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Cellular Physiology > Stem Cells > Flashcards

Stem Cells Flashcards

1
Q

Where can stem cells be found in the body?

A

Both in the embryo and in the adult body

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2
Q

How do we obtain stem cells?

A

Derive from source then can Culture in lab

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3
Q

Define stem cell

A

Undifferentiated cells that are able to develop into many different cell types.

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4
Q

What constitutes a perfect stem cell?

A
  1. It should be a clonogenic cell
  2. Capable of generating at least 1 differentiated cell type
  3. Self-renewing
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5
Q

Define clonogenic cell

A

Cell is able to clone itself and grow into a full colony of cloned cells

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6
Q

Stem cells are able to develop into many different cell types in the body during 2 distinct life stages. Name them and define their stem cell constituents.

A

Early life phase: embryonic stem cells
Growth phase (adulthood): somatic cells

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7
Q

Stem cells serve as an (1) (2) (3). They do this by dividing without (4) to replenish other cells so long as the person/animal is still alive.

A
  1. internal
  2. repair
  3. system
  4. limit
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8
Q

Through what process are stem cells able to renew themselves?

A

Cell division

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9
Q

Where do adult stem cells reside?

(microenvironment)

A

stem cell niche

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10
Q

What happens to stem cells when we place them under certain physiologic/experimental conditions?

A

They become tissue- or organ-specific cells with special functions.

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11
Q

How do stem cells and cancer cells differ?

A
  1. cancer stem cells undergo uncontrolled proliferation
    AND
  2. have a higher degree of dependence on the stem cell niche
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12
Q

wrt telomeres and proliferation ability

Why are embryonic stem cells significant compared to other cells?

A
  • Can renew and proliferate indefinitely
  • Maintain their telomeres in contrast to normal cells where telomeres shorten after each cell division
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13
Q

Define progenitor cells

A
  • Descendants ofstem cellsthat then further differentiate to create specializedcelltypes (that belong to the same tissue or organ)
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14
Q

How many types of progenitor cells exist in the body

A

There are many types ofprogenitor cellsthroughout the human body.

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15
Q

Are stem cells or progenitor cells less specified? Why?

A

Stem cells are less specified than progenitor cells
= precursors, non-specific and multipotent

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16
Q

Define mesenchymal stem cells (MSCs)

A

A population ofnon-hematopoietic, multipotent stemcells

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17
Q

Where can mesenchymal stem cells (MSCs) be found in the body?

A
  1. bone marrow,
  2. adipose tissue,
  3. liver,
  4. amniotic fluid,
  5. embryonic placenta,
  6. umbilical cord blood,
  7. and other tissues.
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18
Q

Define haematopoeitic stem cells

A

Multipotent primitive cells that can develop into all types of blood cells, including myeloid-lineage and lymphoid-lineage cells

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19
Q

How many stem cell types are found in bone marrow? Name them and what they differentiate into.

A

Contains two different stem cell types:
1. Haematopoetic stem cells (differentiates into red and white blood cells)
2. Mesenchymal stem cells (differentiate into osteoclasts, osteoblasts, adipocytes and chondrocytes)

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20
Q

Where in the bone marrow are haematopoietic stem cells found?

A
  • In the endosteal region of the bone marrow
  • Near blood vessels in the bone marrow
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21
Q

What is the only current FDA-approved therapy using bone marrow stem cells?

A

Bone marrow transplant can be used for treatment of blood diseases (like leukemia)

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22
Q

There are 3 different populations in bone marrow stem cells; name them.

A

*Long-term hematopoietic stem cells
*Short-term hematopoietic stem cells
*Multi potent progenitor cells

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23
Q

Between which two structures do bone marrow stem cells reside?

A

between bone marrow itself and blood

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24
Q

Where are mesenchymal stem cells found?

A

In virtually all tissues

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25
Q

What are the three main functions of MSCs?

A
  1. Play a role in homeostasis
  2. Play a role in repair during disease
  3. Important in making and repairing skeletal tissues
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26
Q

Why are MSCs used in regenerative medicine?

A
  1. Easily cultured in vitro
  2. High proliferation rates
  3. Can become many cell types:
    * Osteoblasts
    * Chondrocytes
    * Adipocytes
    * Hepatocytes
    * Neurons and glial cells
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27
Q

Draw a diagram showing HSCs

A
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28
Q

Draw a diagram showing MSCs

A
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29
Q

How are stem cells and cancer cells similar?

A
  1. Both have self-renewal properties
    AND
  2. Are slow cycling.
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30
Q

Define stem cell niche

A

a specialised microenvironment in which stem cells reside

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31
Q

Why are stem cell niches importamt? (2)

A
  1. Play an essential role in maintaining stem cells
  2. Prevent tumorigenesis by providing inhibitory signals for both proliferation and differentiation
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32
Q

What can a loss of the niche lead to?

A

Loss of stem cells, indicating the reliance of stem cells on niche signals.

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33
Q

What happens when cancer stem cells hijack the stem cell niche?

A
  • self-sufficient cell proliferation –> Invasion and metastasis
  • may also involve deregulation or alteration of the niche
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34
Q

What is the probably cause of cancer stem cells?

A

Intrinsic mutation of stem cells

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35
Q

Define telomere

A

The terminal ends of human chromosomes that contain repetitive, non-coding nucleotide sequences which protects the ends of the chromosome from deterioration or from fusion with neighbouring chromosomes

36
Q

Comment on telomere shortening.

A

Telomere shortening limits stem cell function, regeneration, and organ maintenanceduring ageing and is associated with increasing cancer risk.

37
Q

What are synonyms for mesenchymal stem cells?

A

Mesenchymal stem cells OR mesenchymal stromal cells OR bone marrow stem cells OR bone marrow stroma cells

38
Q

Discuss the history of MSCs

A
  • First isolated in 1966 from mice
  • First human isolates in 1999 from pelvic area
  • 2002 isolated form tibia and femur

Cells it can differentiate into: not only bone/connective tissue, but also glial, muscle hepatic cells

39
Q

What can MSCs differentiate into?

A

bone cells, connective tissue cells, glial cells, muscle cells and hepatic cells

40
Q

Discuss the first clinical case involved in MSCs

A
41
Q

Why is umbilical cord blood stored?

A

Routine procedure as a source of stem cells for future disease due to multi-lineage potential

42
Q

What markers do umbilical cord blood derived stem cells have?

A

neuronal, bone and adipose tissue

43
Q

What are umbilical cord blood derived cells referred to as?

A

non-progenitor cells instead of stem cells

44
Q

What properties/functions do umbilical cord blood derived stem cells have?

A
  1. Immunoregulatory properties: they can suppress lymphocyte proliferation and decrease pro-inflammatory cytokine levels

(Reduce inflammation and inflammatory diseases like fibrosis)

45
Q

What is needed before a cell is denoted as a true stem cell?

A

Presence of CD73, CD90 and CD105 on the cell membranes (Use of flow cytometry and fluorescent image stream analyses)

46
Q

What is CD73?

A

Membrane-bound nucleotidase

47
Q

What is another name for CD73?

A

glycosyl phosphatidylinositol (GPI) - linked, membrane-bound glycoprotein

48
Q

What is the function of CD73?

A
  1. Hydrolyses extracellular nucleoside monophosphates into bioactive nucleoside intermediates.
    (EC nucleoside monophosphatases –> bioactive nucleoside intermediates)
  2. Metabolizes (AMP) to adenosine –> when released = can activate one of four types of G-protein coupled, seven transmembrane spanning adenosine receptors or can be internalized through dipyridamole-sensitive carriers
49
Q

What is CD90?

A

A 25-35kD GPI-anchored cell surface protein, (AKA Thy-1)

50
Q

What family does CD90 belong to? What is it heavily glycosylated with?

A
  1. Belongs to Ig superfamily
  2. Heavily N-glycosylated
51
Q

What domain does CD90 have?

A

Has a single V-like immunoglobin domain

52
Q

What was CD90 initially discovered as? Where does this reside?

A
  1. originally discovered as a thymocyte antigen (hematopoetic progenitor cells present in thymus)
  2. Thy-1 is present in outer leaflet of lipid rafts in cell membrane
53
Q

Where is CD90 mainly expressed, and what is it a potential marker for?

A
  1. mainly in leukocytes and is involved in cell–cell and cell–matrix interactions
  2. liver cancer stem cells
54
Q

What is CD105 also known as?

A

Endoglin (ENG)

55
Q

What is CD105?

A
  • Type-1 membrane glycoprotein located on cell surfaces
  • Part of TGF beta receptor complex
56
Q

What does the presence of CD105 indicaate?

A

powerful marker of neovascularization.

57
Q

What are some successfully treated animal stem cell studies?

A

Intervertebral disc regeneration
Myocardial infarction
Encephalitis
Stroke

58
Q

How do we know if the stem cell therapy works?

A

We can monitor stem cell migration in a few different ways:
* Fluorescence (marker binding),
* MRI or transmission electron microscopy
(superparamagnetic iron oxide nanocomposites
that are internalized by the stem cells) –> With MRI, a whole body scan is done
* TEM = parts of tissue removed, prepared, and sectioned as in
micrograph on the right
* Gold nanotracers monitored
* Stem Cells monitored by ultrasound and photo-acoustic methods (in vivo monitoring of AuNT labelled MSCs)

59
Q

Describe what is occurring in this diagram

A
  • Gold nanotracers = AuNT label the MSCs; causes the gold tracer to be incorporated into the cell membrane and the nanotracer is viewed under a microscope as seen in the diagram
60
Q

Describe what is happening in this diagram

A

Stem Cells monitored by ultrasound and photo-acoustic methods (in vivo monitoring of AuNT labelled MSCs)

61
Q

Describe what happens during photo-acoustic spectroscopy

A

non-ionizing lazerpulses are delivered into biological tissues

(whenradiofrequencypulses are used, the technology is referred to asthermoacoustic spectroscopy)

62
Q

List 2 examples of human stem cell successes

A
  1. Cartilage defects: collagen gel or HA-hydrogel infused by bone marrow mesenchymal stem cells
  2. Liver regeneration with bone marrow mesenchymal stem cells into the hepatic artery
63
Q

How to Isolate Cells Directly from Whole Blood - EasySep Direct Protocol, EasyEights Magnet

A
  • EasySep direct is a fast, column free and fully immunomagnetic platform for isolating cells directly
    from whole blood without RBC lysis, density gradient centrifugation or other pre-processing steps
  • Here the general protocol for using EasySep direct with the EasyEights easy set magnet
  • First: add whole blood to a round bottom tube. If you are isolating myeloid cells EDTA must be added to the blood (please refer to the product information sheet for EDTA concentrations)
  • vortex the easy step direct rapid spheres for about 30 seconds to ensure that they are well mixed and evenly suspended
  • Add the EasySep direct isolation cocktails to the blood sample
  • add the EasySep direct rapid spheres to the sample
  • mix using a pipette. Blood is viscous, so using a pipette will ensure that the EasySep reagents and
    sample are well mixed
  • Incubate at room temperature. Most kits will require a 5 minute incubation but check the product
    information sheet for appropriate times.
  • Top up the sample using the recommended medium.
  • For lymphoid isolations we suggest using calcium and magnesium free PBS. For myeloid cell
    isolations use calcium and magnesium free PBS with one millimole EDTA.
  • The final volume will depend on the sample size and the magnet you are using.
  • Please refer to the magnet specific protocol on the PIS mix by gently pipetting up and down two to
    three times.
  • Place the tube into the magnet without a lid.
  • Make sure the tube is seated fully in the indentation. Incubate at room temperature. Check the
    product information sheet for incubation time.
  • During the incubation, the magnetically labelled unwanted cells and red blood cells will be pulled
    to the back and bottom of the tube by the magnet. Because of the volume of red blood cells in
    whole blood you will observe a blood division forming at the bottom of the tube between the red
    blood cells and the clear supernatant.
  • When using the easy eights magnet the clear supernatant containing enriched cell suspension
    must be prepared off, do not pour off.
  • For the five mil side of the magnet use it 2 mil pipette, for the 14 mil side of the magnet used a 10
    mil pipette.
  • For easier visualization, the EasyEights magnet can be placed at a 45 degree angle.
  • touch the tip of the pipette to the front edge of the tube. collect the clear upper fraction of the
    cell suspension from top to bottom as you slowly move down the front side of the tube
  • take care not to disturb the particles on the magnet side of the tube.
  • as you move down the tube, collect the entire upper fraction. The RBC layer will gradually move
    upwards as the upper clear fraction is pipetted off. If you are isolating myeloid cells such as
    monocytes, neutrophils or other granulocytes there is a fixed collection volume.
  • there will be some red blood cells in this fixed volume. this is to ensure optimal recovery
  • please refer to the product information sheet for the volume of cell suspension which should be
    collected
  • if you are isolating lymphoid cells such as T cells, B cells or NK cells collect the entire upper phase
    from top to bottom
  • moving down the front side of the tube also collect a small volume of the RBC pellet up to 10% of
    the original sample volume
  • transfer the cell suspension into a new tube
  • in this first magnetic separation, the cell suspension may appear quite red because of residual red
    blood cells. If you see this do not be concerned, the majority of RBC’s have been depleted and the
    suspension will clear up in subsequent rounds of separation
  • add EasySep direct RapidSpheres to the new tube containing the enriched cell suspension. Mix
    and incubate at room temperature
  • check the product information sheet for incubation times
  • remove the lid and placed the tube containing the enriched cell suspension and RapidSpheres into
    the EasySept magnet for a second round of separation
  • incubate at room temperature
  • check the product information sheet for incubation times
  • collect the enriched cell suspension using a 2 mil or 10 mil pipette depending on whether you’re
    using the 5 mil or 14 mil side of the magnet
  • this time collect the entire cell suspension. Collect the suspension from top to bottom taking care
    not to disturb the particles. Transfer the enriched cell suspension to a new tube
  • once you remove the old tube from the magnet you will see the smear of unwanted cells and
    particles held along the back and bottom of the tube
  • place the sample back into the magnet for a third round of separation
  • there is no need to add more EasySept particles
  • you will see the cell suspension become less red with each magnetic separation. your enrich cells
    are now ready for use.
64
Q

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

A
  • The overall goal of this protocol is to isolate mesenchymal stromal cells from four distinct
    perinatal tissue sources; the umbilical cord lining, Wharton’s jelly, cord-placenta junction, and
    foetal placenta.
  • This method can help advance the field of stem cell biology, alternative medicine by obtaining
    special quality mesenchymal stem cells, non-invasively.
  • The main advantage of this technique is that it productively yields large numbers of high quality
    homogenous cell populations from distinct perinatal tissue sources.
  • Multi-potent cord mesenchymal stromal cells derived from this technique can be used to treat
    various diseases and disorders since they are more primitive than mesenchymal stem cells
    isolated from adult tissues.
  • This method can provide insight into the characteristics of mesenchymal stromal cells from
    different segments and specific niches of the cord and placenta.
  • Begin by placing the sample in a 150 millimetre petri dish on ice in a bio safety cabinet and use a
    needle and syringe to rinse the tissue several times with ice cold PBS.
  • When all the blood clots have been removed, carefully examine the sample to identify the
    different anatomical regions.
  • Then use forceps to grasp the foetal end of the umbilical cord and use scissors to carefully make
    an incision at the top of the cord-placenta junction.
  • Make a second incision below the junction to separate the cord-placenta junction from the
    placenta.
  • And split the separated tissues into individual petri dishes.
  • Next, cut the umbilical cord longitudinally to completely expose the blood vessels and the
    surrounding Wharton’s jelly without disturbing the epithelium. Use a scalpel to scrape the
    Wharton’s jelly away from the blood vessels and interepithelium of the subamnion.
  • Then, remove the blood vessels, transferring any remaining perivascular jelly under and around
    the blood vessels into the Wharton’s jelly dish and place the remaining cord lining tissue in its own
    dish.
  • When all the tissues have been dissected, replace the PBS in each dish with three to five millilitres
    of trypsin and use scissors to cut each tissue sample into one to two millimetre pieces for a 30
    minute incubation at 37 degrees Celsius and five percent carbon dioxide.
  • Use a phase contrast microscope to observe the partial digestion by visualizing the release of cells
    from the tissue.
  • At the end of the partial digestion period, neutralize the trypsin with an equal volume of culture
    medium and transfer the samples into individual 50 millilitre conical tubes.
  • Allow the tissue pieces to settle for three minutes. Then carefully aspirate the supernatant and
    plate 15 to 20 partially digested tissue pieces per sample into individual 75 square centimetre
    tissue culture flasks.
  • Next, add nine millilitres of culture medium for a two to three day incubation at 37 degrees
    Celsius.
  • Change the culture medium after three days and examine the xplants by phase contrast
    microscopy for cell outgrowth.
  • When the cell growth reaches 70 percent confluency, dissociate the cells in one to two millilitres
    of trypsin solution per flask, rotating the flask for an even coating with the enzyme solution and
    incubate the cultures for three minutes at 37 degrees Celsius.
  • Then neutralize the reaction with one to two millilitres of culture medium.
  • Collect the cells by centrifugation, resuspending the pellet in culture medium for subculture at a
    one times ten to the fourth cells per centimetre squared seeding density.
  • The cells from the cord lining and Wharton’s jelly cultures exhibit colony forming efficiency values
    of 59 and 80 colonies respectively.
  • The colony forming efficiency value of the cord-placenta junction cells, however, is similar to that
    observed for bone marrow mesenchymal stem cells suggesting that cord-placenta junction
    derived cells have higher proliferative and self-renewal capabilities.
  • Flow side a metric analysis of the single cell suspensions isolated from these perinatal tissues
    indicates that the percentages of positive cells for specific mesenchymal stem cell markers from all
    four tissue sources are similar to those expressed by standard bone marrow derived mesenchymal
    cells.
  • The median fluorescence intensity ratios, however, indicate that Wharton’s jelly and cord-placenta
    junction derived cells are very similar or higher than the cord lining in foetal placenta derived cells.
  • Interestingly, in spite of their varying colony forming efficiency values, all of the cells from the
    different perinatal sources express similar levels of pluripotency markers by quantitative RTPCR
    analysis except the cord-placenta junction derived mesenchymal stromal cells, which expressed
    the highest levels of expression for all the tested pluripotency markers.
  • Further, mesenchymal stromal cells isolated from all of the perinatal sources readily differentiate
    into adipogenic, chondrogenic, and osteogenic cell types as well as demonstrated trilineage
    differentiation. Although the potential of differentiation varies according to the mesenchymal
    stromal cell source.
  • Once mastered, this technique can be completed in two to three hours if it’s performed efficiently.
  • While attempting this procedure, it is important to practice lateral technique and avoid cross
    contamination between the tissue sources.
  • This technique paved the way for researchers to explore the nature of stem cell niches in perinatal
    tissues.
65
Q

Can stem cell therapy be harmful?

A
  • Have you seen ads or attended a seminar for stem cell therapies that claim to be able to treat diseases like chronic joint pain, Alzheimer’s, cancer and more? Don’t believe it.
  • You may be told stem cells including those from fat, amniotic fluid or umbilical cord like cord blood or Wharton Jelly’s are miracle treatments.
  • You may even hear they are FDA approved, that is not true. None are proven to work to treat
    these conditions and worse some may cause harm.
  • The FDA has not approved stem cell therapies for treating these serious conditions. The FDA has
    only approved a stem cell product for treating certain blood disorders.
  • Beware, unapproved stem cell therapies have led to serious infections blindness and death.
  • If you are considering a stem cell therapy in the US or outside the US ask questions.
  • Researchers are hoping to use stem cells one day to treat disease
66
Q

Adipose tissue can also be used in stem cell therapy in the form of Adipose Mesenchymal Stem Cells. Why are they used?

A
  • Accessible
  • Plastic-adherent
  • Multipotent
67
Q

What are the different terms used for adipose mesenchymal stem cells?

A

Adipose-derived adult stem cells
Adipose-derived adult stromal cells
Adipose-derived stromal cells
Adipose mesenchymal cells
Preadipocytes
Processed lipoaspirate cells
Adipose-derived stromal/stem cells

68
Q

How do we isolate adipose tissue-derived stem cells?

A
  1. Liposuction
  2. Remove fat blocks
  3. Collagenase centrifuge step to separate cells
  4. Adherence of adipose stem cells to plastic

See table 2 in paper Sousa paper for markers characterization

69
Q

What are adipose stem cells? Where can they be found?

A
  • Mostly mature fat cells and a fraction of vascular stroma
  • Found: pre-adipocytes, fibroblasts, smooth muscle cells, endothelial cells, monocytes/macrophages/lymphocytes
70
Q

Describe adipose stem cell morphology after numerous passages.

A

In a cell culture, they show a fibroblastoid morphology.

71
Q

What is the clinical relevance of adipose stem cells?

A

Used to treat muscle disorders (i.e muscular dystrophy) and bone regeneration

72
Q

What stem cells can be derived from the human endometrium?

A

Menstrual-derived mesenchymal stem cells

73
Q

Name the layers of the endometrium

A
  1. Functional layer - constantly undergoing reconstruction
  2. Basal layer - mostly loos connective tissue
74
Q

Why are menstrual-derived mesenchymal stem cells functionally important?

A
  1. Easy adhesion to plastic
  2. Multipotent
  3. Minimally non-invasive
  4. Can become a variety of cells:
    * Cardiomyocytes
    * Respiratory epithelium
    * Neural cells
    * Myocytes
    * Endothelial cells
    * Pancreatic cells
    * Osteocytes
    * Hepatocytes
75
Q

How are stem cells from the endometrium obtained?

A

Hysterectomy
OR
Diagnostic collecting of menstrual blood (in vessels containing heparin and antibiotics)

76
Q

What are placental stem cells?

A
  1. Type of perinatalstem cellderived from theplacentalblood or tissue.
  2. When the baby is born, blood remains in theplacenta.
  3. Both theplacentalblood and tissue are rich sources ofstem cells.
  4. Likestem cellsfrom bone marrow, umbilical cord blood hematopoieticstem cellshave beenused totreat various genetic disorders including leukemia, certain cancers, and some inherited disorders.
  5. In addition to hematopoietic progenitors and HSCs, theplacentaalso enriches MSCs.
77
Q

When is amniotic fluid collected? What 2 stem cell populations does it contain?

A
  1. collected at 12 weeks of pregnancy
  2. Consists of 2 populations: amniotic fluid mesenchymal stem cells, and amniotic fluid stem cells
    * Contains progenitor hematopoietic cells
    * Non-hematopoietic stem cells which can differentiate into myocytes
78
Q

What are Induced-pluripotent stem cells? (IPSCs)

A

= Somatic cells that can be reprogrammed to the pluripotent stage
= Done via ectopic expression of transcription factors (expression of genes at times and locations where the target gene is not known to have a function)
= Adipocyte stem cells can generate IPSCs

79
Q

How are stem cells and cancer cells similar?

A
  1. Both have self-renewal properties
    AND
  2. Are slow cycling.
80
Q

Define stem cell niche

A

a specialised microenvironment in which stem cells reside

81
Q

Why are stem cell niches importamt? (2)

A
  1. Play an essential role in maintaining stem cells
  2. Prevent tumorigenesis by providing inhibitory signals for both proliferation and differentiation
82
Q

What can a loss of the niche lead to?

A

Loss of stem cells, indicating the reliance of stem cells on niche signals.

83
Q

What happens when cancer stem cells hijack the stem cell niche?

A
  • self-sufficient cell proliferation –> Invasion and metastasis
  • may also involve deregulation or alteration of the niche
84
Q

How do stem cells and cancer cells differ?

A
  1. cancer stem cells undergo uncontrolled proliferation
    AND
  2. have a higher degree of dependence on the stem cell niche
85
Q

What is the probably cause of cancer stem cells?

A

Intrinsic mutation of stem cells

Cellular Physiology (4 decks)

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