1. Section #8: An Introduction to Stem Cells
Ward G. Walkup IV

2. Overview of Section #8 Slides
Hi All, below is an overview of my section slides
Slide #4: what a stem cell is
Slides #5-6: adult vs. embryonic stem cells
Slides #7-13: toti, multi and pluripotency of stem cells
Slides #14-15: induced pluripotent stem cells
Slides #16-30: different types of cloning
#17-20: molecular cloning
#21-26: reproductive cloning
#27-30: therapeutic cloning
Slides #31-37: Muotri Paper
Slides #38 on: extra unused stuff on neural differentiation

3. Overview of Section Materials
Todays section material will present an overview of stem cells, covering the following topics:
What a stem cell is
Adult versus embryonic stem cells
Multi, pluri, totipotency and induced pluripotentcy of stem cells
Different types of cloning (Molecular, Reproductive & Therapeutic)
Generation of mice with chimeric brains

4. What is a Stem Cell? Cells that:
Have the capacity for self-renewal and ability to divide for indefinite periods in culture
Can give rise to two or more specialized cells (Unrestricted potential for differentiation)
& Biology 1, 2008 Lecture

5. Embryonic Stem Cells Embryonic stem cells
Primitive (undifferentiated) cells derived from a 5-day preimplantation embryo that have the potential to become a wide variety of specialized cell types
Adult (Somatic) stem cells
An undifferentiated cell found in a differentiated tissue that can:
Renew itself
Differentiate to give rise to all the specialized cell types of tissue from which it originated
Scientists do not agree about whether or not adult stem cells may give rise to cell types other than those of the tissue from which they originate

6. Types of Stem Cells (Adult & Embryonic)
Embryonic stem cell has unrestricted potential for differentiation
Adult stem cells are lineage restricted
ADD SLOW SLIDE

7. Toti, Pluri and Multipotency of Stem Cells
Pluripotent
A stem cell able to give rise to all cell types that make up the body, but not “extra embryonic” tissues such as amnion, chorion and other placental components
An embryonic stem cell is an example of a pluripotent cell
Pluripotency can be demonstrated by:
Ability of the progeny from a single cell to form derivatives of all three embryonic germ layers (Endo-, Ecto- and Mesoderm)
Ability to generate a teratoma after injection into an immunosuppressed mouse

8. Toti, Pluri and Multipotency of Stem Cells
Pluripotent
A stem cell able to give rise to all cell types that make up the body, but not “extra embryonic” tissues such as amnion, chorion and other placental components
An embryonic stem cell is an example of a pluripotent cell
Pluripotency can be demonstrated by:
Ability of the progeny from a single cell to form derivatives of all three embryonic germ layers (Endo-, Ecto- and Mesoderm)
Ability to generate a teratoma after injection into an immunosuppressed mouse

9. Toti, Pluri and Multipotency of Stem Cells
Pluripotent
A stem cell able to give rise to all cell types that make up the body, but not “extra embryonic” tissues such as amnion, chorion and other placental components
An embryonic stem cell is an example of a pluripotent cell
Pluripotency can be demonstrated by:
Ability of the progeny from a single cell to form derivatives of all three embryonic germ layers (Endo-, Ecto- and Mesoderm)
Ability to generate a teratoma after injection into an immunosuppressed mouse

10. Toti, Pluri and Multipotency of Stem Cells
Pluripotent
A stem cell able to give rise to all cell types that make up the body, but not “extra embryonic” tissues such as amnion, chorion and other placental components
An embryonic stem cell is an example of a pluripotent cell
Pluripotency can be demonstrated by:
Ability of the progeny from a single cell to form derivatives of all three embryonic germ layers (Endo-, Ecto- and Mesoderm)
Ability to generate a teratoma after injection into an immunosuppressed mouse

11. Toti, Pluri and Multipotency of Stem Cells
Totipotent
A stem cell able to give rise to all the cell types that make up the body, plus all of the cell types that make up extra embryonic tissues, such as the placenta
A zygote is an example of a totipotent cell
Multipotent
A stem cell able to develop into more than one type of cell in the body
An adult (Somatic) stem cell can be an example of a multipotent cell
can is underlined because this is a conflicting area of research, a true example of a multipotent stem cell would be a tissue specific stem cell
Biology 150
Development Lecture Slides, Fall 2006
Caltech
(Multipotent)

12. Toti, Pluri and Multipotency of Stem Cells
(Multipotent)
Biology 150, Development Lecture Slides, Fall 2006, Caltech

13. Toti, Pluri and Multipotency of Stem Cells
(Multipotent)
Biology 150, Development Lecture Slides, Fall 2006, Caltech

14. induced Pluripotent Stem Cells (iPSCs)
Adult cells (e.g. Fibroblasts) reprogrammed to an embryonic stem cell-like state by forced expression of specific genes
Expression of the transcription factors Oct4, Sox2, c-Myc & Klf4, under lentiviral and retroviral promoters causes reprogramming
Mouse and human iPSCs have been generated
iPSCs have been shown to have important characteristics of pluripotent stem cells:
Expression of stem cell markers
Ability to form tumors containing cells from all 3 germ layers
Ability to contribute to many different tissues when injected into mouse embryos at an early stage in dvelopment

15. Lentiviral & Retroviral
Generating iPSCs
Transfect
Lentiviral & Retroviral
Vectors Containing:

16. Molecular, Reproductive and Therapeutic Cloning
Cloning is an umbrella term traditionally used by scientists to describe different processes for duplicating biological material
A basic understanding of the different types of cloning is paramount for taking an informed stance on current public policy issues
Three types of cloning will be discussed in the upcoming slides:
Recombinant DNA Technology or DNA/Molecular Cloning
Reproductive Cloning
Therapeutic Cloning

17. Molecular Cloning
Refers to the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element
The most common self-replicating genetic element is extra- chromosomal DNA known as a plasmid
Typically a five part process:
A DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes
The gene of interest is united with a plasmid that has been cut using the same restriction enzymes
The gene of interest and plasmid are covalently linked using DNA ligase, to yield a recombinant DNA molecule
The recombinant DNA is moved into a host cell, to allow for propagation using the host cell’s enzymatic machinery
Host cells containing the recombinant DNA are selected for and identified
Plasmid alternatives: YACS (1 MB), BACS ( kb inserts), , cosmids (45 kb), plasmids (20 kb)

18. Molecular Cloning 1.
2. & 3.
Adapted From: Alberts et al. Molecular Biology of the Cell, 2002, Figure 8-30, p 501

19. Molecular Cloning
In this example, #5 is a selection method if you incorporate an antibiotic into the media, when antibiotic resistance is only coded for on the extrachromosomal DNA 4. 5.
Adapted From: Alberts et al. Molecular Biology of the Cell, 2002, Figure 8-31, p 501

20. Molecular Cloning Uses of Molecular Cloning:
Deconvolution of complex biological and biochemical processes by studying components in isolation
Examples: Metabolic pathways, Translation, Transcription
Genetic engineering of organisms
Examples: Bioremediation, crop engineering, heterologous production of pharmaceuticals
Identification of evolutionary relationships
Examples: DNA sequencing of genomes and individual genes

21. Reproductive Cloning
Refers to technology used to generate an animal that has the same nuclear DNA (Somatic cell nucleus) as another currently, or previously existing animal
Can use a technique known as Somatic Cell Nuclear Transfer (SCNT) to perform reproductive cloning*
Genetic material from the nucleus of an adult egg is transfered to an egg whose nucleus, and thus its genetic material, has been removed (enucleated egg)
The reconstructed egg containing DNA from the donor cell is treated with chemicals or electric current to stimulate cell division
Once the embryo reaches a suitable stage, it is transferred to the uterus of a female host, where it continues to grow until birth
Please mention the fact that the animal created using SCNT is not a truly identical clone (Mitochondrial DNA)
* = Initial stage of SCNT (enucleated egg and somatic cell nucleus combination) is the same in both reproductive and therapeutic cloning

22. Stuff

23. Reproductive Cloning
Animals created using nuclear transfer are not truly identical cones of the donor animal!
Only the clone’s chromosomal DNA is the same as the donor
Some of the clone’s genetic material comes from mitochondria located in the cytoplasm of the enucleated egg
Interestingly, acquired mutations in mitochondrial DNA are believed to play a large role in the aging process

24. Reproductive Cloning Uses of Reproductive Cloning:
Production of animals with special qualities
Examples: Models for human disease, drug producing animals
Repopulation of endangered species
Examples: Guars, Sumatran Tigers, Great Pandas

25. Celebrities of Science: Cloned Animals
The sheep is dolly (first cloned animal)
The cat in the middle of the top row was not a clone, but its DNA and enucleated egg were used to clone the kitten below it (Arrow pointing to cloned cat)
to the right is the world’s first cloned mule
bottom left are the worlds first knockout pigs, that lack a gene product that causes a massive immune reaction in humans (first step towards pigs as sources of human organs)
Pigs on right are first pigs cloned
calves are first cows cloned

26. Are Reproductively Cloned Animals Healthy?
Cloning of animals is expensive and inefficient (>90% of attempts fail to produce viable offspring)
While pigs, sheep, cattle, cats, mice, rabbits, goats and a guar have been cloned successfully, attempts to clone monkeys, chickens, horses and dogs have been unproductive
Animals show a multitude of health defects, including:
Compromised immune function
Increased tumor growth and miscellaneous disorders
Growth to abnormally large sizes
Genetic instability (Aberrant gene expression patterns)
Premature and unexplainable death
genetic instability actually does not stem from gene mutation, it is actually due to alteration of normal gene expression profiles (determined by analysis of 10,000 gene expression levels by micro-array)
unexplainable death = autopsy did not reveal any specific or identifiable cause of death

27. Therapeutic Cloning
Refers to combining a patient’s somatic cell nucleus and an enucleated egg, and harvesting of embryonic stem cells from the resulting embryo for treatment of disease or injuries
Therapeutic Cloning has some similarity with Reproductive Cloning
The first step in Therapeutic Cloning is identical to the first step in SCNT (For generation of a blastocyst embryo)
Following generation of the embryo, stem cells are extracted after 5 days of cell division
The stem cell extraction process destroys the embryo, which raises a variety of ethical concerns

28. Therapeutic Cloning Uses of Therapeutic Cloning:
Organ transplants without generating an immune response
Tissue transplants
Repopulation of damaged/apoptotic cells in degenerative diseases
Examples: Parkinson’s, Alzheimer’s, Diabetes

31. Discussion of Muotri et al.
In lecture, the potential for generation of chimeric animals using reproductive cloning was mentioned
In 2005, it was unknown whether human embryonic stem cells (hESCs) could differentiate into authentic human neurons in vivo
Muotri et al. showed that hESCs implanted into the brain ventricles of embryonic mice can differentiate into functional neural lineages and generate mature, active neurons that integrate into the mouse brain
In other words, they generated a mouse with a chimeric brain

32. Experimental Roadmap
The authors first showed in vitro, that their hESCs could differentiate into neurons when given appropriate cues from surrounding cells
The authors then wanted to test if their hESCs could differentiate into functional neurons in vivo
hESC’s were infected with a lentiviral vector designed to express GFP
hESC’s were injected into the lateral ventricle of embryos removed from pregnant mice
After pups were born, they were sacrificed, and their brains were sectioned and examined for the presence of neural and stem cell specific markers, cell morphology, and neural activity
for the in vitro work: the cells are at least neuron like (they just test for morphology and presence or absence of nerual and undifferentiated cell markers, respectively.
note on the last bullet point, specific EGFP neurons were assayed electrophysiologically for activity

33. In Vitro Neruonal Differentiation of hESCs
hESC = human embryonic stem cells
NESTIN = a neuronal precursor marker (e.g. stem cell marker)
MAP2(a+b) = microtubule associated protein, an immature neuronal marker
the main point of this figure is that they used several different neural specific and stem cell specific markers and stains to show that the stem cells can indeed differentiate into neurons by cell co-culture with PA6 cells (cells positive for neuralmarkers, negative for stem cell specific markers)
Muotri AR et al. PNAS (2005) 102, 51:

34. Chimerism of hESC in the Mouse Developing Brain
(Visualized by EGFP Fluorescence)
Shows that hESC can differentiate into different cell types in vivo.
Ki67 = proliferative markers (stem cell markers)
S100-Beta = astrocyte marker (Glial cell marker)
GST-Pi = oligodendrocyte marker
TUJ-1 = neuronal marker
b) 200 laser captured EGFP cells, when PCR’d were positive for only human specific TBP (TATA binding protein gene) sequence, whereas 200 non-EGFP cells were positive only for mouse TBP sequence (Shows no weird fusion events are going on)
c & d) Shows neurons in different areas of the brain, illustrating that the hESCs migrated out of the ventricle and into many different host brain regions
e) EGFP cells are negative for proliferative markers (ki67)
f) EGFP cells express astrocyte specific markers
g) EGFP cells express oligoendrocyte specific markers
h) EGFP celsl express neuron specific markers
Muotri AR et al. PNAS (2005) 102, 51:

35. Results and Conclusions
The Muotri et al. experiment produced several very interesting results:
No teratomas or tumors were observed in any of the cloned mice
No immunological rejection of the hESCs occurred
< 0.1% of the total mouse brain cells were of human origin
hESC derived neural cells showed widespread incorporation into the brain (hippocampus, thalamus, cerebellum, etc.), indicating transplanted cells migrated out from the ventricle and into various brain regions

36. Results and Conclusions (Contd)
hESC derived neurons, oligodendrocytes and astrocytes were the same size, and exhibited the same connectivity patterns as the respective mouse cells, indicating that the hESC derived cells have the ability to adjust their size and connectivity in relation to adjacent cells
hESC derived pyramidal neurons were fully functional in response to electrophysiological stimulation
These two pieces of data point to conserved neural differentiation signals from mouse to man

37. Future Applications
Extend the approach used in the paper to other tissues of the embryo, to evaluate the funcitonal differentiation of hESCs in specific fetal environments
Introduction of hESC-derived neurons into the developing nervous system provides a new approach for the study of neurological disorders
Mainly in cases where animal model does not recapitulate the neurodegenerative process observed in humans
Also applicable in cases where affected individual dies before development of a functional nervous system

38. EXTRA SLIDES

39. Neural differentiation
Reference

40. Stuff
Reference

41. next two slides are on noto-cord development
they just could be used as an example of how turning on a single (or in some cases several) transcription factor(s) can have a huge effect on cell proliferation and differentiation

42. Reference

43. Reference