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Akyash, F., Sadat Tahajjodi, S., Farashahi Yazd, E., Hajizadeh-Tafti, F., Sadeghian-Nodoushan, F., Golzadeh, J., Heidarian Meimandi, H., Moore, H., & Aflatoonian, B. (2019). Derivation of new human embryonic stem cell lines (Yazd1-3) and their vitrification using Cryotech and Cryowin tools: A lab resources report. International Journal of Reproductive BioMedicine (IJRM), 17(12), 891–906. https://doi.org/10.18502/ijrm.v17i12.5808
https://doi.org/10.18502/ijrm.v17i12.5808
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Fatemeh Akyash
Fatemeh Akyash
Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran; Department of Reproductive Biology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran; and Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
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Somayyeh Sadat Tahajjodi
Somayyeh Sadat Tahajjodi
Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran; Department of Reproductive Biology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran; and Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
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Ehsan Farashahi Yazd
Ehsan Farashahi Yazd
1. Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran; Department of Reproductive Biology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
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Fatemeh Hajizadeh-Tafti
Fatemeh Hajizadeh-Tafti
Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
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Fatemeh Sadeghian-Nodoushan
Fatemeh Sadeghian-Nodoushan
Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
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Jalal Golzadeh
Jalal Golzadeh
Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
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Hassan Heidarian Meimandi
Hassan Heidarian Meimandi
Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
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Harry Moore
Harry Moore
Department of Biomedical Sciences, Centre for Stem Cell Biology, University of Sheffield, Western Bank, Alfred Denny Building, Sheffield S10 2TN, UK
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Behrouz Aflatoonian
Behrouz Aflatoonian
Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran; Department of Reproductive Biology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran; and Department of Advanced Medical Sciences and Technologies, School of Paramedicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Published Date
- Dec 31, 2019
Derivation of new human embryonic stem cell lines (Yazd1-3) and their vitrification using Cryotech and Cryowin tools: A lab resources report
Abstract
Background: Cell banking initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws.
Objective: To derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification.
Materials and Methods: Human ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method.
Results: The pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping.
Conclusion: Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient means of preserving hESCs.
Key words: Derivation, Human embryonic stem cells, Human foreskin fibroblast, Microdrop, Vitrification.
References
[1] Evans M. Isolation and maintenance of murine embryonic stem cells. Essentials of Stem Cell Biology; 2009; 345–349.
[2] Solter D, Kowles BB. Immunosurgery of mouse blastocyst. Proc Natl Acad Sci USA 1975; 72: 5099–5102.
[3] Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292: 154–156.
[4] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 1981; 78: 7634–7638.
[5] Bongso A, Fong CY, Ng SC, Ratnam S. Isolation and culture of inner cell mass cells from human blastocysts. Hum Reprod 1994; 9: 2110–2117.
[6] Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 1995; 92: 7844–7848.
[7] Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Hearn JP. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod 1996; 55: 254–259.
[8] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145–1147.
[9] Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci USA 1998; 95: 13726–13731.
[10] Andrews PW. From teratocarcinomas to embryonic stem cells. Philos Trans R Soc Lond B Biol Sci 2002; 357: 405–417.
[11] Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R. Human embryonic stem cell lines derived from single blastomeres. Nature 2006; 444: 481–485.
[12] Omidi M, Aflatoonian B, Thajjodi SS, Khalili MA. Attempts for generation of embryonic stem cells from human embryos following in vitro embryo twinning. Stem Cells Dev 2019; 28: 303–309.
[13] Aflatoonian B, Ruban L, Shamsuddin S, Baker D, Andrews P, Moore H. Generation of Sheffield (Shef) human embryonic stem cell lines using a microdrop culture system. In Vitro Cell Dev Biol Anim 2010; 46: 236–241.
[14] Turetsky T, Aizenman E, Gil Y, Weinberg N, Shufaro Y, Revel A, et al. Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis. Hum Reprod 2008; 23: 46–53.
[15] Giritharan G, Ilic D, Gormley M, Krtolica A. Human embryonic stem cells derived from embryos at different stages of development share similar transcription profiles. PLoS One 2011; 6: e26570.
[16] Ström S, Inzunza J, Grinnemo KH, Holmberg K, Matilainen E, Strömberg AM, et al. Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines. Hum Reprod 2007; 22: 3051–3058.
[17] Ilic D, Genbacev O, Krtolica A. Derivation of hESC from Intact Blastocysts. Curr Protoc Stem Cell Biol 2007: 1A.2.1–1A.2.18.
[18] Hovatta O, Mikkola M, Gertow K, Strömberg AM, Inzunza J, Hreinsson J, et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003; 18: 1404–1409.
[19] Desai N, Ludgin J, Goldberg J, Falcone T. Development of a xeno-free non-contact co-culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line. J Assist Reprod Genet 2013; 30: 609–615.
[20] Assou S, Pourret E, Péquignot M, Rigau V, Kalatzis V, Aït-Ahmed O, et al. Cultured cells from the human oocyte cumulus niche are efficient feeders to propagate pluripotent stem cells. Stem Cells Dev 2015; 24: 2317–2327.
[21] Adewumi O, Aflatoonian B, Ahrlund-Richter L, Amit M, Andrews PW, Beighton G, et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol 2007; 25: 803–816.
[22] Miere C, Wood V, Kadeva N, Cornwell G, Codognotto S, Stephenson E, et al. Generation of KCL037 clinical grade human embryonic stem cell line. Stem Cell Res 2016; 16: 149–151.
[23] Amps KJ, Jones M, Baker D, Moore HD. In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice. Cryobiology 2010; 60: 344–350.
[24] Aflatoonian A, Karimzadeh Maybodi MA, Aflatoonian N, Tabibnejad N, Amir-Arjmand MH, Soleimani M, et al. Perinatal outcome in fresh versus frozen embryo transfer in ART cycles. Int J Reprod Biomed 2016; 14: 167–172.
[25] Draper JS, Pigott C, Thomson JA, Andrews PW. Surface antigens of human embryonic stem cells: Changes upon differentiation in culture. J Anat 2002; 200: 249–258.
[26] Sadeghian-Nodoushan F, Aflatoonian R, Borzouie Z, Akyash F, Fesahat F, Soleimani M, et al. Pluripotency and differentiation of cells from human testicular sperm extraction: An investigation of cell stemness. Mol Reprod Dev 2016; 83: 312–323.
[27] Akyash F, Javidpou M, Sadeghian Nodoushan F, Aflatoonian B. Human embryonic stem cells derived mesenchymal stem/stromal cells and their use in regenerative medicine. J Stem Cell Res Ther 2016; 1: 00047.
[28] Aflatoonian B, Moore H. Pluripotent stem cells in vitro from human primordial germ cells. In: Kaiming Ye, Sha Jin. Human embryonic and induced pluripotent stem cell: Lineage-specific differentiation protocols. USA: Springer Protocols Handbooks; 2012. p. 71–83.
[29] Laowtammathron C, Chingsuwanrote P, Choavaratana R, Phornwilardsiri S, Sitthirit K, Kaewjunun C, et al. High-efficiency derivation of human embryonic stem cell lines using a culture system with minimized trophoblast cell proliferation. Stem Cell Res Ther 2018; 9: 138–147.
[30] First NL, Sims MM, Park SP, Kent-First MJ. Systems for production of calves from cultured bovine embryonic cells. Reprod Fertil Dev 1994; 6: 553–562.
[31] Sherbahn R, Frasor J, Radwanska E, Binor Z, Wood-Molo M, Hibner M, et al. Comparison of mouse embryo development in open and microdrop co-culture systems. Hum Reprod 1996; 11: 2223–2229.
[32] Wakayama S, Hikichi T, Suetsugu R, Sakaide Y, Bui HT, Mizutani E, et al. Efficient establishment of mouse embryonic stem cell lines from single blastomeres and polar bodies. Stem Cells 2007; 25: 986–993.
[33] Obruca A, Strohmer H, Sakkas D, Menezo Y, Kogosowski A, Barak Y, et al. Use of lasers in assisted fertilization and hatching. Hum Reprod 1994; 9: 1723–1726.
[34] Veiga A, Sandalinas M, Benkhalifa M, Boada M, Carrera M, Santaló J, et al. Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote 1997; 5: 351–354.
[35] Cortés JL, Cobo F, Catalina P, Nieto A, Cabrera C, Montes R, et al. Evaluation of a laser technique to isolate the inner cell mass of murine blastocysts. Biotechnol Appl Biochem 2007; 46: 205–209.
[36] Cortés JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martínez-Ramirez A, et al. Whole-blastocyst culture followed by laser drilling technology enhances the efficiency of inner cell mass isolation and embryonic stem cell derivation from good- and poor-quality mouse embryos: new insights for derivation of human embryonic stem cell lines. Stem Cells Dev 2008; 17: 255–267.
[37] Tanaka N, Takeuchi T, Neri QV, Sills ES, Palermo GD. Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model. J Transl Med 2006; 8: 4–20.
[38] Ilic D, Stephenson E, Wood V, Jacquet L, Stevenson D, Petrova A, et al. Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions. Cytotherapy 2012; 14: 122–128.
[39] Stephenson E, Jacquet L, Miere C, Wood V, Kadeva N, Cornwell G, et al. Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product-free environment. Nat Protoc 2012; 7: 1366–1381.
[40] Strelchenko N, Verlinsky O, Kukharenko V, Verlinsky Y. Morula-derived human embryonic stem cells. Reprod Biomed Online 2004; 9: 623–629.
[41] Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R. Derivation of human embryonic stem cells from single blastomeres. Nat Protoc 2007; 2: 1963–1972.
[42] Chung Y, Klimanskaya I, Becker S, Li T, Maserati M, Lu SJ, et al. Human embryonic stem cell lines generated without embryo destruction. Cell Stem Cell 2008; 2: 113–117.
[43] Genbacev O, Krtolica A, Zdravkovic T, Brunette E, Powell S, Nath A, et al. Serum free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil Steril 2005; 83: 1517–1529.
[44] Xu C, Jiang J, Sottile V, McWhir J, Lebkowski J, Carpenter MK. Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells 2004; 22: 972–980.
[45] Akyash F, Sadeghian-Nodoushan F, Tahajjodi SS, Nikukar H, Farashahi Yazd E, Azimzadeh M, et al. Human embryonic stem cells and good manufacturing practice: Report of a 1- day workshop held at Stem Cell Biology Research Center, Yazd, 27th April 2017. Int J Reprod Biomed 2017; 15: 255–256.
[2] Solter D, Kowles BB. Immunosurgery of mouse blastocyst. Proc Natl Acad Sci USA 1975; 72: 5099–5102.
[3] Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292: 154–156.
[4] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 1981; 78: 7634–7638.
[5] Bongso A, Fong CY, Ng SC, Ratnam S. Isolation and culture of inner cell mass cells from human blastocysts. Hum Reprod 1994; 9: 2110–2117.
[6] Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 1995; 92: 7844–7848.
[7] Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Hearn JP. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod 1996; 55: 254–259.
[8] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145–1147.
[9] Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci USA 1998; 95: 13726–13731.
[10] Andrews PW. From teratocarcinomas to embryonic stem cells. Philos Trans R Soc Lond B Biol Sci 2002; 357: 405–417.
[11] Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R. Human embryonic stem cell lines derived from single blastomeres. Nature 2006; 444: 481–485.
[12] Omidi M, Aflatoonian B, Thajjodi SS, Khalili MA. Attempts for generation of embryonic stem cells from human embryos following in vitro embryo twinning. Stem Cells Dev 2019; 28: 303–309.
[13] Aflatoonian B, Ruban L, Shamsuddin S, Baker D, Andrews P, Moore H. Generation of Sheffield (Shef) human embryonic stem cell lines using a microdrop culture system. In Vitro Cell Dev Biol Anim 2010; 46: 236–241.
[14] Turetsky T, Aizenman E, Gil Y, Weinberg N, Shufaro Y, Revel A, et al. Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis. Hum Reprod 2008; 23: 46–53.
[15] Giritharan G, Ilic D, Gormley M, Krtolica A. Human embryonic stem cells derived from embryos at different stages of development share similar transcription profiles. PLoS One 2011; 6: e26570.
[16] Ström S, Inzunza J, Grinnemo KH, Holmberg K, Matilainen E, Strömberg AM, et al. Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines. Hum Reprod 2007; 22: 3051–3058.
[17] Ilic D, Genbacev O, Krtolica A. Derivation of hESC from Intact Blastocysts. Curr Protoc Stem Cell Biol 2007: 1A.2.1–1A.2.18.
[18] Hovatta O, Mikkola M, Gertow K, Strömberg AM, Inzunza J, Hreinsson J, et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003; 18: 1404–1409.
[19] Desai N, Ludgin J, Goldberg J, Falcone T. Development of a xeno-free non-contact co-culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line. J Assist Reprod Genet 2013; 30: 609–615.
[20] Assou S, Pourret E, Péquignot M, Rigau V, Kalatzis V, Aït-Ahmed O, et al. Cultured cells from the human oocyte cumulus niche are efficient feeders to propagate pluripotent stem cells. Stem Cells Dev 2015; 24: 2317–2327.
[21] Adewumi O, Aflatoonian B, Ahrlund-Richter L, Amit M, Andrews PW, Beighton G, et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol 2007; 25: 803–816.
[22] Miere C, Wood V, Kadeva N, Cornwell G, Codognotto S, Stephenson E, et al. Generation of KCL037 clinical grade human embryonic stem cell line. Stem Cell Res 2016; 16: 149–151.
[23] Amps KJ, Jones M, Baker D, Moore HD. In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice. Cryobiology 2010; 60: 344–350.
[24] Aflatoonian A, Karimzadeh Maybodi MA, Aflatoonian N, Tabibnejad N, Amir-Arjmand MH, Soleimani M, et al. Perinatal outcome in fresh versus frozen embryo transfer in ART cycles. Int J Reprod Biomed 2016; 14: 167–172.
[25] Draper JS, Pigott C, Thomson JA, Andrews PW. Surface antigens of human embryonic stem cells: Changes upon differentiation in culture. J Anat 2002; 200: 249–258.
[26] Sadeghian-Nodoushan F, Aflatoonian R, Borzouie Z, Akyash F, Fesahat F, Soleimani M, et al. Pluripotency and differentiation of cells from human testicular sperm extraction: An investigation of cell stemness. Mol Reprod Dev 2016; 83: 312–323.
[27] Akyash F, Javidpou M, Sadeghian Nodoushan F, Aflatoonian B. Human embryonic stem cells derived mesenchymal stem/stromal cells and their use in regenerative medicine. J Stem Cell Res Ther 2016; 1: 00047.
[28] Aflatoonian B, Moore H. Pluripotent stem cells in vitro from human primordial germ cells. In: Kaiming Ye, Sha Jin. Human embryonic and induced pluripotent stem cell: Lineage-specific differentiation protocols. USA: Springer Protocols Handbooks; 2012. p. 71–83.
[29] Laowtammathron C, Chingsuwanrote P, Choavaratana R, Phornwilardsiri S, Sitthirit K, Kaewjunun C, et al. High-efficiency derivation of human embryonic stem cell lines using a culture system with minimized trophoblast cell proliferation. Stem Cell Res Ther 2018; 9: 138–147.
[30] First NL, Sims MM, Park SP, Kent-First MJ. Systems for production of calves from cultured bovine embryonic cells. Reprod Fertil Dev 1994; 6: 553–562.
[31] Sherbahn R, Frasor J, Radwanska E, Binor Z, Wood-Molo M, Hibner M, et al. Comparison of mouse embryo development in open and microdrop co-culture systems. Hum Reprod 1996; 11: 2223–2229.
[32] Wakayama S, Hikichi T, Suetsugu R, Sakaide Y, Bui HT, Mizutani E, et al. Efficient establishment of mouse embryonic stem cell lines from single blastomeres and polar bodies. Stem Cells 2007; 25: 986–993.
[33] Obruca A, Strohmer H, Sakkas D, Menezo Y, Kogosowski A, Barak Y, et al. Use of lasers in assisted fertilization and hatching. Hum Reprod 1994; 9: 1723–1726.
[34] Veiga A, Sandalinas M, Benkhalifa M, Boada M, Carrera M, Santaló J, et al. Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote 1997; 5: 351–354.
[35] Cortés JL, Cobo F, Catalina P, Nieto A, Cabrera C, Montes R, et al. Evaluation of a laser technique to isolate the inner cell mass of murine blastocysts. Biotechnol Appl Biochem 2007; 46: 205–209.
[36] Cortés JL, Sánchez L, Catalina P, Cobo F, Bueno C, Martínez-Ramirez A, et al. Whole-blastocyst culture followed by laser drilling technology enhances the efficiency of inner cell mass isolation and embryonic stem cell derivation from good- and poor-quality mouse embryos: new insights for derivation of human embryonic stem cell lines. Stem Cells Dev 2008; 17: 255–267.
[37] Tanaka N, Takeuchi T, Neri QV, Sills ES, Palermo GD. Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model. J Transl Med 2006; 8: 4–20.
[38] Ilic D, Stephenson E, Wood V, Jacquet L, Stevenson D, Petrova A, et al. Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions. Cytotherapy 2012; 14: 122–128.
[39] Stephenson E, Jacquet L, Miere C, Wood V, Kadeva N, Cornwell G, et al. Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product-free environment. Nat Protoc 2012; 7: 1366–1381.
[40] Strelchenko N, Verlinsky O, Kukharenko V, Verlinsky Y. Morula-derived human embryonic stem cells. Reprod Biomed Online 2004; 9: 623–629.
[41] Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R. Derivation of human embryonic stem cells from single blastomeres. Nat Protoc 2007; 2: 1963–1972.
[42] Chung Y, Klimanskaya I, Becker S, Li T, Maserati M, Lu SJ, et al. Human embryonic stem cell lines generated without embryo destruction. Cell Stem Cell 2008; 2: 113–117.
[43] Genbacev O, Krtolica A, Zdravkovic T, Brunette E, Powell S, Nath A, et al. Serum free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil Steril 2005; 83: 1517–1529.
[44] Xu C, Jiang J, Sottile V, McWhir J, Lebkowski J, Carpenter MK. Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells 2004; 22: 972–980.
[45] Akyash F, Sadeghian-Nodoushan F, Tahajjodi SS, Nikukar H, Farashahi Yazd E, Azimzadeh M, et al. Human embryonic stem cells and good manufacturing practice: Report of a 1- day workshop held at Stem Cell Biology Research Center, Yazd, 27th April 2017. Int J Reprod Biomed 2017; 15: 255–256.