Week 10 - Stem cells and regenerative therapy

Embryonic stem cell

All multicellular organisms arise from one cell

most commonly from inner cell mass of blastocyst

Types:

• Pluripotent (inner cell mass)

  • differentiate ONLY into embryonic cells

• Totipotent (blastomere)

  • can differentiate into placental and embryonic cells

Adult stem cell

replace damaged or dead cells

Types:

• Multipotent

• Oligopotent

• Unipotent

[limited differentiation potential)

Types of self renewal (stem cells)

1. symmetric self-renewal = creates cells that are the same as original

2. asymmetric self-renewal = creates original cell + another cell that is slightly more restricted

3. symmetric differentiation = creates 2 cells that are more restricted

Extraction of embryonic stem cells

from inner cell mass of blastocyst = kills embryo (controversial)

Induced pluripotent stem cells

reprogramming fully differentiated adult cells

= embryonic stem cell like properties

Timeline of stem cell discovery

1. 1962 = frog cloned

2. 1983 = produced multinucleate cells

3. 1987 = trancription factors + mammalian cell cloning

4. 1996 = mammal cloned

5. 1998 = mice cloned

6. 2001 = human embryo cloned

7. 2006 = induced pluripotent stem cells generated

8. 2010 = AID pluripotentcy regulator

Enbryonic stem cell uses (5)

1. study in tissue cultures = to understand factors controlling cell differentiation

2. source of human cells for transplantation = tissue engineering

3. cloning

4. somatic cell nuclear transfer

5. induced pluripotent stem cells (IPSCs)

Somatic Cell nuclear transfer (5 general steps)

1. Egg + somatic cell

2. Egg anucleated

3. Somatic cell nucleus isolated

4. Egg + somatic cell combined

5. Gives rise to totipotent cell

Generates autologous cells (patient’s own cells) which can avoid the immune rejection problem

Why is cloning in humans unacceptable? (4 reasons)

1. ethics

2. Genes vs environment

3. status/quality of DNA ('aged')

4. role of maternal cytoplasmic factors and mtDNA

Bivalent Domains

chromatin regions that contain both activating and repressive marks

= maintains embryonic stem cells in 'poised' state

Adult stem cell uses (3)

1. tissue engineering ex vivo (spare parts)

2. Tissue repair by cell therapy

3. Genetically correct autologous cells = treat disease

Examples of tissue repair by adult stem cell therapy (4)

1. Haematopoetic cells = leukaemia

2. Islet cells = diabetes

3. Heart = after heart attack

4. Neurodegenerative disorders = parkinsons etc.

stem cell niche

environment in which they reside

= dominant part in regulating stem cell activity and behaviour

(adult stem cells are all in a niche)

Satellite cells (location and function)

next to plasma membrane under basal lamina (in skeletal muscle)

• activated by external stimuli = differentiate together + upregulation of MyoD

• upregulation of MyoD = differentiates myoblast → myotube

Haematopoietic stem cells

give rise to many blood cells

(found in bone marrow)

2 regions of the body that undergo Neurogenesis

1. Olfactory Bulb (smell)

• in Subventricular zone (SVZ) of lateral ventrical

2. Hippocampus (learning/memory)

• in Subgranular zone (SGZ) of dentate gyrus, in hippocampus

Induced pluripotent stem cell usecase

Direct reprogramming in vivo via viruses/other molecules

Uses:

• disease modelling

• pharmacological screening + toxicity testing (drug development)

• transplanting cells back into patient

Benefits of iPSCs

1. reduced ethical concenrns

2. disease understanding/treatment

3. patient specificity

3 factors in tissue engineering

1. Cells

2. Growth factors

3. scaffolds

Steps after tissue assembly in Tissue engineering

1. Conditioning

2. Implantation

3. Vascularisation

Ear disease - regenerative therapy

middle: Tympanic membrane tissue engineering

Inner: Cochlear hair cell regeneration and tissue engineering:

- pluripotent stem cell models

Tympanic Membrane

Eardrum (middle ear)

Layers:

- epithelial

- fibrous

- mucousal

Acute middle ear perforation

damaged eardrum

28 days to fully heal

Tympanic Membrane and stem cells

Tympanic membrane has stem cell niche in middle

stem cells (keratinocytes) proliferate, daughter cells growth outwards towards periphery

Keratinocyte biology assays

Assess cell:scaffold interactions

Examples:

- scratch wound assay

- MTT assay

Scratch wound assay

Keratinocytes on top of scaffold

Scratch the keratinocytes

View and analyse migration of keratinocytes (as they fill the gap)

MTT assay (what does it assess)

Assesses cell adhesion and proliferation

Silk scaffolds for chronic Tympanic membrane perforation

Creating a tympanic membrane scaffold from silk:

- 3d print

- electrospin

Laser doppler vibrometer

measures vibrations with laser

can see how eardrum vibrates, to be able to replicate it

Optical Coherence Tomography Imaging

Imaging shape and prototyping tympanic membrane

can view scaffold material as it interacts with different things

Bioink development (requirements for eardrum scaffold printing)

During printing:

- must be viscous, but able to flow

- must have rapid coagulation

After printing:

- low stiffness (for proliferation and migration)

- high enough stiffness to maintain integrity

Bottom up Silk processing (3 steps)

1. degumming

2. dissolving (dissolve silk)

3. dialysis (wash away chemicals)

[used to cast molds for tympanic membrane scaffolds]

Secretome stem cell

wound healing via paracrine factor secretion

paracrine factor = stimulates keratinocyte growth

(applicable to tympanic membrane)