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Molecular Basis of Disease(Sem 4) > Stem Cells and regenerative medicine > Flashcards

Stem Cells and regenerative medicine Flashcards

1
Q

What are stem cells?

A
  • They are cells which can differentiate into many different cell types
  • They can undergo self-renewal through cell division
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2
Q

What are the different types of stem cells?

A

Induced Pluripotent ,Embryonic and Adult

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3
Q

Describe Adult stem cells

ASCs

A
  • These are tissue specific and multipotent which means they can differentiate into set number of cells within a specific location
  • There are exceptions such as adult bone marrow stem cells which have shown variation
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4
Q

Describe embryonic stem cells

ESCs

A
  • These are pluripotent which means they can differentiate into every type of cells and these originate from the blastocyst.
  • This is before implantation when the embryos are just a dew days old.
  • They can form all cells from all three germ layers.
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5
Q

What are the different germ layers?

A

The endoderm (Internal)-Lungs,pancreas,stomach,liver ,germ cells

The ectoderm (External)-Nervous, epithelial and sensory tissues

The Mesoderm(Middle)-Skeletal and cardiac muscle, blood and connective tissues

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6
Q

Describe Induced Pluripotent stem cells (iPSCs)

How are they created and how are they used ?

A

These are induced in the lab by taking normal differentiated tissue and reprogram through exposure to pluripotency factors:
OCT4,Sox2,cMyc,Klf4-Transcription factors
(Yamanka factors)

This produces pluripotent stem cells with similar characteristics to embryonic stem cells -Cell therapy

Crispr Cas -gene editing techniques differentiate to healthy cells in vitro and transplanting back to patient
Reduces graft rejection

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7
Q

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

A 
They can be used as models for research
Grown in layer in dish or 3D organoid model
-Cell differentiation study
-Developmental biology
-Cell replacement therapy
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8
Q

What are organoids?

A

These are tiny self organised three dimensional tissue cultures that can be derived from stem cells .
They can replicate complexity of an organ

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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 stem cells

Pluripotent +(Induced pluripotent ) -These can differentiate into the three germ layers

Multipotent-These will differentiate into tissue specific stem cells which become specialised cell types.

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10
Q

What do tissue specific stem cells require?

A

Microenvironments called stem cell niches (specific anatomical locations )

Required for :

  • Regulation of cell fate (What the cell will become )
  • Protect from depletion and host from excessive proliferation
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11
Q

What are the features of a stem cell niche?

A

-A supportive extracellular matrix
(Collagen ,Fibronectin)

-Secreted soluble signalling factors
(Growth factors, Cytokines

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

-Environmental signals -
(Metabolites, hypoxia ,inflammation)

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12
Q

What is Hedgehog(Hh)and WNT signalling ?

A

These are pathways which direct growth and pattern ins 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

It may cause cancers/tumours

Embryonic stem cells as they have an 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 meaning they can differentiate into any kind of cell
  • Unlimited number of cells due to high cell potency
  • Very low probability of mutation induced damage in DNA (Low spontaneous mutation +high genetic stability)

Cons:
-Higher risk of tumour creation due to uncontrolled stem cell proliferation

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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
  • Less risk of tumour creation

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
  • Limited number can be obtained
  • Less risk of tumour formation

Cons:

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

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

A

Adult stem cells are thought to be 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 in vitro fertilised embryos

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

Outline how the Yamanaka factors and how they induce iPSCs

A

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

•c-Myc promotes DNA replication and relaxes chromatin structure,
•This allows Oct3/4 to access its target genes.
•Sox2 and Klf4 also co-operate with Oct3/4 to activate target genes
-These genes encode transcription factors which establish the pluripotent transcription factor network •This results in the activation of the epigenetic processes (more open chromatin) that establish the pluripotent epigenome.
The iPCS cells have a similar global gene expression profile to that of ES cells.

20
Q

Outline how stem cells can be tracked

A
  • Stem cells can be manipulated in vitro which can make them easier to track in vivo.
  • A reporter gene is inserted (fluorescent )
  • The cells are then transplanted back into pre-clinical animal models + clinical.
  • Non invasive cell tracking can track where stem cells go and how they behave.
  • Development of cell based therapies
21
Q

What takes place in a heart attack

A

The blood supply to a heart muscle is lost. Cardiac muscle will die and is not replaced.
This is because adult cardiomyocyte turnover is low.
There will instead be fibrosis and scarring
=decreased cardiac function and heart failure

22
Q

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

A

It can potentially replace lost cardiac muscle ,increase blood supply.

Useful for
cardiac myopathies -
heart muscle to become enlarged, thick or rigid.

Conduction defects-
Electrical regulation not properly conducted

23
Q

What are two regulation strategies used to treat the heart?

A

Cell based therapies

Cell free therapies

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 delievery
Clinical regulation
25
Q

Expand of cell free therapies

Heart

A

Therapies which involve direct stimulation of endogenous cardiomyocyte production.
Includes reactivation of developmental pathways-
epicardium based on models where there is no scarring and full cardiac regeneration

26
Q

What is neovascularisation?

A

This is improved circulation to an injured area
Paracrine effects improve Cardiac myocyte replacement

-Inhibiting apoptosis+anti-inflammation

27
Q

Explain the cardiac regeneration in adult zebrafish

A

Following injury:

  • Zebra fish can regenerate their heart following injury
  • Apex is removed (Injury)
  • 1 day = there is a large blood filled clot which seals the site
  • 9 days = the apex wound is sealed by fibrin clot
  • 14 days , this is reduced /resolved and starts to be replaced by heart muscle
  • 1 month later =new cardiac wall
  • 2 Months= the entire site is regenerated with cardiac wall
  • We also see re-expression of developmental gene programmes (WT1,RHald H2??)
  • These are found on epicardium (surface level)-causes reactivation
28
Q

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

A

The fibrin clot will not resolve,remodels to form a fibrotic scar

29
Q

What is the epicardium and what is it important for ?

A

It is the surface level of the heart and it contains cells for coronary vessels and signals for cardiac myocyte proliferation.

30
Q

Expand on zebrafish regeneration (cardiac)-Adult

A

There is an injury
Embryonic gene expression
Endocardium activation
Fibrosis degradation

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 two 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 and this will cause oedema + inflammation (poor cardiac repair /function)

If stimulated by form of VEGFC, this will increase the response and reduce oedema and improve cardiac repair and function

34
Q

How are iPSCs specified towards different cardiac lineages ?

A
  • The iPSCS will specify into pre-cardiac mesoderm through inhibition of GSK-3b.
  • Through inhibiting glycogen synthase kinase 3b
  • This kinase acts as a downstream switch for signalling pathways such as WNT signalling.
  • Inhibition of WNT signalling causes differentiation of cardiac progenitor cells
  • These further differentiate into cardiac lineages
35
Q

What are the cardiac lineages?

A 
Epicardial
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
  • Improved cardiac contractile function

-Ventricular arrhythmias were found to be present

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 take place )

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

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 lots after a myocardial infarction
  • If it becomes restored it can promote 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 engineered by treating them using specific factors:
IL7, FLT3L ,SCF = produce T and N killer cells targeted for immunotherapy ( tumour cells).

  • Stem cells can be used to produce anti cancer vaccines
  • 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 the 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(ESCs)- high expansion ability, low immunogenicity (types of immune responses are activated and their magnitude over time.) + secretes bioactive substances such as asFGFS,VEGFs,KGFs (skin cell proliferation + maintenance)

Epidermal stem cells-High proliferation rate, easy access 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.
Delivered through different methods such as dressings

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(focuses light )

-Stem cells at edge of cornea= Limbal stem cells and these make new corneal cells instead of damaged ones.

  • If lost =cornea can’t be repaired.
  • Collected from healthy donor, expanded and transplanted into damaged eye.
  • To avoid rejection ,healthy section of limbus from same patient used
  • Using iPSCs to make corneal epithelial cells for transplant and exposure to right signals = fibroblast cells into 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

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

If not functional -parts of retina can die =loss of vision

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

Retinis 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 cultured using various growth factors )

These are then replaced in the diseased eye leading to restored 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 , reprogrammed and differentiated

Transplanted to the local where they are required.

Cells created -
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 showed NSPC grafts organise into localised active synaptic networks

Optogenetic (biological technique that involves the use of 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 pattenrs resemble the intact spinal cord.

Molecular Basis of Disease(Sem 4) (28 decks)

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