5- Stem Cells and Regenerative Medicine

Question 1

define totipotent stem cells - give an example

Answer 1

can differentiate into any cell type - embryonic and extraembryonic cells

example - cells in the early stages of embryonic development (zygote, first few cells of the morula)

Question 2

define pluripotent stem cells - give an example

Answer 2

can differentiate into cells of all three germ layers - endoderm, mesoderm, and ectoderm - but can’t rise to the extraembryonic tissues

example: embryonic stem cells derived form the inner cell mass of the blastocyst

Question 3

define multipotent stem cells - give an example

Answer 3

can differentiate into a limited range of cell types within a specific tissue or organ system

example: adult stem cells = haemopoietic stem cells in bone marrow give rise to different blood cell types

Question 4

define oligopotent stem cells - give an example

Answer 4

a type of multipotent stem cell - can differentiate into a limited number of cell types

example: lymphoid progenitor cells in bone marrow that give rise to different types of immune cells within the lymphoid lineage

Question 5

define unipotent stem cells - give an example

Answer 5

can only differentiate into one specific cell type

example: spermatogonia stem cells in the testes can only differentiate into sperm cells

Question 6

define a stem cell

Answer 6

an undifferentiated cell of a multicellular organism that can give rise to indefinitely more cells of a cell type through differentiation

Question 7

what are the two main characteristics of a stem cell?

Answer 7

capable of self-renewal through asymmetric and symmetric division

can differentiate into many different types of specialised cells depending on signals they receive

Question 8

what are the 3 main sources of stem cells?

Answer 8

adult
embryonic
induced pluripotent

Question 9

describe adult stem cells - features, functions, sources?

Answer 9

features: rare, tissue-specific and multipotent

function: supply new cells for an organism to grow and replace damaged cells - ability varies with different organs

sources: can be taken from the bone marrow as haemopoietic stem cells or the umbilical cord, though other adult stem cells such as epithelial stem cells exist

Question 10

describe embryonic stem cells - features, functions, sources?

Answer 10

features: pluripotent, proliferate through multiple rounds of cell division before differentiating and giving rise to one of the 3 germ layers

function: supply all of the cells of the developing embryo

sources: blastocyst inner mass cells

Question 11

what are the three germ layers, and what do they develop into?

Answer 11

ectoderm, mesoderm, endoderm

ectoderm = nervous, epithelial and sensory tissues

mesoderm = blood and connective tissues, skeletal and cardiac muscle tissue

endoderm = lungs, pancreas, stomach, liver and germ cells

Question 12

describe induced pluripotent stem cells - features, sources?

Answer 12

features: pluripotent, made in a lab, can be differentiated back into any cell type with exposure to certain signals

sources: differentiated tissues is obtained by skin/ muscle biopsy and reprogrammed by exposure to a specific set of pluripotency factors

Question 13

list the uses of induced pluripotent cells

Answer 13

differentiated back into healthy cells specific to a patient cell type, reduces chance of rejection for treatment like a graft

cell replacement therapy

cell differentiation studies

disease modelling and drug screening

Question 14

what is a stem cell niche?

Answer 14

a specialised supportive microenvironment for tissue-specific stem cells, at specific anatomical locations

Question 15

what are the purposes of a stem cell niche?

Answer 15

interact with stem cells to regulate cell fate

protect them from depletion

protect the host from excessive stem cell proliferation

Question 16

describe the 5 specific features of a stem cell niche

Answer 16

1. support extracellular matrix molecules as biochemical support for cells within niche - e.g. fibrin, actin, collagen
2. secrete soluble signalling factors - e.g. growth factors, cytokines, Wnt
3. interact with tissue-specific surrounding niche cells for exchange of signalling molecules
4. regulate physical parameters that may influence stem cell behaviour - e.g. shear stress, tissue stiffness
5. allow stem cell response to environmental signals - e.g. hypoxia, metabolites, immune cytokines

Question 17

compare the potency and growth potential between adult, embryonic and induced pluripotent stem cells

Answer 17

embryonic = pluripotent, unlimited growth and differentiation potential (can’t differentiate into extraembryonic tissues)

adult = multipotent, limited growth potential

induced pluripotent = less growth potential than embryonic stem cells, still pluripotent

Question 18

why do embryonic stem cells have a higher risk for transplant rejection?

Answer 18

embryonic SCs - more likely to be genetically different

adult and iPSCs will be derived from the patient

Question 19

which stem cell type has a lower mutation risk and why?

Answer 19

embryonic - lower potential mutation rate, higher genetic stability

adult and iPSCs have a higher risk for spontaneous disease-causing mutations

Question 20

what are the four Yamanaka factors for manufacturing iPSCs?

Answer 20

Oct4
Sox2
Klf4
cMyc

other factors play additional roles in reprogramming

Question 21

what does Oct4 do?

Answer 21

prevents differentiation, acts on target gene promoters, maintains pluripotency

Question 22

what do Sox2 and Klf4 do?

Answer 22

work with Oct4 to regulate transcription

Question 23

what does cMyc do?

Answer 23

promotes DNA replication, relaxes chromatin structure

Question 24

describe the mechanism for manufacturing iPSCs with exposure to pluripotency factors

Answer 24

cMyc opens/ relaxes chromatin structure - allows easier access to DNA and target genes

Oct3/4 gains access to target gene promoters, facilitated by Sox2 and Klf4

activate target genes - the Yamanaka factors together work to establish a pluripotency similar to ESCs
- activate certain genes that contribute to pluripotency
- modify epigenetic changes like histone modification or DNA methylations

establish pluripotent epigenome and gene expression profile in iPSCs similar to ESCs, with a more open chromatin structure

Question 25

what is stem cell tracking?

Answer 25

a method of studying the fate and behaviour of transplanted stem cells manipulated beforehand

tracked by inserting a reporter gene with a detectable marker - e.g. GFP protein/ fluorescence - which can be tracked using non-invasive imaging

allows us to monitor cell migration, location and behaviour

Question 26

why are cardiac regeneration strategies needed?

Answer 26

during a heart attack - blood supply to the affected area is lost, cardiac muscle dies and isn’t replaced as cardiomyocytes have a slow turnover rate

leads to reduced cardiac function, potential heart failure = inefficient pumping of blood around the body

Question 27

list the two main cardiac regeneration therapies

Answer 27

stem cell based therapy - stem cell transplantation
- SCs differentiate into new coronary vessels, promote cardiac regeneration and repair

stem cell-free therapy - direct paracrine stimulation of endogenous cardiomyocyte production to improve circulation

Question 28

describe the importance of animal model studies in stem-cell based cardiac regeneration studies

Answer 28

in zebrafish vs larger mammals
- zebrafish had re-expression of developmental gene programming following injury
- fibrin clot formed and then was replaced with new heart muscle/ cardiomyocytes
- however in larger mammals - fibrin clot remodels into a fibrotic scar which contributes to reduced cardiac function and heart wall stiffness

in neonatal vs adult mice
- neonate = embryonic macrophages infiltrated affected site, reduced tissue fluid, oedema and infiltration and encouraged cardiomyocyte proliferation
- adult = increased oedema, tissue fluid from inflammatory cells and inflammation = no cardiomyocyte proliferation

can develop SC therapy ideas/ approaches based on
- developmental gene re-expression
- lymphatic response modulating immune response by stimulating lymphatics with VEGF-C = reduces tissue fluid accumulating, oedema and inflammation to improve cardiac repair and function

Question 29

describe how cardiac cell linages can be made from iPSCs

Answer 29

somatic cells reprogrammed into pluripotency via exposure to Yamanaka factors

directed towards inhibition of glycogen synthase kinase 3B = inhibits Wnt signalling = directs iPSCs towards cardiac progenitor cell differentiation and development

specialised signalling molecules direct further differentiation into more specialised cardiac lineages - e.g. cardiac fibroblasts, smooth muscle, endothelial cells

Question 30

describe the importance of thymosin beta 4 in stem-cell free therapies for cardiac regeneration

Answer 30

thymosin beta-4 is produced by cardiomyocytes - needed for survival, and cell migration to form coronary vasculature

may reactivate epicardial gene reprogramming

re-express Wt1 gene = give rise to cardiac progenitors = can differentiate into de novo cardiomyocytes

Question 31

describe the importance of FSTL1 in stem-cell free therapies for cardiac regeneration

Answer 31

secreted epicardial factor - promotes cardiomyocyte proliferation, expression lost after heart attacks

directly integrating grafted cell sheets soaked in FSTL1 to the myocardium can improve cardiac function

Question 32

describe the use of stem cell therapy for cancer treatment

Answer 32

SC transplantation can re-establish healthy cells after chemo/radio has killed them

HSCs transplantation - replace cancerous blood cells with healthy ones via HSCs

engineering effector T and NK cells for targeted immunotherapy - designed to specifically recognise and kill cancer cells

anti-cancer vaccines using stem cells to stimulate the patient’s immune system to target cancer cells

exosome-mediated drug delivery - exosomes extracted from specific SCs can be used for targeted drug delivery to tumour site = better treatment specificity

in vitro correction of cancer-causing mutations in stem cells before transplantation OR targeting treatment specifically at cancer stem cells

Question 33

describe the use of stem cell therapy for wound healing/ burns

Answer 33

stem cells can speed up the replacement or lost skin cells and improve endogenous healing

1. isolating or producing stem cells - these are amplified in culture
2. stem cells are selected based on various factors - e.g. secreted growth factors and extracellular matrix molecules = important for cell proliferation and regeneration
3. selected cells are delivered through dressings or cell sprays/sheets

various stem cell therapies include - foetal fibroblasts, epidermal and mesenchymal stem cells, and iPSCs
- high proliferation and differentiation potential to improve skin repair and secrete necessary growth factors
- ensure tissue regeneration

Question 34

describe the use of stem cell therapy for cornea regeneration (eye disease/injury) - importance? method?

Answer 34

importance:
the loss of limbal stem cells within the cornea prevents renewal of cornea cells - affects cornea function = loss of vision

method:
1. limbal stem cells can be collected form a healthy donor eye - amplified and transplanted into damaged eye
- avoid immune rejection by tissue compatibility matching and ensuring the cells collected are healthy
2. patient’s fibroblasts can be reprogrammed into iPSCs - then target differentiation into limbal stem cells for transplantation

Question 35

describe the use of stem cell therapy for regenerating the retinal pigment epithelium (eye disease/injury)

Answer 35

importance:
retinal pigment epithelium is part of the retinal - receives light and converts it to neural signals = important in health and function of retina

RPE can be affected by diseases - e.g. age-related macular degeneration, retinitis pigmentosa

method:
- ESCs and iPSCs are cultured with specific growth factors - amplified
- exposed to various growth & differentiation factors
- guided towards differentiating into RPE cells in culture = these are transplanted to replace damaged RPE cells
= restores functionality and vision

Question 36

describe the use of stem cell therapy for spinal cord injury treatment

Answer 36

method:
- somatic cells from patient transformed into iPSCs
- targeted differentiation into neural stem cells = implanted at site of spinal cord injury
- differentiate into a variety of cells for neural regeneration - e.g. neural progenitors, astrocytes, neurons

success:
- neural stem/ progenitor cells can integrate into sites of spinal cord injury and generate neural relays
- exhibit functional connectivity, produce neuronal and synaptic networks and responses with grafts