INTRODUCTION TO

BIOMEDICAL SCIENCE
Nguyen Van Thuan, Ph.D.
School of Biotechnology
International University
nvthuan@hcmiu.edu.vn
Nguyen Van Thuan, DVM, Ph.D
Telephone : 08-37244270 (Ext. 3488)
HP : 0707344522
E-mail : nvthuan@hcmiu.edu.vn
Room number: A1 713
 Nguyen Van Thuan, DVM, Ph.D
Education
1985-1990: Doctor Veterinary Medicine, HoChiMinh University,
Vietnam.
1997-1999: Master course in Reproductive Biotechnology,
Kobe University, Japan.
1999-2002: Ph.D. course in Biosystem Application, Kobe, Japan.
Work positions
1990-1996: Assistant Professor
Faculty of Veterinary Medicine, HoChiMinh Uni.
2002-2007: Research Scientist
Center for Developmental Biology (CDB)
RIKEN Institute, Japan.
2007- 2013: Professor
Department of Animal Biotechnology
Konkuk University, Seoul, Korea.
2013- present: Associate Professor
Department of Biotechnology
International University, Hochiminh City.
Konkuk University, Seoul, Korea
 Stem Cell & Regenerative
Biomedicine LAB (Konkuk Uni. Seoul)
Cellular Reprogramming LAB BT-IU (A1.710)
Cellular Reprogramming LAB BT-IU (A1.710)
Cellular Reprogramming LAB BT-IU (A1.710)
Cellular Reprogramming LAB BT-IU (A1.710)
Applied Biomedical Science for Human
PGD
IVF-ART
Human drugs

Genomic
Reprogramming
Human bio-organs

Genome preservation Gene and Cell therapy

Highest quality
meat and milk
ASSITED REPRODUCTIVE TECHNOLOGY

ART
 ART

Assisted Reproductive Techniques (ART)

- Intracytoplasmic Sperm Injection (ICSI).
- Round Spermatid Injection (ROSI).
- Female and Male Germ Cell Transplantation
 Infertility

Ten to 15% of couples are infertile

inability to conceive after at least 1 year
 The reasons of Infertility

Male Factors Female Factors

low sperm concentration ovulation
low motility tubal or pelvic
poor morphology cervical or uterine
ductal obstruction immunologic
poor DNA integrity nutritional & metabolic

1/5 have >1 cause

15% have unknown cause
 Brief History of IVF and ICSI
1878~ A large number of experiments beginning which contributed to the
first reports of IVF
1951 The discovery of sperm capacitation
1963 The first report of mammalian (hamster) IVF using spermatozoa
capacitated in vitro
1968 The first fetuses by IVF in the mouse
1976 Fertilization after ICSI using a golden hamster model
-
1989 The first live offspring by ICSI in the rabbit
1992 The first human baby by ICSI in the human
1994 The first mice by the electric fusion of oocytes and round spermatids
1995 Practical mouse ICSI and ROSI started with the development of
piezo-driven micromanipulator
Later, offspring were also obtained by ICSI in other mammalian species, cat
(1998), horse (1998), sheep (1998), cattle (1999), monkey (1999) and pig
(2000).
 Overcome Sperm Defects

Assisted Reproductive Techniques (ART)

treatments that bring about conception without
intercourse
increase the number of eggs and sperm
bring gametes into close proximity
Intracytoplasmic Sperm Injection (ICSI)
Injection of a single sperm into the egg
 How to cut the sperm head by piezo-actuated
micromanipulator system
From the tail
Before After
Sperm head Sperm head

Sperm tail Sperm tail

From the head

Sperm head Sperm head

Sperm tail Sperm tail

 Benefic of ICSI
Utilization of infertile or immotile spermatozoa from rare
genetic resources
Utilization of immature sperm cells from testes for fertilization.
New and simple methods for sperm preservation, e.g., Freeze-
drying, without freezing, sperm transportation without freezing
from country to country.
Xenografting of testicular tissues.
Sperm-mediated gene transfer.
Study of fertilization, embryonic development.
Sexual control in animal, provide blastocyst for blastocyst
injection in order to produce chimera animal
 ICSI
When ICSI is use: Treatment for male infertility
Male factor infertility
low sperm concentration
low motility
poor morphology
ductal obstruction
Those factors result in sperm can not enter the
mature egg for fertilization
Therefore Injection of single sperm in to single eggs
in order to get fertilization. This method called
Intracytoplasmic Sperm Injection (ICSI)
In Vitro Fertilization (IVF) &
Intracytoplasmic Sperm Injection (ICSI)

(b) ICSI method

(a) IVF method

 Fertilization: beginning of new organism

Sperm The first

Egg
(oocyte)
differentiation

Fertilization
Fertilization 1-cell embryo
Zygote 8-cell
8-cell embryo
embryo
(Zygote) Blastocyst
Blastocyst

Preimplantation development
 What is Embryo Technology?
Embryo Technology is technology
based on Embryology
Embryological sciences:
Fertilization
Characteristic of embryos development
Zygotic gens activation
Epigenetic reprogramming during embryo development
Differentiation of embryonic cells

To study Embryo Technology

 Fertilization: beginning of new organism
In vivo fertilization
Sperm In vitro fertilization
Egg (IVF)
(oocyte)
Intracytoplasmic
Sperm Injection
(ICSI)
In vitro culture for
development (IVD)
Embryo transfer
Fertilization (ET)
Applications of Reproductive
Biomedicine
Produce next generation from
an infertile male or female
Nguyen Van Thuan, Ph.D.
School of Biotechnology
International University
nvthuan@hcmiu.edu.vn
 Applications of ICSI
Assisted Reproductive Techniques (ART)
Treatment of infertile diseases: In Vitro Growth of
oocytes (IVG), In Vitro Maturation (IVM), In Vitro
Fertilization (IVF), Intracytoplasmic Sperm
Injection (ICSI), In Vitro Development (IVD),
Embryos Transfer (ET).
Sperm preservation

Embryo preservation

Preimplantation genetic diagnosis (PGD)

 Infertile Treatment
Intracytoplasmic Sperm Injection (ICSI)
Preservation for Mouse Spermatozoa
Without Freezing
Nguyen Van Thuan, Ph.D
Department of Animal Biotechnology
Konkuk University
vanthuan@konkuk.ac.kr

Van thuan et al. BOR 2005

 Introduction
Long-term preservation of mammalian spermatozoa:
- Liquid nitrogen (-196 degree C).

However,
Preservation of spermatozoa by the methods described
above still has some difficulties:
Liquid nitrogen is not been readily available to small
animal farms, especially in developing countries.

Inconvenient for transportation from country to country.

Collection and preservation of sperm under field conditions
or from wild endangered animals killed by hunters.
Development of normal mice from oocytes injected
with freeze-dried spermatozoa
Wakayama T, and Yanagimachi R. 1998.

Instant sperm:
Just add water
Freeze-dried sperm were all died
Live/dead cell staining
Fresh sperm

After freeze-dried
Freeze-dried sperm can be activated oocytes
and supported full-term development after ICSI.
%
40

30

20

10
Dry-mon 0
1 day 1 week 1 month 3 months

200 C (Room temperature)

40 C
Wakayama et al. Nat Bio 1998.
 However,

In order to make freeze-dried

sperm, we still need liquid
nitrogen, freeze-
For that reasons

Van Thuan et al., BOR 2005.

 Round Spermatid Injection (ROSI)
Round Spermatid (1N)

ROSI

ICSI

Sperm (1N)
BIOLOGY OF REPRODUCTION 70, 1863 1869 (2004)
Published online before print 25 February 2004.
DOI 10.1095/biolreprod.103.025171

Similar Time Restriction for Intracytoplasmic

Sperm Injection and Round Spermatid Injection
into Activated Oocytes for Efficient Offspring
Production
Satoshi Kishigami, Sayaka Wakayama, Nguyen Van Thuan, Teruhiko Wakayama
Laboratory for Genome Reprogramming, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-
0047, Japan
Embryos Transfer
Applications of embryo technology on
productions of transgenic animals
 What is Transgenic Animal?
A transgenic animal is one that carries
a foreign gene that has been
deliberately inserted into its genome.

In addition to a structural gene, the foreign

gene (DNA) usually includes other sequences
to enable it:
to be incorporated into the DNA of the host and
to be expressed correctly by the cells of the host.
 Historical background
The first chimeric mice were produced by fusion of two different
embryos of different strains to form a single embryo that
subsequently developed into a chimeric adult, exhibiting
characteristics of each strain (Brinster, 1974).

Chimeric mice
 Embryo aggregation
1. Remove zona pellucida by tyrode acid (pH: 2.5)
Before (intact After (without
embryos) zona pellucida)
CZB or Tyrode
KSOM acid

Few
second

Few
second
Tyrode CZB or 4-cell Embryo
acid KSOM

Note: The timing for Tyrode

acid treatment is very
important, just few seconds
enough for zona pellucida
disappear (observe under
stereo microscope) 8-cell Embryo
 Embryo aggregation in the aggregation plate
How to prepare aggregation plate

Use 35 mm culture dish

300 500 micrometer

Success rate more than 30-40%

Embryo aggregation by micromanipulator

Keep few seconds

Keep few seconds

Success rate more than 95%

Embryo aggregation

Chimericmouse
Chimeric mice
 Historical background
DNA microinjection, the first technique to
prove successful in mammals, was first
applied to mice (Gordon and Ruddle, 1981)
Two other main techniques were then
developed: those of retrovirus-mediated
transgenesis (Jaenisch, 1976) and embryonic
stem (ES) cell-mediated gene transfer (Gossler
et al., 1986).
A representative purposes for which transgenic
animals have been used indicates the wide
ranging application of this biotechnology:
 The goals of Transgenic Animals
In medical research: identify the functions of specific
factors in complex homeostatic systems through over- or
under-expression of a modified gene (the inserted transgene).
In molecular biology: analysis of the regulation of gene
expression makes use of the evaluation of a specific genetic
change at the level of the whole animal (for experiments of
the phenotype effects of transgene expression).
In the pharmaceutical industry: targeted production
of pharmaceutical proteins, drug production and product
efficacy testing.
Transgenic animals can be used as bioreactors for the
production of recombinant proteins that have pharmaceutical
applications. Such animals are sometimes called "Pharm
Animals".
 Introduction
Application of transgenic animals
Since the first gene transfers into mice were
successfully executed in 1980, transgenic
animals have allowed researchers to observe
experimentally the roles of genes in
development, physiology and disease.
Recently, transgenic animals are applied
for the purposes of the production of
pharmaceuticals (Human protein drug) .
 Methods of Producing Transgenic Animals via
Micromanipulator Technique

The two principal methods used for the

creation of transgenic animals are
DNA microinjection.
- Injection with sperm via ICSI.
- Injection into pronuclear formation.
embryonic stem cell-mediated gene transfer
- Blastocyst injection.
- Diploid blastocyst
- Tetraploid blastocyst
- 8-cells embryo injection
- Cloning techniques.
Micromanipulator system

1. ICSI
2. Blastocyst injection
3. Enucleation
4. Nuclear transfer
5. Produce transgenic Animal
Piezo-actuated micromanipulator

Piezo-actuated
micromanipulator
 Mammalian Transgenesis
by Intracytoplasmic Sperm
Injection (ICSI)
 DNA microinjection via ICSI
1. Disruption of sperm head membranes

A. Fresh sperm: Intact membranes

B. Whose membranes had been

disrupted by Triton X-100 (B),
C. Whose membranes had been
Disrupted by freeze-thawing.

C. Whose membranes had been

Disrupted by freeze-drying.

a b c d
 DNA microinjection via ICSI

DNA
ICSI

Mixture of DNA & 20 % offspring

membranes expressing the
Sperm membranes disrupted integrated
had been disrupted spermatozoa
transgene.

Mammalian Transgenesis by Intracytoplasmic Sperm Injection

Anthony C. F. Perry, 1* Teruhiko Wakayama, 1 Hidefumi Kishikawa, 1 Tsuyoshi Kasai, 1 Masaru Okabe, 2 Yutaka
Toyoda, 3 Ryuzo Yanagimachi 1 (1999)
 DNA microinjection via ICSI
Transgenic embryos produced by single
shot double transgenesis. Oocytes were
microinjected with spermatozoa that had
been preincubated with DNA.

A. The same embryos are shown (×400) after

3.5 days viewed by Hoffman modulation
contrast microscopy unstained
B. for GFP expression under long-wavelength
(480 nm) UV light,

High GFP expression at the morula and blastocyst stage

after injection of GFP DNA mixed with freeze-dry sperm
visa ICSI technique
 DNA microinjection via ICSI
Sperm treatment Total pups Positive with
GFP (green)
Freeze-dry sperm 14 3 (21%)
Freeze-thaw 12 2 (16%)
Triton X-100 treated sperm 31 6 (19%)
Intact sperm (control) 42 0 (00%)

GFP+ GFP-
Disruption of sperm
head membranes are
very important for
the success of
transgenic animal
visa ICSI
Mammalian Transgenesis
by Pronuclear Injection
 DNA Injection into pronuclear formation
Unsuccessful

Successful

Microinjection of DNA into

pronuclear formation
 Pronuclear injection in pig
Pig zygote
Centrifugation
6,500 g for 10 min

GFP transgenic pig Pronuclear injection

Embryo
transfer
 Cloning Animals

Dolly the Sheep,

the world's first
cloned mammal,

Nature. 1997 Feb 27;385(6619):810-3.

Viable offspring derived from fetal and adult mammalian

cells
Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH.
 The history of cloning animal
The first simple animal cloned animal is a frog. The first
cloned complex animal was Dolly the sheep. After this
many cloned animals have appeared.

1962
1996 Dr. Ian Wilmut
By Dr. John Gurdon
By Dr. Ian Wilmut Nobel prize 2012
in medicine

Oct. 2000
2002-2007:
CDB, RIKEN, Kobe

Laboratory of Genomic Reprogramming, April 2002

After Dolly,

1998 1998 2000 2000

2002 2002 2003 2003 2005

cloned camel

2009
 Procedure of Nuclear Transfer in Mice
Oocyte
donor

Nuclear Activation with

transfer CB for 6 hrs
Oocyte
collection
Enucleation

Subsequently Embryo
culture for 1-2 hr culture

Donor cells Embryo

collection Surrogate transfer
mother
Embryo
culture
Caesarian section at
Remove 19.5dpc
Somatic donor cell Bl
cell nucleus cytoplasm
donor
Cloned mice ntES cell
Cloning Animals

Somatic Cell
Reprogramming

Somatic cell
(Differentiated cell)
? Dolly
cloned sheep
 Cloning Animals
Reproduction 2009
Histone deacetylase inhibitor scriptaid rescues full-term
-1%)
development in cloned inbred mice by enhancing nascent
mRNA production.
Van Thuan Nguyen , Hong-Thuy Bui, Takafusa Hikichi, Sayaka Wakayama, Satoshi Kishigami,
Eiji Mizutani and Teruhiko Wakayama.

We are the first time can increase the success

rate of cloned animals from 1% to 10%
2012

The first The second

Somatic cell generation of generation of
25 generations
cloned mice cloned mice of cloned mice
Cloning Animals
NATURE PROTOCOL, 1, 125-138, 2006
Published online 27 June 2006; doi:10.1038/nprot.2006.21

Production of cloned mice by somatic

cell nuclear transfer
Satoshi Kishigami, Sayaka Wakayama, Nguyen Van Thuan, Hiroshi
Ohta, Eiji Mizutani, Takafusa Hikichi, Hong-Thuy Bui, Sebastian
Balbach, Atsuo Ogura, Michele Boiani & Teruhiko Wakayama
Center for Developmental Biology RIKEN Kobe, 650-0047, Japan. Max Planck Institute for
Molecular Biomedicine, Mendelstrasse 7, 48149 Muenster, Germany. RIKEN
Bioresource Center, Tsukuba, Ibaraki 305-0074, Japan.
Application of Cloning Technique

Production of healthy cloned mice from bodies

frozen at -20 degrees C for 16 years.
Wakayama et al, PNAS 2008
 Induced Pluripotent Stem cell (iPS cells)
Reprogram somatic cell by Induced Pluripotent
Stem Cell method: iPS cells
Cell, 2006
Induction of Pluripotent Stem Cells from Mouse Embryonic
and Adult Fibroblast Cultures by Defined Factors
Kazutoshi Takahashi & Shinya Yamanaka

OCT4 Sox2 c-Myc Klf4

Mouse
fibroblast
cells

Stem Cells and Development. 2012

Differentiation and transplantation of functional
pancreatic beta cells generated from induced pluripotent
stem cells derived from a type 1 diabetes mouse model.
Jeon K, Lim H, Van Thuan N, et al.,
 Offspring derived from iPS cell

Reprogramming by iPS cell

iPS cells will inject into

tetraploid embryos. Can
we get offspring derived
from iPS cells?
Somatic cell Reprogramming:
Applications in Regenerative
Biomedicine and Conservation of Rare Animals

Nguyen Van Thuan, Ph.D.

School of Biotechnology
International University
nvthuan@hcmiu.edu.vn
 Animals PHARMING
The process of using transgenic-
cloned animals to produce human
therapeutic proteins (Human drugs)
200.000.000 USD/Cow
 PHARMING for FARMACEUTICALS
Classic method to produce Novel method to produce
human therapeutic proteins human therapeutic proteins
Dairy cow cells
DNA
Plasmid Transfection
DNA
Bacterial DNA Positive, negative selection
Human proteins
producing genes
Positive
Human proteins Negative (GFP+)
producing genes (GFP-)
Plasmid
DNA cut
with Recombinant
restriction DNA
enzymes Human
proteins Cloned
transgenic
embryo

Cloning animal Embryos

transfer
Recombinant
Bacterium

Fermentation Tank Human Human Transgenic cow

proteins proteins
 Great business?
Therapeutic Proteins Animal $/animal/year
Cow
AAT (alpha-1-antitrysin) 150.000 USD/cow/year
tPA (Tissue plasminogen Cow
700.000 USD/cow/year

Factor VIII or IX or (Blood Cow

200.000 -300.000
USD/cow/year

Lactoferrin 25.000 USD/cow/year Supper dairy cow

CFTR (Cystic fibrosis Cow
800.000 USD/cow/year
transmembrane conductance
regulator)
200.000.000
máu)
Cow 10 milions USD/cow
USD/cow/year
Cow
400.000 USD/cow/year
Glutamic acid decarboxylase Cow
200.000 USD/cow/year

Anpha- Cow
500.000 USD/cow/year
 PHARMING
In 2009, we succeeded transgenic pig
Human Erythropoietin (hEPO) gene,
and that transgenic pig shown to have
a normal reproductive ability
1 2
Full-term development
of human erythropoietin
transgenic cloned pigs
Konkuk, September 24, 2009
 Xenotransplantation

Nhóm nghiên chúng tôi tiên

Hàn thành công ra dòng heo
gen 1,3-galactosyltransferase. quan
dòng heo này có dùng ghép
Organs for human

Generation of RAT pancreas in Pdx1 knockout mice

Kobayashi et al., Cell 2010

Mouse Pdx1 -/-

(Blastocyst) Rat (ES cells)

Chimeric mouse
with rat pancreas

0ur hypothesis: Can we make

human organ from pig cells??
 Our present project (pig product human organs)
Organ related genes
Pig enucleated
knockout cloned pig
blastocyst
oocyte
Embryo
transfer
Pig fibroblasts cells

siRNA
Transfection Lentiviral
vector

Cloned
pig with
Pig knockout human
pancreas, heart, Human organs
iPS cells
? ????
liver, kidney related
genes cells

? ?
? Humans
organs
?
?
Stem cells from Ovary

Development. 2014
Epigenetic reprogramming in somatic cells
induced by extract from germinal vesicle
stage pig oocytes
Bui Hong-Thuy, Van Thuan Nguyen et al.,

Female
germ line Oocytes
stem cells
Ovary
Conservation of Infertile
and Rare Animals
 Generation of infertile age male mouse
via SCNT transfer by Scriptaid treatment

Cloned GFP-ICR
c

2.5 years old GFP-ICR male

Unpublished data
Generation of infertile age female mouse
via SCNT transfer by Scriptaid treatment

Cloned female mice from donor cells of 3

year old ICR female by scriptaid treatment.
 Generation of infertile age mice via
SCNT by Scriptaid treatment

Next generations from male and female infertile

 Offspring derived from dead sperm

BIOLOGY OF REPRODUCTION, 2005

New Preservation
Dead sperm at 4°C for 3 monthsMethod for Mouse Spermatozoa
Without Freezing
Van 83
Thuan Nguyen, Sayaka Wakayama, Satoshi Kishigami and Teruhiko Wakayama.
 Rescue Rare Animals (Bos gaurus)

Ngày 30/5/2014

Can activation oocytes

84
Tinh trùng bò tót
(Spermatozoa of Bos gaurus)
 Rescue infertile Animals via SCNT

( )

Bò cái Wagyu Úc
Bò cái Wagyu Úc
( )
85
 Conclusion

Business of
20 century

Biotechnology
Business of & Biomedical
21 century Science for
human