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. Author manuscript; available in PMC: 2014 Sep 10.
Published in final edited form as: Cancer Lett. 2012 May 29;338(1):158–167. doi: 10.1016/j.canlet.2012.05.028

Table 1.

Studies identifying stem like-cells in osteosarcoma

Isolation Strategy Marker/ Substrate [reference] Source Functional Characterization Comments
Markers CD133 [30,31] Established human cell lines (Saos-2, U-2OS, MG63) 3–5%

  • Higher anchorage-independent growth, proliferation

  • Higher sphere forming ability

  • Side population fraction a small sub-set (0.97%) of the CD133High fraction.

  • Co-enrichment of Oct3/4 and Cd133+ in sarcospheres.
Human Tissue Samples (1.35–7.85% of total population in three osteosarcoma samples).

  • Tumor-initiating capacity of CD133+ sarcosphere fraction

  • Colony-forming ability

  • Multipotency

  • Side population compared to the total adherent population.

  • Both CD133High and CD133Low fractions positive for CD29, CD44 and CD90 antigens

  • Side population fraction a small sub- set (0.97%) of the CD133High fraction.

  • Co-enrichment of Oct3/4 and CD133 in sarcospheres.
Saos-2 human cell line

  • Sphere formation

  • Sarcospheres enriched for Sox2, Oct3/4 and Nanog.

  • SP was positive for CD133.
Stro-1/CD117 double positive (DP) [29] Murine DP cells (1% – 3%) of total monolayer cultures, and 6% – 14% in the sphere fractions, respectively in three murine cell lines) and human (KHOS – (1% DP cells) cell lines

  • Drug resistance (2-fold increase in IC50)

  • tumor initiating capacity (as low as 200 DP cells)

  • Multipotency

  • Stro-1/CD117 useful in both murine and human samples.

  • Higher expression of CXCR4 (metastasis gene) and ABCG2 (drug transporter) in DP population
Sca-1 [17,18,32] Murine osteosarcoma cell lines (30 to 70%) in 4 cell lines

  • Sca-1 and Sox2 expression correlated with increased tumor formation, increased sphere-forming frequency in limiting dilution assay for sphere-forming ability, and multipotency

  • Loss of Sox2 depletes the Sca-1 positive fraction.
Murine osteosarcoma (primary tumors −1.2%) and cell lines established from tumors). Sca-1 expression correlates with tumorigenicity
Intrinsic properties Hoechst 33342-excluding side population (SP) [34,35] Established human cell lines (< 1% – 5.28%) in eight different cell lines.

  • Asymmetric division (SP giving rise to both SP and non-SP populations in culture)

  • Sphere-forming assay (only seen in SP)

  • limiting dilution analysis for tumor initiation

  • Gene expression profiling by microarray analysis (higher expression of drug transporter ABCG2 in SP)
Human tumor samples (3.7% – 10.2% in five human tumor samples)

  • Asymmetric division

  • Limiting dilution analysis for tumor initiation

  • Clonogenicity assay (SP forming colonies under adherent conditions at higher efficiency)

  • Sphere-forming assay

  • SP more drug-resistant with much higher IC50 to doxorubicin, cisplatin and methotrexate

  • Higher expression of ABC transporter, Nanog and Oct4 in SP.

  • Surface marker analysis reveals no difference between SP and non-SP. in expression of CD44, CD117 and CD133
aldehyde dehydrogenase (ALDH1) [36,37] Human cell line (MG63-11%)

  • Sphere-forming ability,

  • drug resistance

  • ALDH1 activity higher in the sphere- forming fraction.
Human cell lines (OS99-1 – 45.07%, Hu09 – 1.84%, Saos-2 – 1.56% and MG63 – 0.59%) OS99-1 xenografts (3.13%)

  • ALDHHi cells from xenografts from OS99-1 cells showed higher tumor initiating frequency in limiting dilution assay, enhanced proliferation and colony-forming ability

  • Increased expression of Oct3/4, Sox2 and Nanog in the ALDHHi cells

  • Authors use the ALDHHi cells (3.13%) from xenografts they injected and not the original OS99-1 cells which showed a higher proportion of ALDHHi cells (45.07%)
Long-term dye retention (Use of PKH26 or PKH67) [33] Human cell lines (8 – 25%)

  • sphere-formation

  • limiting dilution analysis for tumor initiation (27-fold higher than PKH26Low cells)

  • Gene expression analysis of PKH26High cells indicate upregulation of bone development/migration and downregulation of actetylation-related genes,

  • No significant change found in Oct3/4 and Nanog expression between the two fractions.
Expression of exogenous Oct4 promoter-driven GFP transgene [56] Human cell line OS521 (24% of total adherent population)

  • Higher tumor initiation capacity and earlier age of onset in in vivo tumorigenesis assay seen in Oct4- GFPHigh cells,

  • Higher expression of mesenchymal stem cell markers (CD29, CD44, CD56, CD90, and CD105) and no correlation with canonical stem cell markers (CD133, EpCAM, or CD44) seen in the Oct4- GFPHigh cells