Limits...
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

Long-term doxycycline regulation of hematocrit. Mice were transplanted with 50.000 human liver endothelial cells transduced with the autoregulatory Epo expression vector at day 0 (n = 8). Blood was sampled every 2 weeks. Doxycycline was administered in the drinking water at 2, 12, and 20 weeks after cell transplantation and continued for 2 weeks as indicated by the solid bars. Top panel: average hematocrit levels are shown on the Y-axis in PVC (packed cell volume). Up to 16 weeks n = 8, up to 28 weeks, n = 4. Bottom panel: the amount of rat erythropoietin in transplanted mouse serum was determined by ELISA. Following induction by doxycycline administration in the drinking water, the concentration of erythropoietin increased significantly, P < 0.0001. Mean values are shown ± SD. (n = 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4362349&req=5

fig2: Long-term doxycycline regulation of hematocrit. Mice were transplanted with 50.000 human liver endothelial cells transduced with the autoregulatory Epo expression vector at day 0 (n = 8). Blood was sampled every 2 weeks. Doxycycline was administered in the drinking water at 2, 12, and 20 weeks after cell transplantation and continued for 2 weeks as indicated by the solid bars. Top panel: average hematocrit levels are shown on the Y-axis in PVC (packed cell volume). Up to 16 weeks n = 8, up to 28 weeks, n = 4. Bottom panel: the amount of rat erythropoietin in transplanted mouse serum was determined by ELISA. Following induction by doxycycline administration in the drinking water, the concentration of erythropoietin increased significantly, P < 0.0001. Mean values are shown ± SD. (n = 6).

Mentions: We have shown previously in rats that in vivo intramuscular administration of the doxycycline regulated erythropoietin expressing lentiviral vector TREAutoR4rEPO resulted in a doxycycline dependent Epo expression and subsequent regulation of hematocrit in vivo. However, an immune response rapidly cleared the transduced muscle cells. In this study, we examined the use of the TREAutoR4rEPO for ex vivo gene therapy by transducing human liver endothelial cells with this vector followed by intrasplenic transplantation in Rag2−/−γc−/− mice (n = 8). Blood was collected every 2 weeks for hematocrit determination. The hematocrit level of the mice was on average 0.5 ± 0 packed cell volume before transplantation. Two weeks after transplantation, but before starting doxycycline administration, the hematocrit remained at the same level showing tight regulation of background expression of the lentiviral vector. Two weeks after transplantation, the mice received doxycycline in their drinking water for 2 weeks. The hematocrit level increased significantly to 0.7 ± 0 packed cell volume and 0.7 ± 0.1 packed cell volume 2 and 4 weeks after starting the doxycycline water respectively (Figure 2). After withdrawal of doxycycline, the hematocrit returned back to baseline. Second and third rounds of doxycycline administration resulted in similar responses showing robust and long-term (7 months) regulation by this vector system. These results show not only that human liver endothelial cells are capable of engraftment and long-term repopulation following transplantation but also that they can be used for successful regulated gene expression.


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)

Long-term doxycycline regulation of hematocrit. Mice were transplanted with 50.000 human liver endothelial cells transduced with the autoregulatory Epo expression vector at day 0 (n = 8). Blood was sampled every 2 weeks. Doxycycline was administered in the drinking water at 2, 12, and 20 weeks after cell transplantation and continued for 2 weeks as indicated by the solid bars. Top panel: average hematocrit levels are shown on the Y-axis in PVC (packed cell volume). Up to 16 weeks n = 8, up to 28 weeks, n = 4. Bottom panel: the amount of rat erythropoietin in transplanted mouse serum was determined by ELISA. Following induction by doxycycline administration in the drinking water, the concentration of erythropoietin increased significantly, P < 0.0001. Mean values are shown ± SD. (n = 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Long-term doxycycline regulation of hematocrit. Mice were transplanted with 50.000 human liver endothelial cells transduced with the autoregulatory Epo expression vector at day 0 (n = 8). Blood was sampled every 2 weeks. Doxycycline was administered in the drinking water at 2, 12, and 20 weeks after cell transplantation and continued for 2 weeks as indicated by the solid bars. Top panel: average hematocrit levels are shown on the Y-axis in PVC (packed cell volume). Up to 16 weeks n = 8, up to 28 weeks, n = 4. Bottom panel: the amount of rat erythropoietin in transplanted mouse serum was determined by ELISA. Following induction by doxycycline administration in the drinking water, the concentration of erythropoietin increased significantly, P < 0.0001. Mean values are shown ± SD. (n = 6).
Mentions: We have shown previously in rats that in vivo intramuscular administration of the doxycycline regulated erythropoietin expressing lentiviral vector TREAutoR4rEPO resulted in a doxycycline dependent Epo expression and subsequent regulation of hematocrit in vivo. However, an immune response rapidly cleared the transduced muscle cells. In this study, we examined the use of the TREAutoR4rEPO for ex vivo gene therapy by transducing human liver endothelial cells with this vector followed by intrasplenic transplantation in Rag2−/−γc−/− mice (n = 8). Blood was collected every 2 weeks for hematocrit determination. The hematocrit level of the mice was on average 0.5 ± 0 packed cell volume before transplantation. Two weeks after transplantation, but before starting doxycycline administration, the hematocrit remained at the same level showing tight regulation of background expression of the lentiviral vector. Two weeks after transplantation, the mice received doxycycline in their drinking water for 2 weeks. The hematocrit level increased significantly to 0.7 ± 0 packed cell volume and 0.7 ± 0.1 packed cell volume 2 and 4 weeks after starting the doxycycline water respectively (Figure 2). After withdrawal of doxycycline, the hematocrit returned back to baseline. Second and third rounds of doxycycline administration resulted in similar responses showing robust and long-term (7 months) regulation by this vector system. These results show not only that human liver endothelial cells are capable of engraftment and long-term repopulation following transplantation but also that they can be used for successful regulated gene expression.

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