H 1 uman Embryonic Stem Cells 1
hES Cells
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Human Embryonic Stem Cells
October 2, 2007

I. The Breakthrough: Derivation of hES Cells

 

A review: Human embryonic development, days 1-5...

 

Definitions: Please look up before class..(homework!).

Zygote - first diploid cell formed by union of egg and sperm

Morula - day 4 - 5 Solid ball

Blastula - day 5 - 6 Hollow ball with embryonic disc/ inner cell mass (shown here...beautiful!)

Implantation - day 6 - 7 - begin hCG production

Gastrula - day 7 - 10 Early embryo with 3 tissue layers - ecto, endo, meso) (Louis Wolpert, a famous embryologist, once said: "The most important event in your life is not birth, marriage, or death, but gastrulation." ). Best pregnancy tests on the market can give you a yes or no at this date! :)

Days 10 - 14 - Embryo starts to form from embryonic disc, chorion and yolk sac start to form

Day ~14 - first missed menstrual period


The politically charged words...

Embryo: ' b : an animal in the early stages of growth and differentiation; especially : the developing human individual from the time of implantation to the end of the eighth week after conception'

Fetus: 'an unborn or unhatched vertebrate especially after attaining the basic structural plan of its kind; specifically : a developing human from usually three months after conception to birth'

 


Mini-Project: After 2 children via IVF, pair faced stem cell issues

Note to class: Embryo donation is an excellent option! For couples experiencing infertility, this is a lovely, cost-effective gift you can provide that allows an infertile couple to give birth to a baby...not without its psychological costs, but still a wonderful option/

Shown here: George Bush and the "Snowflake Babies" (More on Snowflakes Program)

 

 


Human Embryonic Stem cell research hit the press in November 1998 with the simultaneous publications on the isolation of human embryonic stem (hES) cells derived from two different labs.

 

UW - Madison Press Release: Wisconsin scientists culture elusive embryonic stem cells

James A. Thomson, "Embryonic stem cell lines derived from human blastocysts." Science: 1998 282:1145.

 

What did Thompson report about these cells? Findings [Homework!]:

  1. Source: "Fresh or frozen cleavage stage human embryos, produced by in vitro fertilization (IVF) for clinical purposes,donated by individuals after informed consent and after IRB approval.

  2. Procedure: Embryos were cultured to the blastocyst stage, 14 inner cell masses were isolated, and five ES cell lines originating from five separate embryos were derived

  3. Analysis: Three cell lines (H1, H13, and H14) had a normal XY karyotype, and two cell lines (H7 and H9) had a normal XX karyotype.

    • High levels of telomerase activity (Fig. 2) - replicative life-span /'immortality' in human cell lines.

    • Correct expressed cell surface markers of undifferentiated ES (Fig 3), including stage-specific embryonic antigen (SSEA)-3, SSEA-4, Tumor rejection antigen-1 (TRA-l-60, TRA-1-81), and ALP"

    • Form teratomas when injected into mouse leg muscle: Kinda disgusting, but interesting, AND 'Proof of Pleuripotency' (undifferentiated cells spontaneously differentiate into all three embryonic germ layers and even, to some extent, form complex, organ-like structures) ...YUK!!!

  4. WHY do this? "...A potentially limitless source of cells for drug discovery and transplantation therapies. Many diseases, such as Parkinson's disease and juvenile-onset diabetes mellitus, result from the death or dysfunction of just one or a few cell types. The replacement of those cells could offer lifelong treatment."

  5. Last line: "Supported by the University of Wisconsin (UIR grant 2060) and Geron Corporation (grant 133-BU18)."



Simultaneously, John's Hopkins Press Release: Hopkins Research Team Cultures Long-Awaited Human Embryonic Stem Cells

  • John Gearhart, "Derivation of pluripotent stem cells from cultured human primordial germ cells" Derived hPG / hEG cells (human primordial germ cells / embryonic germ cells) from human fetal tissue (5-9 weeks gestation) PNAS: 1998 95 (23): 13726–13731,

II. Characteristics of Human Embryonic Stem Cells: 4 amazing properties...what are they?

 

1. Pluripotency. hES cells can form virtually any cell in the body. All three germ layers, including the lining of the gut and blood vessels (endoderm); cartilage, bone, and smooth and striated muscle (mesoderm); neural tissue and skin (ectoderm) have been produced.



2. Self-renewing. Using in vitro cell culture, hES cells can propagate indefinitely in the undifferentiated state without losing pluripotency.

3. Express Telomerase (and Oct4). Telomerase is an RNA-dependent DNA polymerase whose function diminishes in aging cells and whose expression, when re-activated in normal cells, allows their continual proliferation. hES cells express telomerase. The continual telomerase expression conveys replicative immortality. Other stem cells express telomerase at low levels and thus stop dividing, age, and die with time. Thompson fig of a Telomerase TRAPEZE assay: active telomerase (in cell or tissue extracts) adds a varied number of telomeric repeats (TTAGGG) onto the 3’ end of the substrate oligonucleotide. PCR amplifies the extension products. The signature telomerase “DNA laddering” results are easily visualized on a native polyacrylamide gel, indicating the presence or absence of telomerase in the experimental sample.


Oct-4 is a "master regulator" of ES cell pluripotency that activates or inhibits a host of target genes and maintains ES cells in a proliferative, non-differentiating state.

4. Normal chromosome structure (karyotype). hES cells maintain a normal set of chromosomes (46, XX or XY) even after prolonged tissue culture. They do not have additions, deletions or rearrangements in their chromosomal structure as is characteristic of cell lines immortalized by viruses.

Question: Are hES cells totipotent? Well, wouldn't we like to know....!
"Non-differentiated embryonic stem cells possessing the ability to grow developing into an entire organism from any single cell ..." .

List some other totipotent cells?!

III. Funding: ...Not the Federal Government (President...?)!

The work for both papers was funded in 1996 by the biotechnology company, Geron, Menlo Park CA.

Pres and CEO Thomas Okarma, MD, PhD, formerly of Stanford University School of Medicine: "Geron is focused on developing products to treat age-related degenerative diseases. The availability of hES cells opens extraordinary opportunities for tissue transplantation, and for developing cell and gene therapy products with breakthrough medical potential. Further, Geron's proprietary telomerase technology for prolonging the replicative lifespan of cells derived from hES cells positions the company to potentially supply the preferred cells for transplantation medicine."

Any Patents held by Geron...oh, just a few here and there......(please look up and list names here)

1. Primate embryonic stem cells US Patent 5,843,780: (12/01/98)

Inventors: Thomson; James A. (Madison, WI)
Assignee: Wisconsin Alumni Research Foundation (WARF, Madison, WI)
Exclusive license: to Geron

2. TelomeraseUS Patent 6,093,809: (7/25/00)

Inventors: Cech; Thomas R. (Boulder, CO) [Nobel Prize winner] et al.
Assignees: University Technology Corporation (Boulder, CO); Geron Corporation (Menlo Park, CA)

3. Oocytes as recipients of nuclear transfer US Patent 6,147,276: (11/14/00)

Inventors: Wilmut; Ian (Midlothian, GB), et al.
Assignee: The Roslin Institute (Edinburgh) (Midlothian, GB); [Oh, and guess what, the Roslin Institute is now owned by...Geron]

4. Methods and Materials for the Growth of Primate-Derived Primordial Stem Cells in Feeder free culture US Patent No: 6,800,480: (10/5/04)

More on Feeder-Free Culture

Well, nice that Geron has the whole field ...

Some other tools in the Geron Pipeline: (Clinical Development)

Cancer programs (Telomerase): Stem Cell Programs
GRN163L: Telomerase Inhibitor
GRNVAC1: Telomerase Cancer Vaccine
Telomerase Plus test: Screening / monitoring test for bladder cancer		 GRNOPC1 (Glial cells) Stem Cell Programs : Spinal Cord Injury
GRNCM1 (Cardiomyocytes) Stem Cell Programs : Heart Disease
GRNIPC1 (Islet Cells) Stem Cell Programs : Diabetes

Other Embryonic Stem Cell Biotech companies Advanced Cell Technology  and Curis

Three main research areas: Stem Cell Differentiation, "Reduced Complexity Program", Cellular Reprogramming AKA therapeutic cloning!

1. Pipeline: Some good patents!
2. Paper to read soon! Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres Nature Letter: Oct 16, 2005

IV a. Potential Benefits of hES to Science and Medicine - Uses of hES cells

1. Human Organ and Tissue Transplantation. The potential medical impact of hES cells is huge because of their capability to produce virtually unlimited quantities of any cell in the body. In addition, they have the potential to be genetically engineered. The kinds of cells already derived from mES cells or hES ells include: (scroll down to Table 2.1)

i. Cardiomyocytes. Possible use: replace heart muscle damaged by injury, ischemia, congestive heart failure.

ii. Hematopoietic stem cells. Possible use: obtain blood-forming cells for BMTs.

iii. Endothelial cells. Possible use: re-line blood vessels to treat arteriosclerosis (650,000 deaths annually in the US) and arterial insufficiency, or for generating new blood vessels in the heart, brain, or extremities.

iv. Islet cells. Possible use: Treatment for the over 1.4 million people in the US with Insulin Dependant Diabetes Mellitus. Should this approach work, it could result in a lifelong cure for this IDDM.

v. Neurons. Possible use: Neurons for the treatment of Parkinson's disease (1 million affected in the US), Stroke (500,000 affected in the US), and Alzheimer's disease (over 4 million affected in the US).

vi. Fibroblast and keratinocyte skin cells. Possible use: wound healing, burn treatment..

vii. Chondrocytes. Possible use: cartilage replacement in osteoarthritis (16 million Americans affected) , or rheumatoid arthritis (2 million Americans affected) .

2. Pharmaceutical research and development. hES cells could provide permanent, stable sources of normal human differentiated cells to be used for drug screening and testing, drug toxicology, new drug target identification or drug development by creating in vivo models of human disease..

3. Human reproductive and developmental biology. hES cells offer a unique way to understand embryonic development, tissue differentiation and repair.

 

Figure 3.2. Biotech Aspect – so what would it take? Major Goals in the Development of Transplantation Therapies from Human ES Cell Line

 

 

International Stem Cell Initiative (ISCI): June 2007: An international survey of human embryonic stem cell lines provides a benchmark for comparing results across laboratories:59 different hES cell lines maintained in 17 laboratories scattered across 11 countries. wow

 

Want to see what research is being done, and recent advances in stem cell research? Take a look!

1. Stem Cell news
2. Stem Cells/Stem Cell Transplantation

 

"Embryonic Human Stem Cells May Help Repair Heart Muscle, Lab Study Shows" Dr. Charles Murry, director of the Center for Cardiovascular Biology and Regenerative Medicine at the University of Washington, in Seattle, NEJM August 27, 2007

 

IV b. Potential Benefits of hES to Science and Medicine - Who's Who!

 

National Stem Cell Centers (not a complete list, and in no particular order...and just a brief overview for now...this will overlap with our next 2 stem cell lectures!)

 

1. University of Wisconsin Stem Cell and Regenerative Medicine Center

2. Case Western Center for Stem Cell and Regenerative Medicine

3. The Indiana University Center for Regenerative Biology and Medicine = Our own department, plus the IU School of Medicine, Lilly, etc.! See all the Way Cool Research here.

 

4. California Institute for Regenerative Medicine (CIRM)

2 Nov 2004: Arnold, Prop 71 (the California Stem Cell Research and Cures Initiative) passes 59% to 41% by statewide ballot.

 

And the premier Stem Cell Institute in the US and worldwide...

 

5. Harvard Stem Cell Institute: 22 April 2004: Harvard Stem Cell Institute formed: "Seven Harvard schools, 100 faculty, emphasis on five types of organ and tissue failure." Co-directors: Doug Melton and Harvard Medical School Professor David Scadden.


 

03 March 2004 Doug Melton, Harvard Professor and HHMI Investigator, derives 17 new hES cell lines using private funds from Harvard, the JDRF, and HHMI and excess IVF blastocysts from Boston IVF. Freely available to all researchers (as long as they aren't using federal funds to do research on these lines). Note: today, 28 stem cell lines - more than the true number of the "Bush 64" cell lines...

20 March 2005 Go-ahead to clone human stem cells approved by Harvard Provost Dr. Steven E. Hyman. Governor (and Presidential Candidate) Mitt Romney and the Massachusetts government are considering new stem-cell legislation to outlaw this research ! Stay tuned!

 

Doug Melton, shown with son Sam

Douglas Melton (By Michael J. Fox)
One of Time Magazine's Top 100 Influential People of 2007: Scientists and Thinkers

 

And tonight's Guest Speaker, direct from Harvard!
Our Own Natasha Arora ! = One of IUPUI's Top 100 Students of 2007 :)

V. Ethical Issues in hES Cells Quotes from AAAS link

Groups Supporting Embryonic Stem Cell Research: No surprises here

Groups Opposed to Embryonic Stem Cell Research: No surprises here either

VI. What about Adult Stem Cells?

 

...This topic will be done next, by Dr. Merv Yoder
NIH - Adult Stem Cells... - and Differences between hES cells and Adult stem cells

 

Biotech companies Researching Adult Stem Cells:

COMPANY LOCATION STEM CELL TYPE
Aastrom Biosciences Ann Arbor, MI Blood
Curis Cambridge, MA Blood, skin, nerve
Dendreon Seattle, WA Immune
Advanced Cell Technology Worcester, MA Blood, organs, (cloning)
Osiris Therapeutics Baltimore, MD Bone marrow
StemCells Sunnyvale, CA Brain, liver, blood, pancreas


Wondering what to do with Baby's Cord Blood? Umbilical stem cells

 

Upcoming readings... The Politics of Stem Cells

1. Oct 13, 2006: California Stem-Cell Institute Unveils 10-Year Plan
2. Embryonic Stem Cell Research: July 14, 2004 HHS and Presiden't Bush's policies
3. The Politics of Stem Cells This is a nice sumary of the history of hES research/ stem cell legislation.
4. 'U.S. federal stem cell legislation'

Objectives:

1. What were the major findings and conclusions from Thonpson's 1998 paper? (Briefly, how was Gearhart's paper different)?
2. What are the 4 characteristics of hES cells that make them a unique and useful human tissue?
3. Explain the role of GERON in the development of hES cells, and describe their 4 major patents.
4. List the 3 main uses of hES cells in medicine and science. Give specific examples of tissue types.
5. Explain the difference between totipotent and pleuripotent. Are hES cells totipotent or pleuripotent?
6.Summarize the importance of Doug Melton - Boston IVF, HHMI, and the HSCI. Be able to explain the major findings of Melton's NEJM paper!

Schedule