Cng ngh Phi & T bo gc Embryo & Stem cells Biotechnology - TM LC

Bui Xuan Nguyen, Ph.D- University Paris VI VAST, USTH Hanoi, Vietnam Buixn(@gmail.com

Embryo
-Etymology : First attested in English in the mid-14c., the word embryon derives from Medieval Latin embryo, itself from Greek (embruon), plural (embrua), lit. "young one - An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination. In humans, it is called an embryo until about eight weeks after fertilization and from then it is instead called a fetus. (wikipedia] - Parthenogenetic haploide, trysomy ???

Embryo development
- Embryogenesis: The development of the embryo - Embryo differentiation: The process by which specialized and diversified structures arise during development of the embryo. The process involves (1) an increase in the number of cell types, and (2) an increase in morphological heterogeneity through the arrangement of cells into increasingly complex structural patterns in the form of tissues and organs. (wikipedia]

Embryo- the start in life- all begins from embryo

Sperm

Oocyte

4 groups connective, muscle, nervous & epithelial tissues

EMBRYO

Fetus

300 different cell types

New organism

Embryo differentiation

Principles of stem cell biology

Stem cell key properties Self-renew & multiply Differentiation , Plasticity

General approaches for stem cell production

1. Adult Stem Cells 2. Embryonic Stem Cells (ES) 3. Induced Pluripotent Stem Cells (iPS)

Fertilization, Embryo, Stem cells & Development

Sperm

Oocyte

Totipotent
EMBRYO

Pluripotent
Multipotent
Cells

Unipotent
New organism

2. Embryonic stem cells ( pluripotent)

From in vitro fertilized embryos (IVF): Mouse (Evan & Kaufman, 1981); Human (Thomson et al, USA, 1998/, Sweden, UK, Japan, China, Korea, Spain, Iran, Israel, Singapore, Thailand) From Parthenotes ( parthenogenetically activated oocytes ): mouse (Cuthbertson,1983), human (Cibelli et al. 2001). From Somatic Cell Nuclear Transfer (SCNT) embryo: Mouse (Wakayama et al, 2001), Bovine, Monkey, Pig, ( Human: Sweden, China)

1.Adult stem cells (multipotent)

"stem cell niche" in brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian Umbilical cord matrix stc : Photo: STEMCELL Technologies Inc Cord Is Richer than Blood: 0.7-2.0 x106cells/cm( Secco et al, 2007) low immunogenicity, lower expression of CD106, HLAABC and HLA-DR, benefit clinical use. (Lu et al, 2006)

3- Induced Pluripotent Stem Cells

First: Takahashi v Yamanaka (2006): exogenous expression of the transcription factors OCT4, SOX2, KLF4, & c-MYC New method single transfer of ESC (embryonic stem cell)derived proteins into fibroblasts, full reprogramming up to the pluripotent state (Chio et al, 2010).

REPRODUCTION

TECHNOLOGY

BIO-MEDICAL APPLICATION

ANIMAL BREEDING INDUSTRY Sperm Oocyte Embryo Transfer

BIODIVERSITY PROTECTION Superovulation

IVM-IVF
38 0C, 5% CO2
EMBRYO

Cryobanking

XX
In vitro Fertilization

TRANSGENIC PHARMACEUTICAL INDUSTRY REGENERATION MEDICINE XENOTRANSPLANTATION GENE THERAPY INFERILITY TREATMENT
Stem cells

PGD
Cells Somatic cloning

New organism Genetic Engineering BXNguyen-Paris VI

Biotechnology: Definition
The fusion of biology and technology. Biotechnology is the application of biological techniques to product research and development. (http://www.medterms.com/ ) Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use. (The United Nations Convention on Biological Diversity ) A field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. (http://en.wikipedia.org/wiki/Biotechnology)

Biotechnology: Definition
Classic Biotechnology arose from the field of zymotechnology, which began as a search for a better understanding of industrial fermentation Modern Biotechnology: The origins of biotechnology culminated with the birth of genetic engineering. -the 1953 discovery of the structure of DNA, by Watson and Crick - 1973 discovery by Cohen and Boyer of a recombinant DNA technique by which a section of DNA was cut from the A plasmid of an E. coli bacterium and transferred into the DNA of another

Embryo & Stem cell

Technology

Life application

Bio-Medical Biotechnology At central first life stage for all life aspects

SCNT- 1. Production of genetic identical stem cells

EMBRYO & STEM CELLS TECHNOLOGY

W Heape, 1890: first animal from

embryo transfer Edward, 1978: first test-tube baby

a dng sinh hc, cong ngh sinh sn- bo tn ex situ-in situ

65 T, nguyn nhn Sao chi

370 T, nguyn nhn ? 440 T, nguyn nhn ?

202 T, nguyn nhn ?

240 T, nguyn nhn ni la

Trong khong 440 triu nm qua c 5 ln sinh vt b tiu dit hng lot (V Qu, Biodiva 2007)

Ln gn y nht, cch nay khoang 65 triu nam l do thin thch ri vo tri t, 20 % s h v 50% s ging b tiu dit, trong c cc loi khng long Hin nay, ang din hin tng cc loi b tiu dit hng lot, nhng nguyn nhn chnh l do hot ng ca loi ngi.
(V Qu, Biodiva 2007)

Kt qu nghin cu ca hn 1500 nh khoa hc trn th gii trong bn Millennium Ecoystem Assessment (2005) th trong khong 400 nm qua c 484 loi ng vt v 654 loi thc b tiu dit, v c khong 30000 loi V v TV ang nguy cp. Tc cc loi b tiu dit nhanh gp 100-1000 ln so vi bnh thng. Trong cc thp k sp ti tc mt cc loi cn cao hn.
(V Qu, Biodiva 2007)

Danh lc Sch ca IUCN (2004): - 23% s loi th (1101 loi) - 12% s loi chim (1213 loi) ang nguy cp - cc nhm khc khong 38% n 86% s loi nguy cp. T 1970 n nay c 58 loi c, 9 loi chim v 1 loi th b tiu dit.
(V Qu, Biodiva 2007)

Nguyn nhn suy thoi cc loi: 1- Ni sinh sng b thu hp li 2- Xm ln ca cc loi ngoi lai 3- nhim mi trng 4- Sn bt qu mc 5- Thay i kh hu ton cu.
(V Qu, Biodiva 2007)

Brief history of embryo freezing

1949 : Polge et al., the successful freezing of chicken sperm by including glycerol 1952: Marden sent rabbit embryos by air back to Cambridge from Worcester, packed with ice balloons in a vacuum flask. 1960: Successful freeze-drying of bull sperm by Meryman 1972: mouse embryos were successfully frozen & transferred to give live young (Whittingham et al.,).

Status of oocyte-embryo cryoconservation: Technical aspects

Slow

freezing (Wittinghqm et al, 1972).

Rapid freezing (Nguyen-JP Renard 1983) Vitrification (Rall & Fahy, 1985).

RT

Rapid freezing
(Nguyen, Renard,1983, 1984)

Sucrose-PROH, 2 min, Room temperature

Clacsic slow programable freezing

-30oC

Rabbit, cattle
Survival in vitro: 60-88% In vivo: 33-81%

-196oC 5 30 300-500

Cloning Cloning by by Somatic SCNT Somatic cell cell nuclear nuclear transfer transfer //SCNT

1997, Doily

clone a woolly mammoth in the next five years. Led by Akira Iritani, Kinki

SCNT : conservation

Wells et al, 1999: Enderby island cattle Robert et al, 2000, Gaur Smith et al, 2001.

25 regions Hot Spots Biodiversity

Vietnam

American Bison - Bison bison European Bison - Bison bonasus Auroch - Bos primigenus Wild Yak - Bos mutus Banteng - Bos javanicus Gaur - Bos gaurus Kouprey- Bos Sauveli African Buffalo - Syncerus caffer Wild Water Buffalo - Bubalus arnee Lowland Anoa - Bubalus depressicornis Mountain Anoa - Bubalus quarlesi Tamaraw - Bubalus mindorensis Saola - Pseudoryx nghetinhensis

Kouprey : Numbers estimated before XX 1938 1940 1970 1975 1986 2003 2000 800 1000 30-70 50 >200 >250 (IUCN 2004)
(V Qu, Biodiva 2007)

Saola protection zone

1. Vu Quang, 1995 2. PuMat- Lao-VN, 1995 3. BachMa, 2008 4. Quang tri, 2010

Nombre et superficie des aires protges au Vietnam

3,000,000 140 120 100 2,000,000 80

2,500,000

1,500,000 60 1,000,000 40 500,000 20 0 Year 1954 Year 1975 Year 1986 Year 1993 Year 2003

Superfice totale (ha)

Nombre des aires protges

Sites

Ha

The saola (Pseudoryx nghetinhensis)

New discovered, but already threathened
1994 - Endangered (Groombridge 1994) 1996 - Endangered (Baillie and Groombridge 1996) 2003 - Endangered (IUCN 2003) 2006 - Critically Endangered (IUCN 2006)

NHAN BAN VO TINH- IN VITRO DEVELOPMENT OF BOVINE-BOVINE AND SAOLA-BOVINE EMBRYOS

Donor cell
Oocyte Fusion Cleaved (%) Morula (%) Blastocyst (%) 21 (20.07)

Cattle

150

91 (60.67)

72 (79.12)

25 (27.5)

Saola

312

166 (50.21)

131 (78.92)

65 (39.15)

46 (27.71)

Nbre cell. totales Nbre cell. ICM Nbre cell. tropho. 88 36 (40.9) 52 (59,1)

Bovine-bovine SCNT D.8

Saola-Bovine SCNT D.8

REGIONAL & COUNTRY EX SITU-INSITU NETWORK International Regional Banking Cryobanking

Informatic connection Equip Mobil Rescue station In situ: reservation1,2.. reintroduction Buffer Zone Economic Support Country Central

Software ART

Bio-Medical application

animals

Samples: semen, egs, cells

CNSS-Ci to nhn ging b tht-sa

VAST Vietnam-Cc mc thnh cng u tin

2003 2001 2000 1999 1996 1994 T bo gc phi nhn bn Chng trnh m ha Nhn bn v tnh: b, kh Xc nh gii tnh phi D cy phi Th tinh ng nghim

1988 Lm ch rng trng trn tru 1986 1983 1978 B cy phi Phng php ng lnh nhanh phi Th cy phi u tien

Ni dung 2: Sn xut phi cao sn c gii tnh xc nh bng k thut PCR

(2) (1)
Vi phu thut PCR
Primer ci Primer c

NST

sRY

(3)
Chn phi
t bo phi ci c

Cy phi

Vn : kh nng phc v cy phi quy m 30-60 phi/ngy, a im xa 70-2000 km) Ch tiu: Thi gian Vi phu thut phi, t l phi sng , thi gian PCR, t l xc nh, mc chnh xc 2 t hp mi : INRA1-148bp-c, INRA2-443 bp-loi, BRY4a-300bp-c, STS -216bp-loi

 phi

FSH PG

PG

phi

PROGESTERON

Estrus-superovulation synchronisation-ET

PG1

PG2

DDuc1

PMSG/ FSH hCG PG3

Dduc2

AI

Embryo

PG1

PG2

DDuc1

PMSG/ FSH hCG PG3

Dduc2

ET

So snh t l cha do cy phi VPT-gii tnh v phi khng VPT

Loi phi

S b nhn (n)

Cha 3 thng n (%) 11 (68,8) 13 (65,0)

n (%) 8 (50,0) 9 (45,0)

B con

B ci n ( %)

GT Khng GT

16 20

11 15

9 (81,8) 6 (40,0)

Tng th
T l cha chun : > 50 % T l cha do cy phi GT: > 50% S b sinh ra : 41 S b ci: 31 T l b ci do cy phi GT: 80% T l b sinh i do cy 2 phi: 50% Thi gian mang thai: 258- 275 ngy Trng lng s sinh: 22-50 kg Tc pht trin: tng ng Holstein

B TTON u tin Vietnam, Vinh Tuong, 11.2002

B sinh do cy phi TTON v xc nh gii tnh u tin Vit nam, 2-2003

so snh s b sa x tiu th sa/ dn s

Php:5000

Thi Lan: 250

VN: 3 2 cc/per
50000 2000 200000 2010 1.5 tr ???

SCNT for animal breeding through ONBS

30 years

1
Sexing Embryo

SCNT

30
A.I

300

300 000 animals

Tip cn 2- cng ngh phi TTON

B H-n Top:

B Holstein >8000 Lit/ck TTON

TRNG
38 0C, 5% CO2

B c sa+Nhit i

(2) (1)
Xc nh gii tnh:VPT phI+PCR

TINH

XX

(3)

(4)
Phi cI cy phi vo b LaiSind

H sinh sn nhn ging M

Cy phi - n thng phm Ngun Gen Trng Tinh cao sn M, Nht Cng nghip Phi Th tinh ng nghim Ngun Gen Tinh hng nhit i Cy phi - n thng phm

Cy phin gc 1 Dc, ci chn lc Sib n gc k tip

c ging phn phi Qung canh Qung canh Qung canh Qung canh Qung canh Qung canh

GENETIC & EPIGENETIC ASPECTS

Inheritance & differentiation

Hypothesis of preformationism, N Hartsoeker, 1694: all organs Were prefigured whithin the sperm

Hypothesis of epigenesis, Each organism develops anew from an undifferentiated condition. - each differentiated cell retain the full complement of genetic information present initially in the zygote? Hans Speman, 1938

Hans Spemann (1869-1941) Nobel award 1935 for discovery of the organizer effect in embryonic development";

1938 the "fantastical experiment" of cloning by nuclear transfer

Stem cell, Life creationone or two directions

Direction : gametes-embryo-cells

Direction : cells-embryo-gametes

Cytogenetic aspects? Molecular aspects?

Shinya Yamanaka et al

C
a

Genomic RNA profiling and the programme controlling preimplantation mammalian development , Christine E. Bell et all, 2008

Epigenetic regulation in mammalian preimplantation embryo development, Lingjun Shi and Ji Wu, 2009

General view of the main epigenetic reprogramming pathways in preimplantation development. (A) DNA methylationreprogramming in the mouse embryo. Active demethylation happens in paternal PN(pronucleus) followed by passive demethylation of the entire genome during cleavage stage.

X- inativation

Mechanisms from initiation of XCI to the shortand long-term maintenance of silencing status. After selection, Xist coats the Xi chromosome to initiate XCI. A ncRNA (RepA) then usher polycomb repressive complex 2 (PRC2) to the Xi chromosome, recruiting histone modification changes such as H2A-K119 ubiquitination (U) and H3K27 methylation (M). Histone modifications bring about short-term silencing followed by histone variant incorporation (macroH2A) and de novo DNA methylation, mediating long-term silencing

Gen expresion, genetic & epigenetic regulation

Direction : gametes-embryo-cells Direction : cells-embryo-gametes Oocyte & embryo -IVM-IVF IVF stc SCNT embryo & embryonic SCNT stc Parthenogenetic Stc iPSc Cancer cells