Identifying Stem Cells and their Niches in Vivo Flashcards
What are the 2 kinds of adult stem cell
- Somatic → set aside during embryonic development to go to particular locations in tisues and organs. Vital for repair and homeostasis
- Germline → needed to produce gametes throughout most of life
- Embryonic stem cells have no relevance to an in vivo situation – they are purely an artiifical construct of cells derived from the blastocyst
What are the properties of adult stem cells
- Undifferentiated
- Low proliferation
- Inhabit a protected niche
- Make up a small proportion of cells in an organ
- Pluripotent – replenishment and repair
How can cell transplantation be used to identify the stem cell niche in the drosophila ovary?
eg) The Drosophila Ovary – A Model System
• Paired structures
• Each ovary comprised of around 15 ovarioles – these make the eggs
Ovariole structure
• Ovarioles comprise of egg chambers, each with a developing egg – germline cell
• There are also other germline cells = nurse cells → nurse cells feed mRNA into the cytoplasm
• Surrounding each egg chamber are follicle cells – these are somatic cells
• Between each egg chamber is a linear array of stalk cells – also somatic cells
• Both the germline and somatic stem cells are located in an anterior structure called the germarium
How did we know this?
Lin and Spradling, 1993
1. Dissect germarium out of ovariole of a fertile female, transplant in a sterile host
2. Transplanted structure made eggs and host was no longer sterile
3. If they used a lazer to ablate the celsl in the germarium, egg production ceased
➢ Cant maintain egg production without germarium cells → proving the location of the stem cells
How can cell transplantation be used to identify the stem cell niche in melanocyte stem cells
- Easy to study as very different from the surrounding epidermis
- Give pigment in the skin/hair
- Located in the follicle bulge – differentiated cells leave the follicle and locate at the base
Transplantation experiment:
- Diffierent sections of follicle (bulb, middle and upper) from a pigmented mouse were transplanted to the skin of an albino mouse
- Only cells form the muddle section (where the bulge is) produces pigmented hairs in the new host
Repopulation assay
• If you take the follicle pieces and transplate into a nude mouse (with no hair at all) you start to get new patches of hair forming
• SO not only can the cells self-renew (maintaining itself in the recipient animal), they can differentiate into the cell types needed to reconstitute the recipient → so it is pluripotent
- Tissue reconstitution is the ‘gold standard’ for isolated stem cells
- Good example medically is the BM transplant
How can negative lineage marking be used to detect germline stem cells in drosophila?
Drosophila Flp-FRT System for Directed Mitotic Recombination
• hsFlp is a gene for a yeast-derived DNA recombinase placed under control of a heat shock responsive enhancer and inserted in a fly
• FRT (Flp recombinase target) DNA sequence recognised by Flp recombinase → promotes DNA strand breakage and rejoining, resulting in recombination between two FRT bearing DNA strands
- Make a transgenic fly heterozygous for b-gal (one allele – the homologous chromosome doesn’t have b-gal) → gives us a reporter
- These homologous chromosomes have the FRT sequence
- hsFlp gene inserted on a different chromosome
- FLP recombinase induced to be expressed in cell undergoing mitosis
- During mitosis, the homologous chromomomes duplicate → now there are 4 copies of the FRT
- If FLP is expressed, it will promote strand exchange and recombination between sister chromatids on adjacent or the same chromosome.
- Homologous chromosomes line up independently on the spindle → depending on orientation, you can get daughter cells identical to the parent (heterozygous for b-gal) or different (one lacking b-gal, and one homozygous for b-gal)
- All other cells in the organism that have not undergone recombination will be heterozygous
- The negative cell (without b-gal) can therefore be used as a marker.
Now we have 3 populations of cells:
- Heterozygoes b-gal
- Homozygous b-gal
- Negative for b-gal
- Mutant clones only induced in mitotically active cells
- In the germarium, this could be germline stem cells, daughter cystoblasts, 2,4 and 8 cell cysts, and follicle stem cells
- Induction is stochastic – only 50% chance
Negative lineage marking for germline stem cells:
• Normally all cells express b-gal
• If you wait 5 days after mutant clone induction, all mutant b-gal negative cells present must belong to a clone that originated during mitosis of a stem cell → all original cells will have passed out of the germarium by then, so all negatively marked cells must have come from a stem cell
• Stem cell clones are ‘permanent’ rather than ‘transient’ as they are maintained by a negatively marked stem cell
• In a germarium, we can see the presence of negatively marked stem cells, and then negatively marked cysts further up → this is tracing the whole lineage
How can positive lineage marking be used to detect follicle stem cells in drosophila?
- Again, use FLP to induce sister chromatid recombination
- This brings together a promoter expressed in all cells, aTub84B – just upstream of b-gal
- This reconstitutes a gene so that b-gal can be expressed
- The other product doesn’t make b-gal
- This generates b-gal expression where you wouldn’t normally see it = positive marking
- If you wait for one week after clone induction, any positively marked cells must have arisen from a marked stem cell
- You see labeling of a follicle cell all the way from the mid-germarium to the late stage egg chamber
- This can only happen if the original cell marked with b-gal was a stem cell
- The location of the most anterior marked cell is the location of the stem cell
Give an example of lineage marking in mammalian tissue
• Cant use heat shock because mammals maintain body temp
• Use CRE recombinase fusion protein, where domain recognizes Tamoxifen
− When Tamoxifen binds the fusion protein, it turns the CRE recombinase on
− CRE recombinase is normally put under the activity of a specific promoter expressed in cells believed to be stem cells
• Also in the mouse is a b-gal reporter construct.
− The b-gal is in front of a promoter that, if active, turns on b-gal constitutively
− However, there is a DNA sequence inbetween the promoter and lacZ that stops transcription
− This sequence is flanked by targets for CRE recombinase
− When you induce CRE, it will cut out the stop sequence and recombine lacZ with its promoter
− This is permanent, because you cant un-do this recombination
− All of the cells derived from this cell will express b-gal
eg) mouse intestine
• 1 day after induction, just 1 cell is marked
• As you move along, you see the progeny of the basal cell expanding up the crypt
• The entire lineage is marked from the base to the tip of the crypt → all marked cells are derived from the single marked cell at the base.
Describe the label retention method of lineage tracing
- Depends on the fact that stem cells are quiescent
- They will divide infrequently to renew themselves and replenish a transit amplifying pool, which does most of the proliferating and differentiating.
DNA synthesis label retention
• BrdU is not present in animals and can be detected using a specific antibody
• BrdU is chemically similar to the base T and is incorporated into the DNA of cells
• Every time a cell divides, it shares its DNA (and so its BrdU label) with its daughters
Describe the template strand retention method of labelling stem cells?
Potten et al, 2002 - Manchester!
‘Identification of intestinal stem cells by template strand retention’
• In the ‘immortal strand’ hypothesis – there is one template strand. This is the strand on which new strands of DNA are built
• In this hypothesis, the template strand is retained in the stem cell, and new strands go into daughter cells
• Any errors occurring during replication will be incorportated into the new strand
• The template strand is the same as before – so the idea is the stem cells will retain the good copy of DNA, and any copies of DNA with errors will end up in the progeny → eventually they will die
It is an attractive way of protecting stem cells, but does it really happen?
Double labeling technique
• Label animals with tritium during developennt, when initial stem cells are being established (symmetrical division(
• Once in the adult tissue, the cells are in asymmetric division – one cell is self renewed, the other is daughter progeny
• During this asymmetric development, the hypothesis is that the template strand is retained, so they retain the tritium
• To show the daughter cells are still dividing, label them with BrdU
1. When the stem cell undergoes its first round of division, you will get a daughter cell labeled with BrdU, and a stem cell labeled with tritium and BrdU
2. After another round of cell division, you get a daughter cell with BrdU and a stem cell that only has the tritum
- Was able to show this in the intestine
- TSR may be a protective mechanism against DNA replication errors
- Controversial as to whether it is a general property of stem cells.
How can specific expression markers be used to show stem cell location?
Drosophila Ovary
• Positively mark them for a cytoskeletal structure called the spectrosome → very characteristic, spherical and located in the cell at the junction with the supporting niche cell
• Negatively mark them for bag of marbles → only stem cells in this region of the germarium don’t express it.
Drosophila Intestine
• Mark them for Delta – only found in intestinal stem cells, not their progeny
• Also antibodies that recognize the stem cell progeny, enteroblasts, enterocytes and enteroendocrine cells – so you can see all their locations.
How can genetic manipulation be used to identify stem cells?
Haematopoeitc Stem Cell Niche in the Mouse
• Stem cell located adjacent to oesteoblasts in the marror
• Osteoblasts suspected of being the niche cells
• HSCs could be labeled using antibodies specific to HSC markers, eg) cKit, Sca-1, Lin-
• Calvi et al, and Zhang use tissue specific gene knockout strategies to alter numbers of osteoblasts by manipulating signals that regulate osteoblast number
• Showed that HSC numbers increased when osteoblast numbers increased
