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Stem Cells > Midterm 1 Lec 1-12 > Flashcards

Midterm 1 Lec 1-12 Flashcards

1
Q

What are the differences between stem cells and differentiated cells? (4)

A
  • Gene expression (Stemness genes, differentiation gene)
  • Specific functions
  • Tissue formation
  • Responses to extracellular stimuli
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2
Q

What are the three germ layers? (name/ layer/ examples of organs/ organ systems)

A
  • Ectoderm: Outer layer; Skin, nervous system
  • Mesoderm: Middle layer; cardiovascular system, muscle, fat, kidneys
  • Endoderm: Inner layer; lungs, digestive system, liver
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3
Q

What are the phases of development from a zygote to a blastocyst? What are the days each stage occurs? What is the potency of each stage?

A
  • Fertilization of egg: Day 0 Totipotent
  • First Cleavage: Day 1 Totipotent
  • 2 cell stage: Day 2 Totipotent
  • 4 cell stage: Day 3-4 Totipotent
  • 8 cell UNCOMPACTED morula: Day 4 Totipotent
  • 8 cell COMPACTED morula: Day 4 Totipotent
  • Early Blastocyst: Day 5 Pluripotent
  • Late stage blastocyst: Day 6-7 Pluripotent
  • Implantation of blastocyst: Day 8-9 Pluripotent
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4
Q

What are the properties of stem cells ?

A
  • They have the ability to self renew and potency
    • Self renewal means that they are able to do cell division/ create daughter cells
    • Potency is the ability/potential to differentiate into a specific cell type (approx 200 different types of differentiated cells
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5
Q

What are the steps needed for a stem cell to become a differentiated cell?

A

Stem cell → progenitor cell (AKA intermediate cell) → specialized/ differentiated cell

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6
Q

What is the difference between a stem cell and a progenitor cell?

A
  • A stem cell has the ability to divide into more stem cells or progenitor cell/specialized cells (Asymmetric or symmetric cell division)
  • A progenitor cell can only divide into cells that are from one tissue or organ meaning they are partially differentiated. (symmetric cell division only)
  • Asymmetric cell division: 1 stem cell and 1 differentiated cell
  • Symmetric cell division: 2 differentiated cells or 2 stem cells
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7
Q

What is necessary for stem cells to become differentiated cells and vice versa?

A
  • Reprogramming of the cell needs to occur. This can be done through gene expression, hormonal signals, and/or environmental signals.
  • In vitro the conditions are important to the outcome/ final product.
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8
Q

How does gene expression dictate whether a cell is a differentiated cell or stem cell?

A
  • When a stem cell → differentiated cell there is an increase in differentiation gene expression and a decrease in stemness genes.
  • When a differentiated cell → Stem cells theres is a decrease in differentiation gene expression and and increase in Stemness genes
    • Stemness genes control self renewal and potency (properties)
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9
Q

How does the expression of stemness genes and differentiation genes change as a cell differentiates?

A
  • Stem cell stage: high Stemness gene expression, low differentiation gene expression
  • Progenitor cells stage: less stemness gene expression, more differentiation gene expression
  • Differentiated cell: Low/no stemness gene expression, high differentiation gene expression
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10
Q

How are stemness genes expressed?

A
  • They are encoded into Transcription factors that bind to promoters to target genes to stimulate mRNA synthesis.
  • The TF sits on the DNA at a transcription binding site in a sequence specific manner. When the transcription factor and the sequence bind together it turns on RNA for that gene. If the sequence isn’t right or mutated it won’t work.
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11
Q

What are the 4 types of tissue and their function/location?

A
  • Muscle Tissue
  • **Epithelial Tissue **: molecules can’t go between the cells, they need to go through. small intestines
    • Multiple organ dysfunction: The barrier function fall apart
  • Nervous Tissue
  • Connective Tissue : have different properties
    • Solid (bones), jelly like (fat), liquid (blood plasma)
    • Mesenchymal stem cells: Found in fat and yellow bone marrow, can form ANY kind of tissue
      • In Vitro can make many different types of cells
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12
Q

What are some limitations of stem cell therapies for multiple sclerosis ?

A

Mice with MS symptoms → human ESC injected into spinal cord → Some improvement, damage to myelin sheath repaired, loss of stem cells
- Using human stem cells in mice doesn’t tell us if it will/won’t work in humans
- “Off target” effect of injecting stem cells
- Potential differentiation of cells into non nerve cells
- Stability of nerve cells made from stem cells

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13
Q

What are the parts of a blastocyst?

A
  • TrophoblastSurrounding
  • Blastocyst cavity/ fluid
  • Inner cell mass
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14
Q

What are the different cell potencies? What can they differentiate into?

A
  • Totipotent : Embryonic and extra embryonic cells (Placenta)
  • Pluripotent : any cell type from 3 germ layers
  • Multipotent : limited to one organ/tissue/1 germ layer
  • Unipotent : limited to one cell type
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15
Q

What potency do embryonic stem cells have?

A
  • Totipotent
  • Pluripotent
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16
Q

What potency does fetal tissue stem cells have?

A

Multipotent

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17
Q

What potency do adult stem cells have?

A
  • Multipotent
  • Unipotent
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18
Q

What are components of the microenvironment?

A
  • Cell-cell interaction
  • Hormone signals
  • Growth factors
  • Ions, pH
  • Extracellular matrix and adhesion molecules
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19
Q

How is a new Human Embryonic Stem Cell line created? (3)

A

1) Inner cell mass is obtained from a donated human embryo
- Usually from extra IVF blastocyst
2) The inner cell mass is separated into individual cells and are grown on ECM in a petri dish
- Contains Cell culture media (nutrients and signals) and Extracellular matrix (collagen)
- Grown at 37c (human body temperature)
3) After a few days the Human Embryonic stem cells divide and grow colonies. The colonies are separated and placed into new petri dishes
- If the cells continue to divide they are health and show the self-renewal (cell division) property of Stem cells

  • When a healthy cell culture is established it is a embryonic stem cell line
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20
Q

How do you test for pluripotency?

A

Check for the formation of teratoma using mice
- Teratoma: Benign tumor that contains tissue from all 3 germ layers (only pluripotent cells can produce cells from all 3 layers)
- Injected under the skin of mice

Differentiate in vitro
- 1) place in minimal media w/ some stem cell signals removed → see if the cells spontaneously differentiate into cell from 3 germ layer
- 2) Direct differentiation: add correct/ known signals to guide differentiation for a specific cell type → check if the intended cells were grown

Test gene expression
- Remove mRNA from human ESC → sequence it (tells what genes are being expressed) → compare it to sequenced lines → Check if the cells are expressing stemness genes

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21
Q

What are the necessary conditions for cell cultures? ( used now?, first use?) (3)

A

Nutrients: Cell culture media (sugars, amino acids, vitamins and other molecules)
- First cultures used serum from cow’s blood → blood was removed and serum was extracted

Signals for cell division: Cell culture media (contains nutrients and signals)
- First used conditioned medium (liquid that embryonal carcinoma cell grow in) → remove cells from embryonal carcinoma and collected extracellular matrix

Extracellular matrix: synthetic ECM, brand name: Matrigel (tissue for the stem cells to attach to and grow on)
- First used a layer of mouse fibroblast cells (secretes extracellular matrix made of collagen)

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22
Q

What are the necessary conditions for cell cultures? ( used now?, first use?) (3)

A

Nutrients: Cell culture media (sugars, amino acids, vitamins and other molecules)
- First cultures used serum from cow’s blood → blood was removed and serum was extracted

Signals for cell division: Cell culture media (contains nutrients and signals)
- First used conditioned medium (liquid that embryonal carcinoma cell grow in) → remove cells from embryonal carcinoma and collected extracellular matrix

Extracellular matrix: synthetic ECM, brand name: Matrigel (tissue for the stem cells to attach to and grow on)
- First used a layer of mouse fibroblast cells (secretes extracellular matrix made of collagen)

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23
Q

What are the properties of adult stem cells? (3)

A
  • Proliferation and self renewal
  • Relatively unspecialized/undifferentiated cells
    • Make stemness genes but they are NOT the same as those expressed by embryonic stem cells
  • Can form specialized/ differentiated cell types
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24
Q

What factors are important in the regulation of stem cell characteristics?

A
  • Cell-cell interactions (between stem cells or stem cells and differentiated cells)
  • Presence of adhesion molecules and extracellular matrix components
  • Hormone growth factors and cytokines that target stem cells
  • Secreted factors from differentiated cells
  • Physicochemical nature of the environment: pH, oxygen tension, ionic strength, ATP
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25
Q

What are 4 ways isolated adult stem cells can be obtained?

A

From the body itself
- Mesenchymal stem cells
- Bone marrow stem cells
From pluripotent stem cells
Transdifferentiation from other adult stem cells
From amniotic fluid/placenta

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26
Q

What role do stem cells have?

A

Maintain and repair tissue
- Though constant cell division + differentiation
Some remain dormant for years, only dividing and generating new cells when they are activated by tissue injury or disease
- In degenerative diseases stem cells are not released quickly enough to repair damaged tissue

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27
Q

What is a stem cell niche?

A
  • A area of a tissue that provides a microenvironment for stem cells in an undifferentiated and self renewable state
  • Each niche promotes a specific microenvironment for a stem cell that regulates the fate of that stem cell
  • Can be In Vivo or In Vitro
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28
Q

What is a stem cell microenvironment? What does it control?

A
  • Surrounding molecules and compounds (nutrients, growth factors signals) surrounding a cell
  • Differences in microenvironment control differentiation
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29
Q

What does a stem cell niche control?

A

It controls whether a stem cell…
- Proliferates (divides)
- Remains dormant
- Participated in tissue generation, maintenance or repair
- Express stemness genes or differentiation genes
- Survival

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30
Q

What interactions do cells have with their microenvironment?

A
  • Dynamic
  • The surrounding microenvironment actively signal to stem cells to promote either self renewal or differentiation to form new tissue
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31
Q

How does a change in the microenvironment affect a stem cell?

A

It can alter the gene expression and properties of stem cells
- Change in microenvironment= change in stem cell lineage
- Stemness genes vs. differentiation genes
- Control lineages by controlling microenvironment ( In Vitro)

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32
Q

How can the In Vivo microenvironment be mimicked in Vitro?

A

Nutrients, hormone signals, grow cell on a specific matrix

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33
Q

How are stem cells obtained from pluripotent stem cells?

A

The pluripotent stem cell’s gene expression, lab cell culture environment, or the tissue microenvironment is altered.

34
Q

How do stem cells from one tissue differentiate into cells of another tissue? What is an example?

A
  • Bone marrow stem cells have been injected into mice with damaged heart, this has repaired the heart
    • It is not known if the results are because of transdifferentiation or the secretion of hormone factors which signal other cells to begin the repair process
35
Q

What are some clinical issues involving adult stem cells?

A
  • Making sure they express the correct characteristics
  • Producing them in large quantities
  • Differentiate into specific tissues
  • Show that stem cells are safe to use
  • The adult stem cells need to stay alive and functional long enough to maintain a healthy tissue/organ
36
Q

What are some experimental ways to confirm that adult stem cells are stem cells?

A

Demonstrate that the isolated stem cells can proliferate and renew in a cell culture

Check that the cells are expressing the correct genes and respond correctly to the environment

Label the isolated stem cells with marker/dye and implant back into the animal to check of the cells replaces their tissue
- If the dye is still present the cell repopulated the tissue
- If not, differentiation could be due to secreted factor

37
Q

What are adult stem cells? Where are they found?

A
  • Somatic stem cells
  • Relatively rare undifferentiated cells that vary in differentiation capacity and are able from many different kinds of tissues and more specialized cells
  • Found in many organs and tissues
38
Q

How can adult stem cells be extracted? What are the benefits?

A

The adult stem cells that come from the patient have no (or reduced) possibility of rejection

By using a patient’s own stem cells it eliminates any ethical or moral issues

Isolate the stem cells from the patient → check gene expression of stem cells→ Culture In Vitro (w/ the correct nutrients, signals and matrix) → Differentiate the adult stem cells (using the appropriate microenvironment) → Check gene expression (look for appropriate differentiation genes) → Transplant back into the patient

39
Q

What are the parts of an intestinal lumen?

A
  • Top half- Villus
  • Bottom Half- crypt
40
Q

What are the cell types of an intestinal lumen? What are examples?

A
  • Bottom: stem cells → Crypt base columnar (CBC), 4+ cells
  • Middle: Progenitor cells
  • Top: Differentiated cells → absorptive cells
41
Q

How are mesenchymal stem cells isolated from fat cells?

A
  • The fat and mesenchymal stem cells are held together by collagen fibers
  • The enzyme collagenase is used to cut the collagen fibers
  • Centrifuge the cells to separate the differentiated fat cells from the stem cells. The differentiated cells will float to the top while the stem cells will sink to the bottom.
  • The stem cells can then be collected, cultured and grown
42
Q

How are Bone Marrow stem cells isolated?

A

Fluorescent Activated Cell Sorting (FACS) is used

Bone Marrow stem cells have a specific marker proteins, CD133

An antibody that recognizes CD133 contains a fluorescent tag

The cells are run through a flow cytometer where a laser deflects cells with the fluorescent tagged antibody
- The deflected cells are the stem cells → They are collected and grown in cell culture

43
Q

Why use adult stem cells?

A
  • Immune compatibility→ autologous
  • Wide variety of cells with less steps needed to differentiate
  • Less expensive
  • Little to no ethical issue
  • Certain tissues are easily obtainable and can be differentiated into many types of cells
44
Q

How is gene expression regulated?

A

The TFs that bind to the gene promoter → Changes in TF binding combinations (TF 1 and TF 2 VS TF 3 and TF 2) can change the magnitude and/or direction (INCREASE/DECREASE) of gene expression

Modification of the protein, line in phosphorylation which is the removal of a phosphate group on ATP which is given to the protein (substrate)
- The process can be activated by extracellular growth factors/hormones present in the microenvironment

45
Q

How are differentiation genes expressed?

A

A co-activator must be binded to the correct transcription factors

46
Q

What is Sox2? What is its function?

A
  • Its a transcription factor that has about 20 gene members
  • It is considered a stemness transcription factor and important for maintaining stem cell properties like pluripotency and self renewal
    • Highly expressed in oocytes, totipotent stem cells and the inner mass cells
47
Q

What is Sox2? What is its function?

A
  • Its a transcription factor that has about 20 gene members
  • It is considered a stemness transcription factor and important for maintaining stem cell properties like pluripotency and self renewal
    • Highly expressed in oocytes, totipotent stem cells and the inner mass cells
48
Q

What happens if you overexpress or delete Sox2?

A
  • Overexpress: Selective differentiation occurs (Induced pluripotent stem cells)
  • Delete: Loss of pluripotency and self renewal
48
Q

What happens if you overexpress or delete Sox2?

A
  • Overexpress: Selective differentiation occurs (Induced pluripotent stem cells)
  • Delete: Loss of pluripotency and self renewal
49
Q

What is a chromatin?

A

A mixture of DNA and proteins(histones) that form the chromosomes

50
Q

What are nucleosomes?

A
  • A basic repeating subunit of chromatin packaged together
    • Composed of histones (+ charged protiens) and a small segment of DNA (- charge)
51
Q

What is a closed/open chromatin structure?

A

Closed: Incapable of transcription (mRNA synthesis)
- Prevents binding of more TF
- TFs CAN NOT find DNA binding sites because the beads are too close together

Open: capable of transcription (mRNA synthesis)
- Allows the binding of TFs, co regulators and transcription machinery
- TFs CAN find their DNA binding sites

52
Q

How do co-regulatoes play a role in maintaining chromatins open or closed?

A
  • Co-activators: Direct the acetylation of histones which reduces the positive charge (more negative) meaning it is open → because DNA is - meaning they don’t repel each other as much
  • Co-repressors: direct deacetylation and keeps the chromosomes closed → + charge means it is repealed by DNA
53
Q

What allows Sox2 to bind to closed chromatin?

A
  • It’s a pioneer factor (TF)
  • Sox2 binds to the closed chromatin and brings in co activators
  • This opens the chromatin structure allowing for other TFs and co-regulators to bind to the chromatin
54
Q

How are stemness genes expressed?

A
  • Stemness gene transcription factors like Sox2 require a transcription factor to regulate gene expression
  • When a co-repressor or co-activator bind to the TF it turn transcription off/on
    • Adult stem cells and ESC express some TFs in common
55
Q

What happens if Sox2 is removed during the process of development?

A
  • The zygotic deletion of sox2 is lethal after implantation
  • It has a critical role in the formation of the inner cell mass → So it would stop development
56
Q

What is gene expression?

A
  • It is the flow of information from DNA to produce function proteins that carry out and control cellular activity.
  • In eukaryotes one gene (info on a section of DNA) encodes for one protein
57
Q

What is a transcription factor?

A
  • Proteins that are need to initiate the transcription process
  • They bind to specific DNA sequences and can bind to other proteins
58
Q

What is a coregulator?

A

They are necessary to the activation of transcription through the transcription factor. They bind to a specific amino acid sequence in the TF and interact with the transcriptional machinery.

59
Q

What is RNA polymerase (and the transcriptional machinery) ?

A
  • It is an enzyme that makes mRNA that is complementary to DNA.
  • It is part of a complex of proteins that help it
  • The transcriptional machinery is a protein complex that includes RNA polymerase
60
Q

What is a gene promoter?

A

A sequence of DNA to which proteins bind to initiate transcription

61
Q

What is a co-activator and a co-repressor?

A
  • Simulated mRNA synthesis
  • Inhibits mRNA synthesis
62
Q

Why is Sox2 important?

A

Helps control pluripotency, differentiation and the silence of differentiation genes

63
Q

How are induced plutripotent stem cells obtained?

A
  • iPSC are retrieved from a patients differentiated cells
  • usually made from skin fibroblasr because they are easy to obtain
64
Q

What are iPSC?

A

They are stem cells that are dedifferentiated from differenitated somatic cells

65
Q

How are iPSC made?

A

Skin Fibroblasts are removed from a patient
- They have stemness genes but don’t express them becasue they are in CLOSED chromatin

Reprogramming factors are used to casue dedifferentiaton → making the cells express stemness genes
- The reprogramming factors include Sox2

66
Q

How are the reprogramming factors introduced into the fibroblast?

A
  • A virus (removed bad genes) with the added DNA for reprogramming factors is used to infect the fibroblast
  • The fibroblast is then able to express reprogramming genes → The Transcription factors are then able to turn on stemness gene expression and turn off expression of genes involved in fibroblast differentiation
67
Q

How does Sox2 play an imporant role in reprogramming the genome?

A

The stemness genes are normally in closed chormatin when the virus is added Sox2 is expressed openning up the stemnesss genes allowing them to be expressed

68
Q

What are some limitations of iPSCs?

A
  • Cancer potential if iPSC aren’t differentiated correctly
  • We don’t know the long term effects
  • its time consuming and expensive
  • They don’t express the same set of stemness genes as ESCs due to Epigenetic memory
69
Q

What are some medical uses for iPSC?

A
  • treating degenerative diseases
  • use them as models of human diseases and drug screenings
    • In Vitro add make the cells have disease and add the correct cells to cure disease with medicine
70
Q

What is CRISPR/Cas 9?

A

It is an enzyme that cuts DNA at a specific location

71
Q

How gene editing work?

A

Gene editing involves 2 processes

1) Cut the DNA in a cell at a specific location (like within a particular gene)

2) the cell then repairs the cut DNA. wehen the DNA is repaired, small deletion can be made OR the DNA sequence can be altered

72
Q

What is CRISPR/Cas 9 role in gene editing? What are the main components?

A
  • It cuts DNA at a precise location
  • 1) Cas9 is an enxyme thaat cuts DNA
  • 2) sgRNA is single guide RNA that guides cas9 to the specific DNA sequence that we want to cut
  • It relies on the fact that an RNA molecule is complementary to a DNA sequence will bind that specific DNA
73
Q

How does CRISPR/Cas 9 actually work to edit DNA?

A
  • Researchers synthesize sgRNA that is complementary to the DNA they want to edit
  • They add the sgRNA and Cas9 into the cell
  • The sgDNA guides cas9 to that one specific sequence in the genome and Cas9 cuts the DNA
74
Q

How does the DNA repair lead to gene editing?

A

once the DNA is cut there are two ways to repair the DNA

  • 1) Impercise method; The two end of the DNA are stuck back together losing a few bases of DNA
    • this can cause the gene to become non functional
    • useful to stop a bad copy of a gene from being expressed
  • 2) Precise method: Cell can use another sequence of DNA asa template for repairing the cut DNA
75
Q

What are some potential problems of uisng CRISPR in humans?

A
  • Off target effects → Not cutting the right sequence in the genome
    • Can lead to new mutations
  • Getting the Cas9 and sgRNA into the human cell
    • Cas9 is a bacterial enzyme not made naturally by humans
76
Q

How is CRISPR being used to treat sickle cell anemia?

A
  • patients w/ sickle cell have their HSC Hb* (mutated cells) removes→ in vitro the cas9, sgRNA complex and template DNA are added
    • The cas9 cuts the mutated gene through a precise edit → the edited HSCs are grown and transplanted back into the patient

→ There is no change in gene expression of offspring because the edits aren’t done on sperm or egg

77
Q

How can CRISPR/Cas9 be used in the genetic editing of embryos?

A

In IVF one cell can be removed to check for genetic abnormalities ← Pre implantation genetic diagnosis

78
Q

What is the problem with editing embryos?

A

mosaicism- some cells are edited and others are not/ edited in different ways

79
Q

What are some ethical issue with the human germline engineering?

A
  • Mosaicism
  • off target effects
  • long term effects → in future generations
  • bad informed consent
  • secrecy
  • no medical reasoning

Stem Cells (3 decks)

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