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#FME - Gene Therapy, RNA Therapeutics and Stem Cells > 4.) Stem Cells - An Introduction to their Biology > Flashcards

4.) Stem Cells - An Introduction to their Biology Flashcards

1
Q

What is a stem cell?

A
  • ‘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)
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2
Q

How do stem cells differ from committed progenitor cells (transit amplifying cells)?

A

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

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3
Q

What is meant by ‘unlimited self-renewal’ of stem cells?

A

• 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

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4
Q

What is meant by differentiation? What is it characterised by?

A

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

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5
Q

What is stem cell potency? What categories are there?

A 
A measure of how many cell types a stem cell can form:
• Totipotent
• Pluripotent (ES/EG)
• Multipotent (adult stem cells)
• Unipotent
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6
Q

What is totipotency? Give examples.

A

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)

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7
Q

What is pluripotency?

A

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

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8
Q

What is the significant difference between embryonic stem cells (ES/EG cells) and iPS (induced pluripotent stem cells)?

A

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

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9
Q

What is multipotency? Give examples.

A

‘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)

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10
Q

What is the advantage of using cord blood stem cells over adult stem cells?

A

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

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11
Q

Briefly outline the concept of stem cell self-renewal and differentiation.

A
  • 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
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12
Q

What are the possible fates of a stem cell?

A

• 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

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13
Q

What factors in the niche/local environment influence stem cell differentiation?

A
  • Cytokines
  • Transcription factors
  • Cell-cell interactions
  • Cell-matrix interactions
  • Nutrient/waste exchange (metabolomics)
  • Oxygen concentration
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14
Q

How has oxygen concentration proved important for stem cell differentiation?

A

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

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15
Q

What is meant by the stem cell controlling axis?

A

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

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16
Q

What arises from unregulated stem cells?

A

Tumour cells e.g. leukaemia

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17
Q

What are the reasons for stem cell therapy research?

A

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

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18
Q

What are the requirements for the repair and regeneration of damaged or diseased tissues?

What are they dependent on?

A
  • 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)

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19
Q

What are the different sources of cells availible in archetyping the repair and regeneration of damaged/diseased tissues?

A
  1. ) Mature (non-stem) cells from patient (transferring tissue/cells e.g. skin graft)
  2. ) ‘Adult’ stem cells from patient (site-specific locations)
  3. ) Cord blood stem cells (HSC/MSC)
  4. ) Embryonic stem cells (ES)/Embryonic germ cells (EG) - reprogramming somatic cells (cloning)
  5. ) Induced pluripotency stem cells (iPS) - reprogramming
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20
Q

Name some examples of sources of ‘adult’ stem cells.

A

• Bone marrow space:

  • HSC (haematopoietic - blood)
  • MSC (mesenchymal - bone/muscle)
  • Gut
  • Skin
  • Brain
  • Muscle
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21
Q

What are the requirements of culture systems in the lab to grow, control and study stem cells?

A
  • 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

22
Q

Outline the advantages and disadvantages of sourcing cells from mature (non-stem) cells for the repair and regeneration of damaged/diseased tissues?

A

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&raquo_space;> pain)
23
Q

What are the advantages of using ‘adult’ stem cells or ES/EG cells for tissue repair?

A
  • 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)
24
Q

What are the specific types of ‘adult’ stem cells and their niches?

A
  • Epidermal
  • Follicular (cosmetic application)
  • Intestinal
  • Neural
  • Haematopoietic
25
Q

What is the basis of leukaemia treatment?

A

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)

26
Q

Mesenchymal stem cells (MSC) form many musculoskeletal tissues. Name some examples, how they arise, and what potency do MSCs have?

A 
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).

27
Q

Outline the advantages and disadvantages of using MSCs (mesenchymal stem cells - bone marrow) for the repair and regeneration of damaged/diseased tissues?

A

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)
28
Q

Why can stem cells (MSCs) be very sparse?

A

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

29
Q

What are cord blood stem cells? Why does it pose great potential?

A

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

30
Q

What is cord blood characterised by?

A
  • 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
31
Q

What are the therapeutic applications of cord blood stem cells?

A

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)

32
Q

At what stage of fertilisation are ES/EG cells isolated? Timeline?

A
  • 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
33
Q

What layers do ES cells mature into initially before differentiating?

A

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

34
Q

What tissues can the ectoderm, mesoderm and endoderm germ layers of ES cells differentiate into respectively?

A

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)

35
Q

Briefly outline the derivation and propagation of ES cells.

A
  • 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
36
Q

What is the role of certain cytokines in the derivation and propagation of ES cells?

A
  • 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
37
Q

Outline the advantages and disadvantages of using ES’ (embryonic stem cells - blastocyst) for the repair and regeneration of damaged/diseased tissues?

A

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)

38
Q

What legislation/ethics surround ES cell research?

A

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

39
Q

Define cell, therapeutic and reproductive cloning.

A

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)
40
Q

Has would reprogramming to achieve ‘totipotent’ cell, achieved? What barrier does this overcome?

A

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

41
Q

Outline the basic concepts of SCNT (somatic cell nuclear transfer), and how it can lead to therapeutic/reproductive cloning.

A

• 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)

42
Q

What are the potential uses for reproductive cloning, even though it is illegal?

A
  • Production of human proteins (clotting factors)

* Conservation of endangered species (e.g. if only one left - no mate for sexual reproduction)

43
Q

How does the direction/induction of fate change between somatic stem cells and different stages? (iPS)

A
  • 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)
44
Q

What are iPS cells?

A
  • 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)
45
Q

What hurdles are there still with iPS technology?

A
  • Relatively low efficiency of reprogramming
  • Use of viruses to introduce genes (x4) associated with pluripotency
    »> Different methods being developed
46
Q

Are ES cells and iPS cells identical?

A

• Both demonstrate pluripotency,
• Resumble ES cells
- But iPs are distinct from ES, have different genotypes from ES cells

47
Q

How can tissue engineering help? Define Autografts, Allografts, Xenografts, and Man-made materials and devices.

A

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

48
Q

Outline the pros and cons of Autografts for tissue engineering.

A

+ Good clinical outcomes, little rejection

- Collection limited (from self), creates 2 wound sites (site of injury, site of biopsy), risk of pain and infection

49
Q

Outline the pros and cons of Allografts for tissue engineering.

A

+ Can restore normal function

- Life-long immunosuppression therapy required, shortage of donors, disease history

50
Q

Outline the pros and cons of Xenografts for tissue engineering.

A

+ Availability

- Rejection, disease transmission (zoonoses e.g. salmonellosis, Ebola), ethics

51
Q

Outline the pros and cons of Man-made materials and devices for tissue engineering.

A

+ Fills immediate short term need

- Material fatigue, toxicity/corrosion

52
Q

Questions on Stem Cells:

A
  • Understanding/defining concepts
  • Differentiation
  • Uses of cell types

#FME - Gene Therapy, RNA Therapeutics and Stem Cells (4 decks)

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