Exam Flashcards
What is the ‘Stimulators of endogenous repair’ approach to Reg Med?
Bone repair: BMP-9 induces osteogenic differentiation of MSCs,
Renal regeneration: use an intra-renal administration of exogenous VEGF as a potential therapy to protect the kidney. VEGF may promote an increase in Akt, a key pro-survival factor, and Ang-1/Tie-2, which together with VEGF play important roles in promoting vascular proliferation and accelerating the maturation of the newly generated vessels
Dystrophic skeletal muscle: VEGF + regenerative capacity of muscle stem cells, by promoting angiogenesis and bone turnover.
What induces bone repair?
Bone repair: BMP-9 induces osteogenic differentiation of MSCs,
Renal regeneration: use an intra-renal administration of exogenous VEGF as a potential therapy to protect the kidney. VEGF may promote an increase in Akt, a key pro-survival factor, and Ang-1/Tie-2, which together with VEGF play important roles in promoting vascular proliferation and accelerating the maturation of the newly generated vessels
Dystrophic skeletal muscle: VEGF + regenerative capacity of muscle stem cells, by promoting angiogenesis and bone turnover.
How can renal regeneration occur?
Bone repair: BMP-9 induces osteogenic differentiation of MSCs,
Renal regeneration: use an intra-renal administration of exogenous VEGF as a potential therapy to protect the kidney. VEGF may promote an increase in Akt, a key pro-survival factor, and Ang-1/Tie-2, which together with VEGF play important roles in promoting vascular proliferation and accelerating the maturation of the newly generated vessels
Dystrophic skeletal muscle: VEGF + regenerative capacity of muscle stem cells, by promoting angiogenesis and bone turnover.
How can VEGF help dystrophic skeletal muscle?
Bone repair: BMP-9 induces osteogenic differentiation of MSCs,
Renal regeneration: use an intra-renal administration of exogenous VEGF as a potential therapy to protect the kidney. VEGF may promote an increase in Akt, a key pro-survival factor, and Ang-1/Tie-2, which together with VEGF play important roles in promoting vascular proliferation and accelerating the maturation of the newly generated vessels
Dystrophic skeletal muscle: VEGF + regenerative capacity of muscle stem cells, by promoting angiogenesis and bone turnover.
What is the xenotransplantation approach to reg med?
What are the benefits? [4]
Downsides? [4]
The transplantation of living cells, tissues or organs from one species to another. For example Pig hearts and Pig kidneys. One way to bridge the gap between available tissues and human need: virtually unlimited supply of organs, scheduling is not dependent on the unpredictable availability of a donor human organ, allowing for both advance planning and the intentionally timed harvesting of an organ for immediate transplantation as well as immunologic pre-treatment of the recipient if needed, the risk of many infections can be reduced by allowing lifelong control of exposure of the source animals to potential infections and extensive pre-screening prior to organ harvest, some animal species are even refractory to infection by certain viruses that persistently infect humans (HIV, Hep B).
Problems: Immune rejection (patients often need to take immunosuppressant drugs for life resulting in many side effects and decreased QoL), life span differences (A pig might only live ten years, a human could live to 80), latent cross-species infections: Nipah virus encephalitis from pigs, hantavirus pulmonary syndrome from mice, and anthrax from cattle (zoonoses, and there is also the risk that the breach of the normal host defense [skin etc] could allow infection by agents that are not normally able to infect humans).
What is an example of cellular therapy as an approach to Reg Med?
Autologous vs allogenic.
Harvest -> Select -> Amplify -> Screen -> Administer.
Example: iPS. 250,000 MEF cells in 100mm dish. Add Sox2, Kif4, c-Myc and Oct4 via viral infection on Day 0. By Day 4 a morphological change is visible. By Day 9, ES-like colonies have formed and by Day 14 the culture is AP test positive. However, oncogenic risk factors result from this induction of pluripotency: integration of gene delivery vectors and transgenes into host cells, chromosomal damage during the reprogramming process, clonal selection for oncogenic or transformed colonies during PSC expansion, Incomplete reprogramming, Failure to silence pluripotency networks in differentiated progeny, DNA damage accumulated during cell culture or resulting from somatic mutations, Aberrant regulation of the imprinting process. End result being tumorgenicity.
What are the risks of cellular therapy?
Autologous vs allogenic.
Harvest -> Select -> Amplify -> Screen -> Administer.
Example: iPS. 250,000 MEF cells in 100mm dish. Add Sox2, Kif4, c-Myc and Oct4 via viral infection on Day 0. By Day 4 a morphological change is visible. By Day 9, ES-like colonies have formed and by Day 14 the culture is AP test positive.
However, oncogenic risk factors result from this induction of pluripotency: integration of gene delivery vectors and transgenes into host cells, chromosomal damage during the reprogramming process, clonal selection for oncogenic or transformed colonies during PSC expansion, Incomplete reprogramming, Failure to silence pluripotency networks in differentiated progeny, DNA damage accumulated during cell culture or resulting from somatic mutations, Aberrant regulation of the imprinting process. End result being tumorgenicity.
What is the gene therapy approach to Reg Med?
This can take two main forms + both employ packaging a therapeutic gene into a delivery vehicle, most commonly a retrovirus. The first technique involves simply injecting this retrovirus into the target organ of a patient. The second approach is more complicated and is called cell- based delivery. This can involve either adult stem cells isolated from a patient and propagated in the lab or genetically modified ES cells from a different source. Whatever the source of the cell they are grown in culture and the retrovirus with the therapeutic gene inside it is mixed in. These genetically modified cells are then reintroduced into the patient.
There are a number of weaknesses with regard to gene therapy including safety (mutagenicity). The problems of targeted delivery and retention can be fixed using novel cell carries/iPS (induced pluripotent cells).
Examples include Advexin for the delivery of WT-p53 to patients with head and neck carcinomas.
What are some examples of biologic-device combinatio products for Reg Med?
Cell-coated vascular stents: The lack of an endothelial layer in prosthetic vascular grafts is a strong stimulus for intimal hyperplasia, which may lead to premature graft failure. Can seed a scaffold with EPCs (a subset of circulating bone marrow-derived stem cells which express CD133 and CD34 and VEGFR1 etc.)
However, this is REALLY hard: alternative method is to cover a stent scaffold in anti-CD34 antibodies so that circulating EPCs become attached to them – varying degrees of success and failure. Also antibodies for vascular endothelial-cadherin.
Artificial blood vessels: poly(L-lactic acid) hollow fibre membranes.
Diabetes: Encaptra Drug Delivery System. Semi-permeable cell containment barrier surrounding a cell therapy e.g. PEC-01 (Pancreatic Endoderm Cells) which allows nutritional inputs of Glucose, O2 and proteins in and allows the therapeutic outputs of INSULIN, amylin, glucagon and others out. Implantable device which is designed to prevent immune rejection and provide long-lasting glucose control for patients.
What are HSCs?
- The stem cells that give rise to all the other blood cells through the process of haematopoiesis: myeloid and lymphoid lineages
- Derived from mesoderm and located in red bone marrow in the core of most bones, although small numbers of them circulate in peripheral blood.
- 1:10,000 of cells in myeloid tissue are HSCs
- Stem and progenitor cells can be taken from the pelvis, at the iliac crest, using a needle and syringe.
- To harvest stem cells from the circulating peripheral blood, blood donors are injected with G-CSF that induces cells to leave the bone marrow and circulate in blood vessels.
What are the markers for HSCs?
Flow cytometry can be used to identify and isolate HSCs.
Lin- (as not mature blood cells)
Low staining with vital dyes such as rhodamine 123.
CD34 – used to isolate HSC for reconstitution of patients who are haematologically incompetent as a result of chemotherapy or disease.
Cluster of differentiation series: CD34, CD38, CD90, CD133, CD105, CD45 and also c-kit.
SLAM code: an alternative method of getting a better harvest: Signalling Lymphocyte Activation Molecule family is a group of more than 10 molecules whose genes are located together. SLAM codes for HSC: CD150+, CD48-, CD244-.
How are HSCs used?
- Autologous HSC transplantation
This involves the extraction (apheresis) of HSCs from the patient and storage of the harvested cells in a freezer. The patient is then treated with high-dose chemotherapy with or without radiotherapy with the intention of eradicating the patient’s malignant cell population at the cost of partial or complete bone marrow ablation. The patients own stored stem cells are then transferred into their bloodstream, where they replace the destroyed tissue and resume the patient’s normal blood cell production.
Autologous transplants have the advantage of lower risk of infection during the immune-compromised portion of the treatment since the recovery of immune function is rapid. - Allogeneic HSC transplantation
This involves two people: a healthy donor and patient. The donor must have the same HLA as the patient and matching is performed on the basis of variability at three or more loci of the HLA gene, and a perfect match at these loci is preferred. Even if there is a good match at these critical alleles, the recipient will require immunosuppressive medications to mitigate graft-versus-host disease.
Donors can be found via a number of programs etc.
Saviour siblings: the process of conceiving a sibling via IVF that is free of an inherited disease or cancer for the purpose of harvesting the HSCs from their umbilical cord/themselves to use to treat the diseased sibling. Popular culture: My Sisters Keeper. Diseases: Fanconi anemia, acute promyelocytic leukemia. Also Debbie Dingle from Emmerdale.
What are saviours siblings?
- Autologous HSC transplantation
This involves the extraction (apheresis) of HSCs from the patient and storage of the harvested cells in a freezer. The patient is then treated with high-dose chemotherapy with or without radiotherapy with the intention of eradicating the patient’s malignant cell population at the cost of partial or complete bone marrow ablation. The patients own stored stem cells are then transferred into their bloodstream, where they replace the destroyed tissue and resume the patient’s normal blood cell production.
Autologous transplants have the advantage of lower risk of infection during the immune-compromised portion of the treatment since the recovery of immune function is rapid. - Allogeneic HSC transplantation
This involves two people: a healthy donor and patient. The donor must have the same HLA as the patient and matching is performed on the basis of variability at three or more loci of the HLA gene, and a perfect match at these loci is preferred. Even if there is a good match at these critical alleles, the recipient will require immunosuppressive medications to mitigate graft-versus-host disease.
Donors can be found via a number of programs etc.
Saviour siblings: the process of conceiving a sibling via IVF that is free of an inherited disease or cancer for the purpose of harvesting the HSCs from their umbilical cord/themselves to use to treat the diseased sibling. Popular culture: My Sisters Keeper. Diseases: Fanconi anemia, acute promyelocytic leukemia. Also Debbie Dingle from Emmerdale.
What is decellularisation?
Decellularization is the process used in biomedical engineering to isolate the extracellular matrix (ECM) of a tissue from its inhabiting cells, leaving an ECM scaffold of the original tissue, which can be used in artificial organ and tissue regeneration. One of the greatest hurdles of organ transplantation derives from organ rejection caused by antibodies of the transplant recipient reacting to donor antigens on cell surfaces within the donor organ. Using DC we can add a patients own cells to the ECM scaffold, bypassing an immune response.
How can decellularisation be achieved? [3]
- Physical treatments
Temperature, force and pressure, and electrical disruption. Temperature methods are often used in a rapid freeze-thaw mechanism. By quickly freezing a tissue, microscopic ice crystals form around the plasma membrane and the cell is lysed. After lysing the cells, the tissue can be further exposed to liquidised chemicals that degrade and wash out the undesirable components. - Chemical treatments
Combination used depends on thickness, extracellular matrix composition, and intended use of the tissue or organ. Enzymes should not be used on a collagenous tissues because they disrupt the connective fibre tissues. However, when collagen is not present in high concentration or needed in the tissue, enzymes can be a vialbe option. Chemicals used: acids, alkaline treatments, ionic detergents, non-ionic detergents, and zwitterionic detergents.
Ionic detergent: SDS (sodium doceyl sulfate) is commonly used as lyses cells without damaging ECM to a significant degree. After SDS lyses the cell membrane, endonucleases and exonucleases degrade the genetic contents, while other components of the cell are solubilised and washed out.
Non-ionic detergent: Triton X-100, disrupts lipid-lipid and lipid-protein. EDTA: Ca2+ chelator, proteins need Ca2+ to interact with eachother. - Enzymatic treatments:
Trypsin acts as a protease to cleave the interactions between proteins – when used in a time sensitive manner.
Dispase is used to prevent undesired aggregation of cells, which is beneficial in promoting their separating from the ECM scaffold.
Collagenase is only used when the ECM scaffold product does not require an intact collagen structure.
Lipases are commonly used when decellularized skin grafts are needed.
What are the main signalling pathways involved in the maintenance of pluripotency?
Wnt pathway activation by 6-bromoindirubin-3-oxime (BIO), a specific pharmacological inhibitor of glycogen synthase-3 (GSK-3), maintains the undifferentiated phenotype and sustains the expression of pluripotent state-specifc transcription factors Oct-3/4, Rex-1 and Nanog.
Wnt signalling is downregulated upon differentiation.
Also BIO-mediated Wnt activation is functionally reversible, as withdrawal of the compound leads to normal multidifferentiation programs in both HESCs and MESCs.
GSK3 inhibitors such as BIO may have practical applications in regenerative medicine.
What is PD03?
PD03 is an inhibitor of MEK which lies downstream of several tyrosine-kinase mediated signalling pathways including the fibroblast growth factor pathway.
What is CHIR?
CHIR inhibits GSK3 and as GSK3-directed phosphorylation and degradation of B-catenin suppresses canonical Wnt signalling, CHIR is a potent agonist of the Wnt signalling pathway.
The MSCs were driven towards neural, muscular, and osteogenic lineages through culture on ____ kPa, ____ kPa, and ____ kPa polyacrylamide gels, respectively.
The MSCs were driven towards neural, muscular, and osteogenic lineages through culture on 0.1–1 kPa, 8–17 kPa, and 25–40 kPa polyacrylamide gels, respectively.
What is the application of muscle stem cells with regard to DMD?
Muscular Dystrophy treatment:
- Duchenne MD the most common and debilitating childhood MD. It is X-linked.
- Signs – muscle weakness and wasting, progressing to all voluntary muscles, diaphragm and heart
- Fatal by 20
- Due to reduced muscle fibre integrity, increased Ca2+ influx, activation of Ca2+ sensitive proteases and necrosis of muscle tissue. On a cellular level, evidence suggests that disease progression is due to a combination of replicative ageing of satellite cells and aberrant differentiation.
Satellite cells – poor migration when systemically delivered – IM every 2mm3. Expansion potential varies esp. in DMD patients. PEricytes and SP cells – a possible alternative.
What is MSI1436B?
Small molecule tyrosine phosphatase inhibitor which stimulates regeneration of the heart and multiple tissues.
As a person ages, the ability of satellite cells to repair damaged muscle declines. Describe how changes in Notch signaling are involved in this reduced healing capacity.
Basically:
Numb = inhibition of nuclear translocation of Notch: inhibits activation of target gene transcription = no repair.
Delta = promotion of Nuclear translocation…
Notch is implicated in satellite cell activation and proliferation. Notch signalling:
Notch ligand Notch receptor proteolytic cleavage and release of the intracellular domain, which enters the cell nucleus to modify gene expression.
Young and old people have similar levels of satellite cells: in resting muscle satellite cells have high levels of Numb and low levels of Delta; this reverses following injury: high levels of Delta and low levels of Numb. Numb inhibits nuclear translocation of Notch to inhibit activation of target gene transcription.
Upregulation of Delta is greatly reduced in old muscle (~25% of cells are activated compared to young muscle).
Activators of Notch signalling applied to muscle are capable of promoting satellite cell activation and proliferation.
Cell surface markers associated with MSCs [4]
CD44
CD90
CD105
N-cadherin
MSCs grown on soft substrates express
Chondrogenic marker collagen-II
Adipogenic marker lipoprotein lipase (LPL)
MSCs grown on stiff substrates have
higher expression of SMC markers alpha-actin and calponin-1.
What is LIF?
What effect does LIF have on STAT3 and on mESC pluripotency?
Leukeami inhibitory factor activates STAT3 and maintains mouse ECS pluripotency.
The following are key features of what?
Ac-LDL intake
Lectin staining
Endothelial marker expression: CD31,CD144, CD146, VEGFR2 (KDR), vWF.
EPC
What are the three main subtypes of EPC?
- early and late EPC
- CFU-Hill
- ECFCs
Only ECFCs have shown convincing ability to incorporate into blood vessels and form blood vessels in different experimental setups. This is possibily due to their high proliferate activity.
What is the only type of EPC to have shown convincing ability to incorporate into blood vessels and form blood vessels in different expirmental setups and why might this be?
Only ECFCs have shown convincing ability to incorporate into blood vessels and form blood vessels in different experimental setups. This is possibily due to their high proliferate activity.
Where can EPCs be obtained from? [2]
Bone Marrow
Peripheral mononuclear cells
What are the main signalling pathways involved in the maintenance of pluripotency?
Wnt pathway more so than Stat-3 signalling.
Wnt activation via BIO (specific inhibitor of GSK-3) maintains the undifferentiated phenotype in both types of ESCs and sustains the expression of the pluripotent state-specific TFs Oct-3/4, Rex-1 and Nanog.
Wnt activated via BIO = expression of Oct 3/4, Rex-1 and Nanog via B-catenin interacting with Oct4; Tcf3 binds to the Sox motif within Oct-Sox composite.
