Lecture 23 Flashcards by sita clark

Developmental Genetics

The field that examine genetic control of processes whereby a one cell embryo gives rise to an entire organism
Many developmental process recapitulated in adult life (harnessed therapeutically to cure/recapitulated as part of body’s response to disease/part of disease itself)
Genetic pathways involved in development also frequently involved in disease (cancer)
Stem cell biology an integgral part of developmental genetics
Understanding genetic programmes for development a prerequisite for understanding and treating human birth defects

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Model orgnaisms

“Model systems” for developmental genetics research
Increasing complexity =increasing technical difficulty
-exponential curve
yeast –> C. elegans –> Drosophilia –> Zebrafish –> mouse
-too hard or not ethically appropriate to be tested on humans
-allows us to do research to understand many features (e.g. stem cells)
-Simpler than humans and not same ethical issues

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Stem cells

Stem cells are self-renewing progenitor cells that can generate one or more specialised cell types
-assymetical division (daughter cell can either differentiate and specialize or become another stem cell and self renew)

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Totipotent cells

Totipotent cells can differentiate into all the cell types in an organism, including the extra-embryonic tissues(part ofplacenta)
-embryonic tissue differentiating feature between totipotent and pleuripotent cells

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Pleuripotent cells

Pleuripotent cells can differentiate into all three embryonic germ layers (endoderm, mesoderm and ectoderm) but NOT into extraembryonic tissues(part of placenta)
2x groups: pluripotent stem cells can differentiate into all cell types in body: 2x groups
1. embryonic stem cells
2. adult stem cells (blood, brain, bone marrow

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Multipotent cells

Multipotent cells can differentiate into cells of multiple, but limited number, of lineages

often in cascades
each type of mutlipotent come from a progenitor cell which had greater diversity into specialization types

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Stem cell development

Totipotent embryonic stem cell

-> Pleuripotent embryonic stem cell (endoderm, mesoderm, ectoderm) human embryonic stem cell. induced pleuripotent stem cells
-> Multipotent stem cells (more specific) (lung, pancreas, heart muscle, RBC, skin, neuron) Adult bone marrow, skin, cord blood, deciduous teeth

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ES cells

Embryonic stem cells
Plueripotent
-not totipotent

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Stem Cell Plasticity

Stem cells in the body are relatively plastic, by turning on different transcription factors inside them, can differentiate into different types of cells
Neural stem cells
–>1. astrocytes, neurons, oligodendrocytes
–>2. All germ layers in chimeric embryos
–>3. Blood blood vessel and muscle. Blood –> Liver
-some stem cells can be induced to differentiate into a range of different cell types
-the closer the cells are to pleuripotent stem cell = more immature stem cell = wider range of cell types it can differentiate into
-Bone marrow/blood stem cells
–can differentiate into liver, muscle or BV cells
– Bone marrow stem cells can derive from neural stem cells

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Background to genetic modification

Type of modification:
-remove genes (gene knockouts in cells and animals)
-change or insert genes (transgenic cells and animals)
Methods:
-homologous recombination (hijacks a DNA repair mechanism)(swapping segments from two pieces of DNA, and artificially remove or add genes)
-Zinc finger nucleases (ZFNs) (specific enzymes to mediate the change, more efficient)
-Transcription-activator like effector nucleases (TALEN) (specific enzymes to mediate the change, more efficient)
-RNA-guided nucleases (CRISPR/Cas9 and now CRISPR/Cpf1) (disruption or change in DNA is guided by a piece of RNA)
-can modify incubated cell. if was pleuripotent cell, can place p cel back into animal, used to generate transcgenic/knockout animal

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Homologous recombination

Gene normal
-want to replace with lab generated in gene
Get segment from lab gene that is same to normal gene
the cells intrinsic crossing over mechanisms can be utilised to swap the gene of interest, with the lab generated gene
-“insert” gene in
-also insert other sequences that can be recognised by other enzymes - can be used at will to swap in and out desired other genes
-powerful systems for generating template which you can conduct a wide range of medical research
“donor plasmid”

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Whole genome mutagenesis screens (e.g. for drug resistance)

GeCKO system
Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells
Use CRISPR-Cas9 genetic modification mechanism to knock out entire genome, -but different gene in every cell
incubate millions of cells
-Can then treat with drugs. Drugs should kill all cells. but if particular gene knocked out that is required for cell killing: Can show which genes are involved in drug resistance
-resistance mechanisms to drugs
-mechanisms that activate pro drugs
-potential biomarkers to better stratify which patients will benefit the most from which drugs

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What is the relationship between technological advances and community knowledge?

development/technical abilities of research is increasing much faster than the public/community discuss and the patient/researcher understnading

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History of stem cells

Pluripotential
ES lines generated from embryonic cells of a blastocyst
Mouse ES cells first described in 1981, which enabled knockouts in mice (Martin Evans) (beginning of acceleration of development)
Human ES cells first described in 1998
(colony of ES cells- green due to genetic modification for expression of a fluorescent protein)
“Feeder layer” of fibroblast cells (to recapitulate the stem cell “niche” - grey= Fibroblasts long thin nuclei - heal wounds
-embryonic fibroblasts which you have to grow the embryonic stem cells on- Major feature: stem cells dont exist well alone, are specialised/evolved to live in specific niches (environments)- this keeps them undifferentiated, (form colonies ontop of these fibroblasts)
Leukemia inhibitory factor (LIF)-cytokine
Feeder layer + LIF = helps cells remain as pleuripotent stem cells, stopping them from differentiating/turning on genetic programmes, and becoming the different tissues of an embryo

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Which cell type stains grey and have long, thin nuclei?

Fibroblasts
“Feeder layer” of fibroblast cells (to recapitulate the stem cell “niche”
heal wounds
-embryonic fibroblasts which you have to grow the embryonic stem cells on- Major feature: stem cells dont exist well alone, are specialised/evolved to live in specific niches (environments)- this keeps them undifferentiated, (form colonies ontop of these fibroblasts)

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Example of gene knockout research

-use of stem cell technology and genetic modification to discover protein function
Apoptosis Regulator Bcl-w is essential for spermatogenesis but appears otherwise redundant
-new protein discovered Bcl-w (part of Bcl-2 family of proteins -which regulates cell death/commit suicide by apoptosis)
-homologous recombination as method to modify genomic DNA inside mouse embryonic stem cells–> implanted into mouse uterus –> offspring carrying modification (knocked out/removed Bcl gene)
Results:
removing the Bclw gene disrupts spermatogenesis (cells were dying as groups, during development in testes, when cels divide remained linked by cytoplasmic bridges, lack of protein reflected throughout entire ball of cells)
-used to better understand male contraceptive and male infertility

An example of transgenic research

Trangenically blocking endothelial cell apoptosis
-development= tissue growth, remodelling/reshaped
-inserting extra gene
-inhibited BV apoptosis (couldn commit suicide) by inserting large amounts anit-apoptotic protein called Bcl2 only in BV cells of embryos
Result: feet webs - if BV didnt regress, the feet webs didnt regress.
BV regression potentially nescessary part of remodelling tissue

Embryoid bodies

Embryoid bodies are used in research
=embrionic stem cells + incubated with series of different growth factors to force to differentiate into different tissues
=these tissues can be used in regenerative medicine
=see BV developing in them
Embryo-like structures differentiating form ES cells
-stirred in spinner cultures = sufficient O2

Stem cell prediction

Stem cells predicted to bring a new age of medicine

better understand processes and how they go wrong during disease
try generate tissue to be used to treat diseases (embryonic stem cells differentiated into BV, neurons for repair of damaged tissue)

After mouse ES cells: Stem cell research preogress (iPS cells)

1997, Dolly the sheep (first cloned animal)
2001, George W. Bush limits federal funding of human ES work (ethical concerned by communities)
2006, Cell reprogrammed Shinya Yamanaka of Kyoto University makes “induced pluripotent stem” iPS cells from adult cells by inserting four genes (oct 3/4, Sox2, c-Myc and Klf4)
“Induction of Pluripotent Stem Cells form Mouse Embryonic and Adult Fibroblast Cultures by defined Factors”
-take normal adult cell, e.g. fibroblast, reprogramme to go back to stem cell, by inserting 4x genes = can generate any type of cells without destroying embryos, instead just taking someones own cells (e.g. own skin fibroblasts)

Induced Pluripotent Stem (iPs) cells

2006
Shinya Yamanaka, Kyoto University
Nobel prize 2012
Healthy or diseased human or mouse –> adult cells from fibroblast –> OCT4, SOX2, KLF4, Myc –> iPS cell (self renewal)

iPS cells forming neurons

After human iPS cells: stem cell research progress

2007 Nobel prize to Evans, Capecchi and Smithies
2009 President Barak Obama lifts 2001
*2010, A person with spinal injury becomes the first to receive medical treatment derived from human ES cells in a trial by Geron, california
2012, Yamanaka and Gurdon Nobel prize PS cells
2013, Shoukhrat Mitalipov produce human ES cells from fetal cells
2014, Charles Vacanti and Jaruko Obokata show that any cell can potentailly be rewound to a pre-embyronic state simply
*2014, The first therapeutic cloning- human ES cells produced from adult cells by Dieter Egli and Young Gie Chung (skin cells from a woman with diabetes (reprogrammed) differentiated into insulin-producing Beta B cells)

IPS cells for research

Patient recruitment
Somatic cell injection
iPSC reprogramming
iPSC differnetiation
Comprehensive drug testing
- taken from patient and reprogrammed via a variety of differnet methods into a variety of different cell types- to identify drugs or diseases (could be from people with disease, used to investigate differnt drugs)

IPS cells for therapy

Isolation of cells from individual

-> cellular reprogramming iPSCs
used to treat someone with genetic abnormality, wiping out genetically abnormal cells in a person and replacing with new cells
-> generate iPSC Biobank
disease modelling, drug discovery and personalised medicine
-> Drugs targeting disease

Spermatogonial stem cell transplantation to preserve male fertility after chemotherapy

Testicular Biopsy
–> 1. Cryopreservation 2. Chemotherpay –> Spermatogonial stem cell transplantion
Cryopreservation of sperm is already in routine clinical practice
Transplantation of SSc was first chown by Hermann et al in Macaque monkeys
The transplanted SSCs can fertilise oocytes
Chemotherapy old fashioned oncology drugs targeting cells that are dividing rapidly/tumour and other cell types
=epithelial cells on surfaces of the body that are rapidly dividing get killed and damaged = toxicity
+
cells in testes and ovaries get killed and damaged as are always very rapidly dividing
- to prevent infertility caused as a side effect of chemotherapy drugs, target, biposy of testes, preserve, and retransplanting human spermatogonial stem cells (or even be transplanted and kept safe in testes of other species.) (put back into testes in order to KEEP STEM CELL NICHE-outside factors required to stop them differentiating and keeping potential)

When will this all become real?

Quest towards limb regeneration
Regenerative engineering approach
-3D printers

Development of stem cell treatments

Stem cell treatments other than those with blood stem cells are still developing their safety and efficacy data
-avoid deterimental consequences
If considering stem cell therapy, look for evidence that:
1. Preclinical studies published , review and repeated by other experts in the field
-Providers have approval from independant committee (Institutional Review Board, Ethics Committee) to ensure risks as low as possible and worth any potential benefits, and rights are being protected (needs to be adequate)
-Providers have approval from regulatory agency for the safe conduct of clinical trials or medical use of a product for the disease
Regulatory safe guards

Potential used of Stem cells

Combination of : induced peripotent stem cells + genetic modificaiton)
Stroke
Traumatic brain injury
Learning defects
Alzheimers disease
Parkinsons disease
Baldness
Blindness
Deafness
Missing teeth
Wound healing
Amyotrophic lateral-sclerosis
Myocardial infarction
Bone marrow transplantation (currently established)
Muscular dystrophy
Spinal cord injuries
Diabetes
Osteoarthritis and Rheumatoid arthritic
Crohn's disease 
Multiple sites: Cancers

What is going to be the massive “catching up” that will occur over the next few years regarding potential uses of stem cells?

Findings are so new

physicians and researchers
field that is developing much faster than the general discussion and understanding of ethical issues
4-5 years will be alot of catchup: discussion and education of public around stem cells and genetic modification issues will be really important

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Lecture 23 Flashcards

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