1a bxn tng2013 slidedaidien

7/28/2019 1a BXN TNg2013 Slidedaidien

1/60

Bui Xuan Nguyen,Ph.D- University Paris VIVAST, USTH Hanoi, VietnamBuixn(@gmail.com

Cng ngh Phi & T bo gcEmbryo & Stem cells

Biotechnology- TM LC


2/60

Embryo

-Etymology : First attested in English in the mid-14c., theword embryon derives from Medieval Latin embryo,itself from Greek (embruon), plural (embrua), lit. "young one

- An embryo is a multicellular diploid eukaryote in itsearliest stage of development, from the time of first celldivision 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 ???


3/60

Embryo development

- Embryogenesis: The development of the embryo

- Embryo differentiation: The process by whichspecialized and diversified structures arise duringdevelopment of the embryo. The process involves (1) anincrease in the number of cell types, and (2) anincrease in morphological heterogeneity through the

arrangement of cells into increasingly complexstructural patterns in the form of tissues and organs.(wikipedia]


4/60

EMBRYO

Fetus

New organism

OocyteSperm

Embryo- the start in life- all begins from embryo

4 groups connective,muscle, nervous &epithelial tissues

300 different cell types


5/60

Embryo differentiation


6/60

Stem cell key properties

Principles of stem cell biology

Self-renew & multiply Differentiation , Plasticity


7/60

General approaches for stem cell production

1. Adult Stem Cells

2. Embryonic Stem Cells (ES)

3. Induced Pluripotent Stem Cells (iPS)


8/60

EMBRYO

New organism

OocyteSperm

Fertilization, Embryo, Stem cells & Development

Cells

Totipotent

Pluripotent

Multipotent

Unipotent


9/60

2. Embryonic stem cells ( pluripotent)

From in vitro fertilizedembryos (IVF):

Mouse (Evan & Kaufman,1981); Human (Thomsonet al, USA, 1998/,

Sweden, UK, Japan,China, Korea, Spain, Iran,Israel, Singapore,Thailand)

From Somatic Cell Nuclear Transfer (SCNT) embryo: Mouse(Wakayama et al, 2001), Bovine, Monkey, Pig, ( Human: Sweden,

China)

From Parthenotes

( parthenogenetically activated oocytes ): mouse (Cuthbertson,1983),human (Cibelli et al. 2001).


10/60

1.Adult stem cells (multipotent)

Umbilical cord matrix stc :

Cord Is Richer than Blood:0.7-2.0 x106cells/cm( Secco et al, 2007)low immunogenicity, lower expression of CD106, HLA-ABC and HLA-DR, benefit clinical use. (Lu et al, 2006)

"stem cell niche" in brain,bone marrow, peripheralblood, blood vessels,skeletal muscle, skin,teeth, heart, gut, liver,ovarian

Photo: STEMCELL Technologies Inc


11/60

3- Induced Pluripotent Stem Cells

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


12/60

TECHNOLOGY

BIODIVERSITY PROTECTION

TRANSGENICPHARMACEUTICALINDUSTRY

REGENERATION MEDICINE

XENOTRANSPLANTATION

GENE THERAPY

INFERILITY TREATMENT

PGD

ANIMAL BREEDING INDUSTRY

BIO-MEDICAL APPLICATIONREPRODUCTION

Superovulation

In vitro Fertilization

Somatic cloning

XX

IVM-IVF38 0C, 5% CO2

Genetic Engineering

Stem cells

Cryobanking

Embryo Transfer

BXNguyen-Paris VI

EMBRYO

New organism

OocyteSperm

Cells


13/60

The fusion of biology and technology. Biotechnology isthe application of biological techniques to productresearch and development. (http://www.medterms.com/)

Biotechnology: Definition

A field of applied biology that involves the use of livingorganisms and bioprocesses in engineering, technology,medicine and other fields requiring bioproducts.(http://en.wikipedia.org/wiki/Biotechnology)

Any technological application that uses biologicalsystems, living organisms, or derivatives thereof, tomake or modify products or processes for specific use.(TheUnited Nations Convention on Biological Diversity)


14/60

Classic Biotechnology arose from the field ofzymotechnology, which began as a search for a betterunderstanding of industrial fermentation

Biotechnology: Definition

A

Modern Biotechnology: The origins of biotechnologyculminated with the birth of genetic engineering.-the 1953 discovery of the structure of DNA, by Watsonand Crick- 1973 discovery by Cohen and Boyer of a recombinant

DNA technique by which a section of DNA was cut fromthe plasmid of an E. coli bacterium and transferred intothe DNA of another


15/60

Technology

Bio-Medical BiotechnologyAt central first life stage for alllife aspects

Embryo &Stem cell

Life application


16/60

SCNT- 1. Production of genetic identical stem cells


17/60

EMBRYO & STEM CELLS TECHNOLOGY

W Heape, 1890: first animal fromembryo transfer

Edward, 1978: first test-tube baby


18/60

a dng sinh hc,cong ngh sinhsn- bo tn ex

situ-in situ


19/60

Trong khong 440 triu nm qua c 5 ln sinh vtb tiu dit hng lot

440 T, nguyn nhn ?

370 T, nguyn nhn ?

240 T, nguyn nhn ni la

202 T, nguyn nhn ?

65 T, nguyn nhn Sao chi

(V Qu, Biodiva 2007)


20/60

Ln gn y nht, cch nay khoang65 triu nam l do thin thch rivo tri t, 20 % s h v 50% sging b tiu dit, trong c cc

loi khng long

Hin nay, ang din hin t-ng cc

loi b tiu dit hng lot, nh-ngnguyn nhn chnh l do hot ngca loi ng-i.



21/60

Kt qu nghin cu ca hn 1500 nh

khoa hc trn th gii trong bnMillennium 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 Vv TV ang nguy cp.

Tc cc loi b tiu dit nhanh gp

1001000 ln so vi bnh th-ng.Trong cc thp k sp ti tc mtcc loi cn cao hn.



22/60

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% n86% s loi nguy cp.

T 1970 n nay c 58 loi c, 9loi chim v 1 loi th b tiu dit.



23/60

Nguyn nhn suy thoi cc loi:

1 Ni sinh sng b thu hp li2 Xm ln ca cc loi ngoi lai3 nhim mi tr-ng

4 Sn bt qu mc5 Thay i kh hu ton cu.



24/60

Brief history of embryo freezing

1949 : Polge et al., the successful freezing of chickensperm by including glycerol

1952: Marden sent rabbit embryos by air back toCambridge from Worcester, packed with iceballoons in a vacuum flask.

1960: Successful freeze-drying of bull sperm by Meryman1972: mouse embryos were successfully frozen &

transferred to give live young (Whittingham et al.,).


25/60

Status of oocyte-embryo cryoconservation:

Technical aspects

Slow freezing (Wittinghqm et al, 1972).

Rapid freezing (Nguyen-JP Renard 1983)

Vitrification (Rall & Fahy, 1985).


26/60

RT

-196oC

-30oC

Clacsic slowprogramable freezing

5 30 300-500

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

Sucrose-PROH, 2 min,Room temperature

Rapid freezing

(Nguyen, Renard,1983, 1984)


27/60

Cloning bySomatic cell nuclear transfer /SCNTCloning byCloning by

Somatic cell nuclear transfer /Somatic cell nuclear transfer /SCNTSCNT

1997, Doily


28/60

clone a woolly mammoth in the next five years.

Led by Akira Iritani, Kinki


29/60

SCNT : conservation

Wells et al, 1999: Enderby island cattle

Robert et al, 2000, Gaur

Smith et al, 2001.


30/60


31/60

American BisonAmerican Bison -- BisonBisonbisonbison

European BisonEuropean Bison -- BisonBisonbonasusbonasus AurochAuroch -- BosBosprimigenusprimigenus Wild YakWild Yak -- BosBosmutusmutus BantengBanteng -- BosBosjavanicusjavanicus

GaurGaur -- BosBosgaurusgaurus KoupreyKouprey-- BosBos SauveliSauveli African BuffaloAfrican Buffalo -- SyncerusSynceruscaffercaffer Wild Water BuffaloWild Water Buffalo -- BubalusBubalusarneearnee

LowlandLowland AnoaAnoa -- BubalusBubalusdepressicornisdepressicornis MountainMountain AnoaAnoa -- BubalusBubalusquarlesiquarlesi TamarawTamaraw -- BubalusBubalusmindorensismindorensis SaolaSaola -- PseudoryxPseudoryxnghetinhensisnghetinhensis


32/60

Kouprey : Numbers estimated- before XX 2000- 1938 800- 1940 1000- 1970 30-70- 1975 50

- 1986 >200- 2003 >250 (IUCN 2004)



33/60

Saola protection zone1. Vu Quang, 1995

2. PuMat- Lao-VN, 19953. BachMa, 20084. Quang tri, 2010

Nombre et superficie des aires protges au Vietnam

0

500,000

1,000,000

1,500,000

2,000,000

2,500,000

3,000,000

Year 1954 Year 1975 Year 1986 Year 1993 Year 2003

Ha

0

20

40

60

80

100

120

140

Sites

Superfice totale (ha) Nombre des aires protges


34/60

1994 - Endangered (Groombridge 1994)1996 - Endangered (Baillie and Groombridge

1996)2003 - Endangered (IUCN 2003)2006 - Critically Endangered (IUCN 2006)

The saola(Pseudoryx nghetinhensis)

New discovered,

but already threathened


35/60

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

Bovine-bovine SCNT D.8 Saola-Bovine SCNT D.8

Nbre cell. totales Nbre cell. ICM Nbre cell. tropho.

88 36 (40.9) 52 (59,1)

Donor cell

Cattle

Blastocyst (%)

Saola

21 (20.07)

46 (27.71)

Oocyte Cleaved (%) Morula (%)Fusion

150

312

91 (60.67)

166 (50.21)

72 (79.12)

131 (78.92)

25 (27.5)

65 (39.15)


36/60

animals

Equip Mobil

Informaticconnection

REGIONAL & COUNTRY EX SITU-INSITU NETWORK

In situ: reservation1,2..

reintroduction

Country Central

Rescue station

Samples: semen, egs, cells

Cryobanking

Software

ART

Bio-Medicalapplication

InternationalRegional

Banking

Buffer ZoneEconomic Support


37/60

CNSS-Ci to nhnging b tht-sa


38/60

VAST Vietnam-Cc mc thnh cng u tin

1978

2000

1988

1986

1983

Chng trnh m ha

Lm ch rng trng trn tru

B cy phi

Phng php ng lnh nhanh phi

Th cy phi u tien

Nhn bn v tnh: b, kh

Xc nh gii tnh phi

Th tinh ng nghim

T bo gc phi nhn bn

2001

19991996

1994

2003

D cy phi


39/60

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 xc2 t hp mi : INRA1-148bp-c, INRA2-443 bp-loi,

BRY4a-300bp-c, STS -216bp-loi

ci ct bo phi

PCR

Primer ciPrimer c

sRY

(2)

(3)Vi phu thut

(1)

NST Y

Chn phi

Cy phi

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


40/60

phi


41/60

phi PROGESTERON

PG

PGFSH


42/60

Estrus-superovulation synchronisation-ET

PG2PG1 DDuc1 hCG PG3PMSG/ FSH EmbryoAIDduc2

PG2PG1 DDuc1 hCG PG3PMSG/ FSH ETDduc2


43/60

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

Loi phi S bnhn

(n)

Cha 3thng

n (%)

n (%)

B con B ci

n ( %)

GT 16 11 (68,8) 8 (50,0) 11 9 (81,8)

Khng GT 20 13 (65,0) 9 (45,0) 15 6 (40,0)


44/60

Tng th

T l cha chun : > 50 %T l cha do cy phi GT: > 50%S b sinh ra : 41S 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 ngyTrng lng s sinh: 22-50 kgTc pht trin: tng ng Holstein


45/60

B TTON u tin Vietnam, Vinh Tuong, 11.2002


46/60

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


47/60

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

VN: 32 cc/per

Thi Lan: 250

Php:5000

50000 200000 1.5 tr

2000 2010 ???


48/60

300 000 animals

300

30

1 SCNT

SCNT for animal breeding through ONBS

Sexing

A.I

Embryo

30 years


49/60

Xc nh giitnh:VPT phI+PCR

B csa+Nhit i

Phi cI

B H-n Top: B Holstein >8000 Lit/ck

Tip cn 2- cng ngh phi TTON

TINHTRNG

XX

T T O N38 0C, 5% CO2

cy phi vo b LaiSind

(1)

(2)

(3)

(4)


50/60

H sinh sn nhn ging M

Ngun Gen Trng Tinhcao sn M, Nht

Cng nghip PhiTh tinh ng nghim

Ngun Gen Tinhhng nhit i

Dc, cichn lc Sib

c gingphn phi

Qungcanh

Qungcanh

Qungcanh

Qungcanh

Qungcanh

Qungcanh

Cy phi - nthng phm

Cy phi - nthng phm

n gck tip

Cy phi-n gc 1


51/60

GENETIC & EPIGENETICASPECTS


52/60

Inheritance & differentiation

Hypothesis of preformationism,N Hartsoeker, 1694: all organsWere prefigured whithin thesperm

Hypothesis of epigenesis,Each organism develops anew from anundifferentiated condition.

- each differentiated cell retain the fullcomplement of genetic informationpresent initially in the zygote? HansSpeman, 1938


53/60

Hans Spemann (1869-1941)

1938 the "fantastical experiment" ofcloning by nuclear transfer

Nobel award 1935 for discovery ofthe organizer effect in embryonicdevelopment";


54/60

Stem cell, Life creation-

one or two directions

Direction : gametes-embryo-cells

Direction : cells-embryo-gametes

Cytogenetic aspects?Molecular aspects?


55/60

Shinya Yamanaka et al


56/60

Ca


57/60

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


58/60

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

General view of the main epigenetic reprogramming pathways in preimplantationdevelopment. (A) DNA methylationreprogramming in the mouse embryo. Active demethylationhappens in paternal PN(pronucleus) followed by passive demethylation of the entire genomeduring cleavage stage.


59/60

X- inativation

Mechanisms from initiation of XCI to the shortandlong-term maintenance of silencing status. After selection, Xist coats the Xichromosome to initiate XCI. A ncRNA (RepA)then usher polycomb repressive complex 2 (PRC2) to the Xi chromosome, recruiting

histone modification changes such asH2A-K119 ubiquitination (U) and H3K27 methylation (M). Histone modifications bringabout short-term silencing followed byhistone variant incorporation (macroH2A) and de novo DNA methylation, mediatinglong-term silencing


60/60

Direction : gametes-embryo-cells

Direction : cells-embryo-gametes

Oocyte & embryo -IVM-IVF IVF stc

SCNT embryo & embryonic SCNT stc Parthenogenetic Stc iPSc Cancer cells

Gen expresion, genetic &

epigenetic regulation

1a bxn tng2013 slidedaidien

Documents