1. SARCOSPHERE METHOD FOR
ISOLATION AND CULTURE
OSTEOSARCOMA CANCER STEM
CELLS
DEDED YUDHA PRANATHA

2. INTRODUCTION
OSTEOSARCOMA PROBLEMS :
METASTASIS, RECCURENCE AND RESISTANT FROM CHEMOTHERAPY
RECENT STUDY CANCER STEM CELLS SELF
RENEWAL, DIFFERENTIATION, PROLIFERATION
OSTEOSARCOMA CSCs RESEARCH HAS NOT BEEN REPORTED
IN INDONESIA
OUR STUDY :
ISOLATION, CULTURE AND CHARACTERIZATION OF HUMAN
OSTEOSARCOMA CELLS
Gibbs CP, et al. Stem-like cells in bone sarcomas: Implications for Tumorigenesis. Neoplasia. 2005;7:11.
Wilson H, Huelsmeyer M, Chun R, Young KM, Friedrichs K, Argyle DJ. Isolation and characterisation of cancer stem cells from
canine osteosarcoma. Veterinary journal. 2008 Jan;175(1):69-75.

3. Research Question
1. ARE OSTEOSARCOMA CSCs
CAN BE ISOLATED AND
CULTURED BY INVITRO
MEANS ?
2. HOW ARE THE
MORPHOLOGY AND
CHARACTERISTIC OF
OSTEOSARCOMA CSCs ?
Research Hypotheses
1. CSCs CAN BE ISOLATED AND
CULTURED FROM
OSTEOSARCOMA CELLS
2. OSTEOSARCOMA CSCs
MORPHOLOGY AND
CHARACTERISTIC
EXAMINATION CAN BE
PERFORMED BY USING
ALIZARIN RED S STAINING,
RT-PCR METHOD, AND IFA

4. Objectives Research Benefits
1. THEORETICAL BENEFITS
– TO PROVE THAT CSCs CAN
BE ISOLATED, CULTURED,
AND CHARACTERIZED BY
IN VITRO MEANS FROM
HUMAN
OSTESOSARCOMA CELLS
2. METHODOLOGICAL BENEFITS
– PROVIDE DONATION IN
PROBLEM SOLVING OF
OSTEOSARCOMA CSCs
STUDY
– HAVING AN INTELLECTUAL
PROPERTY
3. APPLICATIVE BENEFITS
– MAKE A STUDY GUIDE
FOR RESEARCH
DEVELOPMENT OF
HUMAN OSTEOSARCOMA
IN CMH AND UI
1. GENERAL OBJECTIVES
a. TO KNOW WHETHER
OSTEOSARCOMA STEM
CELLS CAN BE
ISOLATED, CULTURED,
AND CHARACTERIZED
BY IN VITRO MEANS
b. TO KNOW THE
MORPHOLOGY AND
CHARACTERISTIC OF
OSTEOSARCOMA CSCs
2. SPECIFIC OBJECTIVES
– TO MAKE THIS STUDY AS
AN INTRODUCTION OF
OSTEOSARCOMA CSCs
STUDY IN CMH AND UI

5. CONCEPTUAL FRAMEWORK
SARCOSPHERE-FORMING ASSAY
ISOLATION AND CULTURE OF CANCER
STEM CELLS
CHARACTERIZATION OF CANCER STEM
CELL
MORPHOLOGY RECULTURIZATION OF ADHEREN
CULTURE
RT-PCR
NANOG CD 133
OCT3/4STAT3
IMMUNOFLUORESCENCE
PHOSPHATASE
ALKALINE
CD133
OSTEOCALCIN
ALIZARIN RED S STAINING
OSTEOSARCOMA CELLS

6. MATERIALS AND METHODS
Inclusion :
age 10-25, CPC,
conventional
Exclusion :
Damage of tissue tumor or
specimen failed to grow
• Number of
samples are
assigned from an
osteosarcoma cell
of predecessor
research
• 2015, CMH and
PSSP Bogor
• Descriptive
Design
Time and
Location
Sample
Characteristics
of Inclusion and
Exclusion

7. RESEARCH PROTOCOL
CHARACTERIZED BY ALIZARIN RED S STAINING, RT-PCR, AND IFA
CSC MORPHOLOGY
SARCOSPHERES FORMING ASSAY
ISOLATION AND CULTURALIZATION OF OSTEOSARCOMA CELL INSIDE OBM + OGM
SINGLEQUOTS MEDIUM, DMEM, THEN CELLS ARE SUPPLEMENTED WITH 10% FBS
AND PS 1% IN 370C, CO2 5%, 95% AIR
CANCER CELL IS OBTAINED BY OPEN BIOPSY AND SURGERY
OSTEOSARCOMA
Gibbs CP, et al. Stem-like cells in bone sarcomas: Implications for Tumorigenesis. Neoplasia. 2005;7:11.
Wilson H, Huelsmeyer M, Chun R, Young KM, Friedrichs K, Argyle DJ. Isolation and characterisation of cancer stem cells from canine
osteosarcoma. Veterinary journal. 2008 Jan;175(1):69-75.

8. RESULTS
STAGE I :
ISOLATION AND CULTURALIZATION OF OSTEOSARCOMA CELLS
STAGE II
SARCOSPHERE FORMING ASSAYS

9. RESULTS
STAGE III CHARACTERIZATION
ALIZARIN RED S STAINING
DAY 3 DAY 7

10. RESULTS
STAGE III CHARACTERIZATION
RT-PCR
NANOG, OCT3/4, STAT3 CD133

11. RESULTS
STAGE III CHARACTERIZATION
IFA
ALKALINE
PHOSPATASE
CD 133 OSTEOCALSIN
NEGATIVE
CONTROL

12. DISCUSSION
GIBBS, ET AL (2005) :
3 human primary
osteosarcoma cells and
1 human osteosarcoma
cell line
WILSON H, ET AL (2007) :
1 human osteosarcoma
cell line and 3
osteosarcoma canine
lines
WANG L, PARK P,
LIN CY (2009):
4 human osteosarcoma
cell lines
NEVES (2011) :
1 human osteosarcoma
cell line
ISOLATION AND CULTURE OSTEOSARCOMA CANCER
STEM CELLS

13. ISOLATION AND CULTURE OF OSTEOSARCOMA
STEM CELL
THOSE FINDINGS WERE APPROPRIATE WITH
OUR STUDY. ABILITY TO PROLIFERATE AND
SELF RENEW WAS SHOWN BY THE ABILITY TO
FORM THE SPHERE ON ANOIKIS CONDITION
AND WHEN THE SPHERE WAS REGROWN
BACK AT THE TISSUE CULTURE PLATE, THE
CELLS COULD RE ATTACH TO THE PLATE
SURFACE
PROVED THAT OUR STUDIED
OSTEOSARCOMA CELLS CONTAINED A
SMALL NUMBER OF PRIMITIVE CELLS
THAT WERE PROBABLY CANCER STEM
CELLS

14. OSTEOSARCOMA STEM CELL
CHARACTERIZATION
DISCUSSION
FIRST TECHNIQUE : ALIZARIN RED S STAINING
SECOND TECHNIQUE : RT-PCR
THIRD TECHNIQUE : IFA

15. OSTEOSARCOMA STEM CELL
CHARACTERIZATION
DISCUSSION
OUR STUDY SHOWED POSITIVE RESULTS, SARCOSPHERE SHOWED BRIGHT RED-
ORANGE COLOR, PROVING A COMPLEX BOND BETWEEN ALIZARIN RED S AND
CALSIUM
FIRST TECHNIQUE : ALIZARIN RED S STAINING
LUK F,YU Y, DONG HT, WALSH WR, YANG
JL (2011):
Alizarin red S staining was used to stain
Osteosarcoma cell line : Sao2-U-2 OS
Cells

16. OSTEOSARCOMA STEM CELL
CHARACTERIZATION
DISCUSSION
SECOND TECHNIQUE : RT-PCR
GIBBS, ET AL (2005) :
Express actived STAT3 and
marker genes of pluripotent
marker, Oct ¾ and Nanog
WILSON H, ET AL (2007) :
Expression of genes
characteristic of Oct4, NANOG
AND STAT3 and Stro-1
WANG L, PARK P,
LIN CY (2009):
Demonstrated Oct ¾ and
Nanog were expresed in all 4
cell lines of human
Osteosarcoma
LOU N,WANG Y, SUN D, ZHAO
J, WANG Y, GAO Z (2010) :
RT-PCR : Oct ¾, Nestin,
Nanog were highly expressed
MG-63-M cells
OUR FINDING SUPPORTED THOSE RESULTS: RT-PCR SHOWED EXPRESSIONS OF OCT
¾, NANOG, STAT3 IN ALL OF THE CELL LINEAGE

17. OSTEOSARCOMA STEM CELL
CHARACTERIZATION
DISCUSSION
SECOND TECHNIQUE : RT-PCR
TIRINO, ET AL (2008) :
Identified CD133+ cells
within osteosarcoma cell
lines
TIRINO, ET AL (2011) :
Show existence of CSCs in
human primary bone
sarcomas and highlight
CD133 as a pivotal marker
for identification
DI FIORE, ET AL (2013):
Demonstrated highly
expresed CD 133 on
3AB-OS Cell Line

18. OSTEOSARCOMA STEM CELL
CHARACTERIZATION
DISCUSSION
THE POSIBILITY OF CD133 WAS NOT DETECTED BECAUSE OF THE NUMBER OF OSTEOSARCOMA
CELLS WERE VERY SMALL IN SARCOSPHERE. THIS WAS ACTUALLY CAN PREVENTABLE BY DOING
FACS ANALYSIS PRIOR SARCOSPHERE TO ENSURE THAT CELLS DERIVED FROM CELL CANCER CELLS
WAS OSTEOSARCOMA CELLS OR BY USING qPCR METHOD
OUR STUDY DEMONSTRATED DIFFRENTIATED RESULTS, CD 133 DID NOT EXPRESS ON RT-PCR
AND WEAK POSITIVE ON IMUNNOFLUORECENCE ANALYSIS.
LOU N,WANG Y, SUN D, ZHAO
J, WANG Y, GAO Z (2010) :
Demonstrated highly
expresed CD 133 MG-63-M
cells
HE, ET AL (2012) :
Demonstrated clinical
relevance expressed CD 133 in
Osteosarcoma Patients with
Pulmonary Metastasis
SECOND TECHNIQUE : RT-PCR

19. OSTEOSARCOMA STEM CELL
CHARACTERIZATION
DISCUSSION
THIS PROVED THAT ON THE SARCOSPHERE THAT WE STUDIED OCCURRED IN VITRO
OSTEOBLAST’S ACTIVITIES. POSITIVE ALKALINE PHOSPHATASE SHOWED PLURIPOTENCY THAT
WAS ALSO PRESENT AT THE SARCOSPHERETHAT WE ANALYZED
OUR RESULTS OF IMMUNOFLUOROSENCE ANALYSIS SHOWED ALKALINE PHOSPHATASE
MARKERS AND OSTEOCALCIN GAVE POSITIVE PROTEIN EXPRESSION RESULTS
SINGH U, ET AL (2012) :
Demonstrated : flurogenic
AP Stain that enables
characterization of ESC
and iPSC
O CONNOR, ET AL (2008) :
Alkaline Phosphatase
(AP+) optimized for
propagating
undifferentiated hESCs.
PALMQVIST, ET AL (2005):
Found : Cells with ability
to develop in vivo
produce alkaline
phosphatase cell colonies
at high frequency
THIRD TECHNIQUE : IFA

20. IN OUR STUDY, OSTEOSARCOMA CSCs
CHARACTERIZATION CAN BE PROVEN BY:
DISCUSSION
• POSITIVE EXPRESSION OF OSTEOBLAST COMPLEX
WITH CALCIUM ON ALIZARIN RED STAINING
• POSITIVE EXPRESSION FROM PLURIPOTENCY
MARKER ON RT-PCR.
• POSITIVE PROTEIN EXPRESSION AS A MARKER FOR
OSTEOBLAST AND SURFACE MARKER ON
IMMUNOPLUOROSENCE ANALYSIS

21. ISOLATION AND CULTURE OF OSTEOSARCOMA CANCER STEM CELLS
OSTEOSARCOMA CSCs COULD BE ISOLATED AND CULTURED IN VITRO FROM OSTEOSARCOMA
CELLS BY METHODS OF SPHERE FORMING ASSAY
MORPHOLOGICALLY , OSTEOSARCOMA STEM CELLS ON THE SARCOSPHERE WAS ROUND SHAPED,
THREE DIMENSION AND DID NOT ADHERE TO THE SUBSTRATE WHEN REPLANTED INTO THE TISSUE
CULTURE PLATE, SARCOSPHERE WAS REATTECHED TO THE SUBSTRATE, FORMING SPINDLE-LIKE
FIBROBLASTS
OSTEOSARCOMA CANCER STEM CELLS CHARACTERIZATION
ALIZARIN RED S STAINING SHOWED POSITIVE EXPRESSION ON OSTEOSARCOMA CSCs
OSTEOSARCOMA CSCs SHOWED GENE EXPRESSION, PROVED BY RT-PCR WITH PROTEIN
EXPRESSION CONFIRMED BY IFA
CONCLUSION

22. ISOLATION AND CELL CULTURE OF OSTEOSARCOMA CANCER STEM CELLS
FACS ANALYSIS IS NEEDED ON SARCOSPHERE STAGE WITH CD133 GENE MARKER
FOR PURIFICATION OF FORMED SACOSPHERE SO THAT CELLS THAT WERE OBTAINED
CAN BE PROVEN AS CD133 POSITIVE
CULTURE OF OSTEOSARCOMA FROM OTHER PATIENT TO STANDARIZED THE
CULTURE PROCEDURE OF CSCs OSTEOSARCOMA
CHARACTERIZATION OF OSTEOSARCOMA CANCER STEM CELLS
qPCR METHOD IS NEEDED FOR CHARACTERIZATION OF OSTEOSARCOMA CSCs
RECOMMENDATION

23. THANK YOU