Abstract
Background:Osteosarcoma cancer stem cells (CSCs) are defined as a subpopulation of osteosarcoma cells, which have the ability of self-renewal, proliferation and differentiation. This study aimed to identify CSCs from human osteosarcomain vitro.Methods:Osteosarcoma CSCs were isolated and cultured with sphere-forming assay technique on an ultra-low well attachment surface plate. After sarcosphere colonies were formed, we conducted reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the expression of genes of embryonic stem cells such asNANOG, Oct3/4, STAT3 and gene of MSC CD133. Immunofluorescence analysis (IFA) of alkaline phosphatase (ALP), osteocalcin, and CD 133 was also performed to see the expression of osteosarcoma CSC surface protein with immuno-enzymatic staining principle. We also performed alizarin red staining to evaluate calcification in osteosarcoma CSCs.Results:The culture sphere-of the osteosarcoma cells showed three dimension round shaped colonies (sarcospheres) in slightly hypoxicand serum free condition which was not attached to the substrate with tight density. RT-PCR demonstrated that sarcospheres expressed genes which encodeNANOG, Oct3/4 STAT 3, but not for CD 133. IFA showed positive protein expression of ALP, osteocalcin and CD 133 which was moderate, strong, and weak positive respectively. Sarcospheres also had a positive reaction toward alizarin red staining.Conclusion:Osteosarcoma CSCs could be isolated from human osteosarcoma by sphere-forming assay technique and characterized by the expression of genes of embryonic stem cells,such asNANOG, Oct3/4, STAT3 and IFA of ALP, osteocalcin, and CD 133.
Highlights
Osteosarcoma is a primary malignant bone tumor, commonly found in children and adolescents, which represents almost 60% of all bone sarcomas
The culture sphere-of the osteosarcoma cells showed three dimension round shaped colonies in slightly hypoxicand serum free condition which was not attached to the substrate with tight density
reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that sarcospheres expressed genes which encode NANOG, Oct3/4 STAT 3, but not for CD 133
Summary
Osteosarcoma is a primary malignant bone tumor, commonly found in children and adolescents, which represents almost 60% of all bone sarcomas. It is an osteoid-matrix-producing solid tumor and mostly develops in long bones, usually on metaphysis close to epiphyseal growth plate [1 - 3]. The incidence of osteosarcoma in all populations is around 4-5 per 1 million people. Incidence estimation of osteosarcoma increases to 8-11 per one million individuals per year at the age of 15-19 years. Osteosarcoma is known to have a bimodal age distribution with first peaked in young adults and the second in the elderly [2, 4 - 7]. This study aimed to identify CSCs from human osteosarcoma in vitro
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