Stem Cells

Question 1

Define stem cells

Answer 1

A cell that can self renew and give rise to more differentiated cell types

Question 2

What are the types of stem cell?

Answer 2

Embryonic stem cells
Cord blood stem cells
Adult blood-derived stem cells
Adult stem cells

Question 3

What cells are the paradigmic stem cells?

Answer 3

Embryonic stem cells

Question 4

What are the stages of ESC isolation?

Answer 4

1. Fertilisation
2. Cleavage
3. Early blastocyst
4. Implantation

Question 5

Describe the cleavage stage of ESC isolation

Answer 5

1. Zygote cleaves into paired blastomeres
2. Blastomeres are contained within the Zona Pellucida
3. Continued cleavge to 16 cells
4. Now called Morula
5. Leaves Fallopian tube and enters uterine cavity

Question 6

Describe the early blastocyst stage of ESC isolation

Answer 6

1. Cell division continues
2. Blastocele cavity forms
3. Cells flatten and compact on the inside of the cavity, zona pellucida remains the same size-blastocyst

Question 7

What are the current isolation protocols?

Answer 7

1. Fresh/Frozen IVF cleavage stage embryos obtained
2. Cultured to blastocyst stage
3. Pronase to remove Zona Pellucida
4. Immunosurgery to remove trophoblast
5. Plated onto combination of collagen IV, fibronectin, laminin and vitronectin
6. Xeno-free medium containing bFGF, LiCl, gamma-aminobutyric acid (GABA), pipecolic acid and TGF beta
7. Mechanical dissociation initially then Dispase to dissociate

Question 8

Define Self-renewal

Answer 8

Cycles of division that repeatedly generate at least one daughter equivalent to the mother cell with latent capacity for differentiation

Question 9

Define pluripotent differentiation potential

Answer 9

Capable of forming all three germ layers of developing embryo

Question 10

How doe ESCs stay immortal?

Answer 10

Endogenous telomerase activity protects telomere end and confers protection from mortality

Question 11

What surface antigens and transcription markers are present on ESCs?

Answer 11

Oct 4, nanog

S SEA3, S SEA4, TRA-1-60, TRA-1-81 (sugar molecules found on the surface of the cell)

Question 12

What does the mesoderm give rise to?

Answer 12

Muscles, blood, blood vessels, connective tissues and the heart

Question 13

What does the ectoderm give rise to?

Answer 13

Brain, spinal cord, nerve cell, hair, skin, teeth, sensory cells of eyes, ears, nose, mouth and pigment cells

Question 14

What does the endoderm give rise to?

Answer 14

The gut, lungs, bladder and germ cells

Question 15

What is the source of MSCs?

Answer 15

Bone marrow- isolated from the superior iliac crest of the pelvis, hip, knee, long bones, vertebrate

Question 16

How are MSCs isolated?

Answer 16

1. Bone marrow scooped/aspirated out of bone cavity. Sides scraped to disrupt and remove cells from all microniches
2. Marrow sample fractionated on density gradient. Purifies nucleated cells
3. Mononuclear fraction plated at low density
4. Recovered over three week period
5. Detached, subcultured, experimentation etc

Question 17

How is an MSC defined?

Answer 17

No single gold standard
CD105 (positive) and CD73 (Negative) commonly used
CFU-fibroblast assay identifies proliferative cells at isolation point
Differentiate into bone, fat and cartilage
Genotype representative of multiple lineages

Question 18

What are the characteristics of MSC proliferation?

Answer 18

MSC are mortal
Telomerase negative
Capable of 20-30 PD in in vitro culture
5-6 log phase passages
Proliferation can be enhanced by low density plating and enrichment

Question 19

What are the primary products in the market in terms of MSC-based therapy?

Answer 19

Osteocel- in market. Osteoinduction/conduction/genesis
Chondrogen (Phase 1/2)- Meniscus regeneration and osteoarthritis prevention
Provacel (Phase1)- Prevention of HF following acute MI
Prochymal (Phase 3)- Treatment of GVHD

Question 20

What are the advantages of MSC’s?

Answer 20

Easy to derive
Easy to culture
Easy to differentiate
Some distinct species are pluripotent
Osiris is already in the market place
Ease of comparison
Immunsuppressive

Question 21

What are the disadvantages of MSCs?

Answer 21

Limited proliferation-mortal
Culture conditions are not optimised
Severe discomfort during removal
Age related decline in number and potentiality
No definite evidence to show that in vivo MSC exist