Hwang and 14 others (2004) Evidence of a pluripotent stem cell line derived from a cloned blastocyst Science 303:1669-1674.

What did the authors report to have done?

What is Cloning?
Somatic Nuclear Transfer (=SNT) Molecular Cloning Cellular Cloning Embryo Twinning (Identical Twins)

How were donor eggs obtained?

Typically only one follicle develops to maturity each month.

Oogenesis in Humans Oocytes in the human ovary complete the rst meiotic prophase before birth. Puberty induces completion of meiosis 1. Each month, ovulation triggers development through metaphase of meiosis 2. Fertilization triggers completion of meiosis 2.
[Gilbert Fig. 19.30]

Cloning Dolly the Sheep

[Campbell Fig. 21.7; See Gilbert Fig. 4.8]

One Method of Nuclear Transplantation [Gilbert 5th ed.]

Cloning Human Stem Cells by Somatic Nuclear Transfer

Development of human embryo [see Gilbert Fig. 11.26]

Cleavage & Compaction in the Mouse Embryo [Wolpert Figs. 2.21 & 8.9]

[See Gilbert Fig. 11.30]

Implantation. A. Mouse blastocysts in uterus. B. Monkey blastocyst starting to implant. [Gilbert Fig.11.30]. Implantation in humans occurs between 7 and 11 days after fertilization.

Stem Cells are Grown on Feeder Cells

[from Gilbert 6th ed. Fig. 4.22]

How did authors show they made stem cells?

Immunostaining for Markers for Stem Cells (Fig. 2)
Alkaline phosphatase Set of markers specic for stem cells
http://www.chemicon.com/Product/ProductDataSheet.asp?Prod uctItem=SCR002

Oct 4 (a Transcription Factor expressed in germ cells)

Immunochemical demonstration of antigen expression [Alberts et al., 4-58]

Embryonic Stem Cells can (1) Retain Pluripotency (2) Differentiate into different cell types depending on chemical signals in the cell culture medium.
[Gilbert Fig. 21-23]

How did the authors demonstrate Pluripotency?

Embryoid Body formation
Representatives of 3 germ layers

Teratoma formation
SCNT-hES-1 cells injected into testis of SCID mice
Formed neuroepithelial rosette, smooth muscle, bone, cartilage, connective tissue, glandular epithelium.

How did authors demonstrate that SCNT-hES-1 cells were true SCNT clones, and not products of parthenogenesis?
DNA ngerprinting (Fig. 4A-C) Based on Short Tandem Repeat polymorphisms Used to verify that DNA of SCNT-hES1 cells was derived from donor. Biparental expression of imprinted genes (Fig. 4D) [Expected expression of maternal genes only; speculate that culture conditions induced expression of paternal genes. Cannot exclude parthenogenesis.]

STR (Short Tandem Repeat) Polymorphisms D7S820

1 aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg actatggagt 61 tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt ttagaacgaa 121 ctaacgatag atagatagat agatagatag atagatagat agatagatag atagacagat 181 tgatagtttt tttttatctc actaaatagt ctatagtaaa catttaatta ccaatatttg 241 gtgcaattct gtcaatgagg ataaatgtgg aatcgttata attcttaaga atatatattc 301 cctctgagtt tttgatacct cagattttaa ggcc

DNA Fingerprinting Method [Pierce Fig. 18.33]

Hwang and 24 others (2005) Patient-specic embryonic stem cells derived from human SCNT blastocysts Science 308:1777-1783.
What did the authors report to have done? Why were these results hailed as seminal breakthroughs in stem cell research?

Why the goal to produce patientspecic SCNT stem cells?

Therapeutic use
To replace non-functional cells/tissues with functional, patient-specic, immune-matched cells.

Why did these results attract international attention?

First documented human SCNT-clone (2004) Patient-specic clones
Relatively high success rates Published in Science magazine

Laws have been passed in the U.S. barring use of federal funds to support research on human cloning and to restrict stem cell research At least one company advertises their human cloning services on the World Wide Web (http://www.clonaid.com)

What caused the scandal?

Oocytes were not donated as claimed
Donors were either pressured or paid

Results from 2004 paper not questioned Patient-specic clones (2005 paper) were fabricated
Few co-authors ever saw stem-cell clones DNA ngerprinting traces came from IVF clinic embryos

Possible sources of Stem Cells

[Gilbert Fig. 21.22]

Are you in favor of SCNT cloning?

What technical problems have to be solved? Who should benet? Does it violate your ethical/ moral principles? Should your tax money be used for SCNT research?