4.) Stem Cells - An Introduction to their Biology Flashcards
What is a stem cell?
- ‘Immature’ (younger compared to differentiated cell), undifferentiated, non-specified cell with the capacity for prolonged/unlimited self-renewal (highly controlled/regulated; unlike tumour cells)
- Can differentiate to produce at least one type (but often many) of cell/tissue e.g. bone, cartilage, skin
- Intermediate formation of committed progenitor cells; more specific than stem cells that are pushed to differentiate into its ‘target’ cell (AKA transit amplifying cells - capacity to increase numbers greatly via this pathway)
How do stem cells differ from committed progenitor cells (transit amplifying cells)?
Committed progenitor cells:
• Can only divide a finite number of times, unlike stem cells
• Highly proliferative (fast dividing), unlike stem cells which proliferate v slowly
• Are pluripotent - can only give rise to a particular number of cell types, unlike totipotency
What is meant by ‘unlimited self-renewal’ of stem cells?
• They can divide and replenish themselves indefinitely
• E.g. can be life long as in ‘adult stem cells’
- Though there comes a point where stem cells will have reduced capacity/reduce in number
What is meant by differentiation? What is it characterised by?
Process whereby a cell acquires distinctive (specific) morphological (form and structure) and functional features:
• Limited ability to proliferate
• Specialised functions
• Determined by genes and environment
»> E.g. cardiac myocytes have specific function to contract muscle
What is stem cell potency? What categories are there?
A measure of how many cell types a stem cell can form: • Totipotent • Pluripotent (ES/EG) • Multipotent (adult stem cells) • Unipotent
What is totipotency? Give examples.
A stem cell that can form ALL tissues of an organism, including extraembryonic membranes and tissues (inc. placenta):
• the fertilised egg; has capacity to make entire organism (totipotency lost upon division > pluripotency)
What is pluripotency?
Stem cell variant that can give rise to MOST tissues of an organism:
• Embryonic stem cells (Embryonic Stem/Germ cells: ES/EG, the spermatozoa/zygote)
• iPS cells: induced pluripotent stem cells
What is the significant difference between embryonic stem cells (ES/EG cells) and iPS (induced pluripotent stem cells)?
Both lab-based technologies:
ES/EG:
• Requires isolation from blastocyst embryonic stage (pre-implantation stage embryo)
• Ethical issue in destroying/manipulating early stage embryo
• So far not been feasible to create patient-matched embryonic stem cell lines
iPS:
• Isolated from adult stem cells; no ethical implication re. destruction of early-stage embryo (blastocyst, pre-implantation) as in ES cells
• Thus can create patient-matched pluripotent stem cell line, reducing risk of immunogenicity
• Introduce genes to program and differentiate cell to target cell type, controlling/mapping cell for particular disease or condition e.g. transplant therapy/neurodegenerative medicine
What is multipotency? Give examples.
‘Adult’ stem cells capable of forming a restricted number of cell types (most tissues in body have adult stem cell population to allow repair, particularly in high attrition tissue e.g. skin/liver):
• Haematopoietic stem cells (HSC) form all blood cells (bone marrow)
• Mesenchymal stem cells (MSC) form many musculoskeletal tissues
• Cord blood stem cells (HSC and MSC - from umbilical cord/placenta)
What is the advantage of using cord blood stem cells over adult stem cells?
Both types of multipotent stem cell:
• Cord blood stem cells are from foetal source thus young AF (from umbilical cord/placenta) thus pose reduced immunogenicity risk in transplant therapy
Briefly outline the concept of stem cell self-renewal and differentiation.
- Asymmetric division - stem cell divides to give 2 daughter cells, where one is identical to the parent cell (i.e. self-renewal) and the other is slightly changed (i.e. differentiating) following transit amplification
- Committed progenitor cells have finite number of times they can divide
- WIth each division comes decreased proliferation potential/ability, but greater differentiation into target cell
What are the possible fates of a stem cell?
• Self-renewal: semi-conservative division, where the stem cell compartment is maintained
> Apoptosis: programmed cell death fundamental to tissue modeling/re-modelling
• Committed progenitor cell (uni/multipotent)
> Apoptosis
• Highly differentiated cells (may arise via several precursors/differentiation)
> Apoptosis
What factors in the niche/local environment influence stem cell differentiation?
- Cytokines
- Transcription factors
- Cell-cell interactions
- Cell-matrix interactions
- Nutrient/waste exchange (metabolomics)
- Oxygen concentration
How has oxygen concentration proved important for stem cell differentiation?
Found that stem cells cultured in lab vs. body oxygen levels produced different results:
• Oxygen tension importance
• Particularly in cartilage - greater capacity to produce stem cells in O2-adjusted lab
What is meant by the stem cell controlling axis?
Renewal of tissues using stem cells upon wear & tear or traumatic injury (e.g. skin, bone, blood etc.):
- Above trigger results in positive feedback to self-renewal of stem cell, differentiation of progenitor cells etc
- Positive feedback at each level of stem cell fate for repair/regeneration
• Stem cells transient in compartment until signalled for; metabolising, but not dividing
What arises from unregulated stem cells?
Tumour cells e.g. leukaemia
What are the reasons for stem cell therapy research?
Potentially unlimited proliferation potential:
• Supply large numbers of cells required for therapies
• Differentiation plasticity, can form many different cell types
• E.g. labs could maintain culture for years, with stem cells retaining functional properties
Developmental biology:
• Cell lineage ontogeny (origination and development of an organism)
• Tissue morphogenesis (formation)
Tissue repair & regeneration:
• Production of desired cell types
• E.g. bone cells - mix with stem cells and scaffold for repair (pushing them down a pathway)
Pharmaceutical testing:
• More accurate physiological models e.g. to test drugs on human models instead of rodents models in drug development
Gene therapy
What are the requirements for the repair and regeneration of damaged or diseased tissues?
What are they dependent on?
- Recapitulate tissue morphogenesis (how a tissue forms)
- Generate adequate cell population/tissue size (need to generate billions for effective repair)
- Differentiate to/maintain specific phenotype and function (function difficult to test)
- Appropriate 3D organisation (ECM/Scaffolds - cells behave differently in 2D e.g. agar dish vs 3D)
- Mechanical/physical integrity (e.g. cartilage/bone)
- Modulation/prevention of immino-rejection (immunogenicity)
- Vascularisation (how to get native blood vessels to grow into new stem cell tissue?)
- Innervation
> > > Dependent on the qualities of the cells (cell source)
What are the different sources of cells availible in archetyping the repair and regeneration of damaged/diseased tissues?
- ) Mature (non-stem) cells from patient (transferring tissue/cells e.g. skin graft)
- ) ‘Adult’ stem cells from patient (site-specific locations)
- ) Cord blood stem cells (HSC/MSC)
- ) Embryonic stem cells (ES)/Embryonic germ cells (EG) - reprogramming somatic cells (cloning)
- ) Induced pluripotency stem cells (iPS) - reprogramming
Name some examples of sources of ‘adult’ stem cells.
• Bone marrow space:
- HSC (haematopoietic - blood)
- MSC (mesenchymal - bone/muscle)
- Gut
- Skin
- Brain
- Muscle
What are the requirements of culture systems in the lab to grow, control and study stem cells?
- Defined conditions (growth factors expected/known to favour growth and differentiation to particular cell type)
- Bioreactors (mass production/scale-up of cell numbers)
- Physical forces/3D interactions/scaffolds (natural/synthetic): cell numbers/density, mechanical forces, oxygen tensions
- Gene transfection: transcription factors
> > > Not every process will have the same parameters
Outline the advantages and disadvantages of sourcing cells from mature (non-stem) cells for the repair and regeneration of damaged/diseased tissues?
Examples: bone, cartilage, skin, liver cells etc. (biopsy)
Advantages:
• Often easily obtained from patient
• No need for immunosuppression if used for re-implantation (in self - body recognises self)
Disadvantages:
- Poor, slow growth, difficult to get enough cells
- May change phenotypic characteristics (particularly the longer outside the body - different microenvironment/stimuli in lab vs. body)
- Tens of millions of cells may be required, taking several WEEKS to grow
- Prolonged patient morbidity and possible mortality
- Creates two wound sites (site of injury, and site of tissue biopsy e.g. skin graft to isolate and grow cells»_space;> pain)
What are the advantages of using ‘adult’ stem cells or ES/EG cells for tissue repair?
- Rapid growth (unlike mature, non-stem cells)
- Plasticity; can from various cell types (both in and ex vivo, provided they’re in appropriate stimuli/environments, with prior knowledge of stem cell biology and target cells)
What are the specific types of ‘adult’ stem cells and their niches?
- Epidermal
- Follicular (cosmetic application)
- Intestinal
- Neural
- Haematopoietic
What is the basis of leukaemia treatment?
Abnormal blood cells produced in the bone marrow, most commonly white blood cells (lymphoblastic) but also RBCs (myeloid):
• Ablate current mutant bone marrow cells (HSC)
• Replace with donor stem cells
(requires immunosuppression)
Mesenchymal stem cells (MSC) form many musculoskeletal tissues. Name some examples, how they arise, and what potency do MSCs have?
Multipotency, target cell type achieved via modulation of culture conditions: • Cartilage • Bone • Fat • Muscle • Haematopoietic support cells • Astrocytes (brain)
> > > MSCs from bone marrow can form blood and brain cells/tissue, as well as musculoskeletal tissues (mesodermal lineage).
Outline the advantages and disadvantages of using MSCs (mesenchymal stem cells - bone marrow) for the repair and regeneration of damaged/diseased tissues?
Advantages:
• Potential to generate various cell types, not just mesodermal lineage (multipotent): bone and cartilage repair, as well as heart tissue
• Harvested from donor and implanted back into donor following expansion & differentiation in vitro: potentially rapid (1 month) unlike v. slow mature non-stem cells, whilst negating immunogenicity issues
Disadvantages:
- Stem cells may be very sparse (1 cell in 100,000)
- Potential to propagate or transmit harmful mutations - allogeneic tx (immunological compatibility issue)
- Numbers & potency diminish with age (e.g. less capacity to repair bone in fracture w/post-menopausal women)
Why can stem cells (MSCs) be very sparse?
1 cell in 100,00:
• Stem cells are NORMALLY very tightly controlled and regulated
• Stem cells transient in compartment until signalled for; metabolising, but not dividing
»> Harvesting enough could pose a challenge
What are cord blood stem cells? Why does it pose great potential?
Multipotent stem cell from foetal source:
• Less immunogenic as foetus has weaker immune system
• Routinely collected from umbilical cord post-birth, posing no risk to baby (placenta/cord often discarded as clinical waste otherwise), minimal ethical issues (informed consent to collect/bank)
• Can also be collected in utero via ultrasound guided needle: potential ethical hurdle, with some risk to baby though a ‘routine’ procedure
What is cord blood characterised by?
- Mainly HSC (haematopoietic stem cells)
- Foetal/newborn cord blood also contains a MSC-like population (mesenchymal)
- Number of cells collected often VARIABLE
- Single (ONE TIME) source - umbilical cord/placenta discarded after extraction
What are the therapeutic applications of cord blood stem cells?
HSC/MSC:
• Less immunogenic/immunomodulatory activity (lower HLA matching - human leukocyte antigen, which immune system recognises/or does not)
• Haematological malignancies (e.g. leukaemias - HSC, though MSC can form blood too)
• Haemoglobinopathies (abnormal structure of Hb e.g. sickle, thalassaemia)
• Bone marrow failures
• Immunodeficiencies (e.g. SCID - Severe Combined ImmunoDeficiency; disturbed development of functional T cells and B cells)
• Bone disorders (e.g. osteogenesis imperfecta ‘brittle bone disease’)
• Various other cell types in vitro (neural, hepatic, cardiac)
At what stage of fertilisation are ES/EG cells isolated? Timeline?
- Pre-implantation blastocyst
- ES cells derived from inner cell mass
- Between 5-7 and 14 days, where the zygote has reached blastocyst development 5 days after fertilisation
What layers do ES cells mature into initially before differentiating?
ES cells are derived from the inner cell mass of the pre-implanted blastocysts, which then go onwards to mature to the 3 germ layers:
• Ectoderm (outside)
• Mesoderm (in between)
• Endoderm
What tissues can the ectoderm, mesoderm and endoderm germ layers of ES cells differentiate into respectively?
They have pluripotency; can generate over 220 cell types in the adult body:
Ectoderm (skin/brain):
• Epidermal cells of skin
• Neurons of brain (CNS)
• Pigment cell (neural crest)
Mesoderm (bone/muscle/blood): • Dorsal (notochord - supports skeleton) • Cardiac muscle • Skeletal muscle cells • Paraxial (bone tissue) • Tubule cell of kidney) • RBCs • Head (facial muscle)
Endoderm (mucosa - gut/airways/pancreas):
• Pancreatic cell
• Thyroid clel
• Lung cell (alveolar)
Briefly outline the derivation and propagation of ES cells.
- ES cells derived from inner cell mass of pre-implantation blastocyst (developing embryo); dissected away from trophectoderm (outer layer which forms placenta etc)
- Inner cell mass cell cultured (now ES cells) under specific conditions e.g. grown-arrested fibroblasts (+feeder cells) w/certain cytokines
- ES cells grow as distinct colonies, collected at a certain size/density, which are then dispersed into small clusters and either re-cultured (on fresh plates) as ES cell repeats, or in the absence of feeders or cytokines and induced to differentiate
- ES cells differentiate via formation of embryoid bodies (3D aggregates), which are either left intact or dispersed and stimulated with growth factors to encourage differentiation of particular cell types
What is the role of certain cytokines in the derivation and propagation of ES cells?
- Added when inner cell mass cells of pre-implantation blastocyst are being cultured
- Work as a ‘brake’ on differentiation during culturing to maintain ES cell pluripotency
Outline the advantages and disadvantages of using ES’ (embryonic stem cells - blastocyst) for the repair and regeneration of damaged/diseased tissues?
Advantages:
• Potential to generate any cell type in the body (pluripotent - differentiate from 3 germ layers)
• Amenable to genetic manipulation: can introduce beneficial/therapeutic genes, and modulate/control immunotolerance (negating need for immunosuppression)
Disadvantages:
- Still difficult to accurately/predictably control differentiation (ES cells not routinely used in clinic yet)
- Ethical concerns: cloning (therapeutic, NOT reproductive e.g. allow Dolly the Sheep), availible donors, destruction of embryo each time ES cells extracted
- Stability (mutations/tumours) and transmission of heritable disease unknown (karyotyping/screening)
> > > Currently may have place as human models for lab testing (instead of rodent models etc)
What legislation/ethics surround ES cell research?
Human Fertility & Embryology Authority (HFEA) and local research ethics committee approval required for use:
• Import & experiment with established hES cell lines (human ES)
• Derive and maintain new hES cell lines (14 days)
• Can perform NT (nuclear transfer) for therapeutic cloning (14 days)
• Research can be funded by Research Councils/Charities
> > > Tightly controlled
Define cell, therapeutic and reproductive cloning.
Cell cloning:
• Creation of a line of cells genetically identical to the originating cell
Therapeutic cloning:
• Reprogramming nucleus of adult cell by transfer to cytoplasm of enucleated oocyte (somatic cell NT)
• Followed by isolating ES cells after formation of blastocysts in vitro
Reproductive cloning:
- Reprogramming nucleus of adult cell by transfer to cytoplasm of enucleated oocyte (somatic cell NT)
- Followed by re-implanting the embryo to enable formation of viable foetus (in surrogate mother e.g. Dolly the Sheep)
Has would reprogramming to achieve ‘totipotent’ cell, achieved? What barrier does this overcome?
Via somatic cell NT:
• Somatic cell nucleus (any cell other than a gamete, germ cell, gametocyte or undifferentiated stem cell) transferred to enucleated donor oocyte
• Cytoplasmic factors (microenvironment) re-programme nucleus to produce ‘totipotent’ cell (able to produce any cell in the body as well as extraembryonic/placental cells)
> > > Reverting mature cells into stem cells, then transforming to desired cell type would overcome issues of cloning
Would require identification & purification of cytoplasmic/nuclear re-programming factors
Outline the basic concepts of SCNT (somatic cell nuclear transfer), and how it can lead to therapeutic/reproductive cloning.
• Chromosomes/DNA removed from donor oocyte (enucleated), whilst intact nucleus of somatic cell is removed
• Somatic cell nucleus is injected into enucleated oocyte, where various cytoplasmic factors present (in cytoplasm) help ‘re-programme’ its DNA
• Electrical/chemical stimulation then allows oocyte-somatic cell nucleus ‘hybrid’ to begin to divide, forming normal developmental process to generate viable embryo
• Inner cell mass cells can then be extracted from the pre-implantation blastocyst, to create ES cells, which are directed to differentiate into specific cell types genetically identical to donor of somatic cell = immunologically compatibility with donor = THERAPEUTIC CLONING
»> If oocyte-somatic cell nucleus hybrid implanted into surrogate mother, this leads to development into intact offspring that is a clone of the donor of the somatic cell = REPRODUCTIVE CLONING (banned/illegal - Dolly the Sheep/Jango Fett, Dolly was the only successful 1 of 100s of embryos, and had many morbidities)
What are the potential uses for reproductive cloning, even though it is illegal?
- Production of human proteins (clotting factors)
* Conservation of endangered species (e.g. if only one left - no mate for sexual reproduction)
How does the direction/induction of fate change between somatic stem cells and different stages? (iPS)
- Induction of fate changes between somatic stem cells and de-differentiation/reprogramming e.g. epimorphic limb regeneration in amphibians
- 4 genes introduced into cell inducing reprogramming - reverting to ES cell (iPS)
What are iPS cells?
- Reprogramming differentiated cells from adult tissues to a less differentiated, pluripotent state
- Introducing (transducing) genes associated with pluripotency (4 genes) into differentiated cells using viruses (via vectors)
What hurdles are there still with iPS technology?
- Relatively low efficiency of reprogramming
- Use of viruses to introduce genes (x4) associated with pluripotency
»> Different methods being developed
Are ES cells and iPS cells identical?
• Both demonstrate pluripotency,
• Resumble ES cells
- But iPs are distinct from ES, have different genotypes from ES cells
How can tissue engineering help? Define Autografts, Allografts, Xenografts, and Man-made materials and devices.
Autografts:
• Using tissues from patients own body for transplanting into another site in the same patient e.g. bone, skin, blood vessels etc
• Self to self
Allografts:
• Using tissue from donor (living or dead) and transplanting into another patient e.g. kidney, heart, lungs, liver, bone marrow, cornea
Xenografts:
• Using tissues and organs from animals for transplantation into human e.g. primate, monkey, porcine organs/tissues
• Animal to human
Man-made materials and devices:
• E.g. artificial hearts, heart valves, prosthetic hips, kidney dialysis, liver support devices etc
Outline the pros and cons of Autografts for tissue engineering.
+ Good clinical outcomes, little rejection
- Collection limited (from self), creates 2 wound sites (site of injury, site of biopsy), risk of pain and infection
Outline the pros and cons of Allografts for tissue engineering.
+ Can restore normal function
- Life-long immunosuppression therapy required, shortage of donors, disease history
Outline the pros and cons of Xenografts for tissue engineering.
+ Availability
- Rejection, disease transmission (zoonoses e.g. salmonellosis, Ebola), ethics
Outline the pros and cons of Man-made materials and devices for tissue engineering.
+ Fills immediate short term need
- Material fatigue, toxicity/corrosion
Questions on Stem Cells:
- Understanding/defining concepts
- Differentiation
- Uses of cell types
