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Osteogenic and Adipogenic Differentiation of Osteosarcoma Cells
骨肉瘤细胞的成骨和生脂分化   

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Abstract

Osteosarcomas are the most common primary, non-hematologic malignant tumors in childhood and adolescence, comprising almost 60% of all bone sarcomas. Although these tumors are osteoblastic in nature, the characteristics of the specific tumor-initiating cells are unclear. Osteosarcomas contain highly proliferative undifferentiated malignant cells with a disrupted bone differentiation program. Cancer stem cells (CSCs) that have tumor-initiating properties and capacity of symmetric and asymmetric division have now been described in many solid tumors. For osteosarcomas, the CSC hypothesis has received support from recently reported findings that both human and murine osteosarcomas contain a sub-population of multipotent cells that that express various mesenchymal stem cell surface markers and are capable of undergoing differentiation in multiple mesenchymal lineages such as osteoblasts and adipocytes. Differentiation into these different lineages can be easily assessed by growing cells in specific medium and assaying for differentiation markers.

Materials and Reagents

  1. Osteosarcoma cells (Basu-Roy et al., 2012)
  2. DMEM - high Glucose from Invitrogen (Life Technologies, Invitrogen™, catalog number: 11330-032 )
  3. Fetal bovine serum (FBS)
  4. Penicillin-streptomycin
  5. Beta-glycerol phosphate (Sigma-Aldrich, catalog number: G9422 )
  6. Ascorbic acid (Sigma-Aldrich, catalog number: A4034 )
  7. Earle's balanced salt solution (EBSS) from Invitrogen (Life Technologies, Invitrogen™, catalog number: 14155-063 )
  8. HCl
  9. Ethanol
  10. DMSO (Sigma-Aldrich, catalog number: D8418 )
  11. Phosphate buffered saline (PBS)
  12. Paraformaldehyde
  13. Isopropanol
  14. Acetone
  15. Double-distilled H2O (ddw)
  16. Citrate concentrated solution (20 ml) (Sigma-Aldrich, catalog number: 854C )
  17. Naphthol AS-MX phosphate alkaline solution, 0.25% (20 ml) (Sigma-Aldrich, catalog number: 855 )
  18. COMPLETE capsule of Fast Blue RR salt (Sigma-Aldrich, catalog number: FBS25-10 CAP )
  19. Oil-Red-O (Sigma-Aldrich, catalog number: O0625 )
  20. Mayer's HemTox stain (Sigma-Aldrich, catalog number: MHS1 )
  21. Alizarin Red S certified by the biological stain commission (Sigma-Aldrich, catalog number: A5533 )
  22. Beta-glycerol phosphate stock solution (see Recipes)
  23. 1,000x ascorbic acid solution (see Recipes)
  24. Dexamethasone (Sigma-Aldrich, catalog number: D4902 ) (see Recipes)
  25. 3-Isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, catalog number: I5879 ) (see Recipes)
  26. Indomethacin (Sigma-Aldrich, catalog number: I8280 ) (see Recipes)
  27. Insulin (recombinant human) (Sigma-Aldrich, catalog number: I2643 ) (see Recipes)
  28. Fixation solution (see Recipes)
  29. Staining solution for osteogenesis (see Recipes)
  30. Oil-Red-O stock solution (see Recipes)
  31. Staining solution for mineralized osteoblasts (see Recipes)

Equipment

  1. Tissue culture set up
  2. Pasteur pipette
  3. Pasteur pipette tip
  4. Light microscope
  5. Pall Life Sciences acrodisc ayringe filter (0.2 μM HT Tuffryn membrane low-protein binding non-pyogenic) (Life Sciences, catalog number: PN 4192 )
  6. 0.2 μM nylon filter
  7. 24-well plates

Procedure

  1. Plating cells for differentiation
    1. Plate 50,000-60,000 osteosarcoma cells per well in 24-well plates in DMEM/10% FBS/ Penicillin-Streptomycin. This can be your cell line of interest too. Change medium according to your cell line of interest.
    2. Next day or when they reach confluence (usually in 36 h), switch medium to differentiation medium. Usually cells will not differentiate unless they are at least 80-90% confluent. All differentiation medium is regular DMEM/10% FBS/PS supplemented with the differentiation cocktail indicated in steps 3 and 4.
    3. For osteogenesis, use 10 mM beta-glycerol phosphate and 100 μg/ml ascorbic acid. These are the final concentrations.
    4. For adipogenesis, use 10 μg/ml Insulin, 100 nM dexamethasone, 250 μM IBMX and 200 μM Indomethacim. These are the final concentrations. How to make stocks is described later.
    5. Feed cells every 2-3 days with fresh differentiation medium made each time during feeding. While feeding make sure Pasteur pipette tip used for aspirating medium does not touch cell layer as cells attach loosely during differentiation, especially during adipogenesis.
    6. Stain cells for osteogenesis and adipogenesis at 2, 5, 8, 10, 12 and 15 days.

  2. Alkaline phosphatase assay to measure osteoblast differentiation.
    This is a colorimetric assay where the yellow substrate. 4-Benzoylamino-2, 5-dimethoxybenzenediazonium chloride hemi (zinc chloride) salt, Azoic Diazo No. 24 is hydrolyzed by alkaline phosphatase to yield a purple dye precipitate.
    Prepare fixation solution and staining solution before starting assay. Protect solutions from light fiter preparation.
    1. Plate required cells in differentiation medium (usually 50,000 cells/well in 24-well plates).
    2. Aspirate off medium from cells plated in differentiation medium at designated time.
    3. Wash once with 1x PBS. Each washing step involved adding 1x PBS followed by aspiration.
    4. Add 1 ml of fixation solution per well and incubate at RT for 1 min.
    5. Wash wells with 1x PBS once.
    6. Add I ml of staining solution per well.
    7. Incubate in the dark for 30 min at RT and check color development. Development of purple-violet stained areas in well indicates positive reaction. If necessary, continue reaction for another 30 min at RT. Record increased incubation time. Do not exceed 1 h.
    8. Stop reaction by washing quickly with dd water or tap water.
    9. Do not forget to photograph plates before proceeding to Alizarin red S staining for mineralization.

  3. Alizarin red S staining to detect mineralization
    Alizarin red S is a calcium-sensing dye. Differentiated osteoblasts deposit large amounts of extracelluar calcium (a hallmark of mineralization) that can be detected by complexing with Alizarin red S. Calcium deposits appear as bright orange-red stained areas.
    1. After photographing the plates stained for alkaline phosphatase, proceed to Alizarin red S staining by washing the stained plated twice with 1x PBS whose pH has been adjusted to 4.2 with 0.1% NH4OH.
    2. Aspirate the PBS and add enough Alizarin red S staining solution to cover the cellular monolayer. Incubate the plate at RT in the dark for 45 min.
    3. Carefully aspirate the Alizarin red S solution and wash the plate fours time wits distilled water.
    4. Extracellular calcium deposits appear as brightly stained orange-red areas.

  4. Oil red O staining assay to measure adipogenic differentiation
    This staining assay is based on the principle that the lipophilic dye Oil Red O is retained in fat globules of adipocytes that can be seen under a microscope.
    1. Aspirate medium and wash cells once in PBS.
    2. Fix cells in 4% PFA at RT for 15 min.
    3. Wash twice with ddH2O for 2 x 5 min.
    4. Stain with Oil-Red-O working solution for 15 min at RT.
    5. Wash with 1x PBS or ddH2O for 3 x 5 min.
    6. Rinse cells with 50% isopropanol once at RT.
    7. Rinse cells with 1x PBS or ddH2O.
    8. The red stained lipid droplets can be visualized by light microscopy. The adipocytes are also readily visualized in normal microscopy through their rounded morphology and markedly enhanced light reflection of larger lipid droplets.
    9. To count total cells, stain with Mayer's HemTox stain for 15 min at RT and wash 2x with dd water.

Recipes

  1. Beta-glycerol phosphate can be prepared by making a 100x stock of 1 M solution. Pre-heat water for ease of dissolution. The formula weight of the compound depends on the degree of hydration and can be determined by using the Lot Number provided on the bottle. Filter using a Pall Life Sciences Acrodisc syringe filter. Make 1 ml aliquots and freeze in -20 °C.
  2. 1,000x ascorbic acid solution can be prepared by dissolving 1 gram of Ascorbic acid in 10 ml of ddw (double-distilled water). Filter using a Pall Life Sciences Acrodisc Syringe filter. Make 1 ml aliquots and freeze in -20 °C.
  3. Dexamethasone (FW 392.5)
    Dissolve 0.0196 g in 10 ml absolute ethanol to yield 5 mM stock; store at -80 °C. Prepare fresh working stock by diluting in 100% ethanol to 100 μM.
  4. 3-Isobutyl-1-methylxanthine (IBMX) (FW 222.2)
    Dissolve 100 mg IBMX in 1.8 ml DMSO and sterile filter through 0.2 μM nylon filter to make 250 mM stock (1,000x). Aliquot and store at -20 °C or below.
  5. Indomethacin (FW 357.8)
    Dissolve 0.5367 g indomethacin in 7.5 ml DMSO, and filter through 0.2 μm nylon filter to sterilize for 200 mM (1,000x) stock. Dispense 1-2 ml aliquots in sterile cryovials, and store in opaque box to protect from light. Store at -70 °C or below.
  6. Insulin, recombinant human
    Dissolve 50 mg insulin in 10 ml EBSS acidified with 50 μl of 0.005 N HCl to yield 500x stock, and sterile filter. Do not forget to use low protein binding filters, as described in equipment. Dispense 1 ml aliquots in sterile cryovials and store at -70 °C or below for up to 2 years. Thawed, working stock can be stored at 4 °C for up to 1 month. Alternatively, sterile-filter acidified EBSS and use the sterile solution to make insulin under sterile conditions.
  7. Fixation solution for osteogenesis
    20 ml dd water + 30 ml acetone + 400 μl citrate concentrated solution.
  8. Staining solution for osteogenesis
    48 ml dd water + 2 ml Naphthol AS-MX phosphate alkaline solution, 0.25% + 1 complete capsule of fast blue RR salt
  9. Oil-Red-O stock solution for staining adipocytes
    Dissolve 500 mg of Oil-Red-O in 100 ml of isopropanol. For working stock, dilute stock as 2 parts dd water + 3 parts Oil Red O stock. Shake, Incubate at RT for 15 min and filter using a Pall Life Sciences Acrodisc Syringe filter. Use within 1 h of preparation.
  10. Staining solution for mineralized osteoblasts
    Dissolve 2 g Alizarin Red S in 100 ml distilled water, mix, and adjust pH to 4.2 with 0.1% NH4OH to prepare the Alizarin Red S staining solution. Filter the dark-brown solution using a using a Pall Life Sciences Acrodisc Syringe filter. And store it in the dark in a foil-covered or amber colored bottle.
    Note: The correct pH of the solution is critical. The solution is stable at RT for 2-3 weeks. If you plan on using it after a month, check pH to ensure pH is still 4.2.

Acknowledgments

This investigation was supported by PHS Grants AR051358 from the NIAMS and DE013745 from the NIDCR, and by an NCI UO1 award (to SHO). UBR is a recipient of a fellowship from The Children's Cancer Research Fund in memory of Dr A Rausen. AM is a recipient of a research grant from St Baldrick's Foundation. JAP is a postdoctoral fellow of the American Cancer Society. SHO is an Investigator of the Howard Hughes Medical Institute. This protocol is associated with the manuscript Basu-Roy et al. (2012).

References

  1. Basu-Roy, U., Seo, E., Ramanathapuram, L., Rapp, T. B., Perry, J. A., Orkin, S. H., Mansukhani, A. and Basilico, C. (2012). Sox2 maintains self renewal of tumor-initiating cells in osteosarcomas. Oncogene 31(18): 2270-2282.

简介

骨肉瘤是儿童和青少年中最常见的原发性非血液恶性肿瘤,占所有骨肉瘤的近60%。虽然这些肿瘤在性质上是成骨细胞的,但是特异性肿瘤起始细胞的特征不清楚。骨肉瘤包含具有破坏的骨分化程序的高度增殖性未分化的恶性细胞。已经在许多实体瘤中描述了具有肿瘤起始性质和对称和不对称分裂能力的癌干细胞(CSC)。对于骨肉瘤,CSC假说已经从最近报道的发现获得支持,即人和鼠骨肉瘤包含表达多种间充质干细胞表面标志物的多能细胞亚群,并且能够在多种间充质谱系例如成骨细胞和脂肪细胞。通过在特定培养基中生长细胞并测定分化标记物,可以容易地评估分化为这些不同谱系。

材料和试剂

  1. 骨肉瘤细胞(Basu-Roy等人,2012)
  2. 来自Invitrogen(Life Technologies,Invitrogen TM,目录号:11330-032)的DMEM-高葡萄糖
  3. 胎牛血清(FBS)
  4. 青霉素 - 链霉素
  5. β-甘油磷酸酯(Sigma-Aldrich,目录号:G9422)
  6. 抗坏血酸(Sigma-Aldrich,目录号:A4034)
  7. 来自Invitrogen(Life Technologies,Invitrogen TM,目录号:14155-063)的Earle's平衡盐溶液(EBSS)
  8. HCl
  9. 乙醇
  10. DMSO(Sigma-Aldrich,目录号:D8418)
  11. 磷酸盐缓冲盐水(PBS)
  12. 多聚甲醛
  13. 异丙醇
  14. 丙酮
  15. 双蒸馏H 2 O(ddw)
  16. 柠檬酸浓溶液(20ml)(Sigma-Aldrich,目录号:854℃)
  17. 萘酚AS-MX磷酸盐碱性溶液,0.25%(20ml)(Sigma-Aldrich,目录号:855)
  18. Fast Blue RR盐(Sigma-Aldrich,目录号:FBS25-10 CAP)的完全胶囊
  19. 油红O(Sigma-Aldrich,目录号:O0625)
  20. Mayer's HemTox染料(Sigma-Aldrich,目录号:MHS1)
  21. 生物染色委员会认证的茜素红S(Sigma-Aldrich,目录号:A5533)
  22. β-甘油磷酸盐储备溶液(见配方)
  23. 1,000x抗坏血酸溶液(见配方)
  24. 地塞米松(Sigma-Aldrich,目录号:D4902)(参见Recipes)
  25. 3-异丁基-1-甲基黄嘌呤(IBMX)(Sigma-Aldrich,目录号:I5879)(参见Recipes)
  26. 吲哚美辛(Sigma-Aldrich,目录号:I8280)(参见Recipes)
  27. 胰岛素(重组人)(Sigma-Aldrich,目录号:I2643)(参见Recipes)
  28. 固定解决方案(参见配方)
  29. 成骨的染色溶液(参见配方)
  30. 油红O储备溶液(见配方)
  31. 矿化成骨细胞染色溶液(参见配方)

设备

  1. 组织培养设置
  2. 巴斯德移液器
  3. 巴斯德吸头
  4. 光学显微镜
  5. Pall Life Sciences acrodisc ayringe filter(0.2μMHT Tuffryn membrane low-protein binding non-pyogenic)(Life Sciences,目录号:PN 4192)
  6. 0.2μM尼龙滤膜
  7. 24孔板

程序

  1. 电镀细胞进行分化
    1. 在DMEM/10%FBS /青霉素 - 链霉素中的24孔板中每孔铺板50,000-60,000个骨肉瘤细胞。 这可能是你感兴趣的细胞系。 根据您的细胞系更换培养基 利息
    2. 第二天或当他们达到汇合(通常在36 h),切换到分化培养基。通常细胞不分化,除非他们至少80-90%汇合。所有分化培养基是补充有步骤3和4中所示的分化混合物的常规DMEM/10%FBS/PS
    3. 对于骨形成,使用10mMβ-甘油磷酸酯和100μg/ml抗坏血酸。这是最终浓度。
    4. 对于脂肪生成,使用10μg/ml胰岛素,100nM地塞米松,250μMIBMX和200μM吲哚美辛。这些是最终浓度。如何制作股票将在后面描述
    5. 每2-3天使用新鲜分化培养基饲养细胞,每次在喂养期间。当喂食时,确保用于吸液介质的巴斯德吸管尖端不会接触细胞层,因为细胞在分化过程中,特别是在脂肪形成过程中松动。
    6. 在2,5,8,10,12和15天,对成骨和成脂细胞进行染色

  2. 碱性磷酸酶测定以测量成骨细胞分化 这是比色测定,其中黄色底物。 4-苯甲酰氨基-2,5-二甲氧基重氮氯化氯半(氯化锌)盐,偶氮二偶氮No.24由碱性磷酸酶水解,得到紫色染料沉淀。
    在开始测定前准备固定溶液和染色溶液。 保护解决方案免受光保护剂制备。
    1. 板在分化培养基中需要细胞(在24孔板中通常为50,000个细胞/孔)
    2. 在指定时间从分化培养基中铺板的细胞中吸出培养基
    3. 用1x PBS洗一次。 每次洗涤步骤包括加入1×PBS,然后吸出
    4. 每孔加入1ml固定液,室温孵育1分钟
    5. 用1x PBS洗孔一次。
    6. 每孔加入1ml染色溶液
    7. 在室温下在黑暗中孵育30分钟并检查显色。 孔中紫色染色区域的发展表明阳性反应。 如果需要,在室温下继续反应另外30分钟。 记录增加的孵育时间。 不超过1小时。
    8. 停止反应,用dd水或自来水快速洗涤。
    9. 在进行茜素红S染色以进行矿化之前,不要忘记拍照板

  3. 茜素红S染色检测矿化
    茜素红S是钙敏感染料。 分化的成骨细胞沉积大量的细胞外钙(矿化的标志),其可以通过与茜素红S络合检测。钙沉积物显示为明亮的橙红色染色区域。
    1. 在拍摄用碱性磷酸酶染色的平板后,通过用1×PBS洗涤染色的平板两次,进行茜素红S染色,所述PBS用0.1%NH 4 OH调节其pH至4.2。
    2. 吸出PBS,并添加足够的茜素红S染色溶液覆盖细胞单层。 将板在室温下在黑暗中孵育45分钟
    3. 小心吸出茜素红S溶液,并洗涤板四秒钟,然后用蒸馏水
    4. 细胞外钙沉积物呈现为明亮染色的橙红色区域

  4. 油红O染色测定脂肪形成分化 该染色测定基于如下原理:亲脂性染料油红O保留在脂肪细胞的脂肪球中,其可以在显微镜下观察到。
    1. 吸出培养基并在PBS中洗涤细胞一次
    2. 在室温下将细胞固定在4%PFA中15分钟
    3. 用ddH 2 O 2洗涤两次,每次2×5分钟
    4. 用油 - 红-O工作溶液在室温下染色15分钟
    5. 用1x PBS或ddH 2 O洗涤3×5分钟
    6. 在室温下用50%异丙醇冲洗细胞一次。
    7. 用1×PBS或ddH 2 O漂洗细胞
    8. 红色染色的脂滴可以通过光学显微镜可视化。 脂肪细胞也可以通过其圆形形态在正常显微镜下容易地显现,并且显着增强较大脂滴的光反射。
    9. 计数总细胞,用Mayer's HemTox染色在室温下染色15分钟,用dd水洗涤2x

食谱

  1. β-甘油磷酸酯可以通过制备100倍的1M溶液的储液来制备。预热水易溶解。化合物的配方重量取决于水合程度,并且可以通过使用瓶上提供的批号来确定。使用Pall Life Sciences Acrodisc注射器过滤器过滤。制成1毫升等分试样,在-20℃下冷冻
  2. 1,000x抗坏血酸溶液可以通过将1克抗坏血酸溶解在10ml ddw(双蒸水)中制备。使用Pall Life Sciences Acrodisc Syringe过滤器过滤。制成1毫升等分试样,在-20℃下冷冻
  3. 地塞米松(FW 392.5)
    将0.0196g溶于10ml无水乙醇中,得到5mM储备液;存储在-80°C。通过在100%乙醇中稀释至100μM制备新鲜的工作原料
  4. 3-异丁基-1-甲基黄嘌呤(IBMX)(FW 222.2)
    将100mg IBMX溶解在1.8ml DMSO中并通过0.2μM尼龙过滤器无菌过滤以制备250mM储备液(1,000x)。分装并储存在-20°C或以下
  5. 吲哚美辛(FW 357.8)
    将0.5367g吲哚美辛溶解在7.5ml DMSO中,并通过0.2μm尼龙过滤器过滤以灭菌200mM(1,000x)原液。分配1-2毫升等分在无菌冷冻管,并存储在不透明的盒子,以保护免受光。储存于-70°C或更低温度
  6. 胰岛素,重组人类
    将50mg胰岛素溶解在用50μl0.005N HCl酸化的10ml EBSS中以产生500x储备液,并进行无菌过滤。不要忘记使用低蛋白结合过滤器,如设备中所述。将1ml等分试样分装在无菌冷冻管中,并在-70℃或更低温度下储存长达2年。解冻后,工作原液可以在4°C下储存长达1个月。或者,无菌过滤器酸化EBSS,并使用无菌溶液在无菌条件下制造胰岛素
  7. 成骨固定溶液
    20ml dd水+ 30ml丙酮+400μl柠檬酸盐浓缩溶液
  8. 成骨染色溶液
    48ml dd水+ 2ml萘酚AS-MX磷酸盐碱性溶液,0.25%+ 1个完整的快蓝色RR盐胶囊
  9. 用于染色脂肪细胞的油红O原液 将500mg油红O溶于100ml异丙醇中。对于工作股票,稀释股票作为2份dd水+ 3份油红O股票。摇动,室温孵育15分钟,使用Pall Life Sciences Acrodisc Syringe过滤器过滤。在1小时内准备使用。
  10. 矿化成骨细胞染色溶液
    将2g茜素红S溶解在100ml蒸馏水中,混合,并用0.1%NH 4 OH调节pH至4.2以制备茜素红S染色溶液。使用Pall Life Sciences Acrodisc Syringe过滤器过滤深棕色溶液。并把它存放在黑暗的箔箔覆盖或琥珀色的瓶子。
    注意:溶液的正确pH是至关重要的。该溶液在室温下稳定2-3周。如果您打算在一个月后使用它,请检查pH值以确保pH仍然为4.2。

致谢

该调查得到来自NIAMS的PHS Grants AR051358和来自NIDCR的DE013745以及通过NCI UO1奖(SHO)的支持。 UBR是来自儿童癌症研究基金的一个研究金的收件人,纪念Dr Rausen博士。 AM是来自圣巴德里克基金会的研究资助的收件人。 JAP是美国癌症协会的博士后研究员。 SHO是霍华德休斯医学研究所的研究员。该协议与Basu-Roy等人的文章(2012)相关联。

参考文献

  1. Basu-Roy,U.,Seo,E.,Ramanathapuram,L.,Rapp,T.B.,Perry,J.A.,Orkin,S.H.,Mansukhani,A.and Basilico,C。(2012)。 Sox2维持骨肉瘤肿瘤起始细胞的自我更新。癌基因 31(18):2270-2282。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Basu-Roy, U., Basilico, C. and Mansukhani, A. (2012). Osteogenic and Adipogenic Differentiation of Osteosarcoma Cells. Bio-protocol 2(24): e308. DOI: 10.21769/BioProtoc.308.
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