Stem Cells Flashcards

Question

What are the three main functions of MSCs?

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

a specialised microenvironment in which stem cells reside

Answer 5

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

Answer 6

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

Answer 7

* self-sufficient cell proliferation --> Invasion and metastasis * may also involve deregulation or alteration of the niche

Answer 8

Intrinsic mutation of stem cells

Answer 9

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

Answer 10

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

Answer 11

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

Answer 12

* 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

Answer 13

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

Answer 14

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

Answer 15

neuronal, bone and adipose tissue

Answer 16

non-progenitor cells instead of stem cells

Answer 17

1. Immunoregulatory properties: they can suppress lymphocyte proliferation and decrease pro-inflammatory cytokine levels (Reduce inflammation and inflammatory diseases like fibrosis)

Answer 18

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

Answer 19

Membrane-bound nucleotidase

Answer 20

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

Answer 21

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

Answer 22

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

Answer 23

1. Belongs to Ig superfamily 2. Heavily N-glycosylated

Answer 24

Has a single V-like immunoglobin domain

Answer 25

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

Answer 26

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

Answer 27

Endoglin (ENG)

Answer 28

* Type-1 membrane glycoprotein located on cell surfaces * Part of TGF beta receptor complex

Answer 29

powerful marker of neovascularization.

Answer 30

Intervertebral disc regeneration Myocardial infarction Encephalitis Stroke

Answer 31

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)

Answer 32

* 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

Answer 33

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

Answer 34

non-ionizing lazer pulses are delivered into biological tissues (when radiofrequency pulses are used, the technology is referred to as thermoacoustic spectroscopy)

Answer 35

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

Answer 36

* 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.

Answer 37

* 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.

Answer 38

* 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

Answer 39

* Accessible * Plastic-adherent * Multipotent

Answer 40

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

Answer 41

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

Answer 42

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

Answer 43

In a cell culture, they show a fibroblastoid morphology.

Answer 44

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

Answer 45

Menstrual-derived mesenchymal stem cells

Answer 46

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

Answer 47

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

Answer 48

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

Answer 49

1. Type of perinatal stem cell derived from the placental blood or tissue. 2. When the baby is born, blood remains in the placenta. 3. Both the placental blood and tissue are rich sources of stem cells. 4. Like stem cells from bone marrow, umbilical cord blood hematopoietic stem cells have been used to treat various genetic disorders including leukemia, certain cancers, and some inherited disorders. 5. In addition to hematopoietic progenitors and HSCs, the placenta also enriches MSCs.

Answer 50

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

Answer 51

= 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

Answer 52