Integrated Biology 3401 Stem Cells

Controlling Differentiation in Bitro

- one of the major challenges for ES cells is to direct them to the cell type you want

Hutchinson-Gilford Progeria Syndrome

-1 in 4 million births -defect in the lamin A gene and causes a change in nuclear structure and function -iPS cell models derived from reprogramming patient somatic cells are being used to study the disease

Martin Evans

-2007 Nobel Prize -introduced specific gene modifications in mice by the use of embryonic stem cells

Adult Stem Cells are found in many organ system. Three we talked about were?

-Brain -Bone Marrow -Gut

Gut Stem Cells

-Clevers Ted Talk -cell marking shows stem cells produces all other cell types (mark stem cell blue-->blue crypt villus) -use a fluorescent protein to mark stem cells -FACS (fluorescent activated cell sorting) to purify -in culture --> minigut fur organoids repair colon in a moues

Aging at the molecular level

-DNA damage -Free Radicals -Telomere shortening

What are the advantages of ES cells when compared to adult stem cells?

-ES cells are easier grown in culture -ES cells grow indefinitely in culture -adult stem cells only give rise to particular lineages (blood and gut epithelium)

1981

-Evans tests the idea that there are cells in a normal embryo that are pluripotent (like EC cells, but not tumorous) -develops successful protocol to grow Embryonic Stem Cells in vitro -make chimeras when transplanted into an embryo (and contribute to germ line) -ES cells combined with methods of genetic modification allow scientists to engineer any desired mutation (homologous recombination, CRISPR)

Is regeneration just a replay of embryogenesis?

-No, if it were all animals could do it equally well -No, in regeneration new tissues have to integrate with old tissues

De-extinction attempy

-Pyrenian ibex, Celia, died when her scull was crushed -eggs preserved via freezing -cloned made using goat surrogate- died at birth with lung problems

Can we manipulate aging?

-Yes, many genes have been discovered that influence aging -Yes, caloric restriction is a well established approach -maybe stem cell therapies could be effective.

Bone Marrow or HSC transplants

-autologous -allogenic -treat a range of diseases

Bone Marrow/Hematopoietic stem cell

-began after bombing in Hiroshima -giving mice whole body irradiation caused death -shielding a single bone (or a spleen) prevented death -whole body irradiated mice could be rescued by injection of bone marrow -1956 it was proven that cells in the bone marrow (and not an indirect effect

Cancer Stem Cell Hypothesis

-cancer stem cells divide SLOWLY and are not killed by standard therapy -rapidly dividing differentiated cells

A Neural Stem Cell should have the following properties:

-capable of extended proliferation -capable of self renewal -capable of generating multiple differentiated cell types -migration to regions of injury or requirement

Adult stem cells are

-capable of proliferation -found in the blood system -multipotenet THEY ARE NOT differentiated cells

Factors in Stem Cell Aging:

-changes in stem cells diminish self-renewal and the ability to produce daughters that differentiate the full range of cell types -aging of the stem cell niche -systemic influences -external influences from the environment

Deeper Insights to Cloning

-cloning potential decreased with donor age -reprogramming a donor nucleus involved reversing effects of differentiation (epigenetic changes)

Age Associated Diseases

-cognitive -neoplastic -immunologic -metabolic

Tumors are complex

-combination of caner cells and the cells of the tumor micro environment

Ethical Issues of hES Cells

-different opinions on whether human embryos can be used for research and medicine -US law currently permits limited research on hES cells (state and private funding can have less stringent regulations) -iPS cells have alleviated some problems associated with the use of hES cells

Honolulu Technique

-donor cell in G1 -donor nucleus injected into egg -chemical activation of the egg -success rate: 3/100

Gurdon frog (1962)

-first animal cloned -proved nuclear equivalence -all genes still present

Mesenchymal Stem Cells

-found in bone marrow -stem cell for bone, fat, and cartilage cells -part of niche for hematopoetic stem cells, which are also in bone marrow -initial experiments suggested MSCs were found in many tissues and could give rise to derivatives of all stem layer -later careful experiment correct this view

Where are adult stem cells found?

-found in special site called a 'stem cell niche' -replenish and repair tissues -multipotent (can differentiate into a few different cell types that make up the tissue)

Neural Stem Cells

-found in specific regions of mammalian brain -differentiate into neurons & glia -cell labeling techniques (in corporation of BrdU during S-phase) showed cells were dividing -2018 repair of spinal cord injury in monkey using human NSCS

Mesenchymal Stem Cell

-found on blood vessels in the bone marrow -bone marrow is a complex microenvironement and MSCs are part of the niche required for HSCs -make fat and bone

Genome damage and aging

-genome damage is accumulated over time (cell dysfunction, apoptosis, cancer) -stem cells are long-lived cells and have the potential to accumulate extensive damage (research is ongoing to understand how aging affects stem cell function)

The Bone marrow has two stem cell types:

-hematopoetic stem cell -mesenchymal stem cell

2013:

-human ES cells derived from cloned human embryos -SCNT used to regenerate embryos from donor nuclei injected into human eggs -hES cell lines derived from SCNT embryos -source of cells for therapeutic cloning b/c hES cells are a match to the patient

Soulutions to iPS mouse problem

-iPS cells without the Myc retrovirus factor -Non-viral delivery methods (plasmids) -DNA-free delivery methods(mRNA/Protein)

Journal Club Article

-in vitro production of hundreds of millions of pancreatic Beta cells offers the chance of curing diabetes type 1 -complex protocols (or inducing differentiation) -test in mouse showed beta cells respond to glucose by secreting insulin and treating diabetes

the adult gut stem cell must have the following properties:

-last for the life time of the animal -make all the cell types in the gut (multipotent)

Cancer cells have multiple mutations including:

-loss of function tumor suppressor genes -gain of function mutations in oncogenes

Problems with iPS cells that produced mice

-mice developed tumors -one of the factors, c-Myc, is an oncogene (cancer risk) -viruses are used to insert the factor genes and can cause mutations (including those that cause cancer)

Recent research on CSCs suggests

-more plasticity in stem cells -other tumor cells can become CSCs -the niche may control cells becoming stem cells -cancer therapies may also need to target niche (TME) as well.

Evan;s potential problems

-numbers of pluripotent cells in an embryo could be small -the time when embryos were competent to produce such cells could be short -the cultured cells may be 'too good' at differentiation and lose pluripotency and the ability to grow in culture

mammalian liver regeneration

-of all body organs, the liver has the greatest capacity to regenerate -not true regeneration because liver shape is not reformed -the mechanism of growth is under intense study because of the medical implications -organoids offer promise of therapy for liver disease and research

Mesenchymal Stem Cells give rise to?

-osteoblasts -adipocytes -chonrocytes

Paneth Cell

-part of stem cell niche -supplies growth factors -secretes antimicrobials and lysozyme

Injection of younf plasma show little or no effect on Alzheimer's disease

-performed slightly better at daily tasks -did no better on cognitive tests

Enteroendocrine Cell

-produces hormones

HSCs can be genetically modified for gene therapy by:

-purify MSCs from patient-->correct gene defect with transgene-->MSCs transplanted back to patient

14 day rule

-red line is prior to the time in development that the nervous system starts to form scientists ethicists decided the red line should be at 14 days of development -red line applies to research on human embryos in the lab

Roslin Technique

-remove oocyte nucleus -put cells near each other and shock them to promote fusion donor cell in G0 -success rate 1/277

Adult Stem Cells

-reside in tissue/organ and replace lost cells (normal wear ot tear, injury, disease) -different rates of activity depending on function

Absorptive cell

-secretes enzymes to fully digest food from stomach -absorbs nutrients

Goblet Cell

-secretes mucus (for lubrication)

Why can planarians regenerate so well?

-simple bilaterian 1/279th fragment can produce a whole new organism

Neoblasts

-stem cells in planaria -pluripotent: irradiation and transplantation experiments shows single neoblast --> every cell type

Aging is a decline in the function of tissues and organs and increased disease such as cancer

-tissues stem cells are required to maintain homeostasis of a tissue -reduction in the number and function of old stem cells is an important component in age-related morbidity

A systematic cellular and genetic analysis of regeneration in planaria

-to describe mechanisms -to find genes involved in the process -the expectation is that we can apply what we learn to other animals including ourselves

Demonstrating HSCs and concept of stem cells i mouse classic experiment

-transplant bone marrow into mouse -host BM destroyed with 3x radiation -not dead due to transplanted HSCs -colonies in spleen with multiple cell types -colony derived from a single HSC can produce all differentiated cell types

Teratocarcinoma

-tumors with differentiated cell types -products of all three germ layers -contain undifferentiated stem cells, called embryonal carcinoma cells

Adult neurogenesis

-was thought that the adult CNS had no dividing cells (all post mitotic) -dividing cells found in rodents then in human -opens the possibility of repairing the nervous system

what mechanisms are occurring in different cases of regeneration?

1. planaria; pluripotenet stem cells called neoblasts are responsible for regeneration 2. tadpole tail regeneration -- shows that tissues only regenerate the same tissue 3. zebrafish heart regeneration-cardiomyocytes dedifferentiate

Which factors worked?

10 and 4

the Yamanaka experiment involved ______ Factors given to ________ cells taht resulted in _______ cells

24, somatic, iPS

How many times did it take to create Dolly?

277

Which factor gave abnormal looking colonies?

3

Somatic Cell Nuclear Transfer

By passing sexual reproduction through animal cloning (reproductive cloning)

when a nucleus from a differentiated cell is injected into an egg, factors in the egg reprogram the nucleus so that it can support development of the embryo. This is called

CLONING

post attempts in making beta cells resulted in cells called polyhormonal cells (PH cells), which got their name becasue they secrete additional hormones such as glucagon. Unlike SC- beta cell sand primary cells, the PH cells...

DO NOT perform glucose-stimulated insulin secretion

Only cancer stem cells express

Dclk1 and Lgr5

determination and differentiation

Determination is FIRST then differentiation

body plan patter formation

Dorsal: back Ventral: front/belly Anterior: head Posterior:tail

1972

Evans establishes cell lines from teratomas called EC cells

2007

Evans gets Nobel Prize

Mouse ES cells

Evans, 1981

the researchers made a gene that combined the Fbx15 gene and resistance to the drug G418. Now any cell that expresses Fbx15 will be resistant to the drug (and survive). Which of the following is true?

Factors that induce pluripotency in somatic cells will cause expression of the Fbxbgeo gene and the cells will survive in G418

Transplanted SC-beta cells alleviate defects in blood glucose levels in diabetic mice. The figure shows the mice produce _________ insulin

HUMAN

1998

Human ES cells first derived (Thomson) -pluripotent -differentiate derivatives of all germ layers in teratomas in mice

the major goal of the study ("Introduction of Pluripotent Stem Cells from mouse embryonic and adult fibroblast cultures by defined factors") was to________

Identify factors that can induce pluripotency in somatic cells

Mechanisms of Development

Intrinsic and Extrinsic

Human Embryonic Stem cells derived by somatic cell nuclear transfer

Mitalipov, 2013

what gene is expressed in INNER cells of the early embryo?

OCT 3/4

The Yamanaka Factors

Oct 3/4 Sox2 Klf4 c-Myc

What type of stem cell is a blastocyst?

Pluripotent (also the inner cell mass)

Hallmarks of Aging

Stem cell exhaustion -diminished self-renewal -aging of the niche -systematic support reduced

pluripotent stem cells

Stem cells that can become almost all types of tissues and cells in the body. (CM cell, ES cell)

Totipotent

Stem cells with the potential to differentiate into any type of cell. (zygote only)

1954

Stevens and Little report spontaneous testicular teratomas in an inbred strain of mice -mouse strain that produces teratomas

Morphogenesis

The process by which an organism takes shape and the differentiated cells occupy their appropriate locations.

Human ES cells

Thomson, 1998

Somatic cells do not normally express Fbx15, but if they are induced to become pluripotent, the Fbx15 gene will be expressed (T/F)

True

Transdifferentiation

a form of wound healing in which mature cells dedifferentiate and produce new cells that are then able to mature into cell types with a completely different function from the originating cells -amphibian eye

what could human ES cells potentially be used for?

a source of cells for wound healing, replacing damaged organs, to cure diabetes

Self-Renewal

a stem cell should remain proliferative for the life time of the organism

your biology professor is 110 years old, according to the recent study, what is the probability she will be alive to teach the class next year?

about 50%

In thinking about the biology of aging in an organism, what cell population do you think could play a critical role in the aging process?

adult stem cells

One criticism of the study is that it is 'biologically implausible.' Why?

because cells such as neurons don't divide and would continue to age and eventually die

RNAi

being used as a tool to yield genes involved in regeneration

the beta cell type is abnormal or missing in diabetes. These cells respond to high blood glucose by secreting ______ insulin

beta

embryonal carcinomas

both teratomas and teracarcinomas are called this

At the DNA level, how is a cancerous cell usually characterized

by at least one active oncogene and the mutation of several tumor-suppressor genes

Confetti Mouse:

cell marking technique that shows stem cells give rise to all the other cells

Extrinsic

cell to cell communication regulating and induction

Methylation

condensed chromatin, represses transcription

the stem cell, which expresses Lgr5, is found in the

crypt

Bicoid RNA

cytoplasmic determinant that control development of anterior patterning in Drosophilia

Gastrulation is a critical period in development when the germ layers form. The germ layers develop into different tissues and organs of the body. How are scientists studying human gastrulation?

'Synthetic' embryos are generated from embryonic stem cells and these undergo processes similar to gastrulation, even through the can't develop into humans if implanted in a surrogate mother.

zebra fish heart was an example of

dedifferentiation (followed by proliferation and redifferentiation)

the problem with EC cells

derived from a tumor and have an abnormal karyotype

the research discussed uses a scaffold on which to rebuild a heart with stem cells. the scaffold, which is comprised of a protein matrix is generated using______

detergents

In normal tissue, the scientists found expression of Dclk1 (marked blue in color) in

differentiated cells

HAVE telomeres

embryonic, stem, cancer, germline

what kinds of cells will be needed to populate the heart scaffold?

endothelial and muscle

Determination

expression of transcription factor (MyoD, Eyl Pax 6)

Does the gut have fast or slow turnover

fast

What can a newt/salamander to, which we can't, that is important for regenerating a limb?

form a blastema

Uses of the word Cloning

gene cloning, therapeutic cloning, reproductive cloning

Differentiation

gene expression initiated so that cells differentiate with a specific structure and function (muscles or eye)

Pleiotropy Theory

genes that are good for the young will be selected even if they are bad for the old

myeloid stem cells

give rise to all other blood cells

lymphoid stem cells

give rise to lymphocytes (B and T cells)

Development of stem cells

growth by cell division (mitosis) to produce the multicellular embryo

Neural Stem Cells

have the expected characteristics and are an example of a stem cell that is often quiescent (not dividing/dormant) for long period of time

what personal interest does the leader of the study (DR.Melton) have in diabetes?

he has two children with type 1 diabetes.

Sharing a blood/plasma supply (parabiosis) or blood factors from a young mouse...

improves stem cell function in old mice

In intestinal cancer, all tumor cells expressed Dclk1 (marked blue). This shows Dclk1 must be expressed

in all cells, including cancer stem cells

Dr. Clevers decided to isolate stem cells from an organ he thought would have the 'champion' of stem cells because cells are replaced every 4 days. This organ is the ______.

intestine

where is the mouse FBx15 gene expressed and is it required for maintenance of pluripotency?

it is expressed in embryos and ES cells, but is it no required for pluripotency

Extrinsic Mortality

it's so dangerous in the wild, mo genes specific to aging have evolved

the researchers found Dclk1 is expressed in cancer stem cells, differentiated normal cells, but not normal stem cells. The means killing Dclk1-expressing cells will____

kill both the tumor and some normal tissue, but not normal stem cells, which will allow regrowth of normal tissues

Evidence for cancer stem cells came from experiments involving

leukemia in mice

Intrinsic

localized determinant (bicoid for head development)

chromosomes from cloned cattle/mice have ________ telomeres than normal

longer

dedifferentiation

loss of differentiated characteristics, return to a more progenitor-like phenotype, proliferation and redifferentiation -- net increase in cell number

niche

microenvironemnt that supports stem cells

24 genes were selected as candidates for encoding factors that induce pluripotency in somatic cells. Why were the genes selected?

other evidence suggested they might play roles in maintaining ES cell pluripotency

Stimulus Triggered Acquisition of Pluripotency Cells were not able to be repeated and fraudulant data was found. They were even reported to make a tissue that even ES cells do not differentiate into which is ____________

placenta

Acetylation

promotes transcription

Chemotherapies and Radiation Target

rapidly proliferating cancer cells but may fail to target slowly dividing cancer stem cells

Isolation

regulation produces 2 complete but smaller animals

What steps will the journal Nature take to make sure fake data are not included in papers?

scan papers for manipulated data

Following amputation, humans eventually heal by forming a _____ rather than regenerating the missing part

scar

How old was Dolly's nuclear donor

six years old

dos the brain have fast or slow turnover

slow

Cancer stem cells may not be affected by therapies because they divide ___________

slowly

Promise of stem cells/therapy

so enormous that it brings dangers (bad science, unethical behavior- specifically MSC)

Stem Cell Transplants:

so far there are only three stem cell therapies that are established and safe -bone marrow -skin -cornea

LACK telomeres

somatic cells

multipotent stem cells

stem cells that can become a limited number of types of tissues and cells types in a lineage (adult stem cell)

Mouse ES cells have their origins from studies of a ...

strange cancer

Stem Cell Definition

the ability to give rise to more stem cells and specialized (differentiated) cells

to purify the stem cells, the researchers inserted a firefly gene into the Lgr5 gene. How was this used to select only stem cells from biopsied crypts

the cells glowed green and could be isolated using a cell sorter

If you amputated a salamander limb close to the body, what would start to develop first?

the digits

Why is understanding development critical for learning how to induce stem cells to differentiate into pancreatic beta cells in a culture dish (in vitro)?

the genes required for normal development of beta cells must be identified first

what is the origin of ES cells in development

the inner cell mass

Mutation accumulation theory:

the lack of selection later in life means that deleterious mutations can accumulate

Dolly was genetically identical to who?

the mammary cell donor's nuclear genome

caloric restriction

the one proven strategy to extend life (in yeast, worms, flies, rodents, dogs, and monkeys) and now promising results for humans -in the group where calories was reduced by 15%, used energy much more efficiently than control group

Targeting a toxin to all ells expressing Dlck1 causes

the tumor to collapse, but normal intestine remains intact

Differentiated liver cells and neurons are different in structure and function. The cells are different because

they express different GENES

what is a major problem with transplanted engineered rat hearts?

they failed to pump sufficient blood

what key observation proved the stress-induced stem cells must be "fakes?"

they had a different genetic background than the body cells they were supposed to be derived from

organoids

tiny versions of organs that can be used in research and medicine (mini brains offers the possibility of modeling human brain diseases)

Transplantation

transplanted cells induce the host (cells) to make an additional body axis (Speman and Mangold 1924)

all organs have stem cells (t/f)

true

teratomas

tumors that differentiate into tissues from all three germ layers

How can HSCs be purified?

using cell surface markers

disposable some theory

using metabolic resources for maintaining the body beyond a certain point is not selected for because life in the wild is short.

Cumulina (mouse) and Dolly were cloned using different approaches. What was a key step in the method that produced Dolly?

using quiescent donor cells (G0)

the main experimental approach in the paper is:

using systematic testing of factors, and, and the order in which they need to be used, to induce human pluripotent stem cells to differentiate into beta cells

Progerias

very rare diseases that cause ACCELERATED AGING -Hutchinson - Gilford -Werner Syndrome

Zhong-Zhong clones

were the first PRIMATES