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[MBD] NoNa > Stem Cells and regenerative medicine > Flashcards

Stem Cells and regenerative medicine Flashcards

1
Q

What are stem cells?

A
  • Undifferentiated cells which can differentiate into many different cell types
  • They self-renew by dividing
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2
Q

What are the different types of stem cells?

A

Embryonic stem cells

Adult stem cells

Induced Pluripotent stem cells

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3
Q

Describe Adult stem cells

(ASCs)

A
  • Tissue specific
  • Multipotent = differentiate into a set number of cells within a specific location
  • There are exceptions - e.g. adult bone marrow HSCs have shown variation
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4
Q

Describe embryonic stem cells

(ESCs)

A
  • Pluripotent = can differentiate into every cell type(all 3 primary germ layers)
  • Originate from the blastocyst = before implantation when embryo is just a few days old.
  • These cells undergo cell division in vivo/in vitro, then differentiate
  • They can form all cells from all 3 embryonic germ layers - ectoderm, mesoderm, endoderm
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5
Q

What are the different germ layers?

A
  • Ectoderm (External) → epithelial tissue, sensory tissue, nervous tissue
  • Mesoderm(Middle) → skeletal muscle, cardiac muscle, blood, connective tissue
  • Endoderm (Internal) → lungs, pancreas, stomach, liver, germ cells
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6
Q

Describe Induced Pluripotent stem cells (iPSCs)
How are they created and how are they used ?

A

-Induced in lab - reprogram normal differentiated tissue(Adult somatic stem cells) by exposing to specific pluripotency factors (Transcription factors - Yamanka factors):

  • OCT4
  • Sox2
  • cMyc
  • Klf4
  • This produces pluripotent stem cells - similar characteristics to embryonic stem cells - Cell therapy
  • Repair mutations by CRISPR/Cas gene editing in vitro = diff. into healthy cells and transplant back into patient
  • Reduces graft rejection - cells specific to patient
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7
Q

How are iPSCs grown and what can this be used for ?

A

Grown in layer in dish/3D organoid model

They can be used as models for research:

  • Cell differentiation study
  • Developmental biology
  • Cell replacement therapy
  • Disease Modelling
  • Drug screening
  • Translational research
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8
Q

What are organoids?

A

Organoids = tiny, self-organised, 3D tissue cultures derived from stem cells.
-Replicate organ complexity.

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9
Q

What are three categories of stem cells in terms of potency ?

A

Totipotent -Undifferentiated , self renew and produce pluripotent embryonic stem cells

Pluripotent +(Induced pluripotent ) -These can differentiate into the 3 embryonic germ layers. Also produce multipotent ASCs

Multipotent (ASCs) -Tissue-specific stem cells which diff. into specialised cell types.

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10
Q

What do tissue-specific stem cells require?

A

Specialised microenvironments (stem cell niches)

Required to:

  • Regulate cell fate (What the cell will become )
  • Prevent stem cell depletion/hyperproliferation

Specific Features of Stem Cell Niches:

  • Supportive ECM molecules - fibronectin/collagen
  • neighbouring niche cells - interactions with tissue-specific surrounding cells
  • secreted soluble signalling factors (e.g. growth factors and cytokines, chemokines, Wnt signalling)
  • physical parameters; shear stress, tissue stiffness, and topography),
  • environmental signals (metabolites, hypoxia, inflammation, etc.) - signals from immune cells recruited during inflammation
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11
Q

What are the features of a stem cell niche?

A

-Supportive ECM
(Collagen, Fibronectin)

-Secreted soluble signalling factors
(Growth factors, Cytokines, chemokines, Wnt signalling)

-Physical parameters-
(Shear stress, tissue stiffness and topography)

-Environmental signals -
(Metabolites, hypoxia ,inflammation) - signals from immune cells recruited during inflammation

-Neighbouring niche cells - interactions with tissue-specific surrounding cells

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12
Q

What is Hedgehog(Hh)and WNT signalling ?

A

Hedgehog(Hh)and WNT signalling = Pathways which direct growth patterns during embryonic development.

Regulate in epithelia of skin and intestine which undergoes constant renewal

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13
Q

What can unregulated stem cell proliferation cause ?
Which stem cells is this most likely to occur in?

A

Cancer/tumour

Embryonic stem cells - have unlimited growth potential

There is a large number of embryonic stem cells which can be used

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14
Q

Outline some pros and cons of embryonic stem cells

A

Pros:

  • Almost unlimited growth potential = can differentiate into any type of cell
  • Unlimited number of cells due to high cell potency
  • Very low probability of DNA mutation damage(Low spontaneous mutation rate + high genetic stability)

Cons:

  • ⬆ tumour risk (uncontrolled stem cell proliferation)
  • ESCs = genetically diff to receipient host = ⬆ rejection risk
  • Ethical - ESCs derived from surplus IVF embryos = parental consent required, strict legal guidelines.
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15
Q

Outline some pros and cons of adult stem cells

A

Pros:

  • Compatible with recipient’s cells -low risk of rejection. bc derived from + for same patient
  • Less risk of tumour creation, low growth potential
  • No ethical issues - patient gives direct consent

Cons-

  • A limited number can be obtained
  • Higher probability of mutation-induced damage in the DNA -risk of disease
  • Limited cell potency
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16
Q

Outline some pros and cons of Induced Pluripotent stem cells

A

Pros:

  • Compatible with recipients cells- low risk of rejection in transplantation, usually derived from + for a specific patient
  • Limited number can be obtained
  • Less risk of tumour formation
  • No ethical issues - patient gives direct consent

Cons:

  • Less growth potential than embryonic stem cells
  • Higher probability of mutation-induced damage in DNA , low genetic stability -risk of disease
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17
Q

Why are Adult stem cells more susceptible to mutations /diseases?

A

Adult stem cells are especially vulnerable to cell cycle mutations since these cells already have the capacity to self-renew and can pass mutations to their daughter cells.

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18
Q

What are the ethical concerns surrounding embryonic stem cells ?

A

These are derived from surplus IVF embryos

  • Parental consent +legal guidelines
  • Unethical to destroy embryos
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19
Q

Outline the Yamanaka factors and how they induce iPSCs

A

The four Yamanaka factors :
-Sox 2, Oct3/4, Klf4, c-Myc

  • c-Myc relaxes chromatin + promotes DNA replication
  • = Oct3/4 can access its target genes.
  • Sox2 + Klf4 co-operate w Oct3/4 to activate target genes
  • These genes encode TFs involved in pluripotent transcription factor network
  • = Activates the epigenetic processes (more open chromatin) that establish the pluripotent epigenome.

The iPSC cells have a similar global gene expression profile to that of ES cells.

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20
Q

Outline how stem cells can be tracked

A
  • Manipulate stem cells in vitro to track them in vivo.
  • Insert a fluorescent reporter gene
  • Then transplant cells back into patient/animal model
  • Non-invasive cell tracking tracks stem cell location + behaviour in vivo

Used to develop cell-based therapies

21
Q

What takes place in a heart attack?

A
  • Loss of blood supply to heart muscle.
  • Coronary vessel is ocluded.
  • Cardiac muscle dies + not replaced (bc low adult cardiomyocyte turnover).
  • Fibrosis + scarring occurs = ⬇ cardiac function + heart failure.
  • doesn’t contract to pump blood around the body
22
Q

What can research help to solve in terms of the heart?

A

Replace lost cardiac muscle, blood supply.
Useful for:
-Cardiac myopathies = heart muscle becomes enlarged/thick/rigid.
-Conduction defects = Dysregulated electrical conduction

23
Q

What are 2 regeneration strategies used to treat the heart?

A

Regenerate cardiomyocytes (neovascularisation) using:

  • Cell-based therapies = transplant, stem cells diff. into new coronary vessels
  • Cell-free therapies = ↑ paracrine stimulation of endogenous cardiomyocytes
24
Q

Expand on cell-based therapies (heart)

A

Cell transplantation

  • Promotes cardiac regeneration and repair
  • Replenishes the lost cardiac myocytes

Challenges:

  • Immune rejection
  • Manufacture/isolation of sufficient cells
  • Mode of delivery
  • Clinical regulation
25
Q

Expand on cell-free therapies

(Heart)

A

-Directly stimulate endogenous cardiomyocyte production.
-Includes reactivation of developmental pathways -
epicardium-based on models where there is no scarring + full cardiac regeneration

26
Q

What is neovascularisation?

A

Neovascularisation = ⬆ circulation to an injured area.

  • Paracrine effects improve cardiomyocyte replacement.
  • Inhibiting apoptosis+anti-inflammation
27
Q

Explain the cardiac regeneration in adult zebrafish

A

Zebrafish can regenerate their heart following injury

  • Apex is removed (Injury)
  • 1 day = A large, blood-filled clot seals the injury site
  • 9 days = Fibrin clot seals the apex wound
  • 14 days = Fibrin clot is resolved and replaced by heart muscle
  • 1 month later = new cardiac wall
  • 2 Months = entire injury site is regenerated with new cardiomyocytes
  • We also see re-expression of developmental gene programmes (WT1,RHald H2??)
  • These are found on epicardium (surface layer of heart)-causes reactivation

•Epicardial activation, then endocardial activation, cardiomyocyte dedifferentiation, form fibrin clot, then degrades as cardiomyocytes proliferate + regenerate heart

28
Q

Explain what’s different in larger mammals in the heart following injury.

A

The fibrin clot will not resolve, but remodels to form a fibrotic scar
-In pigs, humans

29
Q

What is the epicardium and what is it important for ?

A

Epicardium = surface layer of the heart, contains cells for coronary vessels and signals for cardiac myocyte proliferation.

30
Q

Expand on zebrafish regeneration (cardiac)-Adult

A

Injury occurs
Embryonic gene expression
Endocardium activation
Fibrosis degradation

•Epicardial activation, then endocardial activation, cardiomyocyte dedifferentiation, form fibrin clot, then degrades as cardiomyocytes proliferate + regenerate heart

31
Q

Explain the immune response during regeneration of a neonatal mouse heart

A

Infiltration of injury by embryonic macrophages
Revascularisation and global cardiac myocyte proliferation

32
Q

Explain the immune response during regeneration of adult mouse heart

A

Infiltration by monocyte derived macrophages
There is limited revascularisation and no cardiac myocyte proliferation

33
Q

Outline the lymphatic response to cardiac injury.
Explain the 2 different response types

A

It will modulate the immune response during cardiac regeneration.

In normal controlled injury response:
(Endogenous)
-Does not clear excess tissue fluid and inflammatory immune cells efficiently = causes oedema + inflammation (poor cardiac repair/function)

If immune system is stimulated by modified VEGFC, this increases lymphatic response, clears tissue fluid in inflammatory cells, reduces oedema + inflammation + improves cardiac repair/function

34
Q

How are iPSCs specified towards different cardiac lineages ?

A
  • The iPSCs will specify into pre-cardiac mesoderm by inhibiting GSK-3b.
  • Glycogen synthase kinase 3b acts as a downstream switch for signalling pathways e.g. WNT signalling.
  • Inhibit WNT signalling = differentiation of cardiac progenitor cells
  • Specific signalling = cardiac progenitory cells differentiate into specialised cardiac lineages - epicardial cells, cardiac fibroblasts, cardiomyocytes, smooth muscle, endothelial cells
  • iPSCs – cardiac regenerative investigation
  • Form various cardiac lineages from patient somatic tissues
  • Somatic cells are reprogrammed to pluripotency via Yamanaka factors
  • iPSCs are then specified towards precardiac mesoderm by inhibiting glycogen synthase kinase 3b = downstream switch for Wnt signalling
  • Further inhibit Wnt signalling = cardiac progenitor cells diff.
  • Provide specific signalling molecules to diff.
35
Q

What are the specialised cardiac lineages?

A

Epicardial cells

Cardiac Fibroblasts
Cardiomyocytes
Smooth muscle cells
Endothelial cells
Endocardial cells

36
Q

Describe iPSC evidence found in monkey model

A
  • iPSCs used to create patient-specific cardiomyocytes to aid with cardiac repair .
  • Tested on monkey model with identical MHC (Major histocompatibility complex)as humans.
  • They used fibroblast-derived iPSCS then direct intra-myocardial injection to monkeys after myocardial infarction.

Result

  • The graft cardiomyocytes survived for 12 weeks without rejection
  • electrical coupling between host and graft cardiomyocytes
  • Improved cardiac contractile function

-Ventricular arrhythmias were found to be present •iPSC-derived cardiomyocytes contain nodal cardiomyocytes that spontaneously contract faster than normal working cardiomyocytes = post-transplant ventricular tachycardia

37
Q

Describe the paracrine signalling based studies

(Developmental gene reactivation)

A

Myocardial thymosin beta 4 is necessary for epicardial migration, coronary vasculature, cardiomyocyte survival

-If added to adult heart =
*Stimulate epicardial outgrowth + neovascularisation (normally don’t occur)

It reactivated embryonic epicardial gene Wt1
-These activated genes give rise to cardiac progenitors in Myocardial infarction injured adult heart and these could become (new )de novo cardiomyocytes

•Hypothesis: adding Myocardial thymosin β4 reactivates epicardial gene programs

38
Q

Describe how secreted factors from epicardium are important for cardiac regeneration?

A
  • The secreted factor FSTL1 is expressed in epicardium and can promote cardiac myocyte proliferation
  • Epicardial expression is lost after a myocardial infarction
  • If restored, it promotes the regeneration of pre-existing cardiomyocytes in mouse /pig models
39
Q

Outline different ways in which stem cell therapy is used in treating different cancers

A

chemotherapy/radiotherapy kills cancerous cells:

Transplantation of stem cells can reform healthy cells.

-Haematopoietic cell transplantation for blood cells and leukocytes

-iPSCS + ESCs engineered by treating them using specific factors:
IL7, FLT3L ,SCF = produce T and N killer cells targeted for immunotherapy ( target tumour cells).

  • Stem cells can be used to produce anti cancer vaccines - transplanted + target patient’s specific cancer
  • MSC/NSCS (Mesenchymal /neural stem cells) have been used to deliver genes, nanoparticles and oncolytic viruses delivering anti cancer therapy to the tumours
  • Exosomes can be extracted from the culture of drug-priming MSCS/NSCS and can target drugs to tumour sites

Mutation correction in-vitro, drug testing before replacement with in vivo

40
Q

Describe how stem cell-based therapy can be used to treat burns.

A

The skin = largest organ in the body, protects against heat ,light injury and infection

Wound healing of the skin includes:

  • Coagulation
  • Inflammation
  • Proliferation of skin cells
  • Angiogenesis (new blood vessels)
  • Maturation

Foetal fibroblasts(derived from ESCs) - high expansion ability, low immunogenicity (types of immune responses are activated and their magnitude over time.) + secretes bioactive substances e.g. asFGFS, VEGFs, KGFs (-skin cell proliferation + maintenance)

Epidermal stem cells-High proliferation rate, easy to grow, High potency and differentiation potential for long time.

Mesenchymal stem cells -High differentiation potential and plasticity .Migrate to injured tissue ,differentiate and regulate tissue regeneration by producing growth factors, cytokines and chemokines

iPSCS can be differentiated into all cells of skin layers :
Dermal fibroblasts, keratinocytes and melanocytes

Stem cell therapy involves isolating stem cells , selecting and amplifying required phenotype in culture.
Delivered through different methods e.g. dressings, cell spray onto wound

41
Q

What are some complications which can result due to burns ?

A

Sepsis
Fatal shock + dehydration
Scarring

42
Q

Describe how stem cell-based therapy can be used to treat eye injuries

A

Cornea- Protective barrier against injury + outer lens of eye (focuses light)

  • Stem cells at edge of cornea= Limbal stem cells - make new corneal cells to replace damaged cells.
  • If lost =cornea can’t be repaired = vision loss as impairs light entry into eye
  • Limbal stem cells collected from healthy donor eye, expanded and transplanted into damaged eye = cornea repairs, restore vision
  • To avoid rejection, healthy section of limbus from same patient used - to collect limbal stem cells
  • Alternatively, Use iPSCs to make corneal epithelial cells for transplant
  • exposure to specific signals = fibroblast cells -> limbal stem cells
43
Q

What is the retina and how can it be damaged

A

The retinas receives light and converts into neural signals sent to brain
Contains rods and cones photoreceptor cells - respond to light

Retinal pigment epithelium (RPE ) single layer of post mitotic cells which act as selective barrier and regulate the photoreceptor layer above it.

If RPE not functional, parts of retina die =loss of vision

Diseases that damage RPE:
-age related macular degeneration (AMD) (eye disease that can blur the sharp, central vision)

Retinitis pigmentosa
-breakdown and loss of cells in the retina-loss of peripheral vision

Leber’s congenital aneurosis
(severe vision loss)

44
Q

Describe how stem cell-based therapy can be used to treat retina injury

A

Retinal pigment epithelium have been made from ESC and iPSCS (They are grown in culture dish using various growth factors) = diff. into RPE cells

These healthy cells are then replaced into the diseased eye = restore vision

45
Q

Describe how stem cell-based therapy can be used to treat spinal injuries

A

These can be life threatening injuries

Somatic cells are taken from patients -> iPSCs, reprogramme (4 Yamanaka factors) and differentiate into neural stem cells

Transplant to the site of spinal cord injury.

They Then Differentiate into:
Neural progenitor cells
oligodendrocytes (glial cell found in the central nervous system)
Neurons
Astrocytes (glial cells in the central nervous system)
Mesenchymal stromal cells
(multipotent stem cells found in bone marrow)

46
Q

Describe engraftment and evidence of it

A

This is when neural stem /progenitor cell grafts can integrate into sites of spinal cord injury.
(piece of living tissue that is transplanted surgically.)

Calcium imaging of grafts in spinal cord injury sites in vivo and in adult spinal cord slices show NSPC grafts organise into spontaneously active localised synaptic networks

Optogenetic (biological technique that uses light to control neurons) stimulation of host axons produced a neuronal response in the graft.

In vivo imaging showed
that behavioural stimulation elicited focal synaptic responses within grafts

  • Therefore neural progenitor grafts can form functional synaptic subnetworks and the activity patterns resemble the intact spinal cord.
47
Q

Researchers are targeting regions to develop stem cells to develop therapies for diseases+injury:

A
  • Blindness
  • Wound healing (burns)
  • Spinal cord injury
  • Myocardial infarction
  • Cancers
48
Q

Which are the most abundant type of stem cell?

A

Very large numbers of embryonic SCs but more limited iPSCs + ASCs available to work with

[MBD] NoNa (28 decks)

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