Stem Cells and Development Notes

Unit 4: Integration - Exploring Complex Processes

Unit 4 integrates topics from the first three units (DNA, RNA, Protein) in the context of more complex processes.
The first two lectures will focus on normal development, specifically stem cells as fundamental features of development and as tools.
The second two lectures will cover abnormal development, focusing on cancer and how developmental processes can go awry, leading to disease.

Parallels Between Normal and Abnormal Development

Significant parallels exist between normal development and stem cell biology, as well as between abnormal development and cancer cell biology.

Nobel Lecture: Sir Marty Evans

The lecture will cover the work of Sir Marty Evans, along with Mario Capecchi and Oliver Smithies, who won the Nobel Prize for their work on homologous recombination.
The lecture will provide insights into the introduction of specific gene modifications in mice using embryonic stem cells.

Introduction to Gene Targeting in Mice

Mario Capecchi, Martin Evans, and Oliver Smithies were awarded the Nobel Prize for discovering principles for introducing specific gene modifications in mice using embryonic stem cells.
The creation of gene-targeted mice, known as knockout mice, relies on two discoveries:
- Campekki and Smictis demonstrated that homologous recombination could create predetermined genetic changes in cultured mammalian cells.
- Martin Evans isolated and cultured embryonic stem cells.

Homologous Recombination

Homologous recombination involves introducing a short DNA sequence into a mammalian cell, which then finds and interacts with the corresponding DNA sequence on the chromosome to create specific genetic changes.
Strategies were developed to select and isolate clones of cells containing the desired genetic changes.

Embryonic Stem Cells

Embryonic stem cells are derived from pre-implantation embryos and have the potential to differentiate into any cell type in an adult mammalian organism.
These cells can be cultured indefinitely and maintain their full differentiation potential.
When reintroduced into mouse embryos, embryonic stem cells can contribute to the formation of all tissues in an adult mouse.

Process of Creating Transgenic Mice

Fertilized eggs (zygotes or early embryos) are isolated and fragmented into individual cells.
These cells are cultivated indefinitely in culture.
Embryonic stem cells can form every tissue found in the adult animal (mouse).
Injecting embryonic stem cells from one mouse into the blastocyst stage embryo of another mouse allows the implanted cells to integrate and become part of the developing animal.
For example, implanting black six J mouse cells into a BALB C white mouse embryo can result in a white mouse with black patches, where the black patches represent tissues developed from the implanted cells.
If the implanted cells give rise to the germline, the genetic information can be transferred to future generations through breeding.

Transgenic Mouse Technology

Transgenic mouse technology is commonly used to create disease models and study fundamental biological processes.
Martin Evans discovered and isolated stem cells and showed they could be genetically manipulated.
Mario Capecchi and Oliver Smithies developed homologous genetic recombination tools to make specific changes at targeted loci.
Combining these technologies has transformed the field.

Impact of Gene Targeting

Gene targeting became possible by combining homologous recombination (to introduce specific genetic changes into embryonic stem cells) and the derivation of gene-targeted mice from these modified cells.
In the 1980s, most scientists believed creating gene-targeted mice was impossible.
Two of the three Nobel laureates had research grant applications turned down for being unrealistic or overambitious.

Importance of Fundamental Research

The scientists pursued their research because it was personally important to them and they were supported by their institutions.
They were not primarily motivated by wealth, fame, creating biotech companies, or curing disease, but by fundamentally understanding how these processes work.
Fundamental knowledge and creative thinking lead to world-changing discoveries, not just experiments aimed at solving specific problems.

Collaborative Sharing and Interactions

Early development of gene-targeted mice was facilitated by sharing reagents and interacting with each other.
Martin Evans traveled to Oliver Smithies' lab with embryonic stem cells in his pocket.
Mario Capecchi visited Martin Evans' laboratory.

Applications of Gene Targeting

The first gene-targeted mice were born in 1989. It's estimated that at least 10,000 genes in mice have been altered by homologous recombination.
Gene-targeted mice offer opportunities to systematically determine the role of specific genes in development, physiology, disease, and aging.
They are used in embryonic development, immunology, neurobiology, and metabolism research.
The pharmaceutical industry uses gene-targeted mice to develop new drugs.
The creation of gene-targeted mice was a paradigm shift that profoundly changed physiology and medicine.

Nobel Lecture by Martin Evans

Martin Evans discusses the technology that has transformed the understanding of mammalian genetics.
He focuses on mouse embryonic stem cells, their derivation, and their use in genetic manipulation.
His lecture also covers how these cells relate to the normal mouse embryo.

Cell Biology Concepts

The body is made up of a large number of cells that behave as autonomous units or in cooperation with each other.
All cells have a copy of the genetic material inherited from parents.
Cells become specialized and use parts of their genetic material to perform specific jobs.

Cell Specialization and Lineages

Specialization in development involves changes in patterns of gene regulation.
Cells originate as clones from single cells with a single set of genetic information (DNA).
These cells reproduce faithfully to create genetically identical cells.
Changes in gene expression patterns over space and time lead to the diverse cell types that occupy specific positions in the adult body plan.

Cell Differentiation and Lineage Restriction

As cells divide, their gene regulation patterns differentiate, creating lineages of cells pointed towards terminal adult phenotypes.
These lineages typically do not have the ability to change drastically from one type to another (one-way trips).
Under certain conditions, cells can shift their identities through transdifferentiation.

Mutations and Genetic Identity

All cells should have the same gene sequences unless there is a mutation.
Mutations can arise during development or in the adult, changing the identity of a small set of cells.
If a mutated cell establishes itself clonally and expands, it can form a tumor.

Early Development and Cell Potential

At early stages of development, cells must have the ability to go in all different directions.
It is not self-evident that these cells provide or will be a self-renewing population or that they can be extracted.

Mouse Teratocarcinomas

Suggestions about the nature of cells from which the entire organism is derived came from studies on mouse thoracic carcinomas.
Teratocarcinomas are unusual tumors containing different tissues, such as skin, bone, and cartilage.
In the late 1960s, researchers sought a system to study development and restriction of fate in an experimental system in culture.
Leroy Stevens and Barry Pierce established the field by studying a strain of mice (mouse four one two nine) that produced teratomas frequently.

Germ Cells and Teratomas

Roy Stevens found that teratomas come from germ cells, which are the cells that eventually give rise to sperm in the development of the male mouse.
Germline cells have the intrinsic capacity to produce every tissue type and proliferate to create a colony of cells.

Location and Cell Fate

Mislocating germ cells or intact embryos into an environment other than where they are supposed to be (e.g., outside the gonads or fallopian tubes) leads to a loss of proper signaling needed for differentiation.
The ground state of embryonic stem cells or primordial germline cells is to proceed in every direction simultaneously, building up cell mass.
In a regulated environment, these cells can then differentiate to create tissues.
When removed from protective environments, these cells lose instructions to stay STEMI, and their ground state is to differentiate.

Teratomas

Teratomas are disorganized masses of cells.
These are benign tumors that result from dysregulated development due to dislocation of cells rather than genetic changes.
They lack information from niches to maintain specific identity.

Pluripotency

Pluripotency is the potential to form every tissue type in the body.
Teratomas or teratogenic carcinomas result from an embryo trying to form without proper instruction.

Martin Evans' Hypothesis

Given that teratomas start from one cell and form all cell types, Martin Evans hypothesized that they harbor stem cells like embryonic stem cells.
He and others began picking through them to find individual cells capable of becoming every cell type.

Experiments with Embryos

Evans tested whether early embryos could also give rise to teratocarcinomas.
He found that placing an early embryo, not in the uterus but in the kidney of the mouse, could produce a new teratoma.
He shared that these teratomas, which have all the different sorts of tissue, can come from cells with that early ability.

Key Term Definition

He defines an embryonic stem cells: Pluripotential embryonic stem cells, embryonic cells appear to be right both to rapidly differentiating cells and others which like themselves remain. That is the definition of a stem cell, and he therefore is the first to name embryonic stem cells.

Barry Pierce's Work

Barry Pierce, a pathologist, studied the biology of these tumors.
He observed that although these tumors would eventually kill mice, the differentiated tissues (muscle, skin, etc.) were apparently normal and did not grow as a tumor.
Pierce's work led to experiments to determine if all cell types come from a bit of pre-skin, pre-liver, etc., or if they come from cell specialization similar to that in the embryo.

The Hayflick Limit

Terminally differentiated cells have a limited shelf life and can reproduce a limited number of times before they die; this is known as the Hayflick limit.
Embryonic stem cells are immortal, maintaining the potential to regenerate all tissue types.

Cloning Experiment

Fractionate the teratoma into individual cells and you individually implant those individual cells into a naive animal, most of them don't give rise to a teratoma, but some of them do. And you can clone those cells and reproduce them clonally.
- Martin Evans fractionated teratomas into individual cells and implanted them into mice. Most cells did not form a teratoma, but some did.
These cells could be cloned and reproduced clonally, leading to the discovery of pluripotent stem cells, like embryonic stem cells.

Resetting the Hayflick Limit

It is possible to reset the Hayflick limit by inducing pluripotency in terminally differentiated cells.
However, these cells undergo changes that are of interest that scientists continue to study.

Culturing Tumors

Took a tumor from the mice, a teratocarcinoma, minced it up, and grew it in culture.
If you transplant them back into a mouse, you get the keratin carcinoma back again.
Take a single of these cells and get the teratococcus nerve back, proving that he had in culture the pluripotential stem cells.
*If you take tumors, like real tumors, like like transformed cancer tumors, and you fragment those cells and you try to to plate them and reproduce them in tissue culture, most of those cells will divide a certain number of times and die.
*A small fraction of those cells will be immortal.

Chimeric Mice

Embryonic stem cells differentiate in a chimeric embryo.
Putting cells from the tissue culture plate into another early mouse embryo made a mouse with patches of colored hair.
These mice had patches of cells derived from culture all over their bodies. *Making a mosaic, a tissue mosaic by having cells of different origin codeveloping in the same embryo.
This is the beginning of the concept of transgenic mice or transgenesis in general.
These were chimeric mice. Transgenic mice (mosaic like). The tumor line derived was abnormal and never worked.

Normal Chromosome Numbers

The cells needed to be from an authentic embryo, not a teratogenic carcinoma, and have normal chromosome numbers.

Embryoid Bodies

A big conceptual breakthrough on the road to embryonic stem cells was that differentiation was not random. During differentiation the cells were forming a new type of cell on the outside exactly the same was the a normal embryo.
What was happening was that the cells were forming a new type of cell on the outside of little tongue in exactly the same way as a normal embryo, very early on embryo. When it starts to develop, those are the cells in the middle of things we call the inner cell mass cells, and they will start to form a different cell type on the outside.

Growing Embryonic Stem Cells

Delayed blastocysts trick to made it possible. Gave them more cells to start with, flattened out, the cells we wanted beginning to grow out. Inside is the embryoid body maybe.

Embryonic Stem Cell Properties

Recovered normal copy of all the chromosomes.
Can clone them and they differentiate in the tissue calculation. So pluripotent stem cells, when they differentiate spontaneously in culture, they tend to become nerve like first.

Transgenic Mice Creation

Take a male and a female mouse and get them, you know, dim the light, put on some soft music, and then do surgery and remove the early embryos from the pregnant female and then put them in culture, inject the stem cells that you've been propagating in culture. And then, you take another female mouse and we call this pseudo pregnant. You take an irradiated male mouse who's shooting blanks and you take a mouse in in estrus and then they'll get it on and she'll think that she's pregnant. And you implant the embryo in that, and she'll develop the embryo because she's formed a full corpus callum and all that stuff. It's pretty cool, actually.
*What you can do with this information = what you can do with this technology.

Mutagenesis

Make genetic changes (mutagenesis) in the cells in culture and to find some way where we could select a cell that had a useful mutant change in it.
Retroviral vector to put DNA, foreign DNA into the cells in culture, insert randomly.
Making genetic changes (mutagenesis) in the cells in culture and to find some way where we could select a cell that had a useful mutant change in it.

Retro Viral Insertional Mutagen

Alter the gene structure around there and you can detect that using physical means, using southern analysis, using a probe that is specific for the retrovirus.
Total genomic DNA and chop it up restriction enzymes, run it out on an agarose gel, transfer, transfer it onto a membrane, block the membrane, and now you take your radio labeled probe derived from the retrovirus and you probe the membrane, the the probe will stick where the retrovirus is in the DNA.
Grow these clones in culture and if they survive (cannot do anything with them otherwise), then you can use them in transgenesis experiments.

Embryonic Lethality

Make transgenic mice and you ask, do any of the mice have phenotypes that are different than they had beforehand? Then you could surmise it was a consequence of the insertion. And because the insertion is at a specific location and you can determine that, you now are well on your way to discovering the gene that's important for that phenotype.
The first phenotype they looked for was embryonic lethality. genes that are important for early development.

# Morphological changes

There were looking for morphological changes in the mouse. And game called nodal after that, which we found by this method.
Embryonic lethal (Nodal).

Hypomorphic Alles and Transposons

These retroviral mutagenic units can create hypomorphic alleles. In the first case, a disruption mutation by being in the middle of an open reading frame of a gene blocks a gene product from being made, Embryonic lethal. Inserted just upstream of the coding region. Caused premature closure with regulation changes because genes were expressed twice as much because of transposon insertion.

X Linked Genes

HPRT minus mutants can be selected for in cultures that is couple the physical technique of altering the genome to the genetic technique of selection in cultures so that you could go straight to work on a particular gene.
The particular gene they're looking at here is an x-linked gene and so in females, only one of the two X chromosomes is expressed, remember, because of Xist:
Have an inactivated, chromosome that has a mutation, you won't see the mutant phenotype, Right? Because that whole thing is shut down. But if the active x is the one that has the mutant allele, then you see the phenotypic change associated with that.

Reporter Genes

LacZ, which you can stain it blue. When you find out where that insertion has gone, you can find that it's actually in the history of three point three a. *If the reporter hops into a region of the chromosome that has enhancer elements nearby, they will now take over the expression of the reporter gene. In this case, lacZ, it produces beta galactosidase, you can stain the embryo for X gal like stuff and it will turn blue where beta gal is expressed.

BRCA

By sequencing women with breast cancer, they found some that had BRCA mutations. They don't know what they do. It's actually, of course, not. It's a gene that stops you getting breast cancer. We call it a tumor suppressor.
If you take a human mutant that is, like, predisposed to breast cancer and determine the site that has changed in the human mutant, you can then deliberately introduce a change like that into the orthologous gene in the mouse using this transgenic plus homologous recombination technology in stem cells and generate a model for breast cancer in mice and it recapitulates what you see in humans.

End Note

We undersell this to the students, your students, and to the next generation because we talk about the practical aspect. We are not just technicians. We are visionaries and explorers.
On Monday, we're going to have a crash course in everything you need to know about embryonic stem cells, so called adult stem cells that are not pluripotent. But what are their opportunities? And then we'll talk about induced pluripotent stem cells and how to use adult stem cells in therapeutic, situations, and we're in that setting right now.
On Wednesday, we transition to cancer. We'll see another talk by another Nobel laureate. And, and then on the following Monday, we're gonna get into regulation that's gone weird in the form of cancer and see the very striking overlaps with stem cell biology. It's it's hair raising. It's worth thinking about, and we'll just continue along that line. Thanks for showing up.