OVERVIEW 1998 - James Thomson and his colleagues: the first derivation of human ES cells - John Gearhart: the first derivation of human EG cells

Timeline of human ES cell research• 1878: First reported attempts to fertilize mammalian eggs outside the body • 1959: First report of animals (rabbits) produced through IVF in the United States• 1960s: Studies of teratocarcinomas (EC) in the testes of several inbred strains of mice • 1968: Edwards and Bavister fertilize the first human egg in vitro • 1970s: EC cells injected into mouse blastocysts produce chimeric mice• 1978: Louise Brown, the first IVF baby, is born in England• 1980: Australia’s first IVF baby, Candace Reed, is born in Melbourne• 1981: Evans and Kaufman, and Martin derive mouse embryonic stem (ES) cells The first IVF baby, Elizabeth Carr, is born in the United States • 1984-88: Andrews et al., develop pluripotent, EC cells from Tera-2 • 1989: Pera et al., derive a clonal line of human EC cells • 1994: Human blastocysts created for reproductive purposes using IVF • 1995-96: Non-human primate ES cells are derived and maintained in vitro - rhesus monkeys and marmosets • 1998: Thomson et al., derive human ES cells Gearhart and colleagues derive human EG cells • 2000: Pera, Trounson, and Bongso derive human ES cells • 2001: As human ES cell lines are shared and new lines are derived

• 2004: Hwang et al., human reproductive cloning

Numbers of Existing Human Embryonic Stem Cell Lines Reported to NIH

4956342

195

BresaGen, Inc., Athens, Georgia CyThera, Inc., San Diego, CaliforniaKarolinska Institute, Stockholm, SwedenMonash University, Melbourne, AustraliaNational Center for Biological Sciences, Bangalore, IndiaTechnion-Israel Institute of Technology, Haifa, IsraelUniversity of California, San Francisco, CaliforniaGöteborg University, Göteborg, SwedenWisconsin Alumni Research Foundation, Madison, Wisconsin

Institutes Numbers

DERIVATION OF HUMAN EMBRYONIC STEM CELLS

Blastocyst in vitro day 1: 18 to 24 hours after in vit

ro fertilization of the oocyte By day 2 (24 to 25 hours) : 2-cel

l embryo By day 3 (72 hours) : 8-cell emb

ryo (a morula) By day 4 : compaction By day 5 : the cavity of the blast

ocyst is complete.

For deriving ES cell cultures - the trophetoderm is removed: microsurgery or immunosurgery - At this stage, the ICM is composed of only 30 to 34 cells.

Scheme of ESCEstablishment

Frozen and thawed human embryo

Blastocyst culture

Isolation of ICM

Subculture

Identificationand

Characterization

DERIVATION OF HUMAN EMBRYONIC GERM CELLS

- derived from the primordial germ cells in the gonadal ridge, normally develop into mature gamedtes.

- The embryoid body-derived cells: high proliferative capacity and gene expression patterns that are representative of multiple cell lineage.

PLURIPOTENCY OF HUMAN EMBRYONIC STEM CELLS &EMBRYONIC GERM CELLS

- long-term self-renewal in vitro

- retaining a normal karyotype. Human ES cells can proliferate for two years thorough 300 ~ 450 population

doublings. Cultures derived from EB generated by human EG cells have less capacity

for proliferation. Most will proliferate for 40 population doublings. (maximum 70~80 population doubling)

Scheme of EG cell establishment

Isolation of primordial germ cell Feeder cell

Human fetus, 9 weeks

Genital Ridge

Subculture

COMPARISONS BETWEEN HUMAN EMBRYONIC STEM CELLS & EMBRYONIC GERM CELLS

Common point ① the cells replicate for an extended period of time

② show no chromosomal abnormalities

③ generate both XX and XY cultures

④ express a set of markers regarded as characteristic of pluripotent cells

⑤ spontaneously differentiated into derivatives of all three primary germ

Different point ① differ not only in the tissue source from which they derived.

② tvary with respect to their growth characterestics in vitro, and their behavior in vivo

③ human ES cells propagated for two years in vitro, whereas human EG cells maintained

for only 70 to 80 population .

④ human ES cells will generate teratomas containing differentiated cell types, EG cells

will not

POTENCIAL USES OF HUMAN EMBRYONIC STEM CELLS

Table 3.1 Comparison of Mouse, Monkey, and Human Pluripotent Stem Cells

Marker Name Mouse EC/ES/EG cells

Monkey ES cells

Human ES cells

Human EG cells

Human EC cells

SSEA-1 + - - + -

SSEA-3 - + + + +

SEA-4 - + + + +

TRA-1-60 - + + + +

TRA-1-81 - + + + +

Alkaline

phosphatase+ + + + +

Oct-4 + + + Unknown +

Telomerase activity

+ ES, EC Unknown + Unknown +

Feeder-cell dependent

ES, EG, some EC

Yes Yes Yes Some: relatively low clonal efficiency

Growth characteristics

In vitro

Form tight, rounded,

Multi-layer clumps;

Can form EBs

Form flat, loose aggregates; can form E

Bs

Form flat, loose aggregates; can form

EBs

Form rounded multi-layer clu

mps;

Can form EBs

Form flat, loose aggregates; can form

EBs

Teratoma formation

in vivo

+ + + - +

Chimera formation

+ Unknown + - +

POTENTIAL USES OF HUMAN ES CELLS

Using Human Embryonic Stem Cells for Therapeutic Transplants - At this stage, any therapies based on the use of the human ES cells are still hyp

othetical and highly experimental

- Major Goals in the Development of Transplantation Therapies from Human ES Cell Lines.

Parkinson’s disease, diabetes, traumatic spinal cord injury, Purkinje cell degeneration, Duchenne’s muscular dystrophy, heart failure, and osteogenesis imperfecta.

# Treatments for these disease require that human ES cells be directed to differentiate into specific

cell types prior to transplant.

The potential disadvantages of the use human ES cells for transplant therapy induce the propensity of undifferentiated ES cells to induce the formation of tumors (teratomas).

Demonstrate efficacy

Demonstrate efficacy

Figure 3.2 Major Goals in the development of transcription therapies from Human ES Cell lines.

• In rodent models• In non-human primate model• Evaluate integration into host tissue• Recurrent autoimmunity

Human stem CellsHuman stem Cells

Establish pure cultures of specific cell type

Establish pure cultures of specific cell type

Test methods to prevent rejection

Test methods to prevent rejection

▪ ▪ In vitroIn vitro (e.g., stimulated insulin release) (e.g., stimulated insulin release)

Test physiologic functionTest physiologic function

Demonstrate safety

Human trialsHuman trials

• In non-human primate model - show absence of tumor formation - show absence of transmission of infectious agent.

• Multi-drug immunosuppression• Create differentiated cells • Transduce ES cells to express recipient MHC genes• Establish hematopoietic chimera and immunologic tolerance.

• Lineage selection by cell survival or cell sorting• Induce with supplemental growth factor (s) or inducer cell

Immune rejection- the potential immunological rejection of human ES-derived cells might be avoided by followers.

① by genetically engineering the ES cells to express the MHC (major histocompatability ) antigens of the transplant recipient ② by using nuclear transfer technology to generate ES cells ③ by using somatic cell nuclear transfer technology in which the nucleus is removed from one of the transplant patient’s cells

Other Potential Uses of Human Embryonic Stem Cells

to study early events in human development: to identify the genetic, molecular, and cellular events and identify methods for preventing them.

to explore the effects of chromosomal abnormalities in the development of early childhood tumors.

to test candidate therapeutic drugs. to screen potential toxins. to develop new methods for genetic engineering (Genetic manip

ulation of Human Embryonic Stem cells)

A. Genetic manipulation of MHC genes

B. Nuclear reprogramming C. Hematopoietic chimera:complete, mixed, micro

Figure 3.3 Genetic Manipulation of Human Embryonic Stem Cells

배아줄기세포의배아줄기세포의 획득 획득

잉여배아

유산된 태아

핵 이식배아복제

Cloning by Nuclear Transfer and Stem Cell

Embryonic cell in salamander (Spemman et al., 1902)

Intestinal cell in frog (Gurdon et al., 1966)

Embryonic cell nuclear transfer (McGrath and Solter)

Fetal cell nuclear transfer (Willmut and Campbell, 1995)

Roslin technique : Dolly (Wilmut et al., 1997)

Honolulu technique : Cumulina (Wakayama et al., 1998)

Therapeutic cloning for cell and tissue therapy in human (Hwang et al., 2004)