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Stem Cells and Regeneration > Development and Embryonic Stem Cells (Lectures 1,2,3) > Flashcards

Development and Embryonic Stem Cells (Lectures 1,2,3) Flashcards

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)
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2
Q

Cell proliferation

A

increase in number of cells due to cell growth and division

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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
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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
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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
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6
Q

Primary germ layers

A
  • inner layer –> endoderm
  • middle layer –> mesoderm
  • outer layer –> ectoderm
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7
Q

Totipotent

A

potential to give rise to complete organism including placental part that supports development (zygote)

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8
Q

Pluripotent

A

potential to give rise to all cell types found within the embryo (ES cells)

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9
Q

Multipotent

A

potential to give rise to multiple mature cell types (mesenchymal stem cell; haematopoeitic stem cell)

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10
Q

Unipotent

A

tissue-specific progenitors that replenish a single mature cell type

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11
Q

Ectoderm layer development

A

nervous system, skin

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12
Q

Mesoderm layer development

A

muscle, kidneys, heart

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13
Q

Endoderm layer development

A

gut, lungs, liver, intestine

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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
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15
Q

Directed differentiation

A

telling the cells in a dish what to become

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16
Q

How do genetically identical cells become different cell types?

A

epigenetics

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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
Q

Properties of ESC line

A
  • 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
Q

Classic “stemness” genes

A

Nanog, Oct4, Sox2

27
Q

How to make an ESC line

A
  • isolate ICM from blastocyst
  • culture stem cells
  • undifferentiated embryonic stem cells
  • differentiate into cells from all three germ layers
28
Q

Stem cell culture

A
  • mouse embryo fibroblasts (MEFs) were used to support growth as feeder layer
  • used media from teratocarcinoma cultures
29
Q

Teratoma

A

multi-layered benign tumor that contains cells derived from all three germ layers

30
Q

Applications of a stem cell line

A
  • knowledge of human development
  • understand at molecular level what regulates “stemness”
  • transplantation of cell replacement
  • disease modeling
  • drug development
  • organogenesis
31
Q

What facilitated human ESC culture?

A
  • mouse ESC culture
  • primate ESC culture
  • human IVF
32
Q

Chimeras

A
  • showed ESC cultures can generate all tissues

- addition of Leukemia inhibitory factor (LIF) allowed long term culture of mouse ESCs

33
Q

Major hurdles for generating human ESCs

A
  • 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
Q

Definition of ESC to Thomson

A
  • derivation from the pre-implantation embryo
  • prolonged undifferentiated proliferation
  • stable developmental potential to cells of all three germ layers
35
Q

Immunosurgery

A

selectively removing trophoblast cell layer to isolate the ICM

36
Q

End replication problem

A
  • 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
Q

Critique of Thomson study

A

cell lines not cloned from single cell so could be variation in developmental potential among undifferentiated cells (despite homogenous appearance)

38
Q

Teratoma formation

A
  • inject severe combined immunodeficiency (SCID) mice with presumptive ESCs
39
Q

Teratoma formation

A
  • inject severe combined immunodeficiency (SCID) mice with presumptive ESCs
  • remove, section, and histologically stain tumor that forms
40
Q

Passaging

A
  • 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
Q

Blastocoel

A

fluid filled cavity in blastula

42
Q

Core transcriptional network

A
  • 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

Stem Cells and Regeneration (4 decks)

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