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Transfection of Embryoid Bodies with miRNA Precursors to Induce Cardiac Differentiation
用miRNA前体转染拟胚体来诱导心肌分化

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Abstract

In recent years, the utilization of stem cell therapy to regenerate cardiac tissue has been proposed as a possible strategy to treat cardiac damage (Gnecchi et al., 2012, Aguirre et al., 2013; Sanganalmath and Bolli, 2013). Although encouraging results have been obtained in experimental models, the efficiency of cardiac regeneration is very poor and one of the major barriers to progress in the area of cell therapy for damaged heart is represented by the limited capacity of cells to differentiate into mature cardiomyocytes (CMC) (Laflamme and Murry, 2011). Cell manipulation and transfection represent versatile tools in this context (Melo et al., 2005; Dzau et al., 2005). Murine P19 embryonal carcinoma cells are a well-established cell line capable of differentiating in vitro into spontaneously beating CMC. This cell system with its limited cell culture requirements, protocol reproducibility and ease in uptake and subsequent expression of ectopic genetic materials render it ideal for the study of the cardiac differentiation process. P19 cells have been successfully used to gain important insights into the early molecular processes of CMC differentiation (van der Heyden and Defize, 2003; van der Heyden et al., 2003). P19 cells can also be maintained in an undifferentiated state in a monolayer culture when grown in adherence; this condition allows the enrichment of large cell numbers useful for cardiac differentiation protocols (McBurney, 1993). On the other hand, when cultured in bacterial dishes, P19 cells will grow in suspension and generate embryoid bodies (EB). When exposed to dimethyl sulfoxide (DMSO), EB differentiate into spontaneously beating cells, which can be defined as CMC. This definition is based on their gene and protein expression and their electrophysiological properties (Wobus et al., 1994; van der Heyden et al., 2003). In our laboratory, we used this in vitro model to verify whether the over-expression of a defined combination of miRNA can synergistically induce effective cardiac differentiation (Pisano et al., 2015). We used miRNA1, miRNA133 and miRNA499 alone or in combination. Here, we describe how we transiently transfect P19 cells to over-express a single or a combination of miRNA precursors (pre-miRNA).

Keywords: MicroRNA(microRNA), Embryoid bodies(胚状体), Cardiac differentiation(心肌分化)

Materials and Reagents

  1. Bacterial dishes (100 x 15 mm) (Corning, catalog number: 351006 )
  2. Standard culture Petri dishes (100 x 15 mm) (Corning, catalog number: 70165-101 )
  3. 6 multiwell-plates (Corning, catalog number: 353224 )
  4. 50 ml tubes (Falcon®, catalog number: 352098 )
  5. P19 cells (Izsler-Istituto Zooprofilattico Sperimentale, Lombardy and Emilia Romagna “Bruno Ubertini”, Brescia, Italy) (Izsler, catalog number: BS-TCL 206 , Passage 0)
  6. Minimum Essential Medium alpha (α-MEM) (Sigma-Aldrich, catalog number: M8042 )
  7. Penicillin-streptomycin 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  8. L-glutamine 2 nM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
  9. Dimethyl sulfoxide (Sigma-Aldrich, catalog number: D4540-100 ml )
  10. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F6178-50 ml )
  11. Trypsin-EDTA (0.5%), no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15400-054 )
  12. Optimem® (Thermo Fisher Scientific, GibcoTM, catalog number: 31985-070 )
  13. siPORT® NeoFX transfection agent (Thermo Fisher Scientific, Invitrogen™, catalog number: AM4511 )
  14. Pre-miRNA 1 (ID: 000385) (Thermo Fisher Scientific, Applied Biosystems®, catalog number: AM17100 ), Pre-miRNA 133 (ID: 000458) (Thermo Fisher Scientific, Applied Biosystems®, catalog number: AM17100), Pre-miRNA 499 (ID: 001045) (Thermo Fisher Scientific, Applied Biosystems®, catalog number: AM17100) molecules and scramble miRNA (negative control) (Thermo Fisher Scientific, Ambion™, catalog number: AM17110 )
  15. Culture medium (see Recipes)
  16. Standard differentiation medium (see Recipes)
  17. Mix A (see Recipes)
  18. Mix B (see Recipes)

Equipment

  1. Pipetus® Pipettes
  2. Laminar flow-hood (EuroClone S.p.A., model: S@feflow 1.8 )
  3. Humidified cell culture incubator set at 37 °C, 5% CO2 (Panasonic Corporation, Sanyo, model: MCO-18AC )
  4. Inverted bright light microscope equipped with a phase-contrast filter (ZEISS, model: Observer Z1 )

Procedure

  1. Expand the P19 cells form passage 0 to passage 5, in a standard Petri dish and feed the cells with culture medium. When the cells reach 80-90% confluence, harvest the P19 cells using 5% trypsin/EDTA for 5 minutes at 37 °C, 5% CO2 (Figure 1A).
  2. On day 1, transfer 4 x 105 cells to a 100 mm bacterial dish in 8 ml of standard differentiation medium and incubate the cells for 24 h at 37 °C, 5% CO2.
  3. On day 2, add 5 ml of fresh standard differentiation medium (total volume medium: 13 ml, fresh differentiation medium must be prepared just before the use).
  4. On day 3, the morphologic changes of P19 cells start to be visible: some cell clusters sediment at the bottom of the culture dish, become EB and begin to differentiate into CMC. Some isolated cells may remain in suspension: these cells are not committed toward the cardiac lineage and will not adhere at the bottom of the culture dish since bacterial dish are used. These undifferentiated P19 will be discarded when exchanging the cell culture medium.
  5. Replace 5 ml of fresh standard differentiation medium: with a 10 ml pipette gently aspirate 5 ml of medium, avoiding to aspirate also the EB; then, with a new 10 ml pipette, add 5 ml of fresh differentiation medium.
  6. On day 4, the EB are ready for transfection (Figure 1B). Prepare mix A and mix B in two separate 15 ml conical tubes.
  7. Gently add mix B to mix A, invert the tube 3 times and incubate at RT for 10 min.
  8. Using a 25 ml pipette, collect and transfer the EB contained in one bacterial dish to a 50 ml tube and let the EB to sediment.
  9. Aliquot 1 ml of transfection mix into each well of a 6-multiwell-plate; add only standard differentiation medium in those wells that will be used as control.
  10. EB previously collected in the 50 ml tube will now be settled. Carefully aspirate the medium and re-suspend the EB in 14.4 ml of fresh standard differentiation medium.
  11. Transfer 2.4 ml of re-suspended EB to each well and gently rock the multiwell-plate using a 1 ml pipette tip. Now shake back and forth the plate to obtain homogeneous distribution of the cells. Every time you pick an aliquot of the mixture, pipette up and down to re-suspend the EB. Incubate over-night at 37 °C, 5% CO2.
  12. On day 5, EB will have adhered to the bottom of the dish (Figure 1C). You can now exchange the medium with fresh differentiation medium to allow the expansion of EB. This step represents “day 1” of the cardiac induction protocol. A Real-Time PCR can be performed in order to quantify the miRNA transfection efficiency.
  13. On day 5 of the induction protocol, the first contracting EB should appear and will be visible at the microscope (Video 1).

    Video 1. Beating embryoid bodies

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  14. Early cardiac differentiation can be documented by PCR and/or immunocytochemistry starting from day 7 of the induction protocol. More advanced cardiac differentiation will appear starting from day 14 of the induction protocol (Figure 1D) (Pisano et al., 2015 http://onlinelibrary.wiley.com/doi/10.1002/stem.1928/pdf, Figures 2, 3 and 6).


    Figure 1. P19 cells and EB. A. Images of undifferentiated P19 maintained in culture medium; B. EB formation during the cardiac differentiation protocol, day 4; C. When EB are transferred from a bacterial dish to a Petri dish they adhere to the plate; D. After 7 days of cardiac differentiation the attached EB will have expanded and are beating.

Notes

  1. Manipulation of miRNA molecules requires the use of RNAse-free plastic and reagents. Moreover, if the flow-hood used for miRNA transfection is utilized also for other experiments, we suggest to pre-treat the surfaces with 10% NaClO.
  2. During the transfection protocol you must work carefully and aseptically under the flow-hood since Optimem® medium does not contain antibiotics.

Recipes

  1. Culture medium
    Store the medium at 4 °C; when the phenol red contained in the α-MEM turns to pink (or to yellow) discard the medium and use a fresh culture medium.
    α-MEM 500 ml
    FBS 55 ml
    L-glutamine 5.5 ml
    Penicillin-streptomycin 5.5 ml
  2. Standard differentiation medium (prepare fresh before use)
    Culture medium
    DMSO 0.5%
  3. Mix A (prepare fresh before use)
    Dilute the pre-miRNA solution using Optimem®
    We use pre-miRNA1 and pre-miRNA499 at a concentration of 10 nM, and pre-miRNA133 and scramble miRNA at 5 nM.
    Incubate the mix at room temperature (RT) for 10 min
    For one 6-multiwell dish we use 3 ml of Mix A
  4. Mix B (prepare fresh before use)
    Dilute siPORT® 1:50 using Optimem®
    Incubate at RT for 10 min
    For one 6-multiwell dish we use 3 ml of Mix B

Acknowledgments

The development of this protocol was supported by the Fondazione IRCCS Policlinico San Matteo Pavia, Italy; the Fondazione Cariplo (2007-5984) and the Ministero Italiano della Sanità (GR-2008-114278). We want to thank Laurene Kelly for help with editing the manuscript.

References

  1. Aguirre, A., Sancho-Martinez, I. and Izpisua Belmonte, J. C. (2013). Reprogramming toward heart regeneration: stem cells and beyond. Cell Stem Cell 12(3): 275-284.
  2. Dzau, V. J., Gnecchi, M. and Pachori, A. S. (2005). Enhancing stem cell therapy through genetic modification. J Am Coll Cardiol 46(7): 1351-1353.
  3. Gnecchi, M., Danieli, P. and Cervio, E. (2012). Mesenchymal stem cell therapy for heart disease. Vascul Pharmacol 57(1): 48-55.
  4. Laflamme, M. A. and Murry, C. E. (2011). Heart regeneration. Nature 473(7347): 326-335.
  5. McBurney, M. W. (1993). P19 embryonal carcinoma cells. Int J Dev Biol 37(1): 135-140.
  6. Melo, L. G., Pachori, A. S., Gnecchi, M. and Dzau, V. J. (2005). Genetic therapies for cardiovascular diseases. Trends Mol Med 11(5): 240-250.
  7. Pisano, F., Altomare, C., Cervio, E., Barile, L., Rocchetti, M., Ciuffreda, M. C., Malpasso, G., Copes, F., Mura, M., Danieli, P., Viarengo, G., Zaza, A. and Gnecchi, M. (2015). Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation. Stem Cells 33(4): 1187-1199.
  8. Sanganalmath, S. K. and Bolli, R. (2013). Cell therapy for heart failure: a comprehensive overview of experimental and clinical studies, current challenges, and future directions. Circ Res 113(6): 810-834.
  9. van der Heyden, M. A. and Defize, L. H. (2003). Twenty one years of P19 cells: what an embryonal carcinoma cell line taught us about cardiomyocyte differentiation. Cardiovasc Res 58(2): 292-302.
  10. van der Heyden, M. A., van Kempen, M. J., Tsuji, Y., Rook, M. B., Jongsma, H. J. and Opthof, T. (2003). P19 embryonal carcinoma cells: a suitable model system for cardiac electrophysiological differentiation at the molecular and functional level. Cardiovasc Res 58(2): 410-422.
  11. Wobus, A. M., Kleppisch, T., Maltsev, V. and Hescheler, J. (1994). Cardiomyocyte-like cells differentiated in vitro from embryonic carcinoma cells P19 are characterized by functional expression of adrenoceptors and Ca2+ channels. In Vitro Cell Dev Biol Anim 30A(7): 425-434.

简介

近年来,已经提出利用干细胞治疗来再生心脏组织作为治疗心脏损伤的可能策略(Gnecchi等人,2012,Aguirre等人, >,2013; Sanganalmath和Bolli,2013)。虽然在实验模型中已经获得令人鼓舞的结果,但是心脏再生的效率非常差,并且在损伤的心脏的细胞治疗领域中进展的主要障碍之一是细胞分化成成熟心肌细胞的能力有限(CMC )(Laflamme和Murry,2011)。细胞操作和转染在本文中代表多种工具(Melo等人,2005; Dzau等人,2005)。鼠P19胚胎癌细胞是能够在体外分化成自发性搏动CMC的良好建立的细胞系。这种细胞系统具有有限的细胞培养要求,协议重现性和易于摄取和随后的异位遗传材料的表达,使其理想的心脏分化过程的研究。 P19细胞已经成功地用于获得对CMC分化的早期分子过程的重要见解(van der Heyden和Defize,2003; van der Heyden等人,2003)。当在粘附中生长时,P19细胞也可以在单层培养物中维持在未分化状态;这种条件允许富集用于心脏分化方案的大细胞数目(McBurney,1993)。另一方面,当在细菌培养皿中培养时,P19细胞将在悬浮液中生长并产生胚状体(EB)。当暴露于二甲基亚砜(DMSO)时,EB分化成自发性搏动细胞,其可以定义为CMC。该定义基于它们的基因和蛋白质表达及其电生理学性质(Wobus等人,1994; van der Heyden等人,2003)。在我们的实验室中,我们使用这种体外模型来验证定义的miRNA组合的过表达是否可以协同诱导有效的心脏分化(Pisano等人,2015 )。我们单独或组合使用miRNA1,miRNA133和miRNA499。在这里,我们描述我们如何瞬态转染P19细胞过度表达单个或组合的miRNA前体(pre-miRNA)。

关键字:microRNA, 胚状体, 心肌分化

材料和试剂

  1. 细菌培养皿(100×15mm)(Corning,目录号:351006)
  2. 标准培养皿(100×15mm)(Corning,目录号:70165-101)
  3. 6多孔板(Corning,目录号:353224)
  4. 50ml管(Falcon ,目录号:352098)
  5. P15细胞(Izsler,目录号:BS-TCL206,Passage 0)中培养p19细胞(Izsler-Istituto Zoopro fi lattico Sperimentale,Lombardy和Emilia Romagna"Bruno Ubertini",Brescia,Italy)
  6. 最小必需培养基α(α-MEM)(Sigma-Aldrich,目录号:M8042)
  7. 青霉素 - 链霉素10,000U/ml(Thermo Fisher Scientific,Gibco< sup>,目录号:15140-122)
  8. L-谷氨酰胺2nM(Thermo Fisher Scientific,Gibco TM ,目录号:25030-081)
  9. 二甲基亚砜(Sigma-Aldrich,目录号:D4540-100ml)
  10. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F6178-50ml)
  11. 胰蛋白酶-EDTA(0.5%),无酚红(Thermo Fisher Scientific,Gibco TM,目录号:15400-054)
  12. Optimem (Thermo Fisher Scientific,Gibco TM ,目录号:31985-070)
  13. siPORT NeoFX转染剂(Thermo Fisher Scientific,Invitrogen TM,目录号:AM4511)
  14. 将Pre-miRNA 1(ID:000385)(Thermo Fisher Scientific,Applied Biosystems,目录号:AM17100),Pre-miRNA 133(ID:000458)(Thermo Fisher Scientific,Applied Biosystems 分子和scramble miRNA(阴性对照),其中目标编号:AM17100),Pre-miRNA 499(ID:001045)(Thermo Fisher Scientific,Applied Biosystems (Thermo Fisher Scientific,Ambion TM,目录号:AM17110)
  15. 培养基(见配方)
  16. 标准分化培养基(见配方)
  17. 混合A(参见配方)
  18. 混合B(参见配方)

设备

  1. Pipetus ?移液器
  2. 层流罩(EuroClone S.p.A.,型号:S @ flow 1.8)
  3. 设置在37℃,5%CO 2(Panasonic Corporation,Sanyo,型号:MCO-18AC)的加湿细胞培养孵育器
  4. 装备有相位对比滤光器(ZEISS,型号:Observer Z1)的倒置明亮光显微镜

程序

  1. 在标准培养皿中扩增P19细胞,从第0代至第5代,并用培养基饲养细胞。当细胞达到80-90%汇合时,使用5%胰蛋白酶/EDTA在37℃,5%CO 2下收获P19细胞5分钟(图1A)。
  2. 在第1天,将4×10 5个细胞转移到8ml标准分化培养基中的100mm细菌培养皿中,并在37℃,5%CO 2中孵育细胞24小时,/sub>。
  3. 在第2天,加入5ml新鲜标准分化培养基(总体积培养基:13ml,新鲜分化培养基必须在使用前准备)。
  4. 在第3天,P19细胞的形态学变化开始可见:一些细胞簇沉积在培养皿的底部,变成EB并开始分化成CMC。一些分离的细胞可以保留在悬浮液中:这些细胞不向心脏谱系定向,并且将不粘附在培养皿的底部,因为使用细菌培养皿。当交换细胞培养基时,这些未分化的P19将被丢弃
  5. 替换5毫升新鲜标准分化培养基:用10毫升吸管轻轻吸出5毫升培养基,避免吸入EB;然后用新的10ml移液管加入5ml新鲜分化培养基
  6. 在第4天,EB准备用于转染(图1B)。在两个单独的15ml锥形管中制备混合物A和混合物B.
  7. 轻轻加入混合物B混合A,倒置管3次,并在室温下孵育10分钟
  8. 使用25毫升吸管,收集和转移包含在一个细菌盘中的EB到50毫升管,让EB沉淀。
  9. 将1ml转染混合物等分到6多孔板的每个孔中;在那些将被用作对照的孔中仅加入标准分化培养基。
  10. EB先前收集在50ml管中现在将被解决。小心吸出培养基并将EB重新悬浮于14.4ml新鲜标准分化培养基中
  11. 转移2.4毫升重悬EB每个孔,轻轻摇动多孔板使用1毫升吸头。现在摇动板来获得均匀分布的细胞。每次你选择一个混合物的等分试样,吸移管向上和向下重新暂停EB。在37℃,5%CO2下孵育过夜。
  12. 在第5天,EB将粘附在培养皿的底部(图1C)。您现在可以用新鲜的分化培养基交换培养基,以允许EB的扩增。该步骤代表心脏诱导方案的"第1天"。可以进行实时PCR以定量miRNA转染效率
  13. 在诱导方案的第5天,第一次收缩的EB应该出现并且将在显微镜下可见(视频1)。

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    视频1.打败胚状体
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  14. 早期心脏分化可以通过PCR和/或免疫细胞化学从诱导方案的第7天开始记录。更晚期的心脏分化将从诱导方案的第14天开始出现(图1D)(Pisano等人,2015 http://onlinelibrary.wiley.com/doi/10.1002/stem.1928/pdf ,图2,3和6)。


    图1.P19细胞和EB。 A.维持在培养基中的未分化P19的图像; B.心脏分化方案期间的EB形成,第4天; C.当EB从细菌盘转移到培养皿时,它们粘附在板上; D.心脏分化7天后,附着的EB将扩大并叩击

笔记

  1. 操纵miRNA分子需要使用无RNA酶的塑料和试剂。此外,如果用于miRNA转染的流动罩也用于其他实验,我们建议用10%NaClO预处理表面。
  2. 在转染方案中,您必须仔细无菌地在流动罩下工作,因为Optimem ?培养基不含抗生素。

食谱

  1. 培养基
    存储介质在4°C;当α-MEM中包含的酚红变为粉红色(或黄色)时,丢弃培养基并使用新鲜培养基。
    α-MEM 500ml
    FBS 55 ml
    L-谷氨酰胺5.5ml
    青霉素 - 链霉素5.5ml
  2. 标准分化培养基(使用前准备新鲜)
    培养基
    DMSO 0.5%
  3. 混合A(使用前准备新鲜)
    使用Optimem ?
    稀释pre-miRNA溶液 我们使用pre-miRNA1和pre-miRNA499的浓度为10 nM,pre-miRNA133和scramble miRNA的浓度为5 nM。
    在室温(RT)下孵育混合物10分钟
    对于一个6多孔培养皿,我们使用3ml的Mix A
  4. 混合B(使用前准备新鲜)
    使用Optimem ?稀释siPORT ? 1:50
    在RT孵育10分钟
    对于一个6多孔培养皿,我们使用3ml的Mix B

致谢

该协议的开发由意大利圣基茨帕利亚的IRCCS Policlinico支持; Fondazione Cariplo(2007-5984)和意大利部长(GR-2008-114278)。我们要感谢Laurene Kelly帮助编辑手稿。

参考文献

  1. Aguirre,A.,Sancho-Martinez,I.和Izpisua Belmonte,J.C。(2013)。 重新编程朝向心脏再生:干细胞和更远。细胞干细胞 12(3):275-284。
  2. Dzau,V.J.,Gnecchi,M。和Pachori,A.S。(2005)。 通过遗传修饰增强干细胞治疗 J Am Coll Cardiol < em> 46(7):1351-1353。
  3. Gnecchi,M.,Danieli,P。和Cervio,E。(2012)。 间充质干细胞治疗心脏病。 Vascul Pharmacol 57(1):48-55。
  4. Laflamme,M.A。和Murry,C.E。(2011)。 心脏再生。自然 473(7347):326 -335。
  5. McBurney,M.W。(1993)。 P19胚胎癌细胞 Int J Dev Biol 37 (1):135-140。
  6. Melo,L.G.,Pachori,A.S.,Gnecchi,M。和Dzau,V.J。(2005)。 心血管疾病的遗传治疗 Trends Mol Med 11 (5):240-250。
  7. Pisano,F.,Altomare,C.,Cervio,E.,Barile,L.,Rocchetti,M.,Ciuffreda,MC,Malpasso,G.,Copes,F.,Mura,M.,Danieli,P.,Viarengo ,G.,Zaza,A.and Gnecchi,M。(2015)。 miRNA499和miRNA133的组合对心脏分化产生协同效应。干细胞Cells 33(4):1187-1199。
  8. Sanganalmath,S. K.和Bolli,R。(2013)。 心力衰竭细胞治疗:全面概述实验和临床研究,目前的挑战和未来方向。 Circ Res 113(6):810-834。
  9. van der Heyden,M.A。和Defize,L.H。(2003)。 二十年的P19细胞:什么是胚胎癌细胞系教导我们关于心肌细胞分化。 a> Cardiovasc Res 58(2):292-302。
  10. van der Heyden,M.A.,van Kempen,M.J.,Tsuji,Y.,Rook,M.B.,Jongsma,H.J.and Opthof,T。(2003)。 P19胚胎癌细胞:在分子和功能水平进行心脏电生理分化的合适模型系统。/a> Cardiovasc Res 58(2):410-422。
  11. Wobus,A.M.,Kleppisch,T.,Maltsev,V。和Hescheler,J。(1994)。 体外分化的心肌细胞样细胞来自胚胎癌细胞P19的特征通过肾上腺素受体和Ca 2+通道的功能性表达。体外细胞生物学动力学(EMD)30A(7):425-434。
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引用:Pisano, F. and Gnecchi, M. (2016). Transfection of Embryoid Bodies with miRNA Precursors to Induce Cardiac Differentiation. Bio-protocol 6(3): e1726. DOI: 10.21769/BioProtoc.1726.
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serenity Gao
Max plank institute
Hello, Thanks for the protocol. And I have some questions.
Firstly, I am curious about the exact transfection efficiency about this method since you use the EB to be transfected directly if I am right.
Secondly, is the high transfection efficiency just limited to this cell line or the precursor miRNA. I am wondering how is it for the other ESC lines and the general plasmid?
Thirdly, I am wondering what is the difference between the cardiomyocyte differentiated from this embryonal carcinoma cell line and the normal ESC line? If there is no difference, this cell line is really convenient for cardiac differentiation. And how much percentage of the cardiomyocyte in the EBs differentiaed from this cell line?
It would be greatly appreciated if you can give me some idea about these questions. Thank you very much!
3/18/2016 9:22:18 AM Reply
Federica Pisano
Department of Cardiothoracic and Vascular Sciences-Coronary Care Unit and Laboratory of Clinical and Experimental Cardiology-Fondazione IRCCS Policlinico San Matteo, Italy

Firstly, I am curious about the exact transfection efficiency about this method since you use the EB to be transfected directly if I am right.

Yes, you are right. We optimized the standard transfection protocol using EB. To quantify transfection efficiency we performed qPCR on dissociated EB (please, see point 12 of our protocol) and using both a positive control (100% of miRNA expression) and a negative control (P19 naïve cells). RNA was extracted from the isolated cells and RT-PCR and qPCR were performed for miRNA1, miRNA133 and miRNA499. The transfection efficiency ranged 88 to 100%.



Secondly, is the high transfection efficiency just limited to this cell line or the precursor miRNA. I am wondering how is it for the other ESC lines and the general plasmid?

P19 cells are not easy to transfect with liposomes and plasmid. However, the low molecular weight of miRNA precursors (60-80 nucleotides) probably facilitates the transfection efficiency. We initially tried to transfect these cell line with a 10 Kb plasmid but the transfection efficiency was very low. For these reason, we switched to lentivirus to transform P19 cells obtaining high efficiency (please, see Pisano et al., 2015 http://onlinelibrary.wiley.com/doi/10.1002/stem.1928/pdf).
We did not test ESC lines in our studies so we do not have an answer for this specific question.



Thirdly, I am wondering what is the difference between the cardiomyocyte differentiated from this embryonal carcinoma cell line and the normal ESC line? If there is no difference, this cell line is really convenient for cardiac differentiation. And how much percentage of the cardiomyocyte in the EBs differentiaed from this cell line?

As aforementioned, we did not perform the transfection protocol on ESC. Anyway, ESC and P19 behave very similarly and we supposed that the cardiac differentiation efficiency could be similar between these two cell types. Please, refer again to our manuscript (Pisano et al., 2015 http://onlinelibrary.wiley.com/doi/10.1002/stem.1928/pdf).

4/6/2016 12:22:02 AM