Development and Embryonic Stem Cells (Lectures 1,2,3) Flashcards
(42 cards)
1
Q
Stem cell definition
A
- capable of self-renewal through cell proliferation
- stem cells are an unspecialized cell type
- stem cells are capable of differentiating into a specialized cell type
(needs to make 200+ different types of cell types)
2
Q
Cell proliferation
A
increase in number of cells due to cell growth and division
3
Q
Ernst Haeckel
A
- first use of the term stem cell –> “stammzelle” (1868)
- – the fertilized egg that gives rise to the entire animal (the idea that all cells come from this one cell (pluripotent))
- the use of teratorcarcinomas (teratomas) contain undifferentiated cells called embryonal carcinoma (EC) cells –> EC cells are pluripotent with cancer forming properties
- – historically, this was the standard for which pluripotency was studied and allowed the development of culture conditions on ES cells
4
Q
Development
A
- we all come from a single cell, the zygote
- fertilization –> zygote
- cleavage –> blastomeres
- totipotent morula
- blastocyst – pluripotent ICM + trophoblast
- rearrangement of blastocyst into primary germ layers (gastrulation) –> ectoderm + mesoderm + endoderm
- 10 trillion adult cells
5
Q
Amphibian embryo development
A
- sperm enters animal pole
- sperm entry zone determines ventral part of embryo
- egg is polarized with yolk on bottom
- cleavage generates blastomeres
- blastula
- gastrulation begins at the blastopore
6
Q
Primary germ layers
A
- inner layer –> endoderm
- middle layer –> mesoderm
- outer layer –> ectoderm
7
Q
Totipotent
A
potential to give rise to complete organism including placental part that supports development (zygote)
8
Q
Pluripotent
A
potential to give rise to all cell types found within the embryo (ES cells)
9
Q
Multipotent
A
potential to give rise to multiple mature cell types (mesenchymal stem cell; haematopoeitic stem cell)
10
Q
Unipotent
A
tissue-specific progenitors that replenish a single mature cell type
11
Q
Ectoderm layer development
A
nervous system, skin
12
Q
Mesoderm layer development
A
muscle, kidneys, heart
13
Q
Endoderm layer development
A
gut, lungs, liver, intestine
14
Q
Waddington landscape
A
- pluripotent cells lose developmental “plasticity” through the process of differentiation
- going down a mountain is easier than going up (but not impossible)
- – in each split in the trail, you lose potential
- trough for pluripotent cell is super shallow and each time you differentiate the trough gets deeper
15
Q
Directed differentiation
A
telling the cells in a dish what to become
16
Q
How do genetically identical cells become different cell types?
A
epigenetics
17
Q
Epigenetics
A
- specific modifications of DNA or histones that affect the activity state of genes without changing the DNA sequence
- – e.g. DNA methylation, histone modifications, miRNA regulation
18
Q
Signal transduction
A
- transmission of a molecular signal from the cell’s exterior to interior
- signal –> reception –> transduction –> response
19
Q
The tools of developmental/stem cell/regenerative biology
A
- find it
- – determine where a structure or gene of interest is present/expressed in an embryo (transcriptomics, ISH, IHC)
- lose it
- – determine necessity of a structure or gene of interest by removing from an embryo (surgical removal, gene knockout, morpholino knockdown, Crispr/Cas9); also termed a loss of function experiment
- move it
- – determine sufficiency of a structure or gene of interest by moving to a naive area of an embryo (surgical grafting, gene overexpression); also termed a gain of function experiment
- cutting –> loss or ablation of gene function (loss of function); e.g. knocking down gene expression or knocking out gene from genome
- pasting –> express gene extraneously (gain of function); e.g. over-expressing a gene during development
- painting –> express GFP in gene or stain using immunohistochemistry or in situ hybridization; e.g. labeling for specific RNA or protein or tagging of endogenous molecules
20
Q
ICSI
A
- intra-cytoplasmic sperm injection
- used for IVF
- at time of implantation, the mammalian blastocyst has differentiated into three distinct linages (not germ layers)
21
Q
Blastocyst to gastrulation
A
- epiblast gives rise to entire fetus
- trophectoderm/trophoblast gives rise to placenta
- yolk sac supplies nutrients to early embryo
22
Q
Molecular decisions to differentiate ICM from trophectoderm
A
- at 8-cell stage there are low levels of both Cdx2 and Oct4
- at morula stage outer cells express Cdx2 (tophectoderm) and inner cells express Oct4, with low levels of Nanog and Gata6 (ICM)
- the early stage blastocyst ICM cells express Nanog and Gata6
- the late stage blatocyst ICM inner cells express Nanog (epiblast) and the outer cells express Gata6 (primitive endoderm)
23
Q
Hippo/Yap signaling pathway
A
- if cell is on outer edge of morula –> the inactivation of yap turns on Cdx2, inhibiting enhancer of Oct4, turning it into the trophectoderm (yap was in nucleus)
- if cell on inside of morula –> the activation of yap does not turn on Cdx2, not inhibiting Oct4, turning it into the ICM (yap was not in nucleus)
- if yap is activated (ICM) –> Hippo on
- if yap is inactivated (trophectoderm) –> Hippo off
24
Q
Properties of ESCs
A
- derived from pre-implantation embryo
- immortal self-renewal
- ability to differentiate into cell types of all three germ layers
25
Properties of ESC line
- has longterm self-renewal
- expresses classic "stemness" genes Oct4, Nanog, Sox2
- no chromosomal abnormalities
- culture differentiates spontaneously and under directed differentiation
- cell line is capable of generating teratomas
26
Classic "stemness" genes
Nanog, Oct4, Sox2
27
How to make an ESC line
- isolate ICM from blastocyst
- culture stem cells
- undifferentiated embryonic stem cells
- differentiate into cells from all three germ layers
28
Stem cell culture
- mouse embryo fibroblasts (MEFs) were used to support growth as feeder layer
- used media from teratocarcinoma cultures
29
Teratoma
multi-layered benign tumor that contains cells derived from all three germ layers
30
Applications of a stem cell line
- knowledge of human development
- understand at molecular level what regulates "stemness"
- transplantation of cell replacement
- disease modeling
- drug development
- organogenesis
31
What facilitated human ESC culture?
- mouse ESC culture
- primate ESC culture
- human IVF
32
Chimeras
- showed ESC cultures can generate all tissues
| - addition of Leukemia inhibitory factor (LIF) allowed long term culture of mouse ESCs
33
Major hurdles for generating human ESCs
- having experience with primate blastocysts and ICM isolation
- culture conditions a bit different than mouse ESC
- obtaining private funding
- obtaining quality blastocysts from IVF clinics
- being willing to push ethical hurdles of the time
34
Definition of ESC to Thomson
- derivation from the pre-implantation embryo
- prolonged undifferentiated proliferation
- stable developmental potential to cells of all three germ layers
35
Immunosurgery
selectively removing trophoblast cell layer to isolate the ICM
36
End replication problem
- telomeres shorten over time due to cell divisions, and somatic cells go into cell senescence and stop dividing once their telomeres get too short
- ESC lines needed to have telomerase to make sure they would not encounter this problem
37
Critique of Thomson study
cell lines not cloned from single cell so could be variation in developmental potential among undifferentiated cells (despite homogenous appearance)
38
Teratoma formation
- inject severe combined immunodeficiency (SCID) mice with presumptive ESCs
39
Teratoma formation
- inject severe combined immunodeficiency (SCID) mice with presumptive ESCs
- remove, section, and histologically stain tumor that forms
40
Passaging
- ESC colony lives 6-12 months, demonstrating self-renewal
- original passaging was highly inefficient
- now it is slightly better, allowing more people to work with human ESCs
41
Blastocoel
fluid filled cavity in blastula
42
Core transcriptional network
- oct4, sox2, nanog are stemness genes that help regulate self renewal and pluripotency
- in embryonic state, stemness genes have positive transcriptional role
- polycomb group silences the stemness genes since they are not used in differentiation into germ layers
