1. Blastocyst Trophoblast ICM
The stage at which embryonic and extra-embryonic tissues are segregated (about 5 days post fertilization).
Trophoblast
ICM
Consists of:
1. The Inner Cell Mass (ICM) which will give rise to the embryo
2. The trophoblast which will give rise to the placenta

2. Leukemic Inhibitory Factor (LIF) Bone Morphogenetic Protein (BMP)
Culturing ES cells
Human ES cell grown on a monolayer of mouse embryonic fibroblast cells known as “feeder cells”.
Figure 8-32
.    Preparation of embryonic stem (ES) cells
Fertilized mouse eggs divide slowly at first; after 41/2 days, they form the blastocyst, a hollow structure composed of about 100 cells surrounding an inner cavity called the blastocoel. Only ES cells, which constitute the inner cell mass, actually form the embryo. Other cells form the trophectoderm, which gives rise to the membranes (amnion and placenta) by which the embryo is attached to the uterine wall. Embryonic stem cells can be removed from the blastocyst and grown on lethally irradiated “feeder cells.” [See E. Robertson et al., 1986, Nature 323:445.]
Feeder Cells are slowly being replaced by culture media with specific factors required for maintaining ES cell fate:
Leukemic Inhibitory Factor (LIF)
Bone Morphogenetic Protein (BMP)
Both are secreted by feeder cells and/or are present in serum used in media.
Lodish et al., Molecular Cell Biology

3. How can you be sure they are really ES cells on the plate?
Allow the cells to differentiate spontaneously on a Petri plate. They will do this spontaneously when they get crowded.
In liquid suspension, allow the cells to spontaneously form embryoid bodies with three germ layers.
Inject the cells into a mouse (or other mammal) with a suppressed immune system and observe the formation of teratomas.
ES Cell Differentiation (neurons)
Embryoid Bodies
Teratoma

4. Growth factors induce stem cell differentiation
Gilbert, Tyler, Zackin: Bioethics (2005)
Research into…
factors that promote/repress differentiation
…greatly increases the potential for successful stem cell therapy.

5. Embryonic Stem Cells (ES cells)
ICM

6. Embryonic Stem Cells (ES cells)

7. Transgenic Technology using ES Cells
The Knockout Mouse
Transgenic Technology using ES Cells
TLR5 knockout prone to obesity WT

8. Gene Modification in Mice Knockout Mouse Technology
-or-
Knockout Mouse Technology
First transgenic mouse: 1977
First Knockout mouse: 1989
Capecchi had a grant proposal rejected  extremely unlikely
Evans also rejected  too ambitios

9. The Knockout Mouse – targeted gene inactivation
Tsai et al., 2007

10. Targeted Gene Inactivation (Knockout)
Homologous Recombination with Positive/Negative Selection
tk (Herpes Simplex Virus thymidine kinase) phosphorylates ganciclovir and blocks DNA synthesis.
neor confers resistance to an antibiotic (Geneticin, aka G418).
Tsai et al., 2007

11. Griffiths et al., An Introduction to Genetic Analysis
The production of cells that contain a mutation in one specific gene, known as a targeted mutation or a gene knockout. (a) Copies of a cloned gene are altered in vitro to produce the targeting vector. The gene shown here has been inactivated by insertion of the neoR gene (green) into a protein-coding region (blue). The neoR gene will serve later as a marker to indicate that the vector DNA took up residence in a chromosome. The vector has also been engineered to carry a second marker at one end: the herpes tk gene (red). These markers are standard, but others could be used instead. (b) When a vector, with its dual markers, is complete, it is introduced into cells isolated from a mouse embryo. (c) When homologous recombination occurs (top), those regions on the vector (together with any DNA in between) take the place of the original gene, excluding the marker at the tip (red). In many cells, though, the full vector (complete with the extra marker) fits itself randomly into a chromosome (middle) or does not become integrated at all (bottom). (d) To isolate cells carrying a targeted mutation, all the cells are put into a medium containing selected drugs, here a neomycin analog (G418) and ganciclovir. G418 is lethal to cells unless they carry a functional neoR gene, and so it eliminates cells in which there has been no integration of vector DNA (yellow). Meanwhile, ganciclovir kills any cells that harbor the tk gene, thereby eliminating cells bearing a randomly integrated vector (red). Consequently, virtually the only cells that survive and proliferate are those harboring the targeted insertion (green). (After M. R. Capecchi, “Targeted Gene Replacement.” Copyright © 1994 by Scientific American, Inc. All rights reserved.)
Griffiths et al., An Introduction to Genetic Analysis

12. Introduction of Transgene into ES cells in Culture
Transduction: use of a viral vector to insert DNA (complications from virus)
Transfection: use calcium phosphate precipitate of DNA and add to cells in a monolayer, some take up DNA
Lipofection: use liposomes as a vector to introduce DNA, fusion at cell membrane
Electroporation: use electric current to transiently create small pores in cell membrane that DNA can pass through
Cloning Gun for electroporation
Tritech Research

13. Archeology Museum Florence
Chimeric Mice
Archeology Museum Florence
Chimera vs. Mosaic
The offspring will be…

14. Mate with Males (Black a/a)
Strain: 129 (Brown, A/A)
Strain: C57B10/6J (Black, a/a)
Genotype?
Chimera
Lodish et al., Molecular Cell Biology

15. Chimeric mice are mated to Black mice.
ES cell derived progeny are Brown (Brown is dominant to Black).
Not all Brown progeny carry the KO allele.
Screen with…?
How long to get a homozygote?
3 week gestation, 7 weeks until sexually mature
Lodish et al., Molecular Cell Biology

16. Central Nervous System
Knockin Mouse
Tis21::GFP Knockin
Southern Blot
Fig. 5. Generation of Tis21-GFP knock-in mice by homologous recombination in embryonic stem (ES) cells. (a-c) Using the targeting construct shown (a), the protein-encoding portion of exon 1 of the Tis21 gene (b) was replaced (asterisks) by EGFP carrying a C-terminal nuclear localisation signal followed by an simian virus 40 polyadenylation site (GFPpA, green box) and a neomycin cassette (Neo, red box) flanked by lox P sites (yellow triangles). (c) After homologous recombination, the neomycin cassette was excised by Cre-mediated recombination in ES cells in vitro. Ex, exon; shaded boxes, TIS21 ORF; Int, intron; TK, Herpes simplex thymidine kinase gene used for double selection; A, AsnI; B, BamHI; E, EcoRI; H, HindIII. (d) Southern blot of genomic DNA from adult mice [obtained from intercrossing heterozygous (wt/Gfp) animals]. DNA was digested with AsnI and hybridized with the probe indicated by the black bar in (c) (probe A). Arrow, 2.7-kb wild-type (wt) allele; arrowhead, 3.7-kb knock-in allele (Gfp).
Fig. 1. Heterozygous Tis21-GFP knock-in mouse embryos show GFP expression in the developing CNS in temporal and spatial correlation with neurogenesis. (a-d) GFP fluorescence in unfixed whole-mount preparations at various developmental stages. Arrows indicate the ventral midbrain (a and b) or the dorsal telencephalon (c and d); arrowheads the mid-hindbrain boundary (a-c) or the developing cerebellum (d). Note the spreading of GFP expression in the rostral and dorsal directions during development. Asterisks indicate the tailbud. (Scale bars, 500 μm.)
GFP expression in
Central Nervous System
Haubensak et al., 2003, PNAS

17. Somatic Cell Nuclear Transfer (SCNT)
Wilmut et al., 1997
First successful SCNT involving adult mammalian differentiated donor cells  “Dolly”
Early experiments in the 1960’s showed some success with cell reprogramming with amphibians.

18. SCNT – somatic cell was fused with an enucleated oocyte.
“Dolly”
SCNT – somatic cell was fused with an enucleated oocyte.
Egg donor and nucleus donor were from different types of sheep (Blackface and Dorset).
Gilbert, Tyler, Zackin: Bioethics (2005)

19. Cloning Reasons for Cloning
Anne McLaren, 2001 (UK):
One week after the news that a sheep had been cloned, a poll was conducted .
90% of people thought that human cloning should be banned
67% of people thought that cloning animals was unacceptable
56% of people said they would not eat the meat of a cloned animal
Reasons for Cloning
1. Reproductive cloning: creating a genetically identical individual
2. Therapeutic cloning: creating genetically identical ES cells for medical purposes

20. RETRACTED – he fabricated data
Cloning Humans
Hwang Woo-suk and Moon Shin-yong of Seoul National University have developed a line of human Embryonic Stem Cells (ES cells) by cloning adult human somatic cells (Hwang et al., 2004).
RETRACTED – he fabricated data
Many other labs have reported success with SCNT for therapeutic purposes only.

21. Gilbert, Tyler, Zackin: Bioethics (2005)

22. Stem cell therapy
Lewis, Human Genetics

23. March 9, Obama repeals policy preventing federal tax money from funding embryonic stem cell research.
"In recent years when it comes to stem cell research, rather than furthering discovery, our government has forced what I believe is a false choice between sound science and moral values...
In this case, I believe the two are not inconsistent. As a person of faith, I believe we are called to care for each other and work to ease human suffering. I believe we have been given the capacity and will to pursue this research — and the humanity and conscience to do so responsibly.”
- Obama,
At present, research on human SCNT may not be conducted with federal funds but there is no federal ban on privately funded research in this direction.
Some states have issued a ban on human SCNT for reproduction and others have issued the ban for reproduction or therapy.

24. Stem Cells for Treating Disease Parkinson’s Disease Patient
Drugs to treat symptoms
SCNT to create ES cells to differentiate into functional neurons
Parkinson’s Disease Patient
Existing cell lines, aborted embryos, IVF “leftovers”
Gene therapy:
correct genetic defect with various strategies
ES cells differentiate into functional neurons
iPS cells differentiate into functional neurons

25. FDA Approved ES Cell Therapy Clinical Trials
As of Spring 2012…..
2010
Starsgardt and Age-Related Macular Degeneration (cause blindness)
Advanced Cell Technology
12 patients
2009
Spinal Cord Injuries
Geron Corporation (since has dropped stem cell division of company)
10 patients