Stem cells are distinguished from other cell types by two important characteristics:
They are unspecialized cells capable of renewing themselves through cell division
Under certain conditions, they can be induced to become tissue- or organ-specific cells with special functions.
What are unique properties of all stem cells?
Capable of dividing and renewing themselves for long periods
Unspecialized
Can give rise to specialized cell types.
What is an iPSC?
Induced pluripotent stem cell.
Adult (somatic) cells that have been genetically reprogrammed to an embryonic stem cell-like state by being forced to express genes and factor important for maintaining the defining properties of embryonic stem cells.
What is a teratoma?
A multi-layered benign tumor that grows from pluripotent cells.
What are the two cell types and the associated sources.
Primary (direct from tissue) and cell line (immortalized or tumor derived)
Once cells are modified in any manner, they are considered cell lines.
What are the advantages and disadvantages of primary cells?
Advantages - protentially retain all differentiated function
Disadvantages - difficult to maintain viability and differentiated function; may not proliferate
Once cells are modified in any manner, they are considered cell lines.
What are the advantages and disadvantages of cell lines?
Advantages: Relatively easy to maintain; prolifer readily for many generations
Disadvantages: Do not necessarily retain function of normal cells; safety concerns
Once cells are modified in any manner, they are considered cell lines.
What are the three cell sources?
Autologous - patient source
Allogeneic - other human source
Xenogeneic - other animal source
What are some advantages and disadvantages to autologous cells?
No immune rejection
Difficulty/inabilty to grow up cells
Impractical for certain tissues
What are some advantages and disadvantages to allogeneic cells?
Human source
Scarce for some tissues
Immune incompatibility
Transfer adventitious agents
What are some advantages and disadvantages to xenogeneic cells?
Readily available
Immune incompatibility
Function not necessarily identical
Transfer adventitious agents
What are the two responses to foreign materials? What are the acute and late responses?
Toxic response
-Acute
--Hemolysis
--Temperature increase
--Inflammation
--Anaphylactic shock
--Tissue damage
-Late
--Malignant neoplastic formation
--delayed allergy
--teratogenesis
--tissue necrosis
Foreign body response
-Acute
--Complement activation
--Blood coagulation
--Thrombus formation
--Phagocytosis
-Late
--Encapsulation
--Neointima formation
--calcification
--Hyperplasia
--Atrophy
What is mixed chimerism? What are the advantages?
Mixed chimerism involves giving the patient antibodies from the donor to prevent an immune reaction.
Usually chimerism develops accidentally in transplant patients when immature cells from the donor organ are incorporated into the recipient's blood.
This triggers the production of white blood cells matching the donor organ, thus protecting the organ from being destroyed
What are some advantages of established cell lines?
Many kinds of cell lines
Generally easy to grow and manipulate
Proliferate indefinitely
Disadvantages include ploidy problems; loss of biochemical properties of parent tissue.
Describe Primary cell cultures.
Cells derived directly from a tissue
Limited growth potential
Limited Life span
Cells are used within days from culture
May give rise to a cell strain or be immortalized
Cell strain - a lineage of cells originating from one primary culture
Describe transformed cells (cell lines)
Derived from tumors
Arise spontaneously in culture
Primary cells transformed with viruses, radiation etc
Grow indefinitely in culture
Usually unstable complement of chromosomes
Cell line - cells derived from a single parental transformed cell
(T/F) Cells must be grown at the biological pH
True.
(T/F) The medium is selected depending on the cell type.
True.
(T/F) The best growth is not always the fastest and the optimized media for the experiment must be used.
True.
(T/F) CO2 concentration is important for the bicarbonate buffering system in the blood.
True.
Too high in CO2 will change the pH of the cell culture.
Too much oxygen may overwhelm the cells (oxidative stress)
List some pros and cons of using antibotics in cell cultures
Reduce freqnecy of contamination
Encourage development of antibiotic resistant strains
Hide low level cryptic contaminations
Anti-metabolic effect
Encourage poor aseptic technique
Trypsin is a highly active, relatively non-specific, cheap protease derived from pancreas. List some uses in cell cultures.
Used in concentration of 0.05 to 0.25%
Cleaves proteins on the cell surface and extracellular matrix
Detaches cells
Inactivated by serum or soybean trypsin inhibitor
Cells needs to be washed withPBS (phosphate buffered saline) before trypsin treatment to remove serumwhich is a trypsin inhibitor
Over-trypsinization causes cell injury
Trypsin has to be deactivated after use
What are some sources of contamination in cell cultures?
Bacteria, mold, yeast
Mycoplasma
Cross contamination
Describe totipotent stem cells.
Totipotent (a.k.a omnipotent) stem cells can differentiate into embryonic and extraembryonic cell types. Such cells can construct a complete, viable organism.
These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.
Describe pluripotent stem cells.
Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells, i.e. cells derived from any of the three germ layers.
Describe multipotent stem cells.
Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of cells.
Describe oligopotent stem cells.
Oligopotent stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.
Describe unipotent stem cells.
Unipotent cells can produce only one cell type, their own, but have the property of self-renewal, which distinguishes them from non-stem cells (e.g., muscle stem cells).
What are the CD59 and decay accelerating factor (DAF)'s functions?
They mark the cells for destruction.
By inhibiting this protein, the prevention of a response towards foreign materials may be prevented.
(T/F) Pigs are considered clean and have a relatively low risk of viral transmission.
True.
What is the role of cell culture media?
Maintain pH
Provide essential nutrients
Provide energy source
Maintain osmolarity
Why do adult primary cells have a limited lifespan?
Reduction of telomerase activity which then shortens the length of telomers
(T/F) Serum is well defined and the most widely used as an additive to the culture media.
It is not well defined and contains insulin and serotonin. It does not inhibit trypsin
Rmbryonic stem cells are _____ stem cells.
multipotent stem cells.
What are the optimal conditions for cell cultures?
Always keep the cells in low density and keep them attached to each other.
(T/F) Adenovirus can infect both dividing and non-dividing cells, which express CAR.
True
(T/F) Adeno associate virus is larger than adenovirus.
False.
Which of the following many be used to deliver a gene to terminally differentiated neurons?
Lentivirus
Adeno-associated virus
Adenovirus
HSV-1
All of them.
(T/F) Under certain physiological or experimental conditions, stem cells can be induced to become cells with special functions.
True.
What are 4 gene delivery methods?
A gun to shoot gold particples with DNA
Electroporation
Biological vehicles
Liposomes
What is transfection?
Introduction of foreign material into eukaryotic cells
-overexpression
-Downregulation (RNAi, morpholino)
-Protein production (baculovirus, adenovirus)
What are some non-viral methods of DNA delivery?
Chemical (such as calcium)
Liposomes (surround etc)
Electroporation (change electric field outisde the cell membrane to create pores in the membrane)
What are the two transfection categories?
Transient transfection - DNA not inserted in host genome (temporary gene expression, DNA lost after cell division)
Stable transfection - DNA inserted into host genome (Selection pressure to maintain foeign DNA)
What are some methods of gene delivery?
Injection of naked DNA into tumor by simple needle and syringe
DNA coated on the surface of gold pellets which are air-propelled into the epidermis (gene gun); mainly non applicable to cancer
DNA transfer by liposomes (intravascular, intratracheal, intraperitoneal or intracolonic routes)
Biological vehicles (vectors) such as ciruses and bacteria
(T/F) Vector gene delivery should be able to target specific cell types.
True.
Place the following gene delivery methods in decending order for the amount of information they can carry.
Retrovirus
Adenovirus
Adeno-associated
Naked DNA/Liposomes
HISV-1
Naked DNA/Liposomes - unlimited
HISV-1 40 to 150kb
Adenovirus - 30kb
Retrovirus - 8kb
Adeno-associated - 4kb
What are some advantages and disadvantages of the non-viral DNA carrier cationic liposomes?
Positively charge lipids interact with negatively charged DNA
Advantages
Stable complex
Can carry large sized DNA
Can carry to specific cells
Does not induce immunological reactions
Disadvantages
Low transfection efficiency
Transient expression
Inhibited by serum
Some cell toxicity
What are the main limitations of retrovirus and lentivirus (ie HIV)?
Only transduces dividing cells, integration might induce creation of cancer
Main advantage persistent gene transfer in dividing cells
(T/F) Retroviral vectors are able to infect dividing cells only
True.
In dividing cells, nuclear membranes are broken down so viral genome can enter and integrate into the chromosome
THIS IS NOT TRUE OF LENIVIRAL VECTORS WHICH ARE RETROVIRUSES
(T/F) Lentiviral vectors are retroviruses that can infect both dividing and non-dividing cells.
True.
Although retroviruses can infect only dividing cells, lentivuses are apparently the exception.
What are the advantages of lentiviral vectors?
Mobilization of a vector designed to inhibit or prevent HIV
Replication or pathogenesis has been argued to enhance therapeutic effect
Disadvantage
Vector spread beyond the intended target tissue may have safety consequences
Co-packaging of wt-type HIV RNA and vector RNA may result in recombination
(T/F) Adenoviruses can infect both dividing and non-dividing cells (CAR is necessary for inection)
True.
What gene delivery vectors require CAR for attachement and delivery? Can this virus infect both dividing and non-dividing cells?
Adenovirus. Can infect both dividing and non-dividing cells.
(T/F) The Adeno-associated virus (AAV) can infect both dividing and non-dividing cells.
True.
(T/F) The adeno-associated virus (AAV) is the smallest virus and can only carry ~ 4kb of DNA.
True.
The DNA only goes in one spot of the genome.
(T/F) Adeno-associated virus (AAV) does not stimulate inflammation or antibodies in the host.
