RESEARCH ARTICLE


Isolation, Culture and Characterization of Cancer Stem Cells from Primary Osteosarcoma



Achmad Fauzi Kamal1, 2, *, Deded Yudha Pranatha1, Waluyo Sugito1, Faisal Rahman1, Eka Susanto3, Silmi Mariya4, Wei Ming Chen5
1 Department of Orthopaedic & Traumatology, Cipto Mangunkusumo General Hospital, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia
2 Stem Cell Integrated Medical Technology Unit, Cipto Mangunkusumo General Hospital, Jakarta, Indonesia
3 Department of Anatomic Pathology, Cipto Mangunkusumo General Hospital, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia
4 Primate Research Center, Bogor Agricultural Institute, Bogor, Indonesia
5 Department of Orthopaedic Surgery, Taipei Veterans General Hospital, Taipei, Taiwan


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© 2018 Kamal et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Department of Orthopaedic & Traumatology, Cipto Mangunkusumo General Hospital, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia; Tel: (+62) 1511450189; E-mail: fauzikamal@yahoo.com


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 osteosarcoma in 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 as NANOG, 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 encode NANOG, 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 as NANOG, Oct3/4, STAT3 and IFA of ALP, osteocalcin, and CD 133.

Keywords: Osteosarcoma, Cancer stem cells, Sarcosphere, CD133, NANOG, Alkaline phosphate.