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Ex vivo gene transfer into myocardium using replication-defective retrovirus
Cell Transplantation, Vol. 5, No. B-1, pp. S81-S84, 1996 Copyright0 1996 ElsevierScienceInc. Printedin the USA. All rightsreserved 0963.6897/96 $15.00 + .OO ELSEVIER
PII SO963-6897(96)00047-4
Original Contribution
EX VIVO GENE TRANSFER INTO MYOCARDIUM REPLICATION-DEFECTIVE RETROVIRUS SATOSHI GOJO, *’ SOICHIRO KITAMURA,*
WILFRED T.V.
GERMERAAD,~
USING
YOSHITSUGU YOSHIDA,*
KAZUO NIWAYA,*
AND
KANJI KAWACHI* *Department
of Surgery III, Nara Medical College, 840 Shijo-cho, Kashihara, Nara 634, Japan, and TDepartment of Pathology, Children’s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027, USA
lactosidase (B-gal) transgene resulted in the formation of intercalated disks between the grafted fetal cardiac cells and the recipient myocardium (6). The transfer of a therapeutic gene to cells to be grafted may be the factor that brings cardiac cell transplantation from the field of experimentation into the clinical arena. However, it is unknown if genetically engineered cardiomyocytes in vitro can be grafted. We used a recombinant retrovirus carrying the P-gal reporter as the gene transfer vector to examine this question.
0 Abstract -Heart transplantation is the most effective therapy for chronic severe heart failure, but there is an extreme shortage of hearts available. We examined the possibility that cardiomyocytes can be modified genetically prior to being grafted to the heart. We used a replicationdefective retrovirus carrying the P-galactosidase (P-gal) reporter gene. The p-gal gene was transduced into murine fetal cardiac myocytes by culturing a recombinant retrovirus-producing cell line in a Transwell plate hung into the primary cardiomyocyte culture. The cultured cells were stained with the di-R-n-galactopyranoside (FDG) and were sorted by fluorescence-activated cell sorting (FACS). FACS analysis showed that 25.5 f 4.3% of the cardiomyocytes in a primary culture were positive for P-gal activity. These cells were transplanted into the hearts of syngeneic adult mice. Expression of the P-gal gene in the grafted cells was demonstrated by staining with 5bromo-4-chloro-3-indoylP-n-galactoside (X-gal). Gene expression was recognized as long as 6 mo after cell transplantation. Histologic analysis showed neither inflammation nor fibrous scar tissue on the host myocardium. This study demonstrated that genetically modified cardiac cells were transplantable to the heart. 0 Keywords - Cardiac R-Galactosidase.
cell; Gene transfer;
MATERIALS AND METHODS
Mice Adult BALB/c mice 12 to 16 wk old were used as the recipients in the experiments. They were mated overnight and the fertilized females were selected the next morning (after 0.5 embryonic day) on the basis of the presence of a vaginal plug. Fetuses were obtained from timed pregnancies at day 16.5 of gestation.
Retrovirus;
Generation of a Retrovirus Producer Cell Line The retroviral vector W4Z was constructed by ligating the LacZ gene into W5 deleted neo gene (2). Plasmid W4Z was transfected into ecotropic (GP+E86) packaging cells. Virus-containing supernatant from the ecotropic producer cell line was used to infect amphotropic (GPenvAm12) packaging cells. Two days later, supernatant with amphotropic virus was added to a monolayer of GP+E86 cells. This infection cycle was repeated several
INTRODUCTION
Treatments for severely and irreversibly damaged organs include organ transplantation, implantation of artificial organs, and cell transplantation. However, in cardiology, cell transplantation (4,5) has received minimal attention. Recently, it was reported that intracardiac grafting of fetal cardiac cells in transgenic mice carrying the B-ga-
ACCEPTED l/20/96.
Abbreviations-P-gal: P-galactosidase. DMEM: Dulbecco’s modified Eagle’s medium. X-gal: 5-bromo-4-chloro-3indoyl-P-D-galactoside. FDG:di-P-D-galactopyranoside. FACS: fluorescence-activated cell sorting.
‘To
whom correspondence
should be addressed. Satoshi
Gojo, M.D., Department of Surgery III, Nara Medical College, 840 Shijo-cho, Kashihara, Nara 634, Japan. S81
S82
Cell Transplantation
0 Volume 5, Number 5S-1, 1996
times. The ecotropic producer cell line was used in all experiments for gene transduction into cardiomyocytes. No replication-competent virus was detected in the supernatant of W4Z-infected NIH/3T3 cells.
Culture of Murine Fetal Cardiomyocytes and Transduction of the Retroviral Vector into Cardiomyocytes Mouse heart ventricles were removed from BALB/c fetuses at a gestational age of 16.5 days, and were digested for 5 min at 37°C with 0.06% trypsin, using a magnetic stirrer operating at low speed. The suspension was seeded into glass Petri dishes and incubated for 1 h at 37°C in 5% CO,/95% air to exclude nonmyocytes (3). The nonadherent cells were seeded into collagen-coated six-well plastic plates (2 x lo5 cells per well) and incubated at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. Almost all the myocytes became attached to the culture dish and contracted spontaneously after 1 day. On day 1 after seeding, a porous and collagen-coated Transwell plate was hung into each well cultivating cardiomyocytes, and virusproducing cells (5 x lo4 cells) were inoculated into the Transwell (2).
FDG Staining and Fluorescence-Activated Cell Sorting After 3 days of coculture, cardiomyocytes were collected. These cells were incubated in a 37°C water bath for 10 min and mixed gently with 2 mmol/L di-B-Dgalactopyranoside (FDG) in H,O. They were rapidly placed back into the 37°C water bath for 1 min and then chilled at 0°C. Ice-chilled DMEM was added to terminate the enzymatic cleavage of fluorescein FDG. Fluorescence was measured in a flow cytometer. Cells positive for B-gal activity were sorted and collected.
X-Gal and HE Staining of the Heart Some of the animals were sacrificed every week after the cell transplantation. The hearts were dissected and fixed in 4% paraformaldehyde for 1 h, and were then sliced (400 km thick) using a vibratome slicer. The slices were placed on a culture dish, 1 mg/mL 5-bromo-4chloro-3-indoyl-B-D-galactoside (X-gal), 15 mmol/L potassium ferricyanide, 15 mmol/L potassium ferrocyanide, and 2 mmol/L magnesium chloride in phosphatebuffered saline (PBS). The sliced hearts positive for B-gal activity were embedded in paraffin and sections (4 pm) were stained with hematoxylin and eosin (HE). RESULTS
Primary culture of murine fetal cardiomyocytes was performed according to a method reported earlier (3). This procedure can remove almost all fibroblasts. Throughout the cultured period, almost all cells contracted even during an infectious period. Gene transfer was assessed by FDG staining. Fluorescence-activated cell sorting analysis revealed that 25.5 f 4.3% of cultured cells were positive for B-gal activity (n = 10). When the supernatants of the producer cells were added to the cultured cardiomyocytes and incubated for 3 days, the transfer rate to the cardiomyocytes was 7.5 +- 2.7% (n = 5). Gross histological examination of grafted hearts was performed every 7 days following cell transplantation for up to 6 mo (Fig. 1A). HE staining indicated that there was no myocardial fibrosis, capsule formation, or infiltration of monocytes and leukocytes around the grafted cells. These grafted cells, which showed a blue color, aligned themselves circumferentially, parallel to the host myocardium (Fig. IB). DISCUSSION
Cell Transplantation Into Syngeneic Adult Mice The collected cells were washed with DMEM and the final cell density for implantation was 5 x lo7 to 1 x 10s cells per mL. After the recipient mice were anesthesized by an intraperitoneal injection of 0.05 mg pentobarbital per g weight, a transverse incision was made under the arch of a rib. The xiphoid process was inverted so as to point cephalad rather than caudad, and the liver was pushed inferiorly so that the heart became visible through the diaphragm. Immediately before implantation, the cell suspension was drawn up into a 10 FL Hamilton syringe with a 31 gauge needle. Ten microliters of the cell suspension was injected through the diaphragm directly into the ventricular myocardium of syngeneic adult mice (n = 50). The only complication of this procedure was pneumothorax in one animal early in the experiment.
The results presented herein have demonstrated that genetically modified cardiomyocytes were transplantable into the heart. It was previously reported that the Transwell coculture system was effective in improving the rate of gene transfer into hematopoietic stem cells (2). The present study showed that the system was also effective in the case of cardiomyocytes. The implanted cardiac cells survived and expressed the exogenous gene in vivo for as long as 6 mo in the syngeneic host heart. This long and stable expression of recombinant molecules will induce a beneficial effect on cellular transplantation. In addition, no signs of pathological changes were detectable on histologic analysis of the host myocardium. This is an important prerequisite for grafted cells to cooperate with the recipient cardiomyocytes. This intracardiac grafting approach may be of use in replacing the diseased myocardium. In ischemic heart
Ex viva gene transfer into myocardium
??S. GOIO
ET AL
,583
Fig. 1. X-Gal and HE staining of myocardium. Histologic examination of host hearts was carried out every week follow ing implantation. (A) is a macroscopic photograph with X-gal staining taken 6 mo after cell transplantation. The blue areas are gro ups of cells positive for B-gal activity. The bar indicates 1 mm. (B) is a photomicrograph with HE staining 6 mo after implantation. No fibrous scars or infiltration of neutrophils was detectable in any section. In addition, grafted cells aligned themselves with the layrers of the host myocardium. The bar indicates 50 pm.
S84
Cell Transplantation
0 Volume 5, Number X3-1, 1996
disease, the successful implantation of cardiac cells may not be achieved without an adequate vascular support. Grafts, which produce angiogenic factors (1,7), may induce neovascularization of the cell-implanted ischemic area. In the present study, we used P-gal as the reporter gene, but genes of angiogenic factors can also be used with the same system. When this becomes possible in patients, cardiac cell transplantation will have advanced to allow clinical application.
3.
4.
5. Acknowledgments - This work was supported from the Ministry of Health and Welfare, Japan.
in part by a grant
6. REFERENCES 1. Engelmann, blast growth proliferation 1993. 2. Germeraad, Katsura, Y.
G.L.; Dionne, C.A.; Jaye, M.C. Acidic fibrofactor and heart development: Role in myocyte and capillary angiogenesis. Circ. Res. 72:7-19; W.T.V.; Asami, N.; Fujimoto, S.; Mazda, 0.; Efficient retrovirus-mediated gene transduction
7.
into murine hematopoietic stem cells and long-lasting expression using a transwell coculture system. Blood 84:780788; 1994. Goshima, K. Ouabain-induced arrhythmias of single isolated myocardial cells and cell clusters cultured in vitro and their improvement by quinidine. J. Mol. Cell. Cardiol. 9:723; 1977. Koh, G.Y.; Klug, M.G.; Soonpaa, M.H.; Field, L.J. Differentiation and longterm survival of C2Cl2 myoblast grafts in heart. J. Clin. Invest. 92:1548-1554; 1993. Marelli, D.; Desrosiers, C.; El-Alfy, M.; Kao, R.L.; Chiu, R.C.J. Cell transplantation for myocardial repair: An experimental approach. Cell Transplant. 1:383-390; 1992. Soonpaa, M.H.; Koh, G.Y.; Klug, M.G.; Field, L.J. Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 264:98-10 1; 1994. Yanagisawa, A.; Uchida, Y.; Nakamura, F.; Tomaru, T.; Kido, H.; Kamijo, T.; Sugimoto, T.; Kaji, K.; Utsuyama, M.; Kurashima, C.; Ito, H. Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor. Science 257:1401-1403; 1992.
PII SO963-6897(96)00047-4
Original Contribution
EX VIVO GENE TRANSFER INTO MYOCARDIUM REPLICATION-DEFECTIVE RETROVIRUS SATOSHI GOJO, *’ SOICHIRO KITAMURA,*
WILFRED T.V.
GERMERAAD,~
USING
YOSHITSUGU YOSHIDA,*
KAZUO NIWAYA,*
AND
KANJI KAWACHI* *Department
of Surgery III, Nara Medical College, 840 Shijo-cho, Kashihara, Nara 634, Japan, and TDepartment of Pathology, Children’s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027, USA
lactosidase (B-gal) transgene resulted in the formation of intercalated disks between the grafted fetal cardiac cells and the recipient myocardium (6). The transfer of a therapeutic gene to cells to be grafted may be the factor that brings cardiac cell transplantation from the field of experimentation into the clinical arena. However, it is unknown if genetically engineered cardiomyocytes in vitro can be grafted. We used a recombinant retrovirus carrying the P-gal reporter as the gene transfer vector to examine this question.
0 Abstract -Heart transplantation is the most effective therapy for chronic severe heart failure, but there is an extreme shortage of hearts available. We examined the possibility that cardiomyocytes can be modified genetically prior to being grafted to the heart. We used a replicationdefective retrovirus carrying the P-galactosidase (P-gal) reporter gene. The p-gal gene was transduced into murine fetal cardiac myocytes by culturing a recombinant retrovirus-producing cell line in a Transwell plate hung into the primary cardiomyocyte culture. The cultured cells were stained with the di-R-n-galactopyranoside (FDG) and were sorted by fluorescence-activated cell sorting (FACS). FACS analysis showed that 25.5 f 4.3% of the cardiomyocytes in a primary culture were positive for P-gal activity. These cells were transplanted into the hearts of syngeneic adult mice. Expression of the P-gal gene in the grafted cells was demonstrated by staining with 5bromo-4-chloro-3-indoylP-n-galactoside (X-gal). Gene expression was recognized as long as 6 mo after cell transplantation. Histologic analysis showed neither inflammation nor fibrous scar tissue on the host myocardium. This study demonstrated that genetically modified cardiac cells were transplantable to the heart. 0 Keywords - Cardiac R-Galactosidase.
cell; Gene transfer;
MATERIALS AND METHODS
Mice Adult BALB/c mice 12 to 16 wk old were used as the recipients in the experiments. They were mated overnight and the fertilized females were selected the next morning (after 0.5 embryonic day) on the basis of the presence of a vaginal plug. Fetuses were obtained from timed pregnancies at day 16.5 of gestation.
Retrovirus;
Generation of a Retrovirus Producer Cell Line The retroviral vector W4Z was constructed by ligating the LacZ gene into W5 deleted neo gene (2). Plasmid W4Z was transfected into ecotropic (GP+E86) packaging cells. Virus-containing supernatant from the ecotropic producer cell line was used to infect amphotropic (GPenvAm12) packaging cells. Two days later, supernatant with amphotropic virus was added to a monolayer of GP+E86 cells. This infection cycle was repeated several
INTRODUCTION
Treatments for severely and irreversibly damaged organs include organ transplantation, implantation of artificial organs, and cell transplantation. However, in cardiology, cell transplantation (4,5) has received minimal attention. Recently, it was reported that intracardiac grafting of fetal cardiac cells in transgenic mice carrying the B-ga-
ACCEPTED l/20/96.
Abbreviations-P-gal: P-galactosidase. DMEM: Dulbecco’s modified Eagle’s medium. X-gal: 5-bromo-4-chloro-3indoyl-P-D-galactoside. FDG:di-P-D-galactopyranoside. FACS: fluorescence-activated cell sorting.
‘To
whom correspondence
should be addressed. Satoshi
Gojo, M.D., Department of Surgery III, Nara Medical College, 840 Shijo-cho, Kashihara, Nara 634, Japan. S81
S82
Cell Transplantation
0 Volume 5, Number 5S-1, 1996
times. The ecotropic producer cell line was used in all experiments for gene transduction into cardiomyocytes. No replication-competent virus was detected in the supernatant of W4Z-infected NIH/3T3 cells.
Culture of Murine Fetal Cardiomyocytes and Transduction of the Retroviral Vector into Cardiomyocytes Mouse heart ventricles were removed from BALB/c fetuses at a gestational age of 16.5 days, and were digested for 5 min at 37°C with 0.06% trypsin, using a magnetic stirrer operating at low speed. The suspension was seeded into glass Petri dishes and incubated for 1 h at 37°C in 5% CO,/95% air to exclude nonmyocytes (3). The nonadherent cells were seeded into collagen-coated six-well plastic plates (2 x lo5 cells per well) and incubated at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. Almost all the myocytes became attached to the culture dish and contracted spontaneously after 1 day. On day 1 after seeding, a porous and collagen-coated Transwell plate was hung into each well cultivating cardiomyocytes, and virusproducing cells (5 x lo4 cells) were inoculated into the Transwell (2).
FDG Staining and Fluorescence-Activated Cell Sorting After 3 days of coculture, cardiomyocytes were collected. These cells were incubated in a 37°C water bath for 10 min and mixed gently with 2 mmol/L di-B-Dgalactopyranoside (FDG) in H,O. They were rapidly placed back into the 37°C water bath for 1 min and then chilled at 0°C. Ice-chilled DMEM was added to terminate the enzymatic cleavage of fluorescein FDG. Fluorescence was measured in a flow cytometer. Cells positive for B-gal activity were sorted and collected.
X-Gal and HE Staining of the Heart Some of the animals were sacrificed every week after the cell transplantation. The hearts were dissected and fixed in 4% paraformaldehyde for 1 h, and were then sliced (400 km thick) using a vibratome slicer. The slices were placed on a culture dish, 1 mg/mL 5-bromo-4chloro-3-indoyl-B-D-galactoside (X-gal), 15 mmol/L potassium ferricyanide, 15 mmol/L potassium ferrocyanide, and 2 mmol/L magnesium chloride in phosphatebuffered saline (PBS). The sliced hearts positive for B-gal activity were embedded in paraffin and sections (4 pm) were stained with hematoxylin and eosin (HE). RESULTS
Primary culture of murine fetal cardiomyocytes was performed according to a method reported earlier (3). This procedure can remove almost all fibroblasts. Throughout the cultured period, almost all cells contracted even during an infectious period. Gene transfer was assessed by FDG staining. Fluorescence-activated cell sorting analysis revealed that 25.5 f 4.3% of cultured cells were positive for B-gal activity (n = 10). When the supernatants of the producer cells were added to the cultured cardiomyocytes and incubated for 3 days, the transfer rate to the cardiomyocytes was 7.5 +- 2.7% (n = 5). Gross histological examination of grafted hearts was performed every 7 days following cell transplantation for up to 6 mo (Fig. 1A). HE staining indicated that there was no myocardial fibrosis, capsule formation, or infiltration of monocytes and leukocytes around the grafted cells. These grafted cells, which showed a blue color, aligned themselves circumferentially, parallel to the host myocardium (Fig. IB). DISCUSSION
Cell Transplantation Into Syngeneic Adult Mice The collected cells were washed with DMEM and the final cell density for implantation was 5 x lo7 to 1 x 10s cells per mL. After the recipient mice were anesthesized by an intraperitoneal injection of 0.05 mg pentobarbital per g weight, a transverse incision was made under the arch of a rib. The xiphoid process was inverted so as to point cephalad rather than caudad, and the liver was pushed inferiorly so that the heart became visible through the diaphragm. Immediately before implantation, the cell suspension was drawn up into a 10 FL Hamilton syringe with a 31 gauge needle. Ten microliters of the cell suspension was injected through the diaphragm directly into the ventricular myocardium of syngeneic adult mice (n = 50). The only complication of this procedure was pneumothorax in one animal early in the experiment.
The results presented herein have demonstrated that genetically modified cardiomyocytes were transplantable into the heart. It was previously reported that the Transwell coculture system was effective in improving the rate of gene transfer into hematopoietic stem cells (2). The present study showed that the system was also effective in the case of cardiomyocytes. The implanted cardiac cells survived and expressed the exogenous gene in vivo for as long as 6 mo in the syngeneic host heart. This long and stable expression of recombinant molecules will induce a beneficial effect on cellular transplantation. In addition, no signs of pathological changes were detectable on histologic analysis of the host myocardium. This is an important prerequisite for grafted cells to cooperate with the recipient cardiomyocytes. This intracardiac grafting approach may be of use in replacing the diseased myocardium. In ischemic heart
Ex viva gene transfer into myocardium
??S. GOIO
ET AL
,583
Fig. 1. X-Gal and HE staining of myocardium. Histologic examination of host hearts was carried out every week follow ing implantation. (A) is a macroscopic photograph with X-gal staining taken 6 mo after cell transplantation. The blue areas are gro ups of cells positive for B-gal activity. The bar indicates 1 mm. (B) is a photomicrograph with HE staining 6 mo after implantation. No fibrous scars or infiltration of neutrophils was detectable in any section. In addition, grafted cells aligned themselves with the layrers of the host myocardium. The bar indicates 50 pm.
S84
Cell Transplantation
0 Volume 5, Number X3-1, 1996
disease, the successful implantation of cardiac cells may not be achieved without an adequate vascular support. Grafts, which produce angiogenic factors (1,7), may induce neovascularization of the cell-implanted ischemic area. In the present study, we used P-gal as the reporter gene, but genes of angiogenic factors can also be used with the same system. When this becomes possible in patients, cardiac cell transplantation will have advanced to allow clinical application.
3.
4.
5. Acknowledgments - This work was supported from the Ministry of Health and Welfare, Japan.
in part by a grant
6. REFERENCES 1. Engelmann, blast growth proliferation 1993. 2. Germeraad, Katsura, Y.
G.L.; Dionne, C.A.; Jaye, M.C. Acidic fibrofactor and heart development: Role in myocyte and capillary angiogenesis. Circ. Res. 72:7-19; W.T.V.; Asami, N.; Fujimoto, S.; Mazda, 0.; Efficient retrovirus-mediated gene transduction
7.
into murine hematopoietic stem cells and long-lasting expression using a transwell coculture system. Blood 84:780788; 1994. Goshima, K. Ouabain-induced arrhythmias of single isolated myocardial cells and cell clusters cultured in vitro and their improvement by quinidine. J. Mol. Cell. Cardiol. 9:723; 1977. Koh, G.Y.; Klug, M.G.; Soonpaa, M.H.; Field, L.J. Differentiation and longterm survival of C2Cl2 myoblast grafts in heart. J. Clin. Invest. 92:1548-1554; 1993. Marelli, D.; Desrosiers, C.; El-Alfy, M.; Kao, R.L.; Chiu, R.C.J. Cell transplantation for myocardial repair: An experimental approach. Cell Transplant. 1:383-390; 1992. Soonpaa, M.H.; Koh, G.Y.; Klug, M.G.; Field, L.J. Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 264:98-10 1; 1994. Yanagisawa, A.; Uchida, Y.; Nakamura, F.; Tomaru, T.; Kido, H.; Kamijo, T.; Sugimoto, T.; Kaji, K.; Utsuyama, M.; Kurashima, C.; Ito, H. Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor. Science 257:1401-1403; 1992.