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

2. 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

3. Numbers of Existing Human Embryonic Stem Cell Lines Reported to NIH Institutes Numbers BresaGen, Inc., Athens, Georgia CyThera, Inc., San Diego, California Karolinska Institute, Stockholm, Sweden Monash University, Melbourne, Australia National Center for Biological Sciences, Bangalore, India Technion-Israel Institute of Technology, Haifa, Israel University of California, San Francisco, California Göteborg University, Göteborg, Sweden Wisconsin Alumni Research Foundation, Madison, Wisconsin 4 9 5 6 3 4 2 19 5

4. DERIVATION OF HUMAN EMBRYONIC STEM CELLS Blastocyst in vitro • day 1: 18 to 24 hours after in vitro fertilization of the oocyte • By day 2 (24 to 25 hours) : 2-cell embryo • By day 3 (72 hours) : 8-cell embryo (a morula) • By day 4 : compaction • By day 5 : the cavity of the blastocyst 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.

5. Scheme of ESC Establishment Frozen and thawed human embryo Blastocyst culture Isolation of ICM Subculture Identification and Characterization

6. 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)

7. Genital Ridge Human fetus, 9 weeks Isolation of primordial germ cell Feeder cell Subculture Scheme of EG cell establishment

8. 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

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

10. 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 hypothetical 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).

11. Test physiologic function ▪ In vitro (e.g., stimulated insulin release) Demonstrate safety Test methods to prevent rejection • Multi-drug immunosuppression • Create differentiated cells • Transduce ES cells to express • recipient MHC genes • Establish hematopoietic chimera • and immunologic tolerance. • In non-human primate model • - show absence of tumor formation • - show absence of transmission • of infectious agent. Human trials Human stem Cells Figure 3.2 Major Goals in the development of transcription therapies from Human ES Cell lines. • Lineage selection by cell survival • or cell sorting • Induce with supplemental growth • factor (s) or inducer cell Establish pure cultures of specific cell type Demonstrate efficacy • In rodent models • In non-human primate model • Evaluate integration into • host tissue • Recurrent autoimmunity

12. 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 manipulation of Human Embryonic Stem cells)

13. 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

14. 배아줄기세포의 획득 유산된 태아 핵 이식 배아복제 잉여배아

16. 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)