Stem Cells 1 Flashcards
What are stem cells defined by
Function
What are some self renewing organs/tissues
Skin, blood, intestines
Describe skin
Surface of body covered by dead cells
Intact skin for life
Protect it from mechanical force, pathogens and for evaporation fo water
Describe normal blood smear
No nucleus
Need blood forever
Can regenerate
Describe epithelium of small intestine
Structure where cells proliferate - crypts and differentiate as move up and get function required
Digest, absorb, more
Sloughed off ever 5-7 days
Stem cells support cell turnover
Describe stem cells def
Cells that can regenerate and maintain for a life time a complete structure and function of adult tissue
Based on functions
Describe hematopoietic stem cells
Maintenance of stem cell pool and all types of blood cells for a lifetime
Describe stem cells secondary features
Important stem cell markers but secondary to functional definition
Describe tissue specific adult stem cells
Bone marrow- blood
Breast, mammary gland
Brain - nervous system
Fatty tissues
Heart
Pancreas
Skin teeth
Describe tissue specific adult stem cells - other sources
Testis, liver, muscle, intestinal epithelium, retina
Describe stem cells of embryonic origin
Es cells - blastocyst
Artificial in in vitro cells = major focus
Name 3 characteristics of stem cells
Self renewal
Differentiation
Long term
Describe self renewal of stem cells
Reproduce themselves
For maintenance of stem cell pool
Describe differentiation of stem cells
Generate daughter cells that are committed to differentiation
For production of terminally differentiated cells
Describe long term activity of stem cells
Unique to stem cells
Self renewal and differentiation activity are sustained throughout life
Produce all cell types of a given cell lineage
What are stem cells define by
Functions
What are stem cells in cell lineage
Most primitive cell type in cell lineage and support normal homeostasis of tissue and cell lineage
What arises from one stem cell
A series of daughter cells arise from one stem cell
Stem cells produce clonal cell populations
If something happens to stem cell = will inherit problems genetically
Describe terminally differentiated cells
Conduct tissues/organ specie functions
Stem cells = producers not WORKErs
Describe sickle cell anaemia
Adaptation against malaria, point mutation in beta globin gene = globin starts aggregating = deforms rbcs, mutation affect even hematopoietic cells, but phenotype only in rbcs since only cells that express globin
Are there many stem cells
Few
Describe what progeny of stem cells do
Gradually lose differentiation ability
Stem cells = can become all types then gradually lose potency = ability to become diff types of cells
Chooses linage and is restricted to it
What is lineage restriction
Differentiation potential comes restricted
Describe mitotic catastrophe or crisis
Self renewal potential
Will become gradually restricted
Committed cells = with each division = less and less telomeres
Describe stem cell compartment
Hematopoietic stem cells represent only 0.01% of unucleated cells in marrow
Very slowly cell cycling = only 8% of all hematopoietic stem cells enter mitosis daily, much telomeres
<0.007% - nucleated = exclude stem cells
Small fraction of cell lineage
Infrequent division = less likely genetic mutations, helps stem cell keep genetic integrity
How do stem cells support a cell lineage
By their long term, dual function, self renewal and commitement
How are descendents of stem cells produced
In a step wise manner and each step gradually loses their differentiation capacity - lineage restriction
Describe stem cells vs descendants
Stem cells represent small sub pop of a cell linage and dive infrequently
Descendants proliferate more rapidly, compensate stem cells weaknesses
Do stem cells carry out tissue functions
NaaaaaaaaAAhhhhhhhhhhhhhh
Describe totipotent stem cells
They can generate all cell types in an organism, including placenta
Only fertilized egg belongs to this category = zygotes can become all tissues = extraembryonic and embryo
Describe pluripotent stem cells
Cells can generate all cell types in body (also some extra embryonic tissues), NO PLacenta
Equivalent to cells of icm
Ex = embryonic stem cells, embryonic carcinoma cells
Describe unipotent stem cells
These cells produce only one type of terminally diferenatied cells
Ex = spermatogonial stem cells - male germ line stem cells
Describe components of physiological evidence that stem cells exist in male germ line - 3
Life long production of sperm after puberty
Continuous high output sperm production
Regeneration
Describe components of physiological evidence that stem cells exist in male germ line - Life long
Sperm produced lifelong
Picasso last 2 kids born while he was 66 and 68 y/o
Produced terminally differentiated cell
Describe components of physiological evidence that stem cells exist in male germ line - Continuous
High sperm production for life =
> 150 x 106 sperm / day / man
> 5.6 x 106 sperm / day / male mouse >90x106 sperm/day/malerat
> 1,130 x 106 sperm / day / male monkey
1000 sperm/heart beat
Compared to 1egg every 28days in each woman
Describe components of physiological evidence that stem cells exist in male germ line - Regeneration
After chemo patients experience temporal or permanent azoospermia= no sperm in ejaculate
Male fertility can recovery spontaneously
Some cases permanent
Describe spermatogonial transplantation - functional ssc assay
Transgenic mouse with lac z, b-gala - makes blue product (in almost all cells, except fur, b gala breaks Down sugar x-gal and makes it blue)
Mouse soaked in sugar solution
Use donor animal and use digestive enzymes and disperse tissue = make single cell suspension and inject into Testes of recipient mouse (wild type,no lacz, treated with chemo = damaged bone marrow and spermatogensis)
Results = complete regeneration of spermatogenesis, give stem cells environment to do whatever they want and see function (functional detection)
Stem cels = can maintain spermatogenesis
Describe ssc - results of exp
Continuous self renewal and differenairion is a unique functional property of sscs, non stem cells disappear via differentiation or death/mitotic catastrophe
Each blue segment of seminiferous tubules comes from one stem cell, can count each and count number of stem cells
Colony arises from one ssc = colony #=ssc #
A clinical origin of a colony
Describe what type of quantification functional ssc assay was
Retrospective
Do not have cells anymore after exp
Describe detecting stem cells by looking at terminally differentiated cells
See committed cells
The stem cells gone = retrospective detection
These cells are not stem cells retrospectively
Describe functional assay for hematopoietic stem cells
Stem cells = test cells, diff genotype
Compromised, helper cells = differentiated, not stem cells, can support life of recipient mouse until stem cells can make blood, same genotype as recipient
Inject into mouse treated with lethal irradiation = so bone marrow damaged
wait 10 weeks
= take out peripheral blood and examine if Carry genotype of donor animals, quantify stem cells using statistics
If b, t or myeloid cells present = if all 3 can say stem cells present and regenerated
Does self renewal imply proliferation
Nahhhhhh
Can sometimes mean proliferate but in normal steady state = should not proliferate, should be in normal range of size
If no sperm = only stem cells, = cancer? Abnormal since abnormal proliferate
If no stem cells = lots of sperm now but no sperm later = bad
How are stem cells detected
By their regenerative capacity
Most types of stem cells invisible
Describe a way of stem cell detection
Trasnplantation assay
= regeneration and maintanece of cell linage are observed in Vito
Describe what type of exp a transplantation assay is
Functional assay = unequivocal
But detects stem cells retrospectively
- stem cells detected by continuous production of terminally differentiated cells = weakness
Describe neural stem/progenitor cells
Stem cell pop in brain
keep producing olfactory neurons
Exist in specific region of brain = subventricular zone
Adjacent to ventricular
Usually do exp where get rid of function, but cannot, so do in vitro
Describe culture of svz derived cells - exp
Take out region in brain with enzyme and make single cell suspension and put in culture
Culture = must be serum free and have egf or fgf2, also need bottom of culture to be not very adhesive or strong
Most cells die and see nothing but small number of cells stick to dish and proliferate and bulge up = makes ball, bc bottom not adhesive = floating = MAKES NEUROSPHERE
Can dissociate neurosphere w/o enzymes, mechanically
Pick up part of it and replace it and cam be repeated again
Long term self renewal, reproduce and regenerate for long time
What happens when put neural stem cells in diff culture conditions
Spontaneously diferentiate
Ex = can see with immunofloresence staining
1% serum + ecm, so cells can adhere to bottom of well, no growth factor =
Differniaties into oligodendrocytes precursors, Astrocytes, mature oligodendrocytes
Neurons
= MAJOR CELL TYPES OF CNS, can become all cells types
Detect based on functional def
How can functional retrospective detection of stem cells be done
Using in vitro cell cultures
Same experimental concept based on functional defining of stem cells applies to such in vitro assay, renewal and commitment can be identified in vitro, dual functions can contribute for long time
Describe common characteristics of biological assays for stem cells -5
1 - stem cells are functionally defined, need to detect their ability to regenerate and maintain given tissue
2 - approach forces us to observe stem cells retrospectively, they WERE NOT i have them/them here
3 - nobody has seen a stem cell under. A microscope
4 - prospective stem ell enrichment is feasible = increasing concentration of stem cells
5 - significant efforts have been put into purifying (100% enrichment) stem cells but no stem cells have been purified
Describe selecting stem cells using a marker
Enrichment using immunological method
1 = original donor cells mixture, 2 cell types, each express diff antigen
2 = apply antibody to a cell surface antigen, antibody binds to epitope expressed on red cells, must be cell surface maker otherwise would have to damage cells for separation
3 = cell separation, into 2 groups, one with antibody and one without
4 = transplantation or functional biological assay, compare to non selected regional donor cells, if regeneration in one of 2 = can see they are stem cells
Describe ssc cells
Express thy1 antigen
Separate cell pops, with and without selection
Sam number of cells - 7 x 10^4 transpalnted into each testis
See greater number of colonies with selected thy1 positive cells
What does prospective enrichment of stem cells require
Markers
Marker expression must correlate with stem cell activity, measured by functional assay
Often do not know what markers do to stem cells, use as marker but dot know
How can we pull down antigen expressing cells
Immunomagnetic cell sorting= magnet activated, MACS
Fluorescent activated cell sorting (FACS)
How can we pull down antigen expressing cells - MACS
Specific antibody Binds to cell surface antigen and secondary antigen binds to primary with magnetic beads (conjugated to primary antibody, more specific)
= cell pop found wit magnet = bound to antibody
Separate cell then do biological assay
How can we pull down antigen expressing cells - FACS
Fluorescent dye = cell solution after reaction with antibodies,and fluorescent due = put into cell sorter
Vibrates = gravity, falls and makes droplets, each with 1 cell
Laser hit cels with dye - computer tells plates what charge to have and deflects according
+ and - electric field, cells drawn to either
*mammalian cell surface charge is negative
Deepends on colours, could do many colours
Describe human breast tumor cells exp
Contain a stem cell pop
Breast cancer stem cells can be isolated in cd44+ and cd24- cells
Segreagayte cells and transplant into mice, immunodeficient mice or else would reject
All cells are cd44+
See regeneration of same tumour seen in patient
Describe peculiarity about stem cell markers
Stem cell may express a particular molecule that can be used as marker, but not all cells that express marker are stem cells
Describe peculiarity about stem cell markers - EX
Cd44 expressed in normal leukocytes and erythrocytes
Cd24 expressed in Normal B cells and neuroblasts
Markers can be expressed in non stem cells
HOW CAN STEM CELLS BE ENRICHED
Prospectively
Requires stem cell markers
Markers expressed on cell surface useful to harvest target cells live, allowing for a functional assay at subsequent steps
Why are stem cell markers tricky
stem cells may express these markers, but “not” all cells that express stem cell markers are stem cells.
Don’t be fooled by erroneous data interpretation or experimental design (defining a stem cell by expression of an unreliable marker or a single marker expression
What is stem cell niche
Micro environment surrounding stem cells
Which is involved in regulation of stem cell fate control
Describe normal fly Gsc division
Tip of fly testes, =germ line stem cells, surrounded by somatic stem cells (equivalent to sertoli cells)
Hub cell = stem cells, one daughter cell remains associated with hub cell, other one goes away and moves on to spermatognesis = asymmetric division
Describe fly Gsc division after others are killed
Kills hub cells and only one cell remains = changes direction of cel Division, mitotic spindle parallel to surface of hub cells, so 2 daughter cells remain associated with hub stem cells
What does drosophila asymmetric cell division involve
Growth factors, adherens jucntuons and Centrosome
So that stem cell pop doesn’t have substantial growth or expand = balanced
Upd = drosophila homolog of bmp
What does drosophila asymmetric cell division involve - growth factors
Growth factor morphogenetic expressed nay hub cells
Germaine stem cells encourage to remain to stem cells
Gb cells = differenatied and committed = go away so that concentration upd reduced = allows these cells to exit
What does drosophila asymmetric cell division involve - adherens junctions
Mechanical regulation = adherens junction bound to centrosome, tethers, towards hub cells = makes mitotic spindle perpendicular to interface between Germ line stem cell and hub cell so after cell division one cell remains associated with hub cell
Describe balanced and heathy ssc divsion
Sc niche as pocket like in fly
Each unit =pocket provides balance
Healthy, cell turnover can be sustained
Combo of 2 unbalanced ones = makes balanced and healthy, abnormal but if occur at same time = fine
Sc niche not necessarily a pocket, population balance monitoring mechanism, do not know what mechsnaims in mammals in terms of stem cell niche
What are/what can stem cell niches do
Stem cell niches are the microenvironment that encapsulates stem cells
Stem cell niches contribute to fate decision control of stem cells (SC-niche relation is reciprocal)
Stem cell niches can influence stem cell fate control on an individual cell basis or on a population basis.
What makes up neurosphere
Now know that neural stem cells represent only ~2% of neurosphere cells - remaining = committed progenitors
Describe immunological cell sorting
Antibodies against cell surface molecule
If molecule expressed by stem cells = cells selected using the antibodies against molecule should show a higher stem cell activity in functional assay compared to original unsorted cells= positive marker
If stem cell activity reduced in marker positive cells - antibody bound cells = say that molecule is not expressed on stem cells = negative marker
Describe normal gsc divison - orientation
Germline stem cell divides along axis that is perpendicular to plane of hub-germline stem cell interface= one cell remains physically associated with hub cell
Describe when selectively killl stem cells gsc divison - orientation
Remainaing germline stem cells change direction of cell divsion = horizontal along hub-Gsc interface = symmetric cell divsion
Describe how hub cells can lead to stem cell maintenance
Secretion of unpaird = bmp related molecule
Activates jak/stat signalling cascade in gscs
Cells closer to hub cells = higher concentration bmp signaling, ones further from hub cells = lower concentration bmp = leading to lower activation or loss of jak/stat signaling
Describe how hub cells can lead to stem cell maintenance - adherens junctions specifically
Gscs physically attached to hub by localized Cadherins junctions, E cadherin concentrated at gsc cortex adjacent to hub
Beta catenin and apc2 colocalize with e cadeherin at adherens junctions
Provide polarity cue towards which gscs orient throughout cell cycle
G1 phase = single centrosome in each gsc localizes near cell cortex where germ cell attaches to hub
When duplicated centrosome Separate in g2 phase = one stays next to hub and other migrates to opposite ends of cell
Separation of Centrosome occurs early during cell divisions, in male gsc before mitotic spindle built
Apc2 function required fro gscs to maintain proper orientation of centrosome towards the hub, position of centrosome in turn orients mitotic spindle perpendicular to hub-gsc interface, ensures outcome of gsc divsion is asymmetric
Centrosome are not oriented towards niche throughout cell cycle in female gscs = indicates that spindle orientation set up by a different mechanism in fly ovary
Describe multipotent stem cells
Cells generate multiple cell types in one cell linage
Many stem cells found after birth belong to this group
Ex = hematopoietic stem cells-blood, or neural stem cells
Become mutiple cell types
