Chapter 3 Stem Cells and Regenerative Medicine

Question 1

What are the three classes of naturaly occurring stem cells?

Answer 1

• Embryonic
• Fetal/perinatal
• Adult
• (Induced Pluripotent Stem Cells)

Hierarchical nature of stem cell differentiation. Totipotent cells differentiate into all cells of the body, including cells of the fetal membranes. Only the recently fertilized zygote is truly totipotent. Pluripotent cells differentiate into all cells of the body, excluding fetal membranes. Embryonic stem cells (ESCs) are examples of pluripotent cells. Multipotent cells differentiate into a number of specialized cells within a specific lineage (germ layer). Stem cells isolated from adult tissues, such as mesenchymal stem cells (MSCs), are typically considered to be multipotent. Other types of multipotent cells include neural stem cells and hematopoietic stem cells. Using laboratory techniques, terminally differentiated cells or adult stem cells can be genetically altered, or reprogrammed, to regain pluripotency. These cells are termed induced pluripotent stem cells (IPSCs), and they hold much promise for future applications in human beings and veterinary species.

Question 2

Into which four classes can mesenchymal stem cells differentiate?

Answer 2

Bone

Cartilage

Muscle (/Tendon)

Fat

Question 3

Name two concerns regarding embryonic stem cells.

Answer 3

1. Ethical concerns - harvesting results in destruction of embryo
2. Teratoma formation! ESC tend to expand/differentiate in unregulated manner

Question 4

Where is the largest population of neural stem cells located?

What are neural stem cells capable of differentiating into (3 cell types)

Answer 4

Dentate gyrus of hippocampus

Neural stem cells can differentiate into neurons, astrocytes and oligodendrocytes.

Question 5

Name an advantage of induced pluripotent stem cells (IPSC) vs embryonic stem cells (ESC)

Answer 5

IPSC are generated from tissue easily aquired from living donors i.e. avoids ethical concerns

Question 6

Where do mesenchymal stem cells usually reside?

Answer 6

Peri-vascularly (communicate with endothelial cells on one side and tissue on other side (i.e. resident somatic cell population e.g. bone marrow, synovium, adipose tissue)

Question 7

Even when generated from a single cell, cultured mesenchymal stem cells are morphologically diverse - what are the two types of mesenchymal stem cell seen?

Answer 7

• Small, rapidly self-renewing cells (RS cells)
• LArger, more slowly replicating cells

A, In early cultures, mesenchymal stem cells are predominately small, spindle-shaped, rapidly proliferating cells termed RS cells (arrowheads). These cells are believed to be more efficient at differentiation. An RS cell in the final stages of mitosis can be seen in the lower left portion of the image (arrow). B, After five or six passages, cultures begin to be overtaken by large, slowly proliferating cells that are believed to lose their differentiation potential. A large, slowly dividing cell (arrow) can be identified adjacent to a number of RS cells (arrowheads).

Question 8

List the 4 factors used to define mesenchymal stem cells:

Answer 8

1. adhere to tissue culture plastic and exhibit a spindle-shaped or “mesenchymal” appearance
2. form colonies of cells from single parent cells when cultured in low-density “clonal” cultures without media exchange
3. express a specific set of cell surface marker proteins that exclude the cells from hematopoietic lineages
4. possess the ability to differentiate into osteoblast, adipocytes, and chondrocytes (i.e., tri-lineage differentiation) using defined in vitro differentiation assays

Question 9

What are thought to be the two mechanisms of actions of mesenchymal stem cells, broadly speaking

Question 10

List 3 examples of anti-inflammatory agents produced by mesenchymal stem cells

Answer 10

1. TNF-alpha stimulated gene/protein 6 (TSG-6)
2. IL-1 receptor antagonis (IL-1ra)
3. Prostaglanding E₂ (PGE₂)

Summary of some of the antiinflammatory effects of mesenchymal stem cells. 1, Damage-associated molecular patterns induced by sterile injury or pathogen-associated molecular patterns induced by infection activate resident macrophages through pattern recognition receptors (PRRs). 2, Activated macrophages produce inflammatory cytokines, such as IL-1α, IL-1β, or TNF-α, to initiate inflammation. 3, Simultaneously, these cytokines activate mesenchymal stem cells to secrete antiinflammatory factors including TNF-α–stimulated gene/protein 6 (TSG-6), prostaglandin E2 (PGE2), and interleukin-1 receptor antagonist (IL-1ra) that dampen macrophage activation or decrease the downstream effects of the inflammatory cytokines. 4, The net effect is to dampen the amplification of proinflammatory signals by parenchymal cells through secretion of factors that decrease the recruitment of neutrophils, such as IL-6 or CXCL1. CXCL1, Chemokine C-X-C motif ligand 1; IL, interleukin; MSC, mesenchymal stem cell; TNF, tumor necrosis factor.

Question 11

What did Volk et al demonstrate re osteogenic differentiation of canine mesenchymal stem cells

Answer 11

Require BMP-2 supplementation for robust osteogenic differentiation (cf in humans)

Question 12

List the 5 steps of the mesengenic process

Answer 12

1. Proliferation
2. Lineage committment
3. Progression
4. Differentiation
5. Maturation

Question 13

Name two examples of point-of care, commercially available cell therapies

Answer 13

• Stromal vascular fraction
• Bone marrow concentrate

Heterogenous sample and therefore response unpredictable.

Question 14

What does cytotherapy refer to?

Name two disadvantages

Answer 14

Systemic administration of soluble cell suspension.

Disadvantages:

• Only 5% of cells reached reached and survived in site of injury.
• Adverse reactions reported: microvasculature embolization ischaemia. (Dogs = myocardial ischaemia. Cats = vomiting and inc RR).

Chapter authors advise only asdminister systemically if local not possible and full consent obtained.