Stem Cells Flashcards
Define stem cells
A cell that can self renew and give rise to more differentiated cell types
What are the types of stem cell?
Embryonic stem cells
Cord blood stem cells
Adult blood-derived stem cells
Adult stem cells
What cells are the paradigmic stem cells?
Embryonic stem cells
What are the stages of ESC isolation?
- Fertilisation
- Cleavage
- Early blastocyst
- Implantation
Describe the cleavage stage of ESC isolation
- Zygote cleaves into paired blastomeres
- Blastomeres are contained within the Zona Pellucida
- Continued cleavge to 16 cells
- Now called Morula
- Leaves Fallopian tube and enters uterine cavity
Describe the early blastocyst stage of ESC isolation
- Cell division continues
- Blastocele cavity forms
- Cells flatten and compact on the inside of the cavity, zona pellucida remains the same size-blastocyst
What are the current isolation protocols?
- Fresh/Frozen IVF cleavage stage embryos obtained
- Cultured to blastocyst stage
- Pronase to remove Zona Pellucida
- Immunosurgery to remove trophoblast
- Plated onto combination of collagen IV, fibronectin, laminin and vitronectin
- Xeno-free medium containing bFGF, LiCl, gamma-aminobutyric acid (GABA), pipecolic acid and TGF beta
- Mechanical dissociation initially then Dispase to dissociate
Define Self-renewal
Cycles of division that repeatedly generate at least one daughter equivalent to the mother cell with latent capacity for differentiation
Define pluripotent differentiation potential
Capable of forming all three germ layers of developing embryo
How doe ESCs stay immortal?
Endogenous telomerase activity protects telomere end and confers protection from mortality
What surface antigens and transcription markers are present on ESCs?
Oct 4, nanog
S SEA3, S SEA4, TRA-1-60, TRA-1-81 (sugar molecules found on the surface of the cell)
What does the mesoderm give rise to?
Muscles, blood, blood vessels, connective tissues and the heart
What does the ectoderm give rise to?
Brain, spinal cord, nerve cell, hair, skin, teeth, sensory cells of eyes, ears, nose, mouth and pigment cells
What does the endoderm give rise to?
The gut, lungs, bladder and germ cells
What is the source of MSCs?
Bone marrow- isolated from the superior iliac crest of the pelvis, hip, knee, long bones, vertebrate
How are MSCs isolated?
- Bone marrow scooped/aspirated out of bone cavity. Sides scraped to disrupt and remove cells from all microniches
- Marrow sample fractionated on density gradient. Purifies nucleated cells
- Mononuclear fraction plated at low density
- Recovered over three week period
- Detached, subcultured, experimentation etc
How is an MSC defined?
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
What are the characteristics of MSC proliferation?
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
What are the primary products in the market in terms of MSC-based therapy?
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
What are the advantages of MSC’s?
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
What are the disadvantages of MSCs?
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
