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Development of stem cell technologyfor livestock species in Taiwan
Lih‐Ren Chen, PhDLivestock research Institute, Council of Agriculture
Taiwan
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(Winslow, 2001)
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Chen, L. R., J. P. Wang and M. C. Wu, 1991. In vitro culture of mammalian embryonic stem cells : II. In vitro culture of blastocyst-derived cells in mouse and pigs. J. Chin. Soc. Anim. Sci. 20(3):326-339.
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Shim, H., A. Gutierrez-Adan, L. R. Chen, R. H. BonDurant, E. Behboodi and G. B. Anderson. 1997. Isolation of pluripotent stem cells from cultured porcine primordial germ cells.
BOR 57:1089-1095.
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Chen, L. R., Y. L. Shiue, L. Bertolini, J. F. Merdrano, R. H. BonDurant and G. B. Anderson, 1999. Establishment of plurpotent cell lines from porcine preimplantation embryos. Theriogenology 52:195-
212.
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Embryo Splitting
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Micromanipulator
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“The cloning of mammals…………..…. is biologically impossible.”
James McGrath and Davor Solter, Science 1984.
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• Ian Wilmut• Created the
first true clone, the Dorset ewe Dolly
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Dolly, the sheep
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1996 Dolly the sheep
1998 Mouse, Cow
1999 Goat
2000 Pig
2002 Cat, Rabbit, African wild cat
2003 Mule, House, Rat, Banteng,
Deer, Rat
2005 Dog
Somatic clone animals
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C D
A B
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0901/2001, 畜寶 1016/2001 0122/2002
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寶吉與寶祥, 0705/2003
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Ru-Yi, 0227/2003
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0227/2003, 1211/2003, 0116/2003, 0122/2004
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0217/2005
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V.S• Growth
• Reproduction
• Production
• Gene expression
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• Easy to manipulate• Low efficiency
• Variety gene expression level
• nearly impossible for gene targeting
• Complicated manipulation
• Low efficiency
• Uniform gene expression level
• Possible for gene targeting
Vectors for genetic modification
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Preparation of nuclear donor cells
Ear fibroblasts
Frozen
Gene Transfer
Subcloning
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Transfection
Incubation and Passage
Selection
Somatic cells donor
Cloning
Nuclear transfer
Limited passage number
Possible damage in X’some
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The first TGNT goat in TaiwanBao-yu0310/2004
0425/2005
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Stimulators
(Yang et al., 2006)
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Nestin +
NF68 +
MAP2+
ChAT+
TH+
(Yang et al., 2006)
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++++AP activity
++++Oct‐4+++‐TRA‐1‐81+++‐TRA‐1‐60+++‐SSEA‐3/4‐‐‐+SSEA‐1
pEShESrESmESItem
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+??+Chimera formation++++EB formation
Flat, tightaggregates
Flat, loose aggregates
Flat, loose aggregates
Tight, rounded, multi‐layer clumps
Growth characteristics
‐+/‐‐+LIF for selfrenew++++/‐Feeder dependencypEShESrESmESItems
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Cloning ES Tech
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Solter and Gearhart, 1999
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Differentiation induction
Isolation and culture of ES cells
Pig Somatic cell
Therapeutic cloning in pig model
Immunologically compatible transplant for cell therapy
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IVM pig oocyte Enucleation
Transfer of donor cell Electrofusion
LPE-206-1
Donor cell
8(17.3)46(74.1)62(51.6)120
BlastocystCleavedSuccessfully fused
Used for SCNT
Number and(%)of fused embryos
Number and(%)of oocytes
Pig somatic cells
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Dominko et al., 1999. Biol. Reprod. 60:1496-1502.
Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic nuclei from various mammalian species.
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Hybrid Embryo development
Hatching blastocyst at day 7 Hatched blastocyst at day 9
Morula at day 5 Early Blastocyst at day 6
11 (50%)
2‐16 cells
Cleavage (%)(72%)
Adult female buffalo fibroblast
Donor cell
2 (9%)3 (13.6%)
BlastocystMorula22 (84%)2627
fused (%)Nuclear transferoocytes
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Stem cells
Differentiated adult cells
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Human/mouse
somatic cells
streptolysin-O Remove
Stem
cell
extrac
tion
Oocyte/
Stem cell extract
Resealing plasma membrane
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Morphology change of reprogrammed mouse and human fibroblasts
b
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β-actinOct-4
Nanog
mES Reprogrammed hF
β-actin
Oct-4
NanogPECAM-1
PECAM-1
Reprogrammed mF
β-actin
Oct-4Nanog
PECAM-1
hF
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Shiue, Y. L., J. J Tailiu, J. F. Liou, H.T. Lu, C. Tai, J. W. Shiau, and L. R. Chen. 2007. Establishment of the Long-Term In VitroCulture System for Chicken Primordial Germ Cells. Reprod. Dom. Anim. (In press)
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AP+
SSEA-1+
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Bright field
Fluorescence
SSEA-1 SSEA-4 Integrin α6 Integrin β1SSEA-3
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A B
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Acknowledgement• Livestock Research Institute
– Dr. JJ Tailiu– JR Yang– TY Kuo– FH Chu– YH Chen– Dr. JW Shiau
• Buddhist Tzu Chi Medical Center– Dr. SZ Lin– CH Liao
• National Chung HsinUniversity– Dr. CM Chen– Dr. MC Huang
• National Sun Yat‐sen University– Dr. YL Shiue
• National Chung Kung University– Dr. C Tai– CH Kang– FT Lu
• National Taiwan University– Dr. WTK Cheng
• National Pintong Scientific U.– Dr. PC Sheen
• University of Connecticut– Dr. X Yang – Dr. X Tian, – Dr. FL Du
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Formosan rock-monkey (Macaca cyclopis)
Thanks for Your Attention
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Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes.Cell Research 2003; 13(4):251-264
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Fig 4. Morphology and marker expression in ntES cells (A) An ntES cell colony derived from a 42-year-old somatic cell donor. (B) Higher magnification of the same colony shown in (A). (C) A colony derived from a 52-year-old somatic cell donor. (D) Alkaline phosphatase activity in ntES cells after 26 passages. The ntES cells express epitopes recognized by antibodies against (E) SSEA-3,( F) SSEA-4, (G) TRA-1-10, and (H) TRA-1-85. The ntES cells failed to stain for SSEA-1 (data not shown). (Bars in A and C-H = 50 μm; in B = 25 μm)