W4L4 Fri Stem cell and iPS cell

Question 1

Different type of stem cell potentcy

Answer 1

totipotent (i.e. fertilized oocyte and cells after first cleavage divisions; ability to form entire organism)
– pluripotent (i.e. cells of the inner cell mass of the blastocyst; ability to form all three germ layers, but not the extra-embryonic tissue) -embryonic stem cells
– multipotent (i.e. mesenchymal stem cells which can form bone, cartilage and fat; ability to form multiple cell types) – adult stem cells
– unipotent (i.e. a cardiomyocyte within the heart) – differentiated cells with no capacity to differentiate further

Question 2

What defines a stem cell

Answer 2

Self-renewal :The range of developmental options available to a cell
Potency/potential: capacity for differentiation

Question 3

Stem cell renewal characteristic

Answer 3

Ø Indefinite proliferation + replacing stem cell pool
Ø Telomerase activity: enzyme that maintains length of telomeres (protects genome)
Ø Normal karyotype maintained over successive divisions
Ø Marker expression profiles: underpin capacity to self-renew

Question 4

Potancy characteristic

Answer 4

Ø Embryoid body formation: aggregate body with representatives of 3 germ lines
Ø Teratoma formation
Ø Tetraploid complementation

Question 5

Embryo development and stem cell

Answer 5

-ectoderm give rise to neural and skin cell
-mesoderm give rise to muscle and one tissue
-Endoderm give rise to organ cell

Question 6

Limitation of embryonic stem cell derivation

Answer 6

-all human lines are derived from excess IVF embryos (underlying problem),
-Exogenic component as many isolated on to mouse embryonic fibroblast (MEF) feeder layers with bovine serum (undefined conditions, antigens found on stem cells with mouse/bovine origins could illicit human immune response, disease transmission from animal product use),
-culture induces change over time (cells adapt to culture, enzymatic passaging can introduce karyotypic instability)

Question 7

Maintaining Pluripotency

Answer 7

Knock down of Oct4+Nanog= development of an embryo lacking an ICM
Ø These transcription factors are important for driving pluripotency
§ Self-renewal is controlled by interactions b/w Sox2, Oct4+ Nanog – ability to proliferate indefinitely + maintain pluripotency
Ø These 3 transcription factors regulate themselves + other transcription factors responsible for maintaining pluripotency (activate transcription factors) or preventing differentiation (silence transcription factors)

Question 8

Embryonic Stem Cell Characteristics - culture

Answer 8

Differentiate spontaneously in vitro into derivatives of the three germ layers = pluripotent
* Least stringent test
* The expression of differentiation markers is not a test for functionality
* Any changes in culture conditions can stress the cells or induce differentiation

Question 9

Embryonic Stem Cells identification

Answer 9

ESC sare characterised by morphology+ transcription factor expression
Ø Sustaining transcription factors with fluorescent dye to identify activity, disappear once cell differentiation begins

Question 10

ESC potency test

Answer 10

Ø Differentiate spontaneously in vitro into derivates of 3 germ layers (pluripotent)
§ Expression of differentiation markers is not a test for functionality
§ Any changes in culture conditions can stress cells or induce differentiation
Ø Form teratomas when injected into immune-deficient mice
§ Differentiate spontaneously in vivo into derivatives of 3germ layers due to loss of pluripotency+ exposure to signals in new environment that induce differentiation
§ Does not test for ability to promote natural development Ø Chimera formation (ultimate determinant)
§ Derive ESCs from black mouse→inject ESCs from black mouse into blastocyst from white mouse→ ESCs contribute to all tissues of resulting offspring = chimera
§ Can test for germ line competency

Question 11

ESC extraction

Answer 11

1. Inner cell mass is isolated via laser or immuno-surgery to lyse trophectoderm
2. ICM cells plated onto a dish of fibroblast feeder cells which help support ICM cells
3. ICM cells grow + are further isolated with more plates to form several isolated colonies

Question 12

Differentiation of stem cell

Answer 12

Asymmetric division : in response to differentiation cues , stem cell polarises on basement membrane + divides
Ø One cell maintains contact with matrix (stem cell) + the other moves away (daughter progenitor cell)
§ Patterning information from envrionment–positional information
§ Commitment to cell lineage by transcription factor +epigenetic networks
§ Totipotent (zygote)→pluripotent (ICMEScells,EGcells)→multipotent (ASCs)→unipotent(differentiated cell types)

Question 13

ESC epigenetic modification

Answer 13

Ø DNA methylation: gives DNA a ‘memory’, impacts cell’s ability to replicate itself in its current form
§ Signals which genes have been switched on/off
Ø Histone modification: alters extent DNA is wrapped around histones + availability of genes in DNA to be activated
§ Euchormatin:open, active chromatin
§ Heterochromatin:closed, inactive chromatin

Question 14

Epigenetic regulation of differation in stem cell

Answer 14

ICM (pluripotent): DNA is open – transcription of pluripotency genes, repress ‘differentiation’ genes
Ø Epiblast (pluripotent): DNA is open – maintain ‘pluripotency’ genes, repress ‘differentiated’ genes
Ø Ectoderm (committed): DNA has open + closed areas – transcription of ectoderm genes, ‘pluripotency’ genessilenced, ‘mesoderm’ germs silenced
Ø Neural ectoderm: DNA has open + closed areas – transcription of neurectoderm genes

Question 15

What is ASC

Answer 15

-Adult stem cell: undifferentiated cell found amongst differentiated cells in a tissue/organ that can self- renew + differentiate to yield some or all of the majro specialised cell types of the tissue/organ

Question 16

Requirement for ASC

Answer 16

Must be able to self-renew,must be long lived, generally multipotent
Ø Drive renewal of all adult tissues
Ø Recruited continuously or can remain quiescent for long periods
Ø Divide to produce new cells that undergo a robust differentiation program
Ø Repair + regeneration can be limited
Ø In culture: highly resistant to expansion + long-term culture; difficult to isolate homogeneous populations

Question 17

Characteristic of ASC

Answer 17

High cellular turn over+ high regenerative potential ( blood cells, epidermis, gut epithelium)
§ Low cellular turn over+ high regenerative potential ( liver, skeletal muscle)
§ Low cellular turn over+ low regenerative potential (lung parenchyma, brain, kidney, heart)

Question 18

ASC bow marrow stem cell

Answer 18

Bone marrow stem cells (2populations)
Ø Haematopoietic stem cells gives rise to red + white blood cells, platelets
§ Isolated from bone marrow ,peripheral blood ,placenta+umbilicalcord
Ø Bone marrow stromal cells/mesenchymal stem cells give rise to bone, cartilage, fat + stroma
§ Isolated from bone marrow+adipose tissue
Ø Identified by surface marker expression + widely used for transplantation (HLA compatibility)

Question 19

Adult adipose tissue stem cells

Answer 19

-diffenetiad into cardiomyocyte, neural crest

Question 21

Cord blood stem cell

Answer 21

-Use for child transplantation, hampered in adults by low cell dose

Question 22

Induced pluripotency

Answer 22

§ Known that 24 ESC factors were involved in reprogramming, but only 4 found to be critical informing iPS cells
Ø cMyc (oncogene), Klf4, Sox2, Oct4
§ iPSC contributed to all germ layers in vitro+in vivo (pluripotent) but did not contribute to germ line of chimeras
§ Micro array analysis of gene expresion profile of iPS cells compared with ES cell+original fibroblast cells indicate incomplete reprogramming of the iPSC transcriptome
Ø iPS – retention of fibroblast methylation patterns

Question 23

Future of stem cell research

Answer 23

§ In vitro organoid model facilitates an accurate study of arange of invivo biological processes in including tissue renewal, stem cell/niche functions + tissue responses to drugs, mutation or damage
§ Road to therapy: scientific discovery→pre-clinical efficacy in animal model→good manufacturing practices→clinical trials (FDA, TGA, safety trials, protocol optimisation, efficacy trials) → therapeutic / treatment
§ Stem Cell Tourism for unregulated stem cell based treatments: methods not disclosed, possible placebo effect
Ø Pre-clinical data not usually in literature, no peer review, no safety data, no patient follow up