Stem cells Flashcards
Name 5 possible fates of stem cells
- symmetric division –> 2 stem cells (stem cell expansion)
- asymmetric division –> 1 stem cell and 1 differentiated cell (homeostasis)
- symmetric division –> 2 differentiated cells (mature cell expansion and stem cell depletion) –> loss of stem cell pool; cannot do that indefinitely.
- Quiescence (“hang around”)
- Apoptosis (low rate)
What are the phases of mitosis?
G1 phase: Cells increase in size, produce RNA and synthesize different proteins. An important cell cycle control mechanism activated during this period (G1 checkpoint) ensures that everything is ready for DNA synthesis
G0: Cells can leave the cycle and quit dividing (quiescent). This may be temporary resting (e.g. liver cell) or more permanent (e.g. cells that have reached an end stage of development and will no longer divide (e.g. nerve cells in the brain))
Synthesis phase: DNA replication
G2 phase: The cell will continue to grow and produce new proteins for cell division. At the end of this gap is another control checkpoint (G2 checkpoint) to determine if the cell can now proceed to enter mitosis and divide
Mitosis
What is the interphase of mitosis
G1 + S + G2
Name a case in which stem cell differentiation is reversible
Plants, amphibians can grow entire new limbs if they lose them! (e.g. differentiated cells can go back to being stem cells)
Name a case in which differentiation is partially reversible
Humans. Cells can be put under specific conditions in the lab and return to earlier cell type.
Name 4 types of stem cells
- Zygotes (fertilized eggs)
- Embryonic stem cells (Inner cell mass of blastocyst)
- Adult stem cells
- In vitro stem cells
Explain the particularities of zygotes as stem cells
- Totipotent (can make any cell of body)
- 2 divisions (8 cells) required to make an entire human organism
Stops being totipotent at the 4rth cell stage
Explain the particularities of embryonic stem cells
- Pluripotent, self-renewing potential ++
- Blastocysts (Human 4-5 days gestation; mice 3.5 days)
Explain the particularities of adult stem cells
- Multipotent, self-renewing potential
- e.g. hematopoietic stem cells, satellite cells (muscles)
Explain the particularities of organ cells
Limited potential for self-renewal
Progenitor cells –> commited progenitor –> differentiated (no division, functional)
What is plasticity?
The ability of an adult stem cell from one tissue to generate the specialized cell type(s) of another tissue
Differentiate embryonic vs adult stem cells
Embryonic:
- Derived from embryo (blastocyst)
- Most primitive
- Can form most cell types
- Abundant, easy to grow
Adult:
- Derived from adult tissues
- more organ specific (e.g. muscle, brain, blood)?
- Can form only one (tissue of origin) or few cell types (plasticity)
- Rare in tissues, difficult to isolate and grow
Name the genes involved in iPS induction
Oct3/4
Sox2
Klf4
c-Myc, N-Myc, Lin28, Nanog
Name ways in which adult stem cells can cure disease or improve recovery.
- Adult stem cells can promote recovery from spinal cord injury (rat)
- Can repair heart damage (mouse)
- Tissue-engineered autologous bladders for patients needing cystoplasty (human)
- Beta-globin gene transfer to human bone marrow for sickle cell disease
What are induced pluripotent stem cells (iPSCs)?
- Recent discovery; paper published in 2006
- Shinya Yamanaka and John B. Gurdon
- Used a combination of transcription factors (e.g. Oct3/4, Sox2, c-Myc, Klf4) in somatic cells (e.g. muscle, hair..) and created an iPSC.
- Could give rise to different cell types
- Easier to do with adult/progenitor cells vs fully differentiated cells (less steps)
What are the steps in creating a iPSC cell with a somatic cell?
- Inhibition of somatic regulators + induction of proliferation
- Activation of pluripotency loci + inhibition of senescence and apoptosis pathways
- acquisition of factor independance + Immortalization
- complete reprogramming (telomeres, X chromosome, memory erasure)
* Cells eventually become independent of cocktail of transcription factors
Name pros and cons of ESCs
PROS
- Low cost
- Established and characterized
- New lines being made
- Efficient differentiation
- Realistic HLA spectrum
- MHC down-regulation possible
CONS
- Embryo destruction
- Limited: blastocysts days 5-14
- Difficult obtention
- Mutation rate
- Incomplete histories
- Animal pathogen exposure
- Immunosuppressants
- Tissue rejection
- Carcinogenic risk
Name pros and cons of iPSCs
PROS
- No ethical issues
- Any cell type (in theory)
- Easy to obtain
- Blood group compatibility
- HLA histocompatibility
- Disease modelling possible
- Drug/toxicity profiles possible
CONS
- Additional cost
- Retroviral gene delivery
- Oncogene activation risk
- Mutagenesis risk
- Mechanism unknown
- Retention of alterations
- Suboptimal standardization
Name ways in which iPSCs can be used in therapy and research
- Drug screening
- Disease modes and mechanism of disease
- Teratology
- Cell therapy/Tx of patient
Explain the clinical trial that used iPSCs for macular degeneration
Took skin cells and reprogrammed them into iPCS cells –> differentiated into Retinal pigmental epithelium sheet
Removal of Abnormal blood vessels and old retinal pigmental epithelium from eye
–> put new RPE into eyes of donor (autologous RPE transplant)
Explain the clinical trial that used iPSCs for macular degeneration
Took skin cells and reprogrammed them into iPCS cells –> differentiated into Retinal pigmental epithelium sheet
Removal of Abnormal blood vessels and old retinal pigmental epithelium from eye
–> put new RPE into eyes of donor (autologous RPE transplant) = less rejection problem
Explain the self-renewal of satellite cells
Satellite cells are muscle stem cells
If engrafting a donor muscle myofiber with satellite cells, the muscles can regrow.
Which type of stem cell has been the first one to be characterized in details?
Hematopoietic stem cells
Name 2 clinical uses of hematopoietic stem cells
Bone marrow transplantation
Gene therapy
What is an hemangioblast?
The hemangioblast is a transient cell that has the ability to rapidly differentiate into an endothelial or a hematopoietic stem cell
Name 1 positive and 1 negative regulator of the differentiation of a mesoderm cell to an hemangioblast
+: BMP-4
-: Smad5
Name 1 positive and 1 negative regulator of the differentiation of a hemangioblast to an hematopoietic precursor
+: BMP-4
-: Smad5
Name 1 positive regulator of the differentiation of an blastula to a mesoderm cell
+: BMP-4
Name 1 positive and 2 negative regulator of the replication/self-renewal of hematopoietic cells
+: Activin A
-: TGF-alpha, Smad5
What is ldb1 and what is its function?
Lim domain binding protein 1
Binds to the LIM domain of a wide variety of LIM domain-containing transcription factors. Acts with LMO2 in the regulation of red blood cell development, maintaining erythroid precursors in an immature state. Favors hemangioblasts’ development to primitive hematopoietic progenitors vs. endothelial progenitors.
Name transcriptional regulators affected by Ldb1
Gata 1 and 2 Sox7, 17 and 18 Runx1 c-myb Eto 2 etc. \+ many pathways of signalling
What happens in cases of KO ldb1
cannot make hematopoietic stem cells anymore; very important role in hemangioblast formation
Name sites of hematopoiesis in humans
- *Yolk sac (primitive erythroid cells)
- *Fetal liver
- *Bone marrow (second wave of hematopoietic cells)
- Embryo (specific region)
- Placenta (unclear)
MAINLY*
Expain the experiments of Till and McCulloch
1960s
- Isolated bone marrow cells from mice femur
- Injected IV into another, irradiated mouse (could not make its own cells)
- After 10 days, saw growth of colonies with variable size containing differentiated blood cells and a small number of undifferentiated cells (self-renewal)
- smaller colonies = more stem cell-rich
- Showed more injected cells = more colonies per spleen
- Abnormal karyotypes (chromosomes) among CFU-S from donor bone marrow was used to prove clonality
Explain the differences between lineage determination of mice and humans
Cell surface markers are very different in both species; and are very specific for the different types of cells
Humans
- No LT- and ST-HSC (only HSC)
- MLPs can maybe go directly to GMP and make monocytes too
Name techniques to differentiate between different types of cells in the hematopoietic pathway
- Mitochondrial staining - Rh123 (stains active mitochondria) - higher level = more active mitochondria
- Surface markers
(very similar morphologically/under microscope)
Where is the site of hematopoiesis in the bone marrow? Why?
Area close to the bone which harbors the most potent hematopoietic stem cells
Hematopoietic stems cells close to the bone = quiescent (on stem cell niche on the bone)
Once they leave the bone marrow niche, they start to divide more actively, become progenitors and more and more lineage-restricted.
Name different factors that regulate quiescency of HSC
Closeness to the bone (direct interactions)
- Osteoblasts
- Fibroblasts
- Endothelial cells
Others:
- ECM components
- Soluble growth factors
Name cells that contribute to regulating HSCs near the endosteum
Less oxygen, more hypoxia
- reticular cells
- osteoblasts/osteoclasts
- Other cells
Name cells that contribute to regulating HSCs near the perivascular sites
More oxygen, less hypoxia
- reticular cells
- mesenchymal progenitor cell
- Megakaryocytes
- Sinusoidal endothelium
- Other cells
Name molecules that regulate the proliferation of HSC on the niche
SCF (niche) –> c-kit (HSC)
Name molecules that regulate the quiescence and self-renewal of HSC on the niche
Ang-1 (niche) –> Tie2 (HSC)
N-Cad (niche) –> N-Cad (HSC)
Jagged-1 (niche) –> Notch 1 (HSC)
Name molecules that regulate the homing adhesion of HSC on the niche
SDF-1 (niche) –> CXCR4 (HSC)
VCAM (niche) –> Integrin (HSC)
Name 3 sources of hematopoietic stem cells
- Bone marrow
- Mobilized blood
- Cord blood units
How much time does it take before mobilization of blood cells to the blood with G-CSF? What happens in the use of CXCR4 inhibitors? VLA-4 inhibitors?
What are those drugs used for?
- G-CSF = Granulocyte-colony stimulating factor; Usually 4-5 days
- CXCR4 inhibitor: 6-9 hours (Less adhesion)
- VLA-4 inhibitor: hours
- All used to mobilize blood cells faster and help make more blood cells that can then be used for transplantation purposes
Name 3 regulators of SC expansion
- Signalling pathways
- TFs
- Cell cycle regulators
Name Stimulatory and negative regulators (TFs) of stem cell expansion
o Stimulators: HoxB4, Smad7, BMP4, Sonic Hedgehog, Wnt, Notch
o Negative regulators: p21, P18, TGF-Beta1
Over-expression or under-expression of any of those cause stem cell expansion/repression
Name 3 characteristics of quiescent hematopoietic stem cells
- Protection from stress
- Adhesion to niche
- Hypoxic niche
What is the function of the protection from stress in quiescent stem cells?
- Bone shields from UV light and radiation
- High activity of ABC transporter (ABC-G2)
- High activity of aldehyde dehydrogenase (oxidises alkalating agents, which are harmful for cells)
- Regulation of radical oxygen stress (hypoxic environment)
What is the function of the adhesion to niche in quiescent stem cells?
- Low requirement for growth factors
- Anti-apoptotic machinery
- Stem cell/niche interaction (e.g. cadherin/catenin, integrins)
- Slow cycling (e.g. Tie2, p21)
What is the function of the hypoxic niche in quiescent stem cells?
- Osteoblastic zone of trabecular bone
- Low oxidative phosphorylation
- Anaerobic metabolism
Name 4 defense mechanisms in LT-HSC
- Quiescence in cell cycle
- Switch from oxidative phosphorylation to glycolysis under hypoxia
- High levels of ROS scavengers
- High efflux ability (can pump out toxic substances)
Explain somatic cell nuclear transfer/therapeutic cloning.
Take a somatic cell and retain its nucleus –> transfer to another cell to enucleate it (oocyte) –> isolate blastocyst –> take ICM and grow diff types of tissue
Explain what is reproductive cloning
SCNT If used blastocyst to implant into an organism to give rise to offspring (very low efficiency though)
Explain a potential therapeutic use of therapeutic cloning
embryonic stem (ES) cells can made be specifically from, and for, an individual patient to circumvent donor-host immunorejection and disease transmission complications
Explain a recent advancement in therapeutic cloning (2013)
Generation of human embryonic stem cell lines by SCNT (Tachibana, Cell 2013)
Compare the efficiency of embryonic stem cells compared to SCNT for reproductive cloning
ES cells - 15-25%
Cumulus, fibroblasts, sertoli cells, C, T cells, neurons - 0.0001-3%
Explain a study that tested reproductive cloning
to clone the sheep Dolly (1997) researchers used 277 reconstructed eggs to produce one successful pregnancy
Name ethical, legal and social issues in regards to stem cells
Embryonic, adult stem cells & iPSCs
o potential to develop into many different cell types, tissues, (organs)
o ethical controversy: source of stem cells (in case of embryos)
Cloning
o reproductive cloning (Somatic cell nuclear transfer, SCNT)
o therapeutic cloning (SCNT without intention of implanting blastocyst in uterus) e.g. transplant cell/tissue back into same person who donated somatic cell nucleus
Legal issues
o definition of e.g. “embryo”, “fertilization”, “conception”
What are spindle transfer and pronuclear transfer used for?
can be used to generate a baby from 3 parents
Has been used in people who have mutations in the mitochondrial genes
What is pronuclear transfer? what are problems with this technique?
Take pronucleus (consists of 2 nuclei) and implant it in a FERTILIZED, denucleated egg --> implant embryo in host mother - Take a large % of defective mitochondria (stick to pronuclei) – may have contamination of offspring and may have same problems as mother
What is spindle transfer?
Take spindle instead of pronuclei and implant in an UNFERTILIZED egg.
Egg fertilized later, after the transfer.
