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

Section #8: An Introduction to Stem

Cells

Ward G. Walkup IV

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

• 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

Overview of Section Materials

What is a Stem Cell?

http://www.patentbaristas.com/wp/wp-content/uploads/2007/04/stem-cells.jpg & Biology 1, 2008 Lecture

• 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)

http://www.nature.com/ncb/journal/v3/n9/fig_tab/ncb0901-e205_F1.html



• 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

Embryonic Stem Cells

Types of Stem Cells (Adult & Embryonic)

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Embryonic stem cell has

unrestricted potential for

differentiation

Adult stem cells are lineage restricted



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


• Pluripotent






http://www.biology-online.org/articles/embryonic_stem_cells_prospects/figures.html




• Pluripotent






http://erl.pathology.iupui.edu/C604/GENE826.HTM


• 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

(Multipotent)

Biology 150Development Lecture

Slides, Fall 2006Caltech


(Multipotent)

Biology 150, Development Lecture Slides, Fall 2006, Caltech


• 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

induced Pluripotent Stem Cells (iPSCs)

Generating iPSCs

TransfectLentiviral & Retroviral

Vectors Containing:

• 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

Molecular, Reproductive and Therapeutic 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

Molecular Cloning

Molecular Cloning

1.

2. & 3.

Adapted From: Alberts et al. Molecular Biology of the Cell, 2002, Figure 8-30, p 501

Molecular Cloning

4.

5.

Adapted From: Alberts et al. Molecular Biology of the Cell, 2002, Figure 8-31, p 501

• 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

Molecular 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

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


• 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


Celebrities of Science: Cloned Animals

http://www.guardian.co.uk/gall/0,8542,627251,00.html



• 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

Are Reproductively Cloned Animals Healthy?

• 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

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

Therapeutic Cloning

• 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

Discussion of Muotri et al.

• 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

Experimental Roadmap

In Vitro Neruonal Differentiation of hESCs

Muotri AR et al. PNAS (2005) 102, 51: 18644-8

Chimerism of hESC in the Mouse Developing Brain

(Visualized by EGFP Fluorescence)

Muotri AR et al. PNAS (2005) 102, 51: 18644-8

• 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

Results and Conclusions

• 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

Results and Conclusions (Contd)

• 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

Future Applications

EXTRA SLIDES

Neural differentiation

Reference

Stuff

Reference

•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

Reference

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

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