Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

All experimentation using human tissues described herein was approved by the local ethical committee (Rif. N. 141/12). Informed consent for the collection of tissue samples and for the use and storage of the samples was obtained from the donors at AOUC.

1. Preparation for Culture

1. Prepare growth culture medium (GM) by adding 10% Foetal Bovine Serum (FBS), 100 IU/ml penicillin and 100 µg/ml streptomycin to Coon's modified Ham's F12 medium. Filter and sterilize GM using a 0.22 µm filter. Store GM up to 1 month at 4 °C.
2. Prepare collagenase medium (CM) by adding 20% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin and 3 mg/ml collagenase type II to Coon's modified Ham's F12 medium. Filter and sterilize CM using a 0.22 µm filter. Store CM up to 1 month at -20 °C.
3. Prepare medium for cell freezing (FM) by adding 40% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin and 6.5% dimethylsulfoxide (DMSO) to Coon's modified Ham's F12 medium. Filter and sterilize FM using a 0.22 µm filter. Store FM up to 1 month at 4 °C.
4. Prepare medium for CFU assay (CFUM) by adding 20% FBS, 100 IU/ml penicillin and 100 µg/ml streptomycin to Coon's modified Ham's F12 medium. Filter and sterilize CFUM using a 0.22 µm filter. Store CFUM up to 1 month at 4 °C.
5. Prepare trypsin by dissolving 400 mg trypsin, 200 mg EDTA and 1,000 mg D(+)-glucose anhydrous in 1,000 ml Dulbecco's Phosphate-Buffered Saline (DPBS) without Ca²⁺ and Mg²⁺.
   NOTE: Divide fresh trypsin-EDTA into 50 ml aliquots in 25 cm² flasks using a 0.22 µm filter and freeze at -20 °C for up to 3 months. Store thawed filter-sterilized aliquots at 4 °C without loss of activity.
6. Prepare stem cell growth medium (SCGM) by adding 10% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10 ng/ml bFGF (25 µg/ml stock) to Coon's modified Ham's F12 medium. Filter SCGM using a 0.22 µm filter and store SCGM up to 2 weeks at 4 °C.
7. Prepare 2% methylcellulose (MC) by dissolving MC in ultrapure H₂O at 4 °C for 3 d. When MC is completely dissolved, autoclave it and store at 4 °C.
   NOTE: After MC is sterilized, it becomes solid. To bring MC to a liquid state, store at 4 °C.
8. Prepare sarcosphere growth medium (SGM). Prepare this medium fresh (not more than 2 weeks before use) by adding 100 IU/ml penicillin, 100 µg/ml human bFGF (25 µg/ml stock), 20 nM progesterone (10 µM stock), 100 µM putrescine, 30 nM sodium selenite (30 µM stock), 25 µg/ml transferrin (25 mg/ml stock), 20 µg/ml insulin (20 mg/ml stock), and 10 ng/mL human EGF (10 µg/mL stock) to 2X Coon's modified Ham's F12 medium. Filter and sterilize SGM using a 0.22 µm filter. Store for up to 2 weeks at 4 °C.
9. Prepare osteogenic medium (OM) by adding 10% FBS (South American origin), 100 IU/ml penicillin, 100 µg/ml streptomycin, 10 nM dexamethasone (100 µM stock), 0.2 mM sodium L-ascorbyl-2-phosphate (1 M stock), 10 mM β-glycerophosphate (5 mg/ml stock) and 1 µg/ml calcein (200 µg/ml stock) to Coon's modified Ham's F12 medium. Filter and sterilize OM using a 0.22 µm filter. Store at 4 °C.
   NOTE: Store dexamethasone stock solutions under liquid nitrogen to maintain their activity. Prepare fresh OM every 2 weeks to maintain the activity of dexamethasone in order to maintain the differentiation potential of the medium.
10. Prepare erythrocyte lysis buffer (ELB) by dissolving 1.66 mg NH₄Cl, 0.2 mg K₂HPO₄ and 0.007 mg EDTA in 200 ml distilled water (dH₂O). Filter and sterilize ELB using a 0.22 µm filter. Store at 4 °C.
11. Prepare adipogenic medium (AM) by adding 10% FBS (South America origin), 100 IU/ml penicillin, 100 µg/ml streptomycin, 1 µM dexamethasone (1 mM stock), 1 µM bovine insulin (10 mM stock), 0.5 mM isobutylmethylxanthine (IBMX) (500 mM stock), and 100 µM indomethacin (200 mM) to Coon's modified Ham's F12 medium. Filter and sterilize AM using a 0.22 µm filter. Store at 4 °C.
    NOTE: Store dexamethasone stock solutions under liquid nitrogen to maintain their activity. Prepare fresh AM every 2 weeks to maintain the activity of dexamethasone in order to maintain the differentiation potential of the medium.
12. Prepare 2% Bovine Serum Albumin (BSA) in DPBS (BSA/DPBS). Dissolve 10 g BSA in 500 ml DPBS and prepare the stock solution by aliquotting 50 ml stock solution in 50 ml conical tubes. Store at -20 °C.
13. Prepare a solution of 4% paraformaldehyde (PFA) in DPBS (PFA/DPBS). In a chemical hood, dilute paraformaldehyde in DPBS and prepare the stock solution by aliquotting 50 ml stock solution in 50 ml conical tubes. Store at 4 °C.

2. Establishing Primary OS Cell Cultures and OS Finite Cell Lines (OSA)

NOTE: Primary OS cell cultures were prepared from fresh samples of conventional OS biopsies collected at the "Unità Ortopedia Oncologica e Ricostruttiva", AOUC Careggi, Florence. All biopsies, which were obtained by needle aspiration or surgical resection of a small portion of the tumor (Figure 1A, B), were immediately placed in culture medium supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin (pH 7.4) and transported to the laboratory where they were processed. All described manipulations were conducted under aseptic conditions using a laminar flow hood.

1. Isolation of OS cells
   1. Place the biopsy in a 100 mm Petri dish with a small volume of GM. With a sterile lancet and Perry tweezers, mince the OS tissue samples by cutting them into pieces as small as possible (0.5 – 1 mm) (Figure 2A, B) .
   2. Cover the tissue fragments with 10 ml CM for the enzymatic digestion (Figure 2B) and incubate for 3 h in a 37 °C, 5% CO₂ incubator. Gently remove the suspension of fragments using a pipette and transfer it to a 15 ml conical tube for immediate centrifugation (400 x g for 5 min) to pellet the fragments.
      NOTE: After centrifugation, if the biopsies are rich in erythrocytes, a red deposit composed of erythrocytes can be seen over the fragments. Therefore, before proceeding to mechanical dispersion, treat the sample with erythrocyte lysis buffer. Discard the supernatant by aspiration and add 5 ml ELB.
   3. Suspend pellet fragments for 1 min and centrifuge the suspension at 400 x g for 2 min. Remove the supernatant by aspiration. Add 5 ml GM and mechanically disperse the fragments using a 10 ml serological pipette (opening size, 1.5 mm internal Ø) 10 – 20 times.
   4. Centrifuge the suspension at 400 x g for 5 min. Remove the supernatant by aspiration, suspend the cell pellet with 10 ml GM, and then transfer the resulting cell suspension into a 100 mm Petri dish. Incubate the Petri dish in a 37 °C, 5% CO₂ incubator and replace the GM with fresh complete GM every 3 d.
      NOTE: Quite often, OS tissue samples obtained by needle aspiration are very small and contain bone fragments, which are not digested by enzymatic digestion with collagenase. Therefore, after the supernatant has been removed, attempt to separate cells from bone tissue by mashing pellet fragments with a pipette.
2. Subculture
   NOTE: To establish an OSA, when cells reach approximate confluence, remove them from their culture vessel, dilute, and place in a fresh plate to enable further growth. This subculture procedure is achieved using the enzymatic procedure termed trypsinization.
   1. Remove the medium by aspiration. To dissociate the cell monolayer, add 2 – 3 ml trypsin at RT (max 18 – 20 °C), shake gently once, and remove the trypsin immediately by aspiration. Repeat twice. Incubate the cells at 37 °C for 3 – 4 min until they begin to detach.
      NOTE: Good trypsinization can be seen by the experienced user as small holes forming in the slightly opaque monolayer when the dish is held to the light. This dissociation can also be monitored using an inverted microscope; this approach is recommended for beginners.
   2. Stop the trypsin reaction by adding 10 ml GM and wash the dish well using a pipette to detach all the cells. Transfer 1 ml of the suspension to a new 100 mm Petri dish and add 9 ml GM (1/10 split of cells).
      NOTE: The remaining cell suspension can be used for cell expansion (transfer to another 100 mm Petri dish), can be cryopreserved (see Section 2.3), or can be counted and plated in experimental dishes or wells for proliferation assays or other purposes (see below).
3. Cryopreservation of OS primary cultures and OSA
   NOTE: Freeze one confluent 100 mm Petri dish into 4-5 cryovials. The process of cryopreservation must be performed quickly because DMSO, which protects cell membranes during freezing, is toxic to cells at non-freezing temperatures.
   1. Dissociate cell cultures from the monolayer by trypsinization (see Section 2.2), pellet cells by centrifugation at 400 x g for 5 min, remove the supernatant by aspiration, and then quickly suspend the cell pellet in FM.
   2. Aliquot 1 ml of this suspension per cryogenic vial. Immediately place the filled vials in a freezer box with a jacket of 2-propanol and store immediately at -80 °C O/N. Transfer cryovials to liquid nitrogen the following day for long-term storage.
4. Defrost OS cell lines and primary cultures
   1. Defrost OS cell lines from liquid nitrogen storage, thawing cryovials rapidly by placing them in a laboratory water bath at 37 °C. Transfer the contents of the cryovials into a 15 ml conical tube and add approximately 10 ml of GM (See Section 2.3 to remove DMSO). Remove the supernatant by aspiration and suspend the cell pellet in 10 ml GM. Plate the cell suspension in a 100 mm Petri dish and incubate in a 37 °C, 5% CO₂ incubator.

3. Sarcosphere Assay to Isolate OS-CSCs

NOTE: This experiment is performed on OSA. The duration of this experiment is related to the capacity of the cells to form these sphere colonies (sarcospheres), and the range of time is 7, 14, 21, and 28 d.

1. Establishment of sarcosphere assay
   1. Prepare the haemocytometer chamber and all reagents in advance.
   2. Remove the medium by aspiration and dissociate the cells from the monolayer by trypsinization (see Section 2.2). Mix the cell suspension thoroughly, pipetting to disperse any clumps, and collect 10 µL using a 20 µL pipette tip.
   3. Transfer the 10 µL cell suspension immediately to the haemocytometer chamber edge, expel the suspension, and allow it to be drawn under the coverslip by capillary action.
   4. Observe the haemocytometer chamber under phase contrast. Select a 10X objective and focus on the grid lines in the chamber. Count the cells lying in this 1 mm² area using the subdivisions (also bound by three parallel lines) and single grid lines as an aid for counting.
   5. Measure the concentration and apply the following equation: 1 ml = n_*10⁴/z (n: the whole number of cells in all the counted squares 1 mm²; z: the number of counted squares 1 mm²). Calculate the volume of the cell suspension necessary to plate a total amount of 240,000 cells divided into 40,000 cells for each well in the 6-well ultra-low attachment plate.
      NOTE: Be sure to plate the correct number of cells by plating cells into one extra well. Therefore, the total amount of cells to be plated is 280,000 cells.
   6. Prepare 35 ml SGM by adding 50% of 2% MC (SGM-MC) in a 25 cm² flask. Prepare the total volume of SGM considering 5 ml extra in order to plate one extra well.
   7. Add the calculated volume of the cell suspension to the SGM-MC prepared in the flask and mix gently using a pipette to disperse any clumps. Plate the cells in a 6-well ultra-low attachment plate and use an inverted microscope to observe how the cells appear after being plated.
   8. Incubate the cells in a 37 °C, 5% CO₂ incubator. Every 3 d, add fresh aliquots of bFGF and EGF to each well to refresh the concentration of the growth factors. Add 2 µL bFGF and 5 µL EGF to maintain both at the final concentration of 10 ng/ml in each well.
2. Isolation of sarcospheres
   NOTE: Monitor the good progress of the sarcosphere assay at 7, 14, 21, and 28 d to decide when it is necessary to isolate the sarcospheres that formed.
   1. Prepare all the reagents and equipment in the laminar flow hood (Figure 3A, B). Assemble the filtration unit by placing a net filter of 25 mm diameter and a 40 µm mesh into a membrane filter holder; sterilize by autoclaving (Figure 4A-E).
   2. For each well, transfer the medium containing the sarcospheres to a syringe with a sterile membrane filter holder using a sterile pipette with a 1,000 µL tip. After completely removing the medium containing the sarcospheres, wash the well by adding 5 ml GM to be sure to transfer all the spheres to a syringe. Use the microscope to confirm whether all the sarcospheres have been recovered. If not, proceed by repeating this step .
   3. Filter the suspension with a membrane filter holder only by gravity without pressure in order to prevent damaging the spheres and to avoid having sarcospheres crossing through the filter, thereby avoiding the loss of spheres. Remove air bubbles by gently using a sterile glass Pasteur pipette.
      NOTE: Sometimes the filtration is blocked by the presence of an air bubble in the membrane filter holder.
   4. Wash the filtration unit by adding 10 ml GM to the syringe and letting it filter without pressure to be sure to eliminate the single cells. Take apart the filter unit from the syringe and put it into a Petri dish.
   5. Remove the net filter from the membrane filter holder using Perry tweezers and wash it by shaking it gently with the tweezers in a 60 mm Petri dish to release the sarcospheres from the tangle of the membrane. Then, place the membrane in a well and confirm by microscopic observation that there are no more spheres tangled in the membrane. If spheres are still tangled in the membrane, proceed with another wash as described in step 3.2.4.
   6. Observe the released sarcospheres using a microscope. Incubate the released sarcospheres in a 37 °C, 5% CO₂ incubator. Repeat all steps for each well of a 6-well ultra-low attachment plate.

4. OS-CSC Lines

NOTE: OS-CSCs are obtained from the sarcospheres that exhibit adherent expansion by reintroducing and reculturing these cells in a monolayer after they are plated in small 60 mm Petri dishes no longer under ultra-low attachment conditions.

1. OS-CSC cultures
   1. To enable further growth, subculture cells when they reach approximately 90% confluence in a 60 mm Petri dish. Repeat step 2.2.1 to establish OS-CSC cultures.
   2. Stop trypsinization by adding 4 ml SCGM and wash the dish well, using a pipette to detach all the cells. Transfer the cell suspension to a new 100 mm Petri dish and add 6 ml SCGM. When OS-CSCs reach 90% confluence, subculture by repeating section 2.2.
      NOTE: For in vitro analysis to characterize the stem cell-like phenotype of isolated OS-CSCs, cells can be plated in different types of plates.
   3. Preserve the established OS-CSC lines by cryopreservation (repeat all steps of section 2.3). Defrost the cryopreserved OS-CSCs by repeating all steps of section 2.4.

5. In vitro Analysis to Characterize OS-CSCs:

1. Preparation of cells for immunofluorescence
   NOTE: Cells are fixed in paraformaldehyde when they reach the right grade of confluence in each well of a 24-well plate. The grade of confluence is related to the type of experiments.
   1. Plate OS-CSCs in a 24-well plate to study the surface Mesenchymal Stem Cells (MSC) markers by immunofluorescence. Fix cells in each well when they reach 50 – 60% confluence. Remove the SCGM by aspiration and wash twice in a DPBS 24-well plate.
   2. Under a chemical hood, add 500 µL 4% PFA/DPBS to each well. Incubate at RT for 10 min. Remove the PFA/DPBS and wash 3x with ultrapure dH₂O. Allow the 24-well plate to dry in the fume hood.
2. Immunofluorescence for MSC markers
   NOTE: Immunofluorescence staining of the OS-CSCs fixed in 4% PFA/DPBS can be used to investigate the MSC-like phenotype of OS-CSCs using antibodies directed against CD44, Stro-1 and CD105. The following method is used routinely by the authors.
   1. In a chemical hood, permeabilize cells that have been fixed in 4% PFA/DPBS by adding 500 µL 0.2% Triton X-100/DPBS to each well. Incubate the cells at 37°C for 30 min. Gently wash the cells 3x with DPBS. Add 300 µL RNase diluted 1/1,000 with 2% BSA/DPBS to the cells and incubate at 37 °C for 30 min.
   2. Gently wash the cells 3x with 2% BSA/DPBS. Stain the cells for MSC markers. Add the primary antibody only to the wells chosen as positive controls for each antibody.
      1. Add 300 µL anti-CD105 diluted 1/10 with 2% BSA/DPBS, 300 µL anti-CD44 diluted 1/10 with 2% BSA/DPBS, 300 µL anti-Stro-1 diluted 1/10 with 2% BSA/DPBS and only 200 µL 2% BSA/DPBS to the wells chosen as negative controls for each antibody.
      2. Incubate the cells in a humid environment at 4 °C O/N. Wash the wells 3x with DPBS and then twice with 2% BSA/DPBS.
   3. Reveal primary antibodies by adding specific secondary antibodies.
      1. Add 300 µL anti-rabbit IgG (donkey anti-rabbit IgG [H + L]) diluted 1/100 with 2% BSA/DPBS into each well chosen as positive and negative control for CD44 and CD105. Add 300 µL FITC anti-mouse IG (FITC-rabbit anti-mouse IgG [H + L]) diluted 1/100 with 2% BSA/DPBS into each well chosen as positive and negative control for Stro-1. Incubate cells in the dark at RT for 60 min. Wash the wells 6x with DPBS.
   4. Stain MSC marker-positive cells for cytoskeletal actin by adding 300 µL phalloidin diluted 1/100 with 2% BSA/DPBS into each well stained for MSC marker immunofluorescence.
   5. Incubate the cells for 40 min at RT. Wash the wells 3x with DPBS and then wash them twice with ultrapure dH₂O. Proceed to the counterstaining of the nuclei after the immunofluorescence staining described above. Prepare propidium iodide solution 10^-5 M in DPBS (stock 1.5 x 10^-3 M) in a fume hood.
      1. Add 200 µL propidium iodide solution to each well colored as described above. Incubate the cells for 2 – 3 min at RT. Wash the wells twice with ultrapure dH₂O.
         NOTE: Repeat all the steps of sections 5.1 - 5.2 on the cell line HCT8, a primary continuous differentiated colon cancer cell line that is used as a negative control.
3. Osteogenic differentiation assay
   NOTE: Osteogenic differentiation lasts for 20 d.
   1. Plate OS-CSCs in 24-well plates at a cell density of 1 x 10⁴ cells/cm² in SCCGM. Allow the cells to grow in SCGM until they reach 80 – 90% confluency in each well.
   2. Start osteogenic differentiation by changing the SCGM to OM. Allow the cells to grow in OM and refresh medium every 3 – 4 d. Stop the osteogenic differentiation assay at 10 d to evaluate the presence of alkaline phosphatase (ALP).
   3. Fix cells in 4% PFA/DPBS (See section 5.1). Evaluate the osteoblastic phenotype by cytochemical staining for ALP and for HA using Alizarin Red S staining.
   4. ALP cytochemical staining
      NOTE: Prepare the dye mixture in a chemical hood immediately before starting the staining.
      1. Dissolve 40 mg Fast Blue BB or Fast Red Violet LB salt in 50 ml Tris-HCl, pH 9 (Solution A). Dissolve 5 mg naphthol-AS-MX phosphate sodium salt in 1 ml DMSO (Solution B). Add Solution B entirely to Solution A and mix well, obtaining Solution C. Wash cells twice with DPBS.
      2. Add 500 µL – 1 ml Solution C to each well and incubate at 37 °C and 5% CO₂. Monitor the course of staining every 10 min by observing the cells under the microscope.
      3. Stop the staining when ALP-positive cells become intensely colored (blue with the Fast Blue BB salt or red with Fast Red Violet LB salt), which usually occurs within 30 min. Wash the cells 3x with ultrapure dH₂O to remove all residues of Solution C. If a precipitate of the stain is present, wash the cells quickly once with absolute ethanol.
      4. Proceed to the counterstaining of the nuclei after the ALP staining as described in the steps 5.2.5 and 5.2.5.1.
   5. Alizarin Red S cytochemical staining
      NOTE: Prepare the dye mixture before starting the experiment.
      1. Prepare the 2% Alizarin Red S (2 g Alizarin Red S in 100 ml of ultrapure H₂O). Add 2.5% NH₃ to 2% Alizarin Red S to reach pH 6.0. Store 2% Alizarin Red S solution at 4 °C. Wash the cells once with DPBS.
      2. Add Alizarin Red S to the cells for only a few sec.
      3. Wash the wells with ultrapure dH₂O to control the degree of staining; if the HA deposits are not intensely colored, repeat step 5.3.5.2. Stop the staining when HA deposits become intensely red colored, which usually occurs within few minutes. Wash the wells with ultrapure dH₂O.
4. Adipogenic differentiation
   NOTE: Assay duration depends on the OS-CSC lines. The assay can last 14 – 30 d.
   1. Plate OS-CSCs in 24-well plates at a cell density of 1 x 10⁴ cells/cm² in SCGM. Allow the cells to grow in SCGM until they reach 80 – 90% confluency in each well. Initiate adipogenic differentiation by changing the SCGM to AM. Allow the cells to grow in AM and refresh the AM twice a week.
   2. Stop the adipogenic differentiation assay when lipid vesicles are visible. Evaluate the adipogenic phenotype by Oil Red O staining and by haematoxylin counterstaining for nuclei.
   3. Haematoxylin counterstaining for nuclei
      NOTE: Prepare the dye mixture before starting the experiment.
      1. Prepare the 5% haematoxylin solution, called Emallume Carazzi³⁹. Store the solution at 4 °C. Add Emallume Carazzi for only 2 min. Wash wells with ultrapure dH₂O.
5. CFU assay
   NOTE: This experiment must be performed in triplicates.
   1. Plate OS-CSCs in 100 mm Petri dishes at a cell density of 450 cells/cm² in CFUM. Incubate the cells in a 37°C, 5% CO₂ incubator for 4 weeks. Refresh CFUM twice a week.
   2. Stain the CFUs with toluidine blue. Count the colored colonies using an inverted microscope. Calculate CFU efficiency according to the following formula: (number of colonies formed/number of cells seeded)*100.
6. ALDH activity analysis
   NOTE: ALDH activity has been evaluated using an ALDH Activity Colorimetric Assay Kit on the two OS-CSC lines and on a finite fibroblast line, which was used as a negative control. This kit quantifies the ALDH enzymatic activity by absorbance reading at 450 nm. All tests have been performed in triplicates.
   1. Dissociate cell cultures from the monolayer by trypsinization (see Section 2.2). Pellet cells by centrifugation at 400 x g for 5 min. Follow manufacturer's protocol.
7. Flow cytometric analysis:
   NOTE: Single cell suspensions are required for optimal staining of samples for flow cytometry. A single cell suspension must be prepared for each antibody to be tested. Dissociate cell cultures from the monolayer by trypsinization (see Section 2.2).
   1. Place the cell suspension in a conical tube, perform a cell count using the Bürker Counting Chamber, centrifuge cells at 400 x g and resuspend in an appropriate volume of separation buffer to obtain a cell suspension at a final cell concentration of 1 x 10⁵ cells/ml.
   2. Centrifuge the cell suspension for 5 min at 4 °C. Discard the supernatant and wash the pellet with separation buffer. Repeat this step twice.
   3. Stain cells for MSC markers (i.e., CD44, CD105 and Stro-1)⁴⁰.
   4. Analyze positively stained cell suspensions in a flow cytometer. Analyze the marked samples within 1 d. Incubate these samples at 4 °C until analysis.
8. RT-PCR
   1. Extraction and isolation of RNA
      1. Add 1 ml lysis reagent to the cellular frozen packed samples of OS-CSCs and lyse the cells directly in the tube by pipetting the pellet up and down several times.
      2. Centrifuge the samples at 12,000 x g for 1 min at 4 °C. Gently remove the supernatant. Transfer the supernatant to a new tube, add 200 µL chloroform, and cap the tube securely. Shake the tube vigorously for 15 sec and incubate the sample for 5 min at RT.
      3. Centrifuge the samples at 12,000 x g for 15 min at 4 °C. The mixture separates into three different phases. The RNA is exclusively in the colorless upper aqueous phase.
      4. Being particularly careful, remove only the aqueous phase and transfer this phase into a new tube to proceed with RNA isolation. Add 500 µL isopropanol to the tube containing the aqueous phase. Shake the tube gently by hand. Incubate the sample at RT for 10 min. Centrifuge the sample at 12,000 x g for 10 min at 4 °C.
         NOTE: After the sample is centrifuged, it is possible see the RNA, which forms a gel-like pellet on the side and on the bottom of the tube.
      5. Remove the supernatant from the tube, being careful to leave the pellet of the RNA on the bottom. Add 1 ml 75% ethanol to the tube. Vortex the tube by hand for a few sec and then centrifuge the tube at 7500 x g for 5 min at 4 °C.
      6. Discard the ethanol and air dry the RNA pellet. When the pellet is dry, resuspend the RNA pellet in RNase-free water (10 – 50 µL) by pipetting the solution up and down several times.
      7. Determine the yield and purity of the RNA by measuring the absorbance at 260 nm and 280 nm using a spectrophotometer. Evaluate the integrity of the total RNA on standard 1% agarose gel. Store the RNA at -80 °C.
   2. Reverse polymerase chain reaction
      1. Synthesize first-strand cDNAs from 500 ng RNA samples using a reverse transcription kit. Thaw RNA samples on ice and thaw the necessary solutions included in the kit at RT. Proceed to synthesize cDNAs following manufacturer's protocol.
   3. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
      1. Perform all PCRs using 1 µL cDNA for each sample as a template in a final reaction volume of 24 µL. Use the primer sequences listed in Table 1 for the amplification of Nanog, Oct 3/4, SOX2 and CD133 genes.
      2. Separate the RT-PCR products by 1.8% agarose gel electrophoresis and stain with ethidium bromide. Photograph under UV illumination.