Development of stem cell technologyfor livestock species in Taiwan

Lih‐Ren Chen, PhDLivestock research Institute, Council of Agriculture

Taiwan

(Winslow, 2001)

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.

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.

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.

Embryo Splitting

Micromanipulator

“The cloning of mammals…………..…. is biologically impossible.”

James McGrath and Davor Solter, Science 1984.

• Ian Wilmut• Created the 

first true clone, the Dorset ewe Dolly

Dolly, the sheep

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

C D

A B

0901/2001, 畜寶 1016/2001 0122/2002

寶吉與寶祥, 0705/2003

如意, 0227/2003

0227/2003, 1211/2003, 0116/2003, 0122/2004

0217/2005

V.S• Growth

• Reproduction

• Production

• Gene expression

• 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

Preparation of nuclear donor cells

Ear fibroblasts

Frozen

Gene Transfer

Subcloning

寶鈺, 0310/2004

0425/2005

Transfection

Incubation and Passage

Selection

Somatic cells donor

Cloning

Nuclear transfer

Limited passage number

Possible damage in X’some

RA

(Yang et al., 2006)

Nestin +

NF68 +

MAP2+

ChAT+

TH+

(Yang et al., 2006)

++++AP activity

++++Oct‐4+++‐TRA‐1‐81+++‐TRA‐1‐60+++‐SSEA‐3/4‐‐‐+SSEA‐1

pEShESrESmESItem

+??+Chimera formation

++++EB formation

Flat, tightaggregates

Flat, loose aggregates

Flat, loose aggregates

Tight, rounded, multi‐layer clumps

Growth characteristics

‐+/‐‐+LIF for selfrenew

++++/‐Feeder dependency

pEShESrESmESItems

Cloning ES Tech

Solter and Gearhart, 1999

Differentiation induction

Isolation and culture of ES cells

Pig Somatic cell

Therapeutic cloning in pig model

Immunologicallycompatible transplant for cell therapy

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

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.

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

Stem cells

Differentiated adult cells

Human/mouse

somatic cells

streptolysin-O Remove

Stem

cell

extrac

tion

Oocyte/

Stem cell extract

Resealing plasma membrane

Morphology change of reprogrammed mouse and human fibroblasts

b

β-actinOct-4

Nanog

mES Reprogrammed hF

β-actin

Oct-4

NanogPECAM-1

PECAM-1

Reprogrammed mF

β-actin

Oct-4Nanog

PECAM-1

hF

AP+

SSEA-1+

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

Formosan rock-monkey (Macaca cyclopis)

Thanks for Your Attention