1
Q
LO
A
- Describe the fundamental features of stem cells
- Compare the origin of embryonic, adult and induced pluripotent stem cells, and contrast the differences in their developmental potential
- Understand the mechanisms that govern stem cell divisions, self-renewal, potency and differentiation
- Explain the different experimental strategies used by researchers to study development, treat and cure diseases.
- Evaluate the main advantages and limitations of the different types of stem cells for researchers
2
Q
What is a stem cell and its concept?
A
The stem cell concept. A stem cell can make more stem cells while also producing cells committed to undergoing differentiation.
3
Q
What are the criteria that a cell has to meet in order to be classified as a stem cell?
A
- Undifferentiated or unspecified (undifferentiated as not acquired any function, unspecified as not enquired any instructions to enter into differentiation process)
- Have the ability to self-renew
- Mature and differentiate
4
Q
What are the different stem cell division modes?
A
5
Q
A stem cell has the power, or potency to produce many different types of differentiated cells. Tell me about these different potencies and explain each one
A
- Totipotency. The single cell zygote, and the 4 to 8 cells embryo generates the embryo and the extraembryonic tissue.
- Pluripotency. Inner cell mass (ICM)/Embryonic stem cells generate the embryo proper.
- Multipotency. Adult or somatic (resident) stem cells. Fuel organogenesis in the embryo and regeneration in the adult tissue
6
Q
Potency review: progressive differentiation of neurons
A
7
Q
Stem cell regulation
A
8
Q
When referred to as the stem cell niche, what does this mean?
A
Stem cell function and behaviour are influenced by their microenvironment, referred to as the stem cell niche
9
Q
Embryonic stem cells (ES)
What are they? Where do they come from?
What are their properties?
A
- Most scientists use the term pluripotent to describe stem cells that can give rise to cells derived from all three embryonic germ layers—mesoderm, endoderm, and ectoderm. These three germ layers are the embryonic source of all cells of the body
- properties:
Undifferentiated/ non-committed
self-renewel
Pluripotency
10
Q
Tell me how the genes that target ES cells can be a gene for targeting mice, designer animals?
A
- Reliably incorporate ES cells into embryos, resulting in chimeric animals, carrying genetic mutations
- Genetic mutations can be introduced into the mouse germ line through genetically altered ES cells
- Offspring produced by the union of such eggs and sperm held the ES cells’ chromosomes in every cell of their bodies
- Scientists have since created and studied innumerable “designer” animals, including many that mimic aspects of human disorders
11
Q
How is the ICM (inner cell mass) established?
A
12
Q
What are the central pluripotency factors for ICM cells?
A
Nanog, Sox2, Oct4
13
Q
What do Nanog, Sox2, Oct4 drive?
A
Oct4, Sox2 and Nanog drive a pluripotency gene expression network to maintain ICM
14
Q
What must happen to Oct4, Sox2 and Nanog during development?
A
Oct4, Sox2 and Nanog must be differentially repressed during development to allow ICM cells to give the epiblast
15
Q
What is Hippo signalling regulated by?
A
Hippo signaling is regulated by cell density and cell-cell adhesion
16
Q
Tell me the two maintenance in ICM development that involve Hippo signalling?
A
- Maintenance of trophectoderm fate through apical polarity-mediated inhibition of hippo signaling
- Maintenance of ICM through cell-to-cell-mediated activation of hippo signalling
17
Q
Tell me the stages to how Cdx2 is expressed in trophectoderm cells
A
In trophectoderm cells
- Apical localization of the proteins in the partitioning defective (PAR) and atypical protein kinase C (aPKC) families
- Apical polarity proteins recruit and inhibit AMOT
- Inhibition of AMOT leads to Yap/Taz-TEAD-mediated gene expression of Cdx2
18
Q
Tell me the steps to Cdx2 repression and Oct4 activation and how this leads to pluripotency promotion
A
In ICM
- The scaffold protein Angiomotin (AMOT) is phosphorylated and interacts with the E-cadherin-Catenin adherens junction complex
- The Hippo signalling kinase Lats1/2 is then recruited and activated, leading to repression of Yap-Taz-Tead transcriptional complex
- Cdx2 repression and Oct4 activation, which promote pluripotency
19
Q
When were Human ES (hES) cells isolated?
A
1998
20
Q
What are hES cells derived from?
What are they never derived from?
A
- hES cells are derived from embryos that develop from eggs that have been fertilized in vitro.
- hES cells are never derived from eggs fertilized inside of a woman’s body.
21
Q
Protocols for hES cells culture were optimised from what?
A
mouse ES cells
22
Q
Tell me the potency of hES
A
Pluripotent (endoderm, mesoderm and ectoderm)
23
Q
Compare mES cells V hES
A
- mES cells are the most immature, undifferentiated with greatest potential for pluripotency. mES cells are naïve.
- hES cells display some maturation towards the epiblast lineage. hES cells are primed or ready for differentiation.
24
Q
Modelling human development and diseases with ES cells
A
25
What are cerebral organoids useful for?
* Model human brain development.
* Understand the mechanisms underlying human brain expansion:
1. From an evolutionary perspective.
2. Shed light on pathogenesis of neurodevelopmental disorders that affect brain size determination.
3. Tackle difficult questions pertaining to a number of debilitating neurological diseases (autism, schizophrenia…)
* Drug discovery and toxicity testing
26
Tell me the therapeutic potential of hES cells
hES cells have great potential to provide matched cells for “customized” tissue repair in degenerative diseases
* **Alzheimer’** – Forebrain neurons
* **Parkinson’s** – Midbrain neurons
* **ALS** – Motor neurons
* **Cardiovascular diseases** – Cardiac muscle cells
* **Type I diabetes** – Pancreatic β cells
27
Tell me some therapeutic limitations of hES cells
* Difficult to differentiate uniformly and homogeneously into a target tissue.
* Immunogenic – embryonic stem cells from a random embryo donor are likely to be rejected after transplantation.
* Tumorigenic – capable of forming tumors or promoting tumor formation.
* Degenerative diseases are complex genetic disorders involving interactions of many genes with environmental factors.
* Morally objectionable, because the human embryo (life) must be destroyed in order to harvest its stem cells.
28
**Summary I**
* **Stem cells are undifferentiated – They self-renew and differentiate through execution of asymmetric cell divisions**
* **Stem cell function and behaviour are influenced by their microenvironment, referred to as the stem cell niche**
* **The developmental potential of stem cells decreases during development**
* **ES cells are pluripotent and derived from ICM, which is established by asymmetric cell division in trophectoderm**
* **ICM is maintained by cell-cell mediated activation of the Hippo pathway. This in turn activates expression of pluripotency genes (Oct4; Sox2; Nanog)**
* **hES are used in research to model human disease and design customized therapies. However, technical and ethical limitations humper their widespread use**
29
Can we induce pluripotency?
Somatic nuclear transfer and the birth of **cloning** and **reprogramming**
1. Eliminated the nucleus of a frog egg.
2. Replaced it with nucleus from a skin cell taken from a tadpole.
3. The modified egg developed into a normal tadpole.
4. Subsequent nuclear transfer experiments have generated cloned mammals
30
Can iPS cells be propagated?
Like ES cells, iPS cells can be propagated indefinitely.
31
What are iPS cells?
Induced pluripotent stem cells (iPS) cells
32
What can iPS cells form?
Like ES cells, iPS cells can form cell types representative of all three germ layers
33
What can iPS cells generate?
Like ES cells, iPS cells can generate entire embryos.
34
Tell me the potency if iPS cells?
Like ES cells, iPS cells are pluripotent.
35
Can hiPS cells make organoids?
yes
36
Modelling human development and diseases with iPS cells
37
What is microcephaly?
Microcephaly is a congenital disease characterized by a significant reduction in brain size
38
Tell me about microcephaly
* what is it caused by?
* what do neural stem cells exhibit?
* what does it lead to?
* Patient-derived cerebral organoids are smaller.
* Caused by a mutation in the gene for CDK5RAP2, a protein regulating the mitotic spindle function.
* Neural stem cells exhibit abnormally low levels of symmetric divisions.
* Leads to premature neuronal differentiation.
* Depletion of the stem cell pool
39
Tell me how we can apply iPS cells to cure human diseases?
* Generation of autologous iPS cells.
* Correction of the hemoglobin mutation.
* Differentiation of the iPS cells into hematopoietic stem cells (HSCs).
* Transplantation of HSCs in the mouse cured its sickle-cell phenotype.
40
There are therapeutic potential and limitations of hiPS cells
What are the four major medical uses for iPS cells?
1. Making patient-specific iPS cells for modelling diseases (e.g. autism, Dawn syndrome, diabetes…).
2. Combining gene therapy with patient-specific iPS cells to treat diseases.
3. Using patient-specific iPS cell-derived progenitor cells in transplantation medicine without the complication of immune rejection.
4. Using differentiated cells derived from patient-derived iPS cells for screening drugs and toxicity testing.
41
What is the problem with hiPS cells?
Problems, such as tumour formation, associated with using retroviruses and oncogenes for reprogramming need to be resolved before iPS cells can be considered for human therapy.
42
Are adult stem cells differentiated or undifferentiated?
Adult stem cells are undifferentiated cells.
43
Where are adult stem cells found?
They are found in small numbers in most adult tissues.
44
Are adult stem cells finite?
They are finite may not live as long as ES or iPS cells in culture.
45
What another name for adult stem cells?
'somatic stem cells'
46
Tell me about the potency of adult stem cells?
They are multipotent in nature and give rise to unipotent progenitor cells.
47
What do adult stem cells give rise to?
They give rise to a closely related family of cells within the tissue.
48
Tell me about Hematopoietic stem cells (HSCs)
Hematopoietic stem cells (HSCs) are multipotent and give rise to all the various cells
49
There are shifts in sight of hematopoiesis occuring during development.
Tell me the types of hematopoiesis and where they occur?
**Primitive hematopoiesis** in the **embryonic yolk sac.**
**Definitive hematopoiesis** in the **aortic portion of the aorta-gonad-mesonephros (AGM).**
50
HSCs migrate through the developed vasculature to where?
The fetal liver
51
Tell me how homing where HSCs migrate through the circulatory system and find their tissue-specific niche in the developing bones?
* HSCs express CXCL4 receptor sensing the chemokine CXCL12 expressed by osteoblasts and stromal cells.
* Adhesion proteins (e.g. E-selectins and VCAM1 (Vascular Cell Adhesion Molecule 1)), also support HSC homing to the niche.
52
What are the two hematopoietic niches?
the endosteal and the perivascular
53
What are HSCs in the endosteal niche adhered to?
HSCs in the endosteal niche that are adhered to osteoblasts are **long-term HSCs** (purple)
54
Tell me about long term HSCs
Long-term HSCs are typically quiescent. They sustain long-term hematopoiesis
55
What is Quiescence maintained by?
Quiescence is maintained by **Angiopoietin-1 and Thrombopoietin**, secreted by osteoblasts
56
In the perivascular niche, what are the short-term active HSCs (red) associated with?
In the perivascular niche, short-term active HSCs (red) can be seen associated with blood vessels (green) at oxygen-rich pores
57
What do short-term HSCs interact with?
Short-term HSCs interact directly with stromal cells including the CAR cells (yellow; CXCL12-abundant reticular cells) and mesenchymal stem cells
58
Short-term HSCs and their derived progenitor cells are mobile and migrate into where?
Short-term HSCs and their derived progenitor cells are mobile and migrate into the blood stream, which can be stimulated by sympathetic connections
59
The hematopoietic niche
60
Tell me to the stages of the HSC transplanation therapy of autologous HSCs transplanation?
61
Tell me some reasons why adult stem cells matter in therpy
* Adult stem cells naturally exist in our bodies, and they provide a **natural repair mechanism** for many tissues.
* They are **somewhat** **specialized** – their inducement may be simpler.
* They belong in the microenvironment **“niche”** of an adult body, so they tend less then ES or iPS cells to cause tumors.
* They are **not immunogenic** – recipients who receive the products of their own stem cells will not experience immune rejection.
* **Relative ease of procurement** – some adult stem cells are easy to harvest (skin, muscle, marrow, breast).
62
**Summary II**
* **Somatic cells can be reprogrammed into pluripotent stem cells by overexpression of pluripotency genes (Oct4-Sox2-Nanog): iPS cells**
* **Patient-specific iPS cells represent a powerful tool to model disease and design personalised therapies. However, iPS cells can generate tumours**
* **Multipotent Adult stem cells naturally exist in our bodies, and they provide a natural repair mechanism for many tissues**
* **In regenerative therapy adult stem cells “remember” the history of their microenvironment “niche”, so they tend less then ES or iPS cells to cause tumors**
* **HSCs are specialised adult multipotent stem cells that give rise to the myriad of cell type that comprises our blood. They arise and differentiate following three important processes during development: 1) primitive haematopoiesis; 2) definitive haematopoiesis; 3) homing**
* **HSCs are located in two niches in the bone marrow (endosteal vs perivascular), where several mechanisms control their self-renewal and differentiation potentia**l
BIOL2045
flashcards
Decks in class (14)
# Cards
-
Introduction to the main cellular components of development53
-
Model organisms26
-
Gametogenesis and Sex determination38
-
Sex determination61
-
Fertilisation41
-
Stem cells62
-
Cleavage to implantation107
-
Vertebrate gastrulation66
-
Extraembryonic membranes66
-
Body plans and segmentation42
-
Neural tube formation43
-
Neural Crest development33
-
Epigenetic control of development95
-
Limb development100
