Abstract
Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76–80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11–13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells.
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Ambrosi DJ, Tanasijevic D, Kaur A, Obergfell C, O’Neill R, Krueger W, Rasmussen TP (2007) Genome-wide reprogramming in hybrids of somatic cells and embryonic stem cells. Stem Cells 25:1104–1113
Constantinescu D, Gray HL, Sammak PJ, Schatten GP, Csoka B (2006) Lamin A/C expression is a marker of mouse and human embryonic stem cell differentiation. Stem Cells 24:177–185
Cowan CA, Atienza J, Melton DA, Eggan K (2005) Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309:1369–1373
Eakin GS, Behringer RR (2003) Tetraploid development in the mouse. Dev Dyn 228:751–766
Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M, Rideout WM III, Yanagimachi R, Jaenisch R (2001) Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci USA 98:6209–6214
Han DW, Do JT, Gentle L, Stehling M, Lee HT, Schöler HR (2007) Pluripotential reprogramming of the somatic genome in hybrid cells occurs with the first cell cycle. Stem Cells 26:445–454
Hogan B, Beddington R, Constantini F, Lacy E (1994) Manipulating the mouse embryo, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Matveeva NM, Shilov AG, Kaftanovskaya EM, Maximovsky LP, Zhelezova AI, Golubitsa AN, Bayborodin SI, Fokina MM, Serov OL (1998) In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes. Mol Reprod Dev 50:128–138
Matveeva NM, Pristyazhnyuk IE, Temirova SA, Menzorov AG, Vasilkova A, Shilov AG, Smith A, Serov OL (2005) Unequal segregation of parental chromosomes in embryonic stem cell hybrids. Mol Reprod Dev 71:305–314
Nagy A, Rossant J (1993) Production of completely ES-derived fetuses. In: Joyner A (ed) Gene targeting: a practical approach. IRL Press, Oxford, pp 147–179
Nagy A, Gocza E, Diaz EM, Prideaux VR, Ivany M, Markkula M, Rossant J (1990) Embryonic stem cells alone are able to support fetal development in the mouse. Development 110:815–821
Pells S, Di Domenico AI, Gallagher EJ, McWhir J (2002) Multipotentiality of neuronal cells after spontaneous fusion with embryonic stem cells and nuclear reprogramming in vitro. Clon Stem Cells 4:331–338
Pralong D, Lim ML, Vassiliev I, Mrozik K, Wijesundara N, Rathjen P, Verma PJ (2005) Tetraploid embryonic stem cells contribute to the inner cell mass of mouse blastocysts. Clon Stem Cells 7:272–278
Pratt T, Sharp L, Nichols J, Price DJ, Mason JO (2000) Embryonic stem cells and transgenic mice ubiquitously expressing a tau-tagged green fluorescent protein. Dev Biol 228:19–28
Pristyazhnyuk IE, Temirova SA, Menzorov AG, Kruglova AA, Matveeva NM, Serov OL (2005) Visible and “cryptic” segregation of parental chromosomes in embryonic stem hybrid cells. Russian J Dev Biol 36:119–126
Tada M, Takahama Y, Abe K, Nakatsuji N, Tada T (2001) Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr Biol 11:1553–1558
Tada M, Morizane A, Kimura H, Kawasaki H, Ainscough JFX, Sasai Y, Nakatsuji N, Tada T (2003) Pluripotency of reprogrammed somatic genomes in embryonic stem hybrid cells. Dev Dyn 227:504–510
Talbot NC, Rexroad CE, Pursel VG, Powell AV (1993) Alkaline phosphatase staining of pig and sheep epiblast cells in culture. Mol Reprod Dev 36:139–147
Vasilkova AA, Kizilova HA, Puzakov MV, Shilov AG, Zhelezova AI, Golubitsa AN, Battulin RN, Vedernikov VE, Menzorov AG, Matveeva NM, Serov OL (2007) Dominant manifestation of pluripotency in embryonic stem cell hybrids with various numbers of somatic chromosomes. Mol Reprod Dev 74:941–951
Wang L, Jaenisch R (2004) At most three ES contribute to the somatic lineages of chimeric mice and of mice produced by ES-tetraploid complementation. Dev Biol 275:192–201
Wernig M, Meissner A, Foreman R, Brambrinck T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318–327
Yang X, Smith SL, Tian XC, Lewin HA, Renard J-P, Wakayama T (2007) Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning. Nat Genet 29:295–302
Ying Q-L, Nichols J, Evans EP, Smith AG (2002) Changing potency by spontaneous fusion. Nature 416:545–547
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A. A. Kruglova and E. A. Kizilova contributed equally to this work.
This study was financially supported by grants from the Russian Academy of Sciences, Siberian Branch 5.2 and 14.0.
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Kruglova, A.A., Kizilova, E.A., Zhelezova, A.I. et al. Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras. Cell Tissue Res 334, 371–380 (2008). https://doi.org/10.1007/s00441-008-0702-9
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DOI: https://doi.org/10.1007/s00441-008-0702-9