Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes.
hMSCs derived from bone marrow are useful as a species-specific cell culture system for studying cell lineage differentiation and tissue remodeling. However, hMSCs usually have a short in vitro life span due to replicative senescence. We therefore used a high dose of retroviral vector LXSN-16E6E7 to transduce hMSCs of an aging donor and obtained an actively proliferating cell line, designated KP-hMSCs, which expressed HPV16 E6/E7 mRNA. Whereas parental hMSCs ceased to grow after 30 PDs, KP-hMSCs could be propagated beyond 100 PDs. With culture procedures to avoid selection pressure and crowded cell growth, KP-hMSCs showed no signs of neoplastic transformation as examined by soft-agar anchorage-independent growth and NOD-SCID mouse tumorigenicity assays. KP-hMSCs gave similar cytofluorimetric profiles of 31 CD markers to those of the parental primary hMSCs, except with some morphologic changes and expansion of an originally very minor CD34(dim)CD38(+)CD50+ cell population. Upon exposure to specific stimulating conditions in vitro, KP-hMSCs could respond and differentiate along the mesenchymal (bone, fat and cartilage) and nonmesenchymal (neuron) cell lineages. Our results indicated that hMSCs could be immortalized by transduction with HPV16 E6/E7, maintained without neoplastic transformation by careful culture procedures and thus useful for stem cell research and clinical application.
Hung
Shih-Chieh
SC
Department of Orthopedics and Traumatology, Veterans General Hospital-Taipei, Taipei, Taiwan, ROC. hungsc@vghtpe.gov.tw
Yang
Den-Mei
DM
Chang
Ching-Fang
CF
Lin
Ruey-Jen
RJ
Wang
Jih-Shiuan
JS
Low-Tone Ho
Larry
L
Yang
Wen K
WK
D036541:ADP-ribosyl Cyclase / Q000096:biosynthesis; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D018952:Antigens, CD34 / Q000096:biosynthesis; D015815:Cell Adhesion Molecules; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D019070:Cell Lineage; D002471:Cell Transformation, Neoplastic; D005260:Female; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D008562:Membrane Glycoproteins; D008648:Mesoderm / Q000166:cytology*; D051379:Mice; D016513:Mice, SCID; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology*; D013997:Time Factors
D015703:Antigens, CD; D018952:Antigens, CD34; D015815:Cell Adhesion Molecules; C068651:E6 protein, Human papillomavirus type 16; C483616:ICAM3 protein, human; D008562:Membrane Glycoproteins; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1