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Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells

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Abstract

The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2–5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.

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References

  • Anand T, Kumar D, Singh MK, Shah RA, Chauhan MS, Manik RS, Singla SK, Palta P (2011) Buffalo (Bubalus bubalis) embryonic stem cell-like cells and preimplantation embryos exhibit comparable expression of pluripotency-related antigens. Reprod Domest Anim 46:50–58

    Article  CAS  Google Scholar 

  • Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R (2003) Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 17:126–140

    Article  CAS  Google Scholar 

  • Bao L, He L, Chen J, Wu Z, Liao J, Rao L, Ren J, Li H, Zhu H, Qian L, Gu Y, Dai H, Xu X, Zhou J, Wang W, Cui C, Xiao L (2011) Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors. Cell Res 21:600–608

    Article  CAS  Google Scholar 

  • Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA (2005) Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122:947–956

    Article  CAS  Google Scholar 

  • Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113:643–655

    Article  CAS  Google Scholar 

  • Dattena M, Chessa B, Lacerenza D, Accardo C, Pilichi S, Mara L, Chessa F, Vincenti L, Cappai P (2006) Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts. Mol Reprod Dev 73:31–39

    Article  CAS  Google Scholar 

  • Dutta R, Malakar D, Khate K, Sahu S, Akshey Y, Mukesh M (2011) A comparative study on efficiency of adult fibroblast, putative embryonic stem cell and lymphocyte as donor cells for production of handmade cloned embryos in goat and characterization of putative ntES cells obtained from these embryos. Theriogenology 76:851–863

    Article  Google Scholar 

  • Dyban AP, Sorokin AV (1983) Comparison of the size of paternal and maternal homologous chromosomes during the first 2 cleavage divisions in mouse embryos. Ontogenez 14:238–246

    CAS  Google Scholar 

  • Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156

    Article  CAS  Google Scholar 

  • Fong H, Hohenstein KA, Donovan PJ (2008) Regulation of self-renewal and pluripotency by Sox2 in human embryonic stem cells. Stem Cells 26:1931–1938

    Article  CAS  Google Scholar 

  • Giles JR, Yang X, Mark W, Foote RH (1993) Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetuses following injection into blastocysts or morulae. Mol Reprod Dev 36:130–138

    Article  CAS  Google Scholar 

  • Gjorret JO, Maddox-Hyttel P (2005) Attempts towards derivation and establishment of bovine embryonic stem cell-like cultures. Reprod Fertil Dev 17:113–124

    CAS  Google Scholar 

  • Graves KH, Moreadith RW (1993) Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos. Mol Reprod Dev 36:424–433

    Article  CAS  Google Scholar 

  • Guest DJ, Allen WR (2007) Expression of cell-surface antigens and embryonic stem cell pluripotency genes in equine blastocysts. Stem Cells Dev 16:789–796

    Article  CAS  Google Scholar 

  • Hall V (2008) Porcine embryonic stem cells: a possible source for cell replacement therapy. Stem Cell Rev 4:275–282

    Article  Google Scholar 

  • Hart AH, Hartley L, Ibrahim M, Robb L (2004) Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human. Dev Dyn 230:187–198

    Article  CAS  Google Scholar 

  • Hatano SY, Tada M, Kimura H, Yamaguchi S, Kono T, Nakano T, Suemori H, Nakatsuji N, Tada T (2005) Pluripotential competence of cells associated with Nanog activity. Mech Dev 122:67–79

    Article  CAS  Google Scholar 

  • Hatoya S, Torii R, Kondo Y, Okuno T, Kobayashi K, Wijewardana V, Kawate N, Tamada H, Sawada T, Kumagai D, Sugiura K, Inaba T (2006) Isolation and characterization of embryonic stem-like cells from canine blastocysts. Mol Reprod Dev 73:298–305

    Article  CAS  Google Scholar 

  • He S, Pant D, Schiffmacher A, Bischoff S, Melican D, Gavin W, Keefer C (2006) Developmental expression of pluripotency determining factors in caprine embryos: novel pattern of NANOG protein localization in the nucleolus. Mol Reprod Dev 73:1512–1522

    Article  CAS  Google Scholar 

  • Huang B, Xie TS, Shi DS, Li T, Wang XL, Mo Y, Wang ZQ, Li MM (2007) Isolation and characterization of EG-like cells from Chinese swamp buffalo (Bubalus bubalis). Cell Biol Int 31:1079–1088

    Article  CAS  Google Scholar 

  • Huang B, Li T, Wang XL, Xie TS, Lu YQ, da Silva FM, Shi DS (2010) Generation and characterization of embryonic stem-like cell lines derived from in vitro fertilization buffalo (Bubalus bubalis) embryos. Reprod Domest Anim 45:122–128

    Article  CAS  Google Scholar 

  • Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, DeCoste C, Schafer X, Lun Y, Lemischka IR (2006) Dissecting self-renewal in stem cells with RNA interference. Nature 442:533–538

    Article  CAS  Google Scholar 

  • Kim HS, Oh SK, Park YB, Ahn HJ, Sung KC, Kang MJ, Lee LA, Suh CS, Kim SH, Kim DW, Moon SY (2005) Methods for derivation of human embryonic stem cells. Stem Cells 23:1228–1233

    Article  Google Scholar 

  • Lee JB, Song JM, Lee JE, Park JH, Kim SJ, Kang SM, Kwon JN, Kim MK, Roh SI, Yoon HS (2004) Available human feeder cells for the maintenance of human embryonic stem cells. Reproduction 128:727–735

    Article  CAS  Google Scholar 

  • Lindvall O, Kokaia Z (2005) Stem cell therapy for human brain disorders. Kidney Int 68:1937–1939

    Article  Google Scholar 

  • Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78:7634–7638

    Article  CAS  Google Scholar 

  • Masui S, Nakatake Y, Toyooka Y, Shimosato D, Yagi R, Takahashi K, Okochi H, Okuda A, Matoba R, Sharov AA, Ko MS, Niwa H (2007) Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol 9:625–635

    Article  CAS  Google Scholar 

  • Mitalipova M, Beyhan Z, First NL (2001) Pluripotency of bovine embryonic cell line derived from precompacting embryos. Cloning 3:59–67

    Article  CAS  Google Scholar 

  • Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113:631–642

    Article  CAS  Google Scholar 

  • Munoz M, Diez C, Caamano JN, Jouneau A, Hue I, Gomez E (2008) Embryonic stem cells in cattle. Reprod Domest Anim 43(Suppl 4):32–37

    Article  Google Scholar 

  • Navara CS, Redinger C, Mich-Basso J, Oliver S, Ben-Yehudah A, Castro C, Simerly C (2007) Derivation and characterization of nonhuman primate embryonic stem cells. Curr Protoc Stem Cell Biol 1:1A.1.1–1A.1.21

    Google Scholar 

  • Notarianni E, Laurie S, Moor RM, Evans MJ (1990) Maintenance and differentiation in culture of pluripotential embryonic cell lines from pig blastocysts. J Reprod Fertil Suppl 41:51–56

    CAS  Google Scholar 

  • Notarianni E, Galli C, Laurie S, Moor RM, Evans MJ (1991) Derivation of pluripotent, embryonic cell lines from the pig and sheep. J Reprod Fertil Suppl 43:255–260

    CAS  Google Scholar 

  • Passier R, Mummery C (2003) Origin and use of embryonic and adult stem cells in differentiation and tissue repair. Cardiovasc Res 58:324–335

    Article  CAS  Google Scholar 

  • Pawar SS, Malakar D, De AK, Akshey YS (2009) Stem cell-like outgrowths from in vitro fertilized goat blastocysts. Indian J Exp Biol 47:635–642

    CAS  Google Scholar 

  • Piedrahita JA, Moore K, Oetama B, Lee CK, Scales N, Ramsoondar J, Bazer FW, Ott T (1998) Generation of transgenic porcine chimeras using primordial germ cell-derived colonies. Biol Reprod 58:1321–1329

    Article  CAS  Google Scholar 

  • Pouton CW, Haynes JM (2007) Embryonic stem cells as a source of models for drug discovery. Nat Rev Drug Discov 6:605–616

    Article  CAS  Google Scholar 

  • Rathjen J, Lake JA, Bettess MD, Washington JM, Chapman G, Rathjen PD (1999) Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors. J Cell Sci 112:601–612

    Google Scholar 

  • Roach M, Wang L, Yang X, Tian XC (2006) Bovine embryonic stem cells. Methods Enzymol 418:21–37

    Article  CAS  Google Scholar 

  • Saito S, Ugai H, Sawai K, Yamamoto Y, Minamihashi A, Kurosaka K, Kobayashi Y, Murata T, Obata Y, Yokoyama K (2002) Isolation of embryonic stem-like cells from equine blastocysts and their differentiation in vitro. FEBS Lett 531:389–396

    Article  CAS  Google Scholar 

  • Saito S, Sawai K, Ugai H, Moriyasu S, Minamihashi A, Yamamoto Y, Hirayama H, Kageyama S, Pan J, Murata T, Kobayashi Y, Obata Y, Yokoyama KK (2003) Generation of cloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells. Biochem Biophys Res Commun 309:104–113

    Article  CAS  Google Scholar 

  • Saito S, Yokoyama K, Tamagawa T, Ishiwata I (2005) Derivation and induction of the differentiation of animal ES cells as well as human pluripotent stem cells derived from fetal membrane. Hum Cell 18:135–141

    Article  Google Scholar 

  • Sritanaudomchai H, Pavasuthipaisit K, Kitiyanant Y, Kupradinun P, Mitalipov S, Kusamran T (2007) Characterization and multilineage differentiation of embryonic stem cells derived from a buffalo parthenogenetic embryo. Mol Reprod Dev 74:1295–1302

    Article  CAS  Google Scholar 

  • Stice SL, Strelchenko NS, Keefer CL, Matthews L (1996) Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer. Biol Reprod 54:100–110

    Article  CAS  Google Scholar 

  • Stojkovic M, Lako M, Stojkovic P, Stewart R, Przyborski S, Armstrong L, Evans J, Herbert M, Hyslop L, Ahmad S, Murdoch A, Strachan T (2004) Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture. Stem Cells 22:790–797

    Article  Google Scholar 

  • Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147

    Article  CAS  Google Scholar 

  • Vaags AK, Rosic-Kablar S, Gartley CJ, Zheng YZ, Chesney A, Villagomez DA, Kruth SA, Hough MR (2009) Derivation and characterization of canine embryonic stem cell lines with in vitro and in vivo differentiation potential. Stem Cells 27:329–340

    Article  CAS  Google Scholar 

  • Verma V, Gautam SK, Singh B, Manik RS, Palta P, Singla SK, Goswami SL, Chauhan MS (2007) Isolation and characterization of embryonic stem cell-like cells from in vitro-produced buffalo (Bubalus bubalis) embryos. Mol Reprod Dev 74:520–529

    Article  CAS  Google Scholar 

  • Wang XY, Liu B, Yuan CH, Yao HY, Mao N (2003) Effect of bone marrow mesenchymal stem cells on hematopoietic differentiation of murine embryonic stem cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi 11:329–334

    Google Scholar 

  • Wang L, Duan E, Sung LY, Jeong BS, Yang X, Tian XC (2005) Generation and characterization of pluripotent stem cells from cloned bovine embryos. Biol Reprod 73:149–155

    Article  CAS  Google Scholar 

  • Wheeler MB (1994) Development and validation of swine embryonic stem cells: a review. Reprod Fertil Dev 6:563–568

    Article  CAS  Google Scholar 

  • Yadav PS, Kues WA, Herrmann D, Carnwath JW, Niemann H (2005) Bovine ICM derived cells express the Oct4 ortholog. Mol Reprod Dev 72:182–190

    Article  CAS  Google Scholar 

  • Yu X, Jin G, Yin X, Cho S, Jeon J, Lee S, Kong I (2008) Isolation and characterization of embryonic stem-like cells derived from in vivo-produced cat blastocysts. Mol Reprod Dev 75:1426–1432

    Article  CAS  Google Scholar 

  • Zwaka TP, Thomson JA (2003) Homologous recombination in human embryonic stem cells. Nat Biotechnol 21:319–321

    Article  CAS  Google Scholar 

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Acknowledgments

The study was supported by research grant from Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India. The authors thank The Director, Indian Veterinary Research Institute for providing necessary facilities for carrying out research work.

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The authors indicate no potential conflicts of interest.

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Authors and Affiliations

  1. Division of Animal Genetics and Breeding, Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, India

    R. Kumar & S. P. S. Ahlawat

  2. Division of Physiology and Climatology, Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, India

    M. Sharma, O. P. Verma & G. Taru Sharma

  3. Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, India

    G. Sai Kumar

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  1. R. Kumar
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Kumar, R., Ahlawat, S.P.S., Sharma, M. et al. Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells. Cytotechnology 66, 239–250 (2014). https://doi.org/10.1007/s10616-013-9561-7

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