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Immortalization of Mesenchymal Stem Cells From Bone Marrow of Rhesus Monkey by Transfection With Human Telomerase Reverse Transcriptase Gene
Immortalization of Mesenchymal Stem Cells From Bone Marrow of Rhesus Monkey by Transfection With Human Telomerase Reverse Transcriptase Gene K. Gao, Y.R. Lu, L.L. Wei, X.F. Lu, S.F. Li, L. Wan, Y.P. Li, and J.Q. Cheng ABSTRACT Introduction. Our previous experiments indicated that bone marrow mesenchymal stem cells of rhesus monkey (RhBMSCs) have a low proliferative ability with a finite life span, which will hamper their application in biomedical research. Establishing an immortalized RhBMSC lineage might solve the problem. Methods. RhBMSCs isolated from the bone marrow of rhesus monkeys using density gradient centrifugation were purified using adherence separation. Then, the cells were steadily transfected by plasmid containing human telomerase reverse transcriptase gene (pCI-neo-hTERT). We analyzed expression of hTERT, proliferation, phenotype (SH-2, SH-3, SB-10, CD29, CD34, CD45, and HLA-DR), differentiation toward osteogenic lineage, karyotype, and tumorigenesis of transfected cells. Results. After transfection, the RhBMSCs proliferated vigorously, undergoing more than 50 population doublings (PDs). Apoptotic rate of transfected RhBMSCs at PD40 was only 4.5%, versus untransfected RhBMSCs at PD15, which was more than 33.5%. Compared with normal RhBMSC, the life span of transfected RhBMSCs was prolonged, retaining similar morphology, karyotype, and potential to differentiate into an osteogenic lineage. More than 99% of transfected RhBMSCs were positive for stem cell markers, including SH-2, SH-3, SB-10, and CD29, and negative for CD34, CD45, and HLA-DR. Furthermore, the transfected cell line was benign in nude mice tumor formation. Conclusion. Our results demonstrated that hTERT gene had been transfected into RhBMSCs. The transfected RhBMSCs proliferated vigorously. Phenotype, differentiation, and karyotype of transfected RhBMSC showed no significant difference from untransfected cells. The transfected RhBMSCs are a potential cell source for transplantation as well as tissue engineering.
B
ONE marrow stem cell (BMSC) is a kind of selfrenewing marrow cell that provides progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells.1 It is a good choice for cell transplantation and tissue engineering due to its capacity for multilineage differentiation and low immunogenicity.2 The rhesus monkey has been extensively and largely used in medical research because of evident similarities to humans in genetics, physiology, immunology, and developmental perspectives. In a preclinical study of pig-to-human xenotransplantation, the rhesus monkey can play a transitional role for pig-to-monkey xenotransplantation. Thus, bone marrow mesenchymal stem cells of rhesus monkeys (RhBMSCs) may be valuable for xenotransplantation and 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.053 634
From the Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China. Supported by the National High Technology Research and Development Program of China (No. 2006AA02A117), National Basic Research Program of China (No. 2003CB515504), Program for Changjiang Scholars and Innovative Research Team in University, Ministry of Education, and National Basic Research Program of China (No. 2004CCA01800). Address reprint requests to Jingqiu Cheng, Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, P.R. China. E-mail: [email protected] © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 634 – 637 (2008)
MESENCHYMAL STEM CELLS
635
other biomedical research that use rhesus monkeys as their animal model. But BMSCs have a low proliferative ability with a finite life span. Our previous experiments indicated that normal RhBMSCs undergo growth arrest when the population doubling (PD) number exceeded 20, a limited proliferative capacity. We sought to establish an immortalized RhBMSC lineage to solve the problem.
2 or 3 days for 2 weeks. Specimens were stained for minerals using alizarin red. Karyotype. Metaphase preparations were used according to the method described by Moore et al.5 Then, the cells were fixed using fresh stationary liquid (methanol:glacial acetic acid ⫽ 3:1), spread on slides, stained with Giemsa solution, and observed under an immersion objective.
MATERIALS AND METHODS Animals
Proliferation and Growth Analysis of Transfected RhBMSCs
All healthy rhesus monkeys used in this study were males, 3 years old of 3– 4 kg. They were housed at the National Chengdu Center for Safety Evaluation of Drugs. All procedures in this study were in accordance with the Guide for the Care and Use of Laboratory Animals.
Growth curve analysis. Transfected RhBMSCs at PD32 and untransfected cells at PD10 were plated into 96-well plates (2 ⫻ 105/mL). After 24 hours the proliferation of cells was measured using a MTS assay (cell Titer 96 Aqueous One Solution cell proliferation Assay, Promega, USA). A standard curve was obtained to display the relationship between absorbance and cell numbers. Then, cells (1 ⫻ 104/mL) were plated in 96-well plates, over the following 7 days 3 random wells were evaluated using MTS assays each day. Finally, the growth curve was drawn according to the standard curve. Apoptosis analysis. For apoptosis analysis of transfected RhBMSCs at PD40 and untransfected cells at PD15, the DNA content was measured using means of flow cytometry using the conventional procedure.
Isolation and Culture of RhBMSCs Mononuclear cells were isolated using density gradient centrifugation from bone marrow that was aspirated from the anterior iliac crest of the pelvis. The harvested cell pellets were cultured in DMEM supplemented with 10% fetal bovine serum, 37°C, 5% CO2 for 24 hours for further purification by adherence separation. Nonadherent cells were removed the next day. Before cells reached 80%–90% confluence, the medium was replaced every 2 or 3 days.
Transfection With hTERT Gene
Tumor Formation by Inoculating Cells Into Nude Mice
The plasmid pCI-neo-hTERT was prepared using Endofree Plasmid Maxi Kit (Qiagen, Gibco, USA). Then the RhBMSCs at PD2 were transfected with pCI-neo-hTERT by Effectene Transfection Reagent Kit (Qiagen, Germany) and followed by selection with G418 (200 g/mL; GIBCO). After 2–3 weeks of selection, we isolated the surviving clones.
Three four-week-old BALB/c nu/nu mice were subcutaneously injected each with 0.1 mL (5 ⫻ 106 cells/mL) PD40 transfected cell suspension at their oxters, and the other 3 mice were injected with 0.1 mL (5 ⫻ 106 cells/mL) SMMC-7721 cell suspension. The mice were observed weekly for 1 and a half month.
Detection of Transcription of hTERT Gene Using Reverse Transcriptase Polymerase Chain Reaction Following the harvest of PD20 transfected with hTERT gene and PD10 untransfected RhBMSC, total RNA was extracted using Trizol reagent (Invitrogen, USA). Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed using RNA PCR Kit (Takara, Japan). The hTERT gene in messenger RNA (mRNA) was detected with -actin as control. The specific primers were as follows: hTERT forward (5=-CATCGCCAGCATCATCAAA-3=) and reverse (5=-CAAGAAATCATCCACCAAACG-3=); -actin forward (5=-ACTCTTCCAGCCTTCCTTC-3=) and reverse (5=GTCACCTTCACCGTTCCA-3=). The expected sizes for hTERT and -actin PCR products were 558 base pairs (bp) and 514 bp.
Identification of Transfected RhBMSCs Surface molecules were detected with monoclonal antibodies CD29-PE, CD34-PE, CD45-PE-Cy5, and HLA-DR-FITC (Becton Dickinson, USA)3 for the corresponding antigens of RhBMSCs using flow cytometry according to standand protocols. Monoclonal antibodies SH-2, SH-3, and SB-10 were used to detect surface molecules on cells using immunofluorescence staining. Osteogenic differentiation. Osteogenic differentiation of transfected RhBMSCs was performed in DMEM supplemented with dexamethasone (0.1 mol/L), ascorbic acid (0.05 mmol/L), and -glycerophosphate (10 mmol/L).4 The medium was replaced every
RESULTS Transcription of hTERT Gene
mRNA expression of hTERT gene was detected using RT-PCR with specific hTERT primers. For transfected cells, 558-bp–specific amplification bands of hTERT were detected, whereas for untransfected cells, no specific band was detected. However, 514-bp–specific amplification band of -actin was detected. These experiments proved that the hTERT gene was integrated into the genomic DNA of RhBMSCs and transcribed into mRNA. Identification of Transfected RhBMSCs
The results revealed that transfected RhBMSCs retained their typical morphology. Their karyotype corresponded to the rhesus monkey normal chromosomal complement, namely, diploid number 42.6 For cell phenotype, more than 99% of transfected RhBMSCs were positive for stem cell markers, including SH-2, SH-3, SB-10, and CD29, and negative for CD34, CD45, and HLA-DR (Fig 1A–G). The result of osteogenic differentiation showed that the morphology of transfected cells cultured with osteogenic medium changed from spindle to cuboidal. Many large calcium nodules were formed after 2 weeks induction, which stained
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Fig 1. Identification of transfected RhBMSCs: (A–D) expression of surface molecules (CD29, CD34, CD45, and HLA-DR) on transfected RhBMSCs detected using flow cytometry; (E–G) expression of surface molecules (SH-2, SH-3, and SB-10) on transfected RhBMSC detected using immunofluorescence; and (H) calcium nodules stained with alizarin red.
with alizarin red (Fig 1H). Transfected RhBMSCs had the ability to differentiate into osteogenic lineages.
Proliferation and Growth Analysis of Transfected RhBMSCs
Cell growth curves showed the population doubling times of transfected cells at PD32 and untransfected cells at PD10 were approximately 40 hours and 70 hours, respectively (Fig 2). The results of flow cytometry revealed that the apoptotic rate of transfected RhBMSCs at PD40 was only 4.5%, versus untransfected RhBMSCs at PD15, which was more than 33.5%, which was the typical “apoptosis peak,” whereas an apoptosis peak was not observed in the former setting.
Tumorigenesis Assay
The 3 nude mice injected with SMMC-7721 cells developed tumors, whereas the other 3 injected with transfected RhBMSCs did not. Malignant transformation did not take place in transfected cells.
DISCUSSION
Our results showed that the untransfected RhBMSCs had no hTERT expression, and showed expected slowing of growth that is associated with aging in vitro, they failed to proliferate after 20 PDs. In contrast, after transfection with pCI-neo-hTERT, RhBMSC-hTERTs showed strong proliferation. They have undergone more than 50 PDs so far and still have the potential to divide further. Moreover, RhBMSC-hTERTs maintained normal cell characteristics in morphology, phenotype, karyotype, and function, and did not form tumors. Our results verified that immortalized RhBMSCs were strongly positive for the crucial markers of BMSCs, such as SH-2, SH-3, SB-10, and CD29, and negative for CD34, CD45, and HLA-DR (MHC class II), which are usually used in the identification of the phenotype of mesenchymal stem cells.7 It is well known that BMSCs are progenitor cells that are able to differentiate into osteogenic lineages.8 When cultured in induction media, RhBMSC-hTERTs form osteoblast retaining the potential to differentiate. In conclusion, this study demonstrated that the life span of RhBMSCs was prolonged by transfection with hTERT gene. hTERT-immortalized cells retained typical RhBMSC characteristics without malignant transformation. hTERTimmortalized RhBMSCs are an immortalized cell line of rhesus monkey BMSCs with potential as a cell source for transplantation as well as tissue engineering.
REFERENCES
Fig 2.
Cell growth curve.
1. Barry FP, Murphy JM: Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 36:568, 2004 2. Uccelli A, Pistoia V, Moretta L: Mesenchymal stem cells: a new strategy for immunosuppression. Trends Immunol 28:219, 2007 3. Reimann KA, Waite BCD, Lee-Parritz DE, et al: Use of human leukocyte-specific monoclonal antibodies for clinically immunophenotyping lymphocytes of rhesus monkeys. Cytomerty 17: 102, 1994
MESENCHYMAL STEM CELLS 4. Coelho MJ, Fernandes MH: Human bone cell culture in biocompatibility testing. Part II: effect of ascorbic acid, betaglycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials 21:1095, 2000 5. Moore CM, Janish C, Eddy CA, et al: Cytogenetic and fertility studies of a rheboon, rhesus macaque (Macaca mulatta) ⫻ baboon (Papio hamadryas) cross: further support for a single karyotype nomenclature. Am J Phys Anthropol 110:119, 1999
637 6. Soares MBM, Armada JLA, da-Silva VF, et al: Standardization of the karyotype of the rhesus monkey, macaca mulatta, and interspecitle homologies with human chromosomes. J Hum Evol 11:291, 1982 7. Pittenger MF, Martin BJ: Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 95:9, 2004 8. Catherine MK, Elizabeth C, Rocky ST: Biology of adult senchymal stromal cells: regulation of niche, self-renewal and differentiation. Arthrit Res Ther 9:204, 2007
B
ONE marrow stem cell (BMSC) is a kind of selfrenewing marrow cell that provides progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells.1 It is a good choice for cell transplantation and tissue engineering due to its capacity for multilineage differentiation and low immunogenicity.2 The rhesus monkey has been extensively and largely used in medical research because of evident similarities to humans in genetics, physiology, immunology, and developmental perspectives. In a preclinical study of pig-to-human xenotransplantation, the rhesus monkey can play a transitional role for pig-to-monkey xenotransplantation. Thus, bone marrow mesenchymal stem cells of rhesus monkeys (RhBMSCs) may be valuable for xenotransplantation and 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.053 634
From the Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China. Supported by the National High Technology Research and Development Program of China (No. 2006AA02A117), National Basic Research Program of China (No. 2003CB515504), Program for Changjiang Scholars and Innovative Research Team in University, Ministry of Education, and National Basic Research Program of China (No. 2004CCA01800). Address reprint requests to Jingqiu Cheng, Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, P.R. China. E-mail: [email protected] © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 634 – 637 (2008)
MESENCHYMAL STEM CELLS
635
other biomedical research that use rhesus monkeys as their animal model. But BMSCs have a low proliferative ability with a finite life span. Our previous experiments indicated that normal RhBMSCs undergo growth arrest when the population doubling (PD) number exceeded 20, a limited proliferative capacity. We sought to establish an immortalized RhBMSC lineage to solve the problem.
2 or 3 days for 2 weeks. Specimens were stained for minerals using alizarin red. Karyotype. Metaphase preparations were used according to the method described by Moore et al.5 Then, the cells were fixed using fresh stationary liquid (methanol:glacial acetic acid ⫽ 3:1), spread on slides, stained with Giemsa solution, and observed under an immersion objective.
MATERIALS AND METHODS Animals
Proliferation and Growth Analysis of Transfected RhBMSCs
All healthy rhesus monkeys used in this study were males, 3 years old of 3– 4 kg. They were housed at the National Chengdu Center for Safety Evaluation of Drugs. All procedures in this study were in accordance with the Guide for the Care and Use of Laboratory Animals.
Growth curve analysis. Transfected RhBMSCs at PD32 and untransfected cells at PD10 were plated into 96-well plates (2 ⫻ 105/mL). After 24 hours the proliferation of cells was measured using a MTS assay (cell Titer 96 Aqueous One Solution cell proliferation Assay, Promega, USA). A standard curve was obtained to display the relationship between absorbance and cell numbers. Then, cells (1 ⫻ 104/mL) were plated in 96-well plates, over the following 7 days 3 random wells were evaluated using MTS assays each day. Finally, the growth curve was drawn according to the standard curve. Apoptosis analysis. For apoptosis analysis of transfected RhBMSCs at PD40 and untransfected cells at PD15, the DNA content was measured using means of flow cytometry using the conventional procedure.
Isolation and Culture of RhBMSCs Mononuclear cells were isolated using density gradient centrifugation from bone marrow that was aspirated from the anterior iliac crest of the pelvis. The harvested cell pellets were cultured in DMEM supplemented with 10% fetal bovine serum, 37°C, 5% CO2 for 24 hours for further purification by adherence separation. Nonadherent cells were removed the next day. Before cells reached 80%–90% confluence, the medium was replaced every 2 or 3 days.
Transfection With hTERT Gene
Tumor Formation by Inoculating Cells Into Nude Mice
The plasmid pCI-neo-hTERT was prepared using Endofree Plasmid Maxi Kit (Qiagen, Gibco, USA). Then the RhBMSCs at PD2 were transfected with pCI-neo-hTERT by Effectene Transfection Reagent Kit (Qiagen, Germany) and followed by selection with G418 (200 g/mL; GIBCO). After 2–3 weeks of selection, we isolated the surviving clones.
Three four-week-old BALB/c nu/nu mice were subcutaneously injected each with 0.1 mL (5 ⫻ 106 cells/mL) PD40 transfected cell suspension at their oxters, and the other 3 mice were injected with 0.1 mL (5 ⫻ 106 cells/mL) SMMC-7721 cell suspension. The mice were observed weekly for 1 and a half month.
Detection of Transcription of hTERT Gene Using Reverse Transcriptase Polymerase Chain Reaction Following the harvest of PD20 transfected with hTERT gene and PD10 untransfected RhBMSC, total RNA was extracted using Trizol reagent (Invitrogen, USA). Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed using RNA PCR Kit (Takara, Japan). The hTERT gene in messenger RNA (mRNA) was detected with -actin as control. The specific primers were as follows: hTERT forward (5=-CATCGCCAGCATCATCAAA-3=) and reverse (5=-CAAGAAATCATCCACCAAACG-3=); -actin forward (5=-ACTCTTCCAGCCTTCCTTC-3=) and reverse (5=GTCACCTTCACCGTTCCA-3=). The expected sizes for hTERT and -actin PCR products were 558 base pairs (bp) and 514 bp.
Identification of Transfected RhBMSCs Surface molecules were detected with monoclonal antibodies CD29-PE, CD34-PE, CD45-PE-Cy5, and HLA-DR-FITC (Becton Dickinson, USA)3 for the corresponding antigens of RhBMSCs using flow cytometry according to standand protocols. Monoclonal antibodies SH-2, SH-3, and SB-10 were used to detect surface molecules on cells using immunofluorescence staining. Osteogenic differentiation. Osteogenic differentiation of transfected RhBMSCs was performed in DMEM supplemented with dexamethasone (0.1 mol/L), ascorbic acid (0.05 mmol/L), and -glycerophosphate (10 mmol/L).4 The medium was replaced every
RESULTS Transcription of hTERT Gene
mRNA expression of hTERT gene was detected using RT-PCR with specific hTERT primers. For transfected cells, 558-bp–specific amplification bands of hTERT were detected, whereas for untransfected cells, no specific band was detected. However, 514-bp–specific amplification band of -actin was detected. These experiments proved that the hTERT gene was integrated into the genomic DNA of RhBMSCs and transcribed into mRNA. Identification of Transfected RhBMSCs
The results revealed that transfected RhBMSCs retained their typical morphology. Their karyotype corresponded to the rhesus monkey normal chromosomal complement, namely, diploid number 42.6 For cell phenotype, more than 99% of transfected RhBMSCs were positive for stem cell markers, including SH-2, SH-3, SB-10, and CD29, and negative for CD34, CD45, and HLA-DR (Fig 1A–G). The result of osteogenic differentiation showed that the morphology of transfected cells cultured with osteogenic medium changed from spindle to cuboidal. Many large calcium nodules were formed after 2 weeks induction, which stained
636
GAO, LU, WEI ET AL
Fig 1. Identification of transfected RhBMSCs: (A–D) expression of surface molecules (CD29, CD34, CD45, and HLA-DR) on transfected RhBMSCs detected using flow cytometry; (E–G) expression of surface molecules (SH-2, SH-3, and SB-10) on transfected RhBMSC detected using immunofluorescence; and (H) calcium nodules stained with alizarin red.
with alizarin red (Fig 1H). Transfected RhBMSCs had the ability to differentiate into osteogenic lineages.
Proliferation and Growth Analysis of Transfected RhBMSCs
Cell growth curves showed the population doubling times of transfected cells at PD32 and untransfected cells at PD10 were approximately 40 hours and 70 hours, respectively (Fig 2). The results of flow cytometry revealed that the apoptotic rate of transfected RhBMSCs at PD40 was only 4.5%, versus untransfected RhBMSCs at PD15, which was more than 33.5%, which was the typical “apoptosis peak,” whereas an apoptosis peak was not observed in the former setting.
Tumorigenesis Assay
The 3 nude mice injected with SMMC-7721 cells developed tumors, whereas the other 3 injected with transfected RhBMSCs did not. Malignant transformation did not take place in transfected cells.
DISCUSSION
Our results showed that the untransfected RhBMSCs had no hTERT expression, and showed expected slowing of growth that is associated with aging in vitro, they failed to proliferate after 20 PDs. In contrast, after transfection with pCI-neo-hTERT, RhBMSC-hTERTs showed strong proliferation. They have undergone more than 50 PDs so far and still have the potential to divide further. Moreover, RhBMSC-hTERTs maintained normal cell characteristics in morphology, phenotype, karyotype, and function, and did not form tumors. Our results verified that immortalized RhBMSCs were strongly positive for the crucial markers of BMSCs, such as SH-2, SH-3, SB-10, and CD29, and negative for CD34, CD45, and HLA-DR (MHC class II), which are usually used in the identification of the phenotype of mesenchymal stem cells.7 It is well known that BMSCs are progenitor cells that are able to differentiate into osteogenic lineages.8 When cultured in induction media, RhBMSC-hTERTs form osteoblast retaining the potential to differentiate. In conclusion, this study demonstrated that the life span of RhBMSCs was prolonged by transfection with hTERT gene. hTERT-immortalized cells retained typical RhBMSC characteristics without malignant transformation. hTERTimmortalized RhBMSCs are an immortalized cell line of rhesus monkey BMSCs with potential as a cell source for transplantation as well as tissue engineering.
REFERENCES
Fig 2.
Cell growth curve.
1. Barry FP, Murphy JM: Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 36:568, 2004 2. Uccelli A, Pistoia V, Moretta L: Mesenchymal stem cells: a new strategy for immunosuppression. Trends Immunol 28:219, 2007 3. Reimann KA, Waite BCD, Lee-Parritz DE, et al: Use of human leukocyte-specific monoclonal antibodies for clinically immunophenotyping lymphocytes of rhesus monkeys. Cytomerty 17: 102, 1994
MESENCHYMAL STEM CELLS 4. Coelho MJ, Fernandes MH: Human bone cell culture in biocompatibility testing. Part II: effect of ascorbic acid, betaglycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials 21:1095, 2000 5. Moore CM, Janish C, Eddy CA, et al: Cytogenetic and fertility studies of a rheboon, rhesus macaque (Macaca mulatta) ⫻ baboon (Papio hamadryas) cross: further support for a single karyotype nomenclature. Am J Phys Anthropol 110:119, 1999
637 6. Soares MBM, Armada JLA, da-Silva VF, et al: Standardization of the karyotype of the rhesus monkey, macaca mulatta, and interspecitle homologies with human chromosomes. J Hum Evol 11:291, 1982 7. Pittenger MF, Martin BJ: Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 95:9, 2004 8. Catherine MK, Elizabeth C, Rocky ST: Biology of adult senchymal stromal cells: regulation of niche, self-renewal and differentiation. Arthrit Res Ther 9:204, 2007