stem cell and regenerative medicine

Question 1

What are biological functions of stem cells?

Answer 1

• can differentiate into many different cell types
• capable of self renewal via cell division to provide new cells as organism grows
• replace cells that are damaged or lost
• targeted by researchers for their therapeutic potential

Question 2

What are 3 main stem cell sources?

Answer 2

1. IPSC = induced pluripotent cells
2. ESCs = embryonic stem cells
3. ASCs = adult stem cells

Question 3

What are the different types of stem cells?

Answer 3

1. Totipotent embryonic stem cells
2. Pluripotent embryonic stem cells
3. multipotent stem cells

Question 4

What is stem cell niches?

Answer 4

• tissue specific stem cells are maintained in specific supportive microenvironments called stem cell niches.
• support ECM
• neighbouring niche cells
• secreted soluable signalling factors
• physical parameters
• environmental signals (metabolites,hypoxia, inflammation)

Question 5

how do scientists modify normal cells to reprogram them?

Answer 5

By inducing pluripotency factors to reprograme them
-Kfl4, SOX2, Oct3/4, cMyc

1. CMyc promotes DNA replication and relaxes chromatin structures allowing Oct3/4 to enter and access target gene
2. Target gene are activated by oct3/4 which work with co factors Klf4 and SOX2
3. Target gene encode transcription factors which establish pluripotent transcription

Question 6

Why do scientists modify stem cells?

Answer 6

1. model for basic and translational studies
2. disease modelling
3. drug screening
4. cell differentiation
5. developmental biology

Question 7

How do we generate iPSCs?

Answer 7

c-Myc factpr promotes DNA replication and relaxes chromatin structure enabling Oct 3/4 to enter and bind to target gene.
Oct 3/4 works with Klf4 and SOX2 to bind to target gene activating it.
Activated target gene encode transcription factors which establish pluripotent transcription network.

Question 8

Why cardiovascular disease need stem cells and regenerative medicine?

Answer 8

• low turnover of cardiomyocytes
• so damage leads to fibrosis and scarring and decreased cardiac function
• research into therapies to replace lost cardiac muscle and improve blood supply to affected regions = stem cells and regenerative medicine
• neovascularisation = improved circulation to injured area, paracrine effects improving cardiomyocyte replacement.

Question 9

what is lymphatic response of cardiac regeneration?

Answer 9

-lymphatic system doesn’t drain leading to odema

Question 10

What is immune response to cardiac regeneration modelled by neonatal and adult mice?

Answer 10

1. neonatal mice heart:
   - infiltration of injury by embryonic macrophages
   - revascularisation
   - global cardiomyocyte proliferation
2. adult mice heart
   - infiltration by monocyte derived macrophages
   - limited revascularisation
   - no cardiomyocyte proliferation

Question 11

What did research on induced pluripotent stem cells vs embryonic stem cells of monkeys find?

Answer 11

monkey study
1. IPSCs from normal monkey cells induced to cardiomyocytes (both host an recipient from same species)
-monkey model with identical MHC to humans
-fibroblast derived iPSC from MHC haplotype HT4
-injected into heterozygous monkeys after myocardial infarction
Findings:
-GFP+ survived 12 weeks with no evidence of rejection
1. Improved cardiac contractile function at 4-12 weeks
2. ventricular tachycardia increased compared to control

2. human embryonic stem cells injected into monkeys to make cardiomyocytes
   - 1 month after implantation of embryonic stem cells left ventricular ejection improved
   - grafts formed electricomechanical junctions with host heart and 99% ventricular myocytes made.
   - ventricular arrythmia increased

Question 12

What are two molecules important for genetic reactivation for CV disease

Answer 12

1. thymosin beta 4
   - necessary for epicardial migration
   - coronary vasculature
   - cardiomyocyte survival
2. re-expression of key embryonic epicardial gene Wt1
   - give rise to cardiac progenitors in the MI injured adult heart
   - these can differentiate into de novo cardiomyocytes

Question 13

Stem cell- based therapy for cancer

Answer 13

1. chemo can kill healthy cells, stem cell transplantation to replace these lost cells.
2. immunotherapy from cancer requires more T cells and NK cells (effector immune cells) which can be made from ESCs/IPSCs
3. used to produce anti-cancer vaccines
4. deliver genes, nanoparticles, and oncolytic viruses to tumour niche
5. exosomes can target the drugs to tumour sites

Question 14

stem cell - based therapy for burns

Answer 14

1. fetal fibroblast (from ESCs) = high expansion ability , low immunogenicity. secrete bioactive substances
2. epidermal stem cells = high proliferation rate and easy acess and keep potency and differentiation potential for long periods.
3. mesenchymal stem cell = high differentiation potential and a certain degree of plasticity , produce growth fcators, cytokines, chemokines
4. iPSCs = can be differentiated into dermal fibroblasts, keratinocytes, and melanocytes.

Question 15

stem cell - based therapy for eye injury/disease - limbial cells

Answer 15

• limbial stem cells = responsible for amking new corneal cells to replace damaged ones.
• if limbial stem cells are lost due to disease corneal cells cant be replaced which affects the ability of light to enter eye = loss of vision
• limbial cells collected from healthy donors and transplanted can repair cornea and permanently restore vision.
• iPSCs can transform fibroblast cells into limbial stem cells.

Question 16

stem cell - based therapy for eye injury/disease - retinal pigment epithelium (RPE)

Answer 16

• act as selective barrier and overlay photoreceptor layer
• role in maintaining retina, lack of functional RPE leads to loss of vision
• RPE cells can be damaged in a variety of disease such as age - related macular degeneration(AMD), retinitis, pigmentosa and Leber’s congenital aneurosis
• RPE made from both ESC and iPSC

Question 17

stem cell - based therapy for spinal injury

Answer 17

1. take somatic stem cells biopsy from patients
2. transform these into pluripotent stem cells via 4 reprogramming factors (cMyc, Oct3/4, Klf4, SOX2)
3. these pluripotent cells can differentiate into many different neuronal cells.

=> neural stem/progenitor cells (NSPC) grafts can integrate into sites of spinal cord injury (SCI) and generate neuronal relays across lesions.
=> calcium imaging of NSPC grafts organise into localised and spontaneously active synaptic networks.
=> optigentic stimulation of host axons produce neuronal response in graft

Question 18

The process of making cardiac lineage from iPSCs

Answer 18

1. Somatic cells reprogrammed to make induced pluripotent stem cells
2. IPSCs undergo specification to make pre cardiac mesoderm
3. Pre cardiac mesoderm undergo differentiation (aided by Wnt) cardiovascular progenitors cells
4. These progenitor cells can divide in a number of different cardiac cells including epicardium, cardiomyocytes etc.