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Molecular Mechanisms of Disease > Week 6: Stem Cells > Flashcards

Week 6: Stem Cells Flashcards

1
Q

What are the original 4 properties of stem cells?

A
  1. Ability to self-renew (to duplicate themselves and maintain themselves in an undifferentiated state)
  2. Asymmetry of daughter cell fate
  3. Undirectionality of cell differentiation
  4. Resident within a supportive niche
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2
Q

What are the revised stem cell properties?

A
  1. Continual and stochastic stem cell turn over
  2. Competition of niches by motile stem cells (clonal drift)
  3. Context-dependent stem cell potential (a cell may have stem cell properties in a damaged tissue but not a homeostatic tissue)
  4. Reprogramming capabilities
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3
Q

Describe the potency of stem cells:

A
  • Potency refers to the amount of potential for differentiation the cell has
  • Stem cells in some tissues are unipotent (will give rise to one kind of terminally differentiated cell) but in other tissues there may be multipotent stem cells (they give rise to multiple types of terminally differentiated cells)
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4
Q

Describe the Linear Hierarchy of Stem Cells:

A
  1. Quiescent stem cells
  2. Stem cells biased towards self-renewal
  3. Stem cell primed towards differentiation
  4. Intermediate progenitors
  5. Terminally differentiated cells
  • There is forward and reverse movement between populations 1,2,3 (and 4)
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5
Q

What are the two strategies for stem cell division asymmetry?

A
  1. Single cell level: one daughter cell will always be a stem cell whilst the other daughter cell goes on to differentiate
  2. Population level: this means that on average half the divisions produce differentiating cells and half the divisions produce stem cells. E.g. a single stem cell may divide to produce 2 identical daughter stem cells but on the population level the balance is still maintained
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6
Q

What are the two molecular mechanisms for regulating stem cell division?

A
  1. Internal regulation: cell-autonomous cues

2. External regulation: environmental cues (from niche)

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7
Q

What characteristics do tumours and normal stem cells share?

A
  • Solid tumors arise in tissues with stem cell populations
  • Cancers also exhibit cellular hierarchy, as within tumours there will be cancer-initiating cells (CICs) at the apex that are able to self renew and also partially differentiate to form the bulk of the tumour
  • Generally CICs are stem cells that have undergone oncogenic mutations in genes such as Wnt, Shh, Notch etc.
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8
Q

What are some unique features of germline stem cells?

A
  • They are unipotent (as opposed to stem cells in the epidermis, intestine etc.)
  • The terminally differentiated cells produced by the GSCs can produce a totipotent zygote
  • The genomic quality of the differentiated cells produced is essential
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9
Q

Where are GSCs found?

A
  • It is contentious as to whether GSCs still exist in the adult ovaries
  • GSCs are definitely found within the testes inside the basal compartment of the seminiferous tubules
  • The GSCs in the testis are known as undifferentiated spermatogonia
  • Spermatogonia divide in a syncytial manner to allow for the coordination of differentiation
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10
Q

Describe the hierarchy of GSC differentiation in rodent testes?

A
  • The undifferentiated spermatogonia can exist as single cells (As), pairs (Apr) or in short chains (Aal)- in order of decreasing stem cell capacity
  • Note: The Aal cells are primed to differentiate as they express NGN3
  • As the stem cells begin to differentiate there is the emergence of heterogeneous morphology and gene expression including the upregulation of c-KIT
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11
Q

Describe the hierarchy of GSC differentiation in primates:

A
  • In primates the undifferentiated spermatogonia are comprised of Adark and Apale- in order of dereasing stem cell capacity
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12
Q

Describe the regulatory markers within mammalian spermatogonia:

A 
Stem Cells (undifferentiated)
1. GFRa1 
2. PLZF 
(primed to differentiate) 
3. NGN3
(differentiation has begun) 
4. c-KIT
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13
Q

How is the presence of stem cells functionally tested for?

A
  • This is done by transplanting stem cell populations into a niche in a recipient organism
    e. g. transplantation of spermatogonia into the testis of an irradiated recipient mouse- proves they are stem cells as they will repopulate
  • In terms of taking context dependent stem cell function into account, experiments can be done using fate mapping
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14
Q

Describe the development process of spermatogonia:

A
  1. Primordial germ cells are specified in the post-implantation embryo
  2. Primordial germ cells migrate to the developing gonads as gonocytes
  3. Gonocytes in the male enter mitotic arrest prior to birth (G0)
  4. After birth the gonocytes (prospermatogonia) resume proliferation and migrate from lumen of the developing seminiferous tubule to the basement membrane
  5. Gonocytes generate the nascent spermatogonial pool and a population of GSCs (this occurs 8-12 weeks after birth in humans)
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15
Q

Are GSCs in the testis essential for initial sperm production?

A
  • No, the first round of spermatogenesis is from gonocytes
  • This is confirmed as this first round of spermatocytes do not express Ngn3- which is expressed on the differentiating spermatogonia derived from the GSCs
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16
Q

What are the main intrinsic factors needed for GSC stem maintenance?

A
  1. PLZF:
    - functional GSCs are contained within the PLZF+ spermatogonial population
    - PLZF promotes stem cell maintenance by blocking pathways needed for differentiation such as Kit gene expression
  2. Nanos2:
    - Is an RNA binding protein that inhibits the translation of mRNAs required for GSC differentiation
    - It is expressed primarily in GRFA1+ cells (As and Apr) but it is also expressed in Aal
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17
Q

What are the components of the GSC niche?

A
  1. Sertoli cells
  2. Basement membrane
  3. Peritubular myoid cells
  4. Leydig cells
  5. Endothelial cells of blood vessels
  6. Macrophages
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18
Q

What are the main extrinsic factors needed for GSC maintenance?

A
  1. GDNF:
    - Sertoli cell derived
    - The receptor for GDNF is only expressed in undifferentiated spermatogonia, mainly As and Apr
    - It drives self-renewal
    - Decreased GDNF expression causes GSC loss (GDNF levels are decreased in the aging male)
  2. FGF2:
    - Drives GSC self renewal
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19
Q

What factors promote GSC differentiation?

A
  1. Sohlh1
  2. Solhlh2
  3. Retinoic Acid stimulation
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20
Q

How was it proven that Aal and Apr are not committed progenitors, but rather undifferentiated spermatonia primed toward differentiation?

A
  • This was done using lineage tracing
  • This showed that these Aal and Apr cells not only produced differentiating cells but also contributed to the long-lived stem cell pool
  • Interestingly, during tissue regeneration or upon transplantation the proportion of NGN3+ Aal cells that contribute to the stem cell pool is increased
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21
Q

What is the cell of origin in testicular cancers?

A
  • Embryonic gonocytes that fail to mature into spermatogonia
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22
Q

How does knowledge of GSCs have therapeutic applications?

A
  • High dose chemotherapy often causes infertility as it destroys GSCs
  • By understanding the GSC niche it may be possible to remove GSCs prior to cancer treatment and then expand the population in vitro so they can be transplanted back in after treatment is completed
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23
Q

What are the features of the Integumentary system?

A
  • The integumentary system is made up of:
  1. Skin:
    - epidermis, dermis and hypodermis
  2. Skin appendages:
    - hair follicles
    - sebacious glands
    - sweat glands
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24
Q

Describe epidermal stem cells:

A
  • Epidermal stem cells are found in the basal layer of the epidermis within the interfollicular epidermis as well as within hair follicles
  • It is thought that they undergo asymmetric cell division at an individual cell level
  • p63 and Notch signalling is essential for the epidermal stem cells to differentiate
25
Q

What types of stem cells are found within the epidermis/hair follicle?

A
  • Multipotent bulge stem cells give rise to:
    1. Bulge stem cells
    2. Isthmus stem cells
    3. Junctional stem cells
26
Q

What is the multipotent bulge stem cell niche?

A
  • The dermal papilla
27
Q

How the stem cell nature of epidermal stem cells functionally determined?

A
  • This can be determined through skin reconstitution assays performed in mice
  • Epidermal stem cells can be isolated from donor tissue and combined with dermal cells and transplanted onto a recipient mouse
  • The stem cell capacity of isolated epidermal cells is assessed by their ability to generate hair follicles
28
Q

What is the role of epidermal stem cells in wound healing?

A
  • Wound healing involves 3 phases: inflammation, tissue formation and tissue remodeling
  • Following inflammation, bulge cells from the hair follicles are recruited into the inter-follicular epidermis to help restore barrier function
  • Growth factor signalling promotes proliferation of epidermal stem cells during the tissue formation phase
29
Q

What are the therapeutic applications of epidermal stem cells?

A
  • Using patient-derived epithelial stem cells, cultured epithelial autografts can be generated
  • These autografts can be used to treat severe burns or chronic wounds
  • The grafted skin restores epidermal barrier function but does not have many features of normal skin e.g. hair, glands, sensation
30
Q

Describe the relevance of epidermal stem cells to:

  1. Basal cell carcinoma
  2. Squamous cell carcinoma
A
  1. Basal cell carcinoma
    - The most common type of skin cancer
    - Causes by Ptch1 mutation resulting in aberrant Hedgehog signalling in basal cells
    - Ptch1 and p53 mutations lead to BCC tumorigenesis
    - The cell of origin of BCCs are the epidermal stem cells of the inter-follicular epidermis as well as the hair follicles
    - Prognosis of BCC is good following removal- they rarely metastasise but can recur
  2. Squamous Cell Carcinoma:
    - Accounts for 20% of non-melanoma skin cancer
    - Can be removed but can also metastasise
    - Ras and p53 mutations are required for SCC formation
    - The K15+ hair follicle bulge stem cells are likely the cellular origin of SCCs
31
Q

Describe human embryonic stem cells:

A
  1. Derived from the inner cell mass of the blastocyst
  2. Can be grown indefinitely in an upspecialised state
  3. They are pluripotent- can differentiate into tissue and cells of all 3 germ layers
  4. ESCs are extracted by growing embryos in vitro culture and exposing them to growth factors such as FGF2 and activin nodal signalling (these growth factors are species specific)
32
Q

Describe the 2 “promises” of Human ES cells:

A
  1. Differentiating human ES cells under appropriate conditions (by exposing them to certain factors) can allow researchers to develop them into a target cell type which can be transplanted back into a patient
    e. g. differentiating ES cells into cardiomyocytes in vitro and then transplanting tissue pieces back into the heart
  2. Using Human ES cells to screen for therapeutic compounds and then developing treatment drugs to use on a patient
    - More effective than screening on model organisms
33
Q

What are the limitations of human ES cells?

A
  1. Ethical issues:
    - Due to the destruction of embryos
  2. Patient Immune response to the ES derived cells if transplanted
    - ES cell derived tissue can be rejected if not very well matched to the recipient
    - Immunosuppressive agents need to be taken
    - Ideally using the ES cells of a patient on that patient
34
Q

How do the different cells of the body express different phenotypes when they all have the same genetic makeup?

A
  • It is known that all cells of the body have the same genetic makeup and genetic information is NOT lost during development
  • Adult nuclei within individual cells have all the genetic information required to produce a complete embryo (Gurdon 1958)
  • Cell differentiation is possible because of epigenetics
  • Epigenetic regulation allows certain subsets of genes to be expressed or not expressed in different cells
  • The subset of genes that are expressed in a cell define the cell’s function
35
Q

Describe the main layers of epigenetic regulation:

A
  1. DNA (cytosine) methylation:
    - High DNA methylation = inactive gene transcription
    - Low DNA methylation = active gene transcription
  2. Nucleosome rearrangement:
    - Nucleosome depletion at the start site = active gene transcription
  3. Histone variant replacement:
    - Different histones are associated with different transcriptional states (active or inactive)
  4. Post-translational modification of histone:
    E.g. histone 3 can be modified by K9 methylation = inactive gene expression
    E.g. Histone 3 can be modified by K4 methylation = active gene expression
  5. Chromatin packaging:
    - HP1
    - PRC1
36
Q

How can epigenetic regulation and thus the fate of a cell be altered?

A
  • Cell fate can be altered by introducing/removing transcription factor activators/repressors that alter the epigenome
37
Q

What is the basis of nuclear reprogramming?

A
  • Nuclear reprogramming involves introducing transcription factors (proteins that bind to gene promoters and regulate the expression of genes) into one cell type causing it to transdifferentiate into another cell type
    e. g. Introducing MyoD transcription factor into fibroblasts converts them into skeletal myocytes
  • The same transcription factor nuclear reprogramming has been used to reprogram differentiated cells back into progenitor states
    e. g. introduction of Yamanaka’s TFs can revert terminally differentiated cells back into a pluripotent state
38
Q

How induced pluripotent stem cells made?

A
  1. Somatic cells from a patient are isolated
  2. The Yamanaka TFs (Oct3/4, Sox2, Klf4 and c-Myc) are introduced into these terminally differentiated cells e.g. by retrovirus
  3. The 4 TFs epigenetically activate/inactive certain genes within the genome
  4. This results in a small amount of the somatic cells becoming pluripotent (0.1%)
  5. Induced pluripotent stem cells (iPS cells) are formed
39
Q

How are Transfactors delivered into the genome of somatic cells?

A
  • Various delivery mods can be used, with different efficacy and safety
  • The most commonly used strategies are Sendai Viruses and RNA
  1. Retroviruses
    - Very efficient but have some risks
    - Are RNA viruses that use reverse transcriptase to convert their RNA into DNA which is then integrated into the host cell genome in a semi-RANDOM manner
    - The semi-random nature of DNA integration can have unwanted consequences e.g. mutations that might cause cancer
  2. RNA:
    - Very safe and effective but not stable
    - Must be done multiple times
  3. Protein:
    - Very safe but not efficient
40
Q

How is the pluripotency of iPS cells determined?

A
  1. Chimera formation:
    - Gold standard
    - Inject the iPS cells into an early embryo and assess whether they contribute to all cell types of the body
    - These iPS cells are tagged e.g. with GFP, and the organism will be assessed for fluorescence after development has occurred
    - The organism will be a mixture of iPS and host cells
  2. Tetraploid Complimentation:
    - After a zygote has undergone cleavage to form 2 cells, the cells are shocked so they fuse back into one single cell (but this single cell has twice the amount of DNA as normal- is tetraploid)
    - These tetraploid cells can only give rise to extraembryonic tissues- will not form an embryo
    - If iPS cells are injected into the tetraploid cell and a mouse embryo develops, this confirms that the entirety of the embryo is formed from the iPS cells
  3. Teratoma assay:
    - The iPS cells are injected into the flank of a mouse
    - The iPS cells should keep proliferating for a while before
    - This results in the formation of a teratoma (a benign tumour)
    - The teratoma can then be isolated and histologically assessed for derivatives of all 3 germ layers
  4. In vitro differentiation assay:
    - exposing the iPS cells to TFs to cause them to differentiate into all 3 germ layers
41
Q

Do different species have different factors that generate iPS cells?

A
  • No

- The yamanaka factors: Oct3/4, Sox2, Klf4 and c-Myc are highly conserved

42
Q

What are the mechanisms that generate iPS cells?

A
  • During nuclear reprogramming the epigenome is reset to an ESC stage
    e. g. Oct4 (a pluripotency gene) is normally hypermethylated (inactive) in differentiated cells. In both ES and iPS cells Oct4 is hypomethylated which allows these cells to be pluripotent
  • Global histone profiles of ES and iPS cells are similar
43
Q

How can reprogrammed iPS cells be used in research?

A

Pros:

  • Useful for diseases with an underlying genetic cause, as the epigenome is rest but the genome remains the same
  • Can allow for patient specific drug screening

Cons:

  • Cannot be used for diseases with an epigenetic basis
  • Their implantation can give rise to tumours
  • The stability of the genes in the cells must be ensured
44
Q

What organisms are used as models for tissue regeneration?

A
  1. Zebrafish:
    - Have one nephron
    - If this is injurted hey can repair it and regain kidney function
    - The endogeneous stem cell population has been located
45
Q

What tissues are humans able to regenerate?

A
  • In humans the ability to regenerate tissue is limited to tissues such as skin and organs such as the liver
  • Currently there are no identifiable population of stem cells in the kidney
46
Q

Why are kidneys difficult to regenerate with tailored organs?

A
  • 3D printing can produce decellularised solid organs that can provide scaffolds using seeded cells
  • The kidney has a number of complex cell types which makes this a challenge
  • Stem cells that can differentiate into all the kidney cells may be a source of replacement
47
Q

How can iPS cells be used to help treat kidney disease?

A
  1. Drug development and toxicology screening:
    - Kidney disease patients currently have their drugs tested for toxicology on cancer cell lines which are irrelevant to them
    - iPS cell banks from kidney disease patients are now used to screen drugs
    - Genome editing can be used to create cell lines from iPS cells
  2. Transplanting corrected cells into patients
    - Genome editing
48
Q

How are human iPS cells generated for the kidney?

A
  • The iPS cells are generated from human kidney mesangial cells
  • Mesangial cells are fibro-blast like cells within the kidney
  • The mesangial cells are treated with the Yamanaka factors (Oct4, Sox2, Klf4 and c-Myc) to generate iPs cells
  • The pluripotency of cells can be confirmed with qPCR (assessing gene expression), immunostaining and teratoma assay
49
Q

What are kidney organoids?

A
  • Kidney organoids are structures formed from iPS cells that have >500 nephrons surrounded by epithelia
  • They are transcriptionally similar to a human foetal kidney
  • They can be used for drug screening but cannot be transplanted
50
Q

How are iPS derived kidney organoids used to model PKD?

A
  • PKD = polycystic kidney disease
  • Nephron organoids made from human iPS cells have been used to model PKD
  • CRISPR/Cas9 was used to artifically cause mutations in the PKD1/PKD2 genes in the iPS cells
  • The iPS cells hten went on to form large cyst-like structures
51
Q

What is the importance of podocyte replacement in the treatment of end stage renal disease?

A
  • 2/3 of patients with chronic kidney disease who progress to having ESRD have glomerular abnormalities
  • There is a leakage within the filtration barrier in the kidneys and a loss of podocytes (cells within the Bowman’s capsule) that leads to a loss of kidney function and secondary tubulointerstitial damage
  • Podocyte depletion precedes sclerosis and nephron loss
  • iPS cells are being used to try and model podocyte pathogenesis and possibly replace podocytes
  • Podocytes have a limited capacity to divide in vivo and in vitro
  • At birth there is a reduction in the proliferation of podocytes and they become quiescent
  • Postnatally podocytes become highly specialised and terminally differentiated
  • There is research being done as to whether damaged podocytes in patients with kidney disease can be reprogrammed back to an immature podocyte form (direct reprogramming)
52
Q

How are iPS cells used to model inherited kidney disease?

A
  • iPS modelling of kidney disease can be done by taking somatic cells from patients with the genetic mutation and the generation of iPS cells with that mutation
  • The iPS cells can then be used to model the disease in vitro
  • They can be used for disease modelling, toxicology screening
  • See an individual response to intervention

E.g. Alport syndrome iPS cells

  • iPS cells are generated from the effected patient
  • The Alport syndrome iPS cells can be exposed to factors specific for podocyte formation
  • The Alport syndrome iPS derived podocytes can have toxicology screens performed on them or may be modified for cell replacement
53
Q

What studies have shown that human podocytes have been generated from iPS cells:

A
  1. GFP-Nephrin study:
    - Human iPS cells were generated that expressed GFP in the NPHS1 locus
    - This locus encodes nephrin which is a gene specific to podocytes
    - Therefore it was observed that nephrin + cells (podocytes) were formed in vitro
    - These nephrin + GFP+ cells could be isolated using flow cytometry
    - A key difference between iPS derived podocytes and normal podocytes is that the iPS podocytes can be maintained indefinitely- so it is important that the cells are stable and karyotypically normal
54
Q

What cells can be used to:

  1. Replace cells in the kidney
  2. Reduce inflammation in the kidney
A
  1. iPS cells

2. Mesenchymal stem cells

55
Q

What are mesenchymal stem cells?

A
  • Mesenchymal stem cells are multipotent stem cells
  • They are a heterogeneous subset of stromal stem cells that can be isolated from many adult tissues
  • They contribute to the maintenance, formation and survival of haemopoietic stem cells
  • Mesenchymal stem cells express CD73, CD90 and CD105
56
Q

How do mesenchymal stem cells function in the kidney?

A
  • Mesenchymal stem cells enter the kidney via the blood stream and roll and tether in response to chemoattractant and cell death signals
  • The mesenchymal stem cells then release a range of factors e.g. I-4 and VEGF at the site of the injury
  • They are able to:
  1. Reduce inflammation
  2. Repair tubular damage
    - By changing the microenvironment in the kidney to one that promotes cell growth and repair
  3. Reduce vascular damage
    - Generate large range of angiogenic factors that promote blood vessel recovery and endothelial cell proliferation
  • These mainly aim to protect against acute kidney injury but may also be used to prevent chronic inflammation and immune rejection of kidney transplants
57
Q

How do mesenchymal stem cells reduce inflammation and suppress the immune system?

A
  1. Secretion of immunosuppressive factors
  2. Direct interference with effector functions of dendritic cells
  3. Direct interference with T cell function
    - Via IL-12 which promotes Treg expansion
  4. Arresting B cell maturation and Proliferation
  5. Changing the phenotype of macrophages
    - So the macrophages are programmed for wound repair
58
Q

How can mesenchymal stem cells be used to transfer small molecules?

A
  • Mesenchymal stem cells transfer small molecules via exosomal trafficking
  • MSCs can be engineered to migrate to the kidney and exocytose microRNAs important for anti-fibrosis
  • Researchers are looking as to how to load MSCs with certain microRNAs and then extract the exosomes and use them as a treatment

Molecular Mechanisms of Disease (12 decks)

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