression vector.Our study shows the potential of human liver endothelial cells for long-term regulated gene therapy.

View Article: PubMed Central - PubMed

Affiliation: Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, University of Amsterdam , Amsterdam, The Netherlands.

ABSTRACT
Possible risks and lack of donor livers limit application of liver transplantation. Liver cell transplantation is, at this moment, not a feasible alternative because engraftment in the liver is poor. Furthermore, there is also shortage of cells suitable for transplantation. Fetal liver cells are able to proliferate in cell culture and could therefore present an alternative source of cells for transplantation. In this study, we investigated the utility of human fetal liver cells for therapeutic protein delivery. We transplanted human fetal liver cells in immunodeficient mice but were not able to detect engraftment of human hepatocytes. In contrast, transplantation of human adult hepatocytes led to detectable engraftment of hepatocytes in murine liver. Transplantation of fetal liver cells did lead to abundant reconstitution of murine liver with human endothelium, indicating that endothelial cells are the most promising cell type for ex vivo liver cell gene therapy. Human liver endothelial cells were subsequently transduced with a lentiviral autoregulatory erythropoietin expression vector. After transplantation in immunodeficient mice, these cells mediated long-term regulation of murine hematocrits. Our study shows the potential of human liver endothelial cells for long-term regulated gene therapy.

No MeSH data available.


Related in: MedlinePlus

Histological staining for hematopoietic and endothelial markers in murine liver transplanted with endothelial cells expressing Epo. (a) Sections of paraplast embedded human fetal livers or (b) murine livers transplanted with endothelial cells transduced with an autoregulatory Epo expression vector were stained with a specific antihuman Lyve 1 antibody. The murine liver was harvested 3 months after transplantation in a period the mouse did not receive doxycycline water. In the human fetal liver (gestation 14–18 weeks) that is shown as a positive control, Lyve1-positive sinusoidal endothelium lines islands of hematopoietic cells and hepatoblasts (arrow). In the transplanted murine liver, clusters of hematopoietic cells can be seen showing extramedullary hematopoiesis (asterisk). Occasionally, Lyve 1 human liver endothelial cells were also detected, (arrow). (c) Liver tissue from mice with a humanized immune system that is shown as a positive control or (d) mice transplanted with endothelial cells transduced with an autoregulatory Epo expression vector were stained for human CD45. The mouse transplanted with endothelial cells was harvested 4 months after transplantation in a period the mouse received doxycycline water. Human CD45-positive hematopoietic cells were detected in mice with humanized immune systems but not in mice transplanted with endothelial cells.
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fig3: Histological staining for hematopoietic and endothelial markers in murine liver transplanted with endothelial cells expressing Epo. (a) Sections of paraplast embedded human fetal livers or (b) murine livers transplanted with endothelial cells transduced with an autoregulatory Epo expression vector were stained with a specific antihuman Lyve 1 antibody. The murine liver was harvested 3 months after transplantation in a period the mouse did not receive doxycycline water. In the human fetal liver (gestation 14–18 weeks) that is shown as a positive control, Lyve1-positive sinusoidal endothelium lines islands of hematopoietic cells and hepatoblasts (arrow). In the transplanted murine liver, clusters of hematopoietic cells can be seen showing extramedullary hematopoiesis (asterisk). Occasionally, Lyve 1 human liver endothelial cells were also detected, (arrow). (c) Liver tissue from mice with a humanized immune system that is shown as a positive control or (d) mice transplanted with endothelial cells transduced with an autoregulatory Epo expression vector were stained for human CD45. The mouse transplanted with endothelial cells was harvested 4 months after transplantation in a period the mouse received doxycycline water. Human CD45-positive hematopoietic cells were detected in mice with humanized immune systems but not in mice transplanted with endothelial cells.

Mentions: Four months after transplantation, the mice were sacrificed and sections of spleen and liver were embedded in paraffin and stained for human Lyve 1 (Figure 3b). Interestingly, the transplanted mice showed signs of extramedullary hematopoiesis in liver en spleen (data not shown). For comparison, in Figure 3a, a section of human fetal liver with Lyve1-positive endothelium surrounding islands of hepatoblasts and hematopoiesis is shown. To determine whether the hematopoietic cells in the liver were of human or murine origin, we used an antibody specific for the human hematopoietic marker CD45. No CD45-positive cells were detected in our transplanted mice (Figure 3d). As a control, positive CD45 staining in a mouse with a humanized immune system is shown (Figure 3c). No other gross abnormalities were observed.


Human fetal liver cells for regulated ex vivo erythropoietin gene therapy.

El Filali E, Duijst S, Hiralall JK, Legrand N, van Gulik T, Hoekstra R, Seppen J - Mol Ther Methods Clin Dev (2014)

Histological staining for hematopoietic and endothelial markers in murine liver transplanted with endothelial cells expressing Epo. (a) Sections of paraplast embedded human fetal livers or (b) murine livers transplanted with endothelial cells transduced with an autoregulatory Epo expression vector were stained with a specific antihuman Lyve 1 antibody. The murine liver was harvested 3 months after transplantation in a period the mouse did not receive doxycycline water. In the human fetal liver (gestation 14–18 weeks) that is shown as a positive control, Lyve1-positive sinusoidal endothelium lines islands of hematopoietic cells and hepatoblasts (arrow). In the transplanted murine liver, clusters of hematopoietic cells can be seen showing extramedullary hematopoiesis (asterisk). Occasionally, Lyve 1 human liver endothelial cells were also detected, (arrow). (c) Liver tissue from mice with a humanized immune system that is shown as a positive control or (d) mice transplanted with endothelial cells transduced with an autoregulatory Epo expression vector were stained for human CD45. The mouse transplanted with endothelial cells was harvested 4 months after transplantation in a period the mouse received doxycycline water. Human CD45-positive hematopoietic cells were detected in mice with humanized immune systems but not in mice transplanted with endothelial cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362349&req=5

fig3: Histological staining for hematopoietic and endothelial markers in murine liver transplanted with endothelial cells expressing Epo. (a) Sections of paraplast embedded human fetal livers or (b) murine livers transplanted with endothelial cells transduced with an autoregulatory Epo expression vector were stained with a specific antihuman Lyve 1 antibody. The murine liver was harvested 3 months after transplantation in a period the mouse did not receive doxycycline water. In the human fetal liver (gestation 14–18 weeks) that is shown as a positive control, Lyve1-positive sinusoidal endothelium lines islands of hematopoietic cells and hepatoblasts (arrow). In the transplanted murine liver, clusters of hematopoietic cells can be seen showing extramedullary hematopoiesis (asterisk). Occasionally, Lyve 1 human liver endothelial cells were also detected, (arrow). (c) Liver tissue from mice with a humanized immune system that is shown as a positive control or (d) mice transplanted with endothelial cells transduced with an autoregulatory Epo expression vector were stained for human CD45. The mouse transplanted with endothelial cells was harvested 4 months after transplantation in a period the mouse received doxycycline water. Human CD45-positive hematopoietic cells were detected in mice with humanized immune systems but not in mice transplanted with endothelial cells.
Mentions: Four months after transplantation, the mice were sacrificed and sections of spleen and liver were embedded in paraffin and stained for human Lyve 1 (Figure 3b). Interestingly, the transplanted mice showed signs of extramedullary hematopoiesis in liver en spleen (data not shown). For comparison, in Figure 3a, a section of human fetal liver with Lyve1-positive endothelium surrounding islands of hepatoblasts and hematopoiesis is shown. To determine whether the hematopoietic cells in the liver were of human or murine origin, we used an antibody specific for the human hematopoietic marker CD45. No CD45-positive cells were detected in our transplanted mice (Figure 3d). As a control, positive CD45 staining in a mouse with a humanized immune system is shown (Figure 3c). No other gross abnormalities were observed.

Bottom Line: Liver cell transplantation is, at this moment, not a feasible alternative because engraftment in the liver is poor.Human liver endothelial cells were subsequently transduced with a lentiviral autoregulatory erythropoietin expression vector.Our study shows the potential of human liver endothelial cells for long-term regulated gene therapy.

View Article: PubMed Central - PubMed

Affiliation: Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, University of Amsterdam , Amsterdam, The Netherlands.

ABSTRACT
Possible risks and lack of donor livers limit application of liver transplantation. Liver cell transplantation is, at this moment, not a feasible alternative because engraftment in the liver is poor. Furthermore, there is also shortage of cells suitable for transplantation. Fetal liver cells are able to proliferate in cell culture and could therefore present an alternative source of cells for transplantation. In this study, we investigated the utility of human fetal liver cells for therapeutic protein delivery. We transplanted human fetal liver cells in immunodeficient mice but were not able to detect engraftment of human hepatocytes. In contrast, transplantation of human adult hepatocytes led to detectable engraftment of hepatocytes in murine liver. Transplantation of fetal liver cells did lead to abundant reconstitution of murine liver with human endothelium, indicating that endothelial cells are the most promising cell type for ex vivo liver cell gene therapy. Human liver endothelial cells were subsequently transduced with a lentiviral autoregulatory erythropoietin expression vector. After transplantation in immunodeficient mice, these cells mediated long-term regulation of murine hematocrits. Our study shows the potential of human liver endothelial cells for long-term regulated gene therapy.

No MeSH data available.


Related in: MedlinePlus