Unsolved Problems: Regeneration of tissues and organs.

Maybe if we understood how salamanders regenerate their legs (and also some other organs) then that knowledge could be used to replace human arms and legs. This hope is called "Regenerative Medicine."

But very little success has been achieved. Science is no closer to a method for replacing legs or arms. A related hope is that stem cells (undifferentiated, but susceptible to being stimulated to differentiate) can be . cultured from early embryos, and used to rebuild or regenerate organs.

Bone marrow transplants are Blood Stem Cell Transplants, and are a well established medical procedure.

Skin transplants likewise depend on not-yet-fully-differentiated skin stem cells.

Why shouldn't it be practical to graft stem cells for every kind of differentiated cells, maybe grown in tissue culture? (Or at least many more different kinds of differentiated cells?) cartilage, bone, kidney, liver, nerves?

A possible reason why not is the need for complex geometric arrangements, for kidney, liver, cartilage? Geometric arrangements take care of themselves for skin and bone marrow transplants.

Hydra can regenerate any part of their body. This is by rearrangement of differentiated cells. Their cells rearrange all the time. For example, tentacles are continually formed by geometric rearrangement of cells of the trunk, and so are asexual buds. Cells constantly fall off the tips of tentacles. The same occurs in the villi of human intestines.

Sponges re-form functional anatomy even after being dissociated into single cells, randomly mixed together. This was discovered by H. V. Wilson in 1907, and is one of the most famous and important discovery ever made in the UNC Biology Department (or, arguably, in any UNC department).

Wilson mistakenly guessed that sponge cells must switch from one differentiated cell type to another, based on their new locations. Later he decided that undifferentiated "archeocytes" probably provide the cells for the newly re-formed sponges. He was probably wrong, however. You can easily watch or make time-lapse videos of sponge cells rearranging. Unfortunately, Wilson never had access to time-lapse filming apparatus, or even good enough microscopes.

Julian Huxley discovered the true explanation, which is rearrangement of cells according to differentiated cell type. Calhoun Bond discovered that sponge cells rearrange constantly, from day to day, or even from hour to hour, even when not disturbed

Planaria (flatworms) can regenerate the whole body from fragments as small as one three-hundredth of the body; but (I have read) such regeneration occurs only those species of flatworms that reproduce asexually by budding new individuals from their rear end.

It is not really known to what degree flatworm regeneration is produced by rearrangement of cells, as opposed to differentiation of stem cells. Historically, the latter tends to be assumed, unless the former can be proven. I don't know why most people are so strongly biased in favor of changes in differentiation instead of changes in locations and geometric arrangements; But in my experience this bias is extremely strong. This bias is itself an unsolved problem.

Salamanders (especially the sub-group of salamanders called newts) can regenerate their legs, tails, retina, lenses of their eyes, and lower jaw (And I don't know what other organs and tissues).

Salamander lens regeneration is especially unusual in that the new lens differentiates from pigmented retina cells. For one differentiated cell type to be converted into another is unusual.

Some frogs can regenerate their lenses; but this occurs by infolding of the cornea (NOT from the retina).

Salamanders are the only vertebrates that can regenerate legs (not counting younger stages of frog tadpoles).

What are some possible reasons?
1) Maybe only salamander cells can rearrange enough?
2) Maybe all other vertebrates need an Apical Ectodermal Ridge to stimulate leg formation (i.e. only salamanders can make a new leg without an AER?)
3) Maybe only salamander skeletal muscles can separate into individual multi-nucleated cells?
4) Maybe only salamanders continue to have long-range signaling molecules.
5) Maybe salamanders are the only vertebrate whose adult cells continue to be sensitive to long-enough range signaling molecules.

6) Students: Please invent five more possible explanations.

Except for salamanders, during embryonic development, all vertebrates have an "Apical Ectodermal Ridge" (AER) along the outer edge of their arms, legs, wings or fins. If the AER is surgically removed from embryos, then distal structures (hands, wrists, arms) do not form. One of the "Fibroblast Growth Factors" causes limbs to develop even if the AER is removed, and can cause extra legs to develop along the flank

During regeneration, a thickening in the epidermis forms at the tip of salamander limb stumps. This "Apical Ectodermal Cap" is necessary for regeneration; if surgically removed, regeneration ceases, at least until a new AEC is regenerated. It is a riddle why these ectodermal thickenings are needed for limbs to develop (and especially why the one kind of vertebrate that doesn't form an AER is the only kind that can regenerate legs; and DOES form an ectodermal thickening)

A central question is whether cells switch from one cell type to another during regeneration. Apparent de-differentiation occurs in salamander leg regeneration; for example chondrocytes and muscle cells become indistinguishable. Skeletal muscles are syncytial, but subdivide back into single uninucleate cells during this dedifferentiation

By labeling cartilage and muscle nuclei (with radioactive hydrogen and phosphate, with green fluorescent protein, and with triploid nuclei) many researchers have studied whether chondrocytes ever re-differentiate into muscle cells, or vice versa. They have all concluded that this interconversion never occurs. Although muscle and cartilage cells become dedifferentiated in the sense that you can't tell them apart, both remain loyal to their former differentiated cell type. In other words, former muscle cells always redifferentiate back to being muscle cells, and former cartilage cells always redifferentiate back to being chondrocytes

The one change in cell type (besides lenses) is conversion of many dermal fibroblasts into chondrocytes. In fact, as many as half the cartilage cells in the regenerated skeleton are descended from dermal fibroblasts

Despite this unanimity of results, that dedifferentiating cartilage and muscle cells then redifferentiate back into the same cell type as they were before the amputation, few researchers think of regeneration as a process of geometric rearrangement of differentiated cells into new spatial arrangements

Much research funding is being invested in the idea of undifferentiated "stem cells" being able to create missing organs and structures. The intense religious arguments about the morality of stem cells has somehow prevented serious consideration about whether the method can succeed, even in animals. Salamander legs don't develop from stem cells, but much funding has been based on the conclusion that they do. (Read between the lines in the concluding pages of Stocum's big review paper.)

Some odd and interesting facts about salamander limb development:

I) The AER, and all that. Why have a thickening in the skin control leg formation? And what causes this thickening, anyway? An increased contraction? A decreased contraction? A directional contraction? Think of some possible answers?

Is there any way to discover the cause of any phenomenon other than by guessing what might be the cause, then figuring out what the theory predicts (weird, surprising, predictions are the best) and then testing or otherwise observing whether the prediction is true?

II) Imagine an experiment in which one "hand" of a salamander is amputated, and then grafted to the elbow of another salamander's "arm", or if the hand were grafted to the shoulder, or anywhere along the length of the "arm"

Would you expect anything in particular to happen?

What does happen is that the grafted hand moves very slowly, day by day, out the length of the "arm", toward the normal hand, until it is connected at the wrist, at the location next to the normal hand

This was discovered by Nardi and Stocum at the University of Illinois at Urbana, where they have an axolotl colony. The reverse experiment has also been done, with analogous results

Nardi and Stocum have hypothesized that limbs have some kind of adhesion gradient, with distal (hand) cells being more strongly adhesive than shoulder cells, and elbow cells having intermediate adhesiveness

They got this idea from Malcolm Steinberg's "Differential Adhesion Hypothesis", of which I have been the main critic for 30+ years, so my explanation may be biased. Other researchers have discovered direct evidence of gradients of cell surface properties along salamander legs

Nardi and Stocum dissected bunches of cells from wrist, elbow and shoulder areas of salamander legs, and put these masses of cells in contact with each other, and in nutrient saline, in each possible pairwise combination

e.g. Shoulder next to wrist; Shoulder next to elbow; Shoulder next to Shoulder; Elbow next to wrist; Elbow next to elbow

In each case, the more distal (distal means far from the main body axis) tissue was engulfed by the more proximal (from nearer the body axis) tissue. {Unless I have remembered it backwards} According to Steinberg's theory, cells that get engulfed are more adhesive than the cells that do the engulfing. My theory (and Wayne Brodland's) is that engulfment results from stronger surface ("interfacial") contraction by whichever cells get engulfed.

III) If regenerating cells remain "loyal" to their original differentiated cell types
     (muscle -> muscle; cartilage -> cartilage)
does that mean that regeneration of legs is caused by rearrangement of cells?



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