embryology lecture 2 and 3

8/3/2019 Embryology Lecture 2 and 3

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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?


2/27

What is Cloning?

Somatic Nuclear Transfer (=SNT)

Molecular Cloning

Cellular Cloning

Embryo Twinning (Identical Twins)


3/27

How were donor eggs obtained?


4/27

Typically only one follicle develops to maturity each month.


5/27

Oogenesis in Humans

Oocytes in the human

ovary complete the first

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]


6/27


7/27

Cloning Dolly the Sheep

[Campbell Fig. 21.7; See Gilbert Fig.

4.8]


8/27

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


9/27

Cloning Human Stem Cells by Somatic Nuclear Transfer


10/27

Development of human embryo [see Gilbert Fig. 11.26]


11/27

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


12/27

[See Gilbert Fig. 11.30]


13/27

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.


14/27

Stem Cells are Grown on

Feeder Cells

[from Gilbert 6th ed. Fig. 4.22]


15/27

How did authors show they made

stem cells?

Immunostaining for Markers for Stem Cells (Fig. 2)

Alkaline phosphatase

Set of markers specific for stem cells

http://www.chemicon.com/Product/ProductDataSheet.asp?ProductItem=SCR002

Oct 4 (a Transcription Factor expressed in germ cells)


16/27


17/27

Embryonic Stem

Cells can

(1) Retain

Pluripotency

(2) Differentiate

into different

cell typesdepending on

chemical signals

in the cell

culture medium.

[Gilbert Fig. 21-23]


18/27

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.


19/27

How did authors demonstrate that

SCNT-hES-1 cells were true SCNTclones, and not products of

parthenogenesis?

DNA fingerprinting (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.]


20/27

STR (Short Tandem Repeat) Polymorphisms

D7S820

1 aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg actatggagt

61 tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt ttagaacgaa121 ctaacgatagatagatagat agatagatag atagatagat agatagatag atagacagat

181 tgatagtttt tttttatctc actaaatagt ctatagtaaa catttaatta ccaatatttg

241 gtgcaattct gtcaatgagg ataaatgtgg aatcgttata attcttaaga atatatattc

301 cctctgagtt tttgatacct cagattttaa ggcc


21/27

DNA Fingerprinting Method [Pierce Fig. 18.33]


22/27

Hwang and 24 others (2005)

Patient-specific 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?


23/27

Why the goal to produce patient-

specific SCNT stem cells?

Therapeutic use

To replace non-functional cells/tissues with

functional, patient-specific, immune-matched

cells.


24/27

Why did these results attract

international attention? First documented human SCNT-clone (2004)

Patient-specific clones

Relatively high success rates

Published in Science magazine

Laws have been passed in the U.S. barring use of federalfunds to support research on human cloning and to restrictstem cell research

At least one company advertises their human cloningservices on the World Wide Web(http://www.clonaid.com)


25/27

What caused the scandal? Oocytes were not donated as claimed

Donors were either pressured or paid

Results from 2004 paper not questioned

Patient-specific clones (2005 paper) werefabricated

Few co-authors ever saw stem-cell clones DNA fingerprinting traces came from IVFclinic embryos


26/27

Possible

sources of

Stem Cells[Gilbert Fig.

21.22]


27/27

Are you in favor of SCNT

cloning?

What technical problems have to be solved?

Who should benefit?

Does it violate your ethical/ moralprinciples?

Should your tax money be used for SCNTresearch?

embryology lecture 2 and 3

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