REGENERATION

Regeneration is the reactivation of development in post-embryonic life to

restore missing or damaged tissues.

A very “unhuman” phenomenon which led humans to wonder why we cannot regrow our arms and legs.

What gives other animals the ability to regenerate tissues while we cannot?

Three 18th century naturalists namely Tremblay (Hydra), Réaumur (crustaceans) and Spallazini (salamanders) set forth to

answer these very questions.

Their work has given rise to the field of experimental biology and set the standards for experimental research as well as

discussion of one’s data.

More than two centuries later we are now at the point where we can answer

some of these questions.

We are now much closer to the objective of being able to alter the human body such that our limbs, nerves and organs

can be regenerated.

Regeneration can occur in four major ways:1. Stem cell mediated regeneration: Stem cells are the source of the regenerating the lost tissue or organ e.g. hair shaft regrowth from stem cells in the hair bulge and replacement of blood cells from hematopoietic stem cells.

2. Epimorphosis: Adult cells in some species can dedifferentiate into an undifferentiated mass which then redifferentiates to form new structures e.g. in regenerating amphibian limbs.

3. Morphallaxis: Regeneration occurs through re-patterning of existing tissues and there is little new growth e.g. the regeneration seen in Hydra.

4. Compensatory regeneration: Here differentiated cells divide but maintain their differentiated function e.g. that observed in the mammalian liver.

Stem cell mediated regeneration in flatworms

Planarian flatworms reproduce by binary fission, splitting the body from top to bottom and regenerating the right and left halves. The cells capable of doing these are pluripotent stem cells which are also used to repair and replace body parts.

From 1700 it is known that when planarians are cut in half each half regenerates the missing piece

Thomas Hunt Morgan and C.M. Child realized that such polarity indicated an important principle of development.Morgan pointed out that when head and tail were both cut off the middle piece would always generate a head from the former anterior region and a tail from the former posterior region, never the reverse. If however the middle piece was too small then the regenerating portions would be abnormal.Based on these observations they postulated that a gradient of anterior producing material was emanating from the head. The middle segment would be told what to generate at the two ends by concentration gradient of these materials.

Which cells formed the new head or tail?

For many years it was believed that the old cells dedifferentiated at the cut ends of the plananria to form a regeneration blastema, a collection of relatively undifferentiated cells that can be organized into new structures by paracrine factors located at the wound site.

However in 2011 a series of experiments done by Wagner and colleagues provided evidence that de-differentiation does not occur.

The regeneration blastema forms from pluripotent stem cells called clonogenic neoblasts, a set of pluripotent stem cells in flatworms that serve as stem cells to replace the aging cells of the adult body.

The picture shows neoblasts in the planaria flatworm Schmidtea mediterranea, stained red by antibodies to phorpho histone 3, which marks mitotic cells. All nuceli are marked in blue. Neoblasts are scattered throughout the body posterior to the eyes but are absent from the centrally located pharynx.

Clonogenic neoblasts can migrate into a wound site and regenerate tissues.Wagner and colleagues showed that if planaria are irradiated with 1750 rads which kills nearly all dividing cells (neoblasts) keeping a few or one alive. This would be sufficient to produce the cells of all three germ layers indicating that they are indeed pluripotent stem cells.Next the same researchers irradiated the planaria with 6000 rads which killed all neoblasts. These planaria died because of failed tissue replacement. However, the introduction of a single clonogenic neoblast into such worms could in some cases restore all the cells of the organism. When this worm was split inot pieces each piece regenerated the missing parts and cells were all of the genotype of the donor neoblast.This confirmed that regeneration takes place from pluripotent stem cells.

Questions that remain:First line of questions regarding the clonogenic neoblasts:

1. Are all neoblasts pluripotent? 2. What is the embryonic origin of these clonogenic neoblasts?3. What is the hierarchy by which these clonogenic neoblasts generate the 30 or so cell

types of the adult planarian?

These questions are currently being investigated.

Second line of questions involving polarity:

4. How does the flatworm tell the anterior blastema to become the head and the posterior blastema to become the tail?

5. How do the blastema derived cells retain their dorsal ventral polarity?

Polarity establishment in the flatwormFlatworms establish the AP and DV polarity using the same factors and processes employed by vertebrates

BMP expression defines the dorsal region in flatworms. BMP inhibitors such as Noggin are produced in the ventral side.

Anterior-posterior polarity is determined by Wnt signaling, with Wnt produced in the posterior and Wnt antagonists made in the anterior.

AP polarity during regeneration in the flatworm

Normally Wnts are produced in the posterior blastema which then forms a tail but if RNAi is used to target either Wnt1 or β-catenin the posterior blastema forms a head.

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Show movie of flatworm regeneration

Epimorphic regeneration of salamander limbs• When a salamander limb is amputated the

remaining cells regenerate a new limb which is complete with all the differentiated cells arranged in proper order.

• Remarkably the limb generates only the missing structures and no more.

• In some ways the salamander limb knows the point in the proximo-distal axis where it has been severed and regenerates from that point onwards and no more.

• The salamander accomplishes epimorphic regeneration by cell dedifferentiation to form a regeneration blastema- an aggregation of relatively undifferentiated cells derived from the originally differentiated tissue- which then proliferates and redifferentiates into the new limb parts.

Formation of the apical ectodermal cap and the regeneration blastema When a salamander limb is

amputated a plasma clot forms.

Within 6-12 hrs epidermal cells from the remaining stump migrate to cover the wound surface forming a wound epidermis.

In contrast to wound healing in mammals no scar forms and the dermis does not move with the epidermis to cover the wound.2 days after amputation the

skin and muscle have retracted from the bone surface At day 5 a thin accumulation

of blastema cells is seen under the apical ectodermal cap (AEC)

At day 7 a large number mitotically active blastema cells are seen distal to the humerus

At day 8 blastema elongatesBy mitotic activity and much dedifferentiation has occured

At day 9 early redifferentiation can be seen. A marks apical mesenchyme of blastema, H marks the proximal part of regenerating humerus, U and R are precatilagous condensations for ulna and radius. P represents the stump where the amputation was done.

At day 10, C marks the precartilagenous condensations of the carpal bones (ankle) and D1 and D2 mark the same for the first tweo digits.

In the 4 days following the amputation the extracellular matrices of the tissue beneath the wound epidermis are degraded by proteases liberating single cells and undergo dramatic dedifferentiation.Bone, muscle, cartilage cells and fibroblasts lose the differentiated cell characteristics. Differentiated cell markers are downregulated and increased expression of genes such as msx1 associated with the proliferating progress zone mesenchyme in the embryonic limb is observed.This cell mass is the regeneration blastema and these cells will continue to proliferate and will redifferentiate to form new structures of the limb.The wound epidermis thickens to form the apical epidermal cap (AEC) similar to the AER during limb development.

• Bone dermis and cartilage just beneath the site of amputation contribute to the regeneration blastema as do satellite cells from the nearby muscles.

• Unlike the flatworm where the regeneration blastema tissue comes from pluripotent stem cells, in the salamander most of the regeneration blastema appears to arise from dedifferentiation of adult cells followed by cell division and redifferentiation of those cells back into their original cell types.

• However, there is still controversy about whether this is “true” dedifferentiation of normally postmitotic cells or whether much of the limb blastema is formed from the activation of uncommitted stem cells residing within the adult tissues.

Annu Rev Cell Dev Biol. 2011;27:409-40. doi: 10.1146/annurev-cellbio-092910-154115. Epub 2011 Jul 29.

Limb regeneration: a new development?Nacu E1, Tanaka EM.

A major question is: do the blastema cells keep a “memory” of what they have been?

Kragl and colleagues in 2009 found that the blastema is not simply a collection of homogenous fully dedifferentiated cells. Rather in the regenerating limb of the salamander muscle cells arise from previous muscle cells, dermal cells from only old dermal cells and cartilage cells can arise from only old cartilage cells or old dermal cells.

Thus the blastema is not simply a collection of unspecified multipotent progenitor cells. Rather the cells retain their specification and the blastema is a henterogenous assortment of restricted progenitor cells.

Blastema cells retain their specification even though they differentiate

Cartilage from a specific position in the limb of a salamander expressing GFP is transplanted into an equivalent position in the cartilage of a salamander limb that does not express GFP. The grafted tissue integrates with the host tissue.Amputation is performed in the middle of the grafted tissue. The blastema forms containing GFP expressing cells. The regenerated limb is studied to see if the GFP expressing cells are present only in regenerated cartilage or other tissues

Longitudinal section of a regenerated limb 30 days after amputation. Muscle cells are stained in red, nuclei are stained blue. The majority of GFP positive cells (green) are seen in the regenerated cartilage and not in the regenerated muscle.

Nature. 2009 Jul 2;460(7251):60-5. doi: 10.1038/nature08152.Cells keep a memory of their tissue origin during axolotl limb regeneration.Kragl M1, Knapp D, Nacu E, Khattak S, Maden M, Epperlein HH, Tanaka EM.

Proliferation of the blastema cells: requirement of the nerves and the AECThe AEC stimulates the growth of the blastema by secreting Fgf8 just like the AER does during normal limb development.But the effect of the AEC is only possible when nerves are present. Singer (1954) demonstrated that a minimum number of nerve fibres must be present for regeneration to take place. The neurons are believed to secrete factors necessary for the proliferation of the blastema cells. Among the many candidate for the nerve derived blastema mitogens, the best is newt anterior gradient protein (nAG).This protein permits normal regeneration in limbs that have been denervated.

Curr Opin Genet Dev. 2008 Aug;18(4):381-6. doi: 10.1016/j.gde.2008.06.008. Epub 2008 Aug 11.

New regulators of vertebrate appendage regeneration.Yin VP1, Poss KD.

The creation of the amphibian regeneration blastema may also depend on maintaining ion currents driven through the stump; if this electric field is suppressed, the regeneration blastema fails to form.

Skin flaps inhibit both the current of injury at the amputation surface and regeneration of that limb in newtsAlicia M. Altizer, Sarah G. Stewart, Brian K. Albertson and Richard B. Borgens*

Article first published online: 29 AUG 2002, DOI: 10.1002/jez.10141

Such fields have been shown to be necessary for regeneration of tails in the frog Xenopus laevis.

In this frog the V-ATPase proton pump is activated within six hours after tail amputation, changing the membrane potential and establishing the flow of protons through the blastema.

If this proton pump is inactivated either by mutation or by drugs, the depolarization of the blastema cells fails to occur and there is no regeneration.

Development 134, 1323-1335 (2007) doi:10.1242/deH+pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regenerationDany S. Adams, Alessio Masi and Michael Levin*