1. There are four main types of regeneration: stem cell mediated, epimorphosis, morphallaxis, and compensatory regeneration. 2. Epimorphosis involves de-differentiation of cells forming a blastema which then re-differentiates, as seen in salamander limb regeneration. 3. Morphallaxis involves re-patterning of existing tissues with little new growth, as seen when hydra fragments regenerate entire organisms.

1. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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Regeneration
Regeneration is the reactivation of development in postembryonic life to restore missing
tissues. Regeneration can occur in four major ways:
1. Stem-cell mediated regeneration: Stem cells allow an organism to regrow certain
organs or tissues that have been lost; for example: the continual replacements of
blood cells from the hematopoietic stem cells (that perform hematopoiesis/formation
of blood cells) in the bone marrow.
2. Epimorphosis: In some species (amphibians, etc.), adult structures can undergo de-
differentiation to form a relatively undifferentiated mass of cells that then re-
differentiate (differentiate again) to form the new structure. For example: planarian
flatworm regeneration and regenerating amphibian limbs.
3. Morphallaxis: In morphallaxis, regeneration occurs through the re-patterning of
existing tissues, and there is little new growth. Such regeneration is seen in Hydra (a
cnidarian).
4. Compensatory regeneration: In compensatory regeneration, the differentiated cells
divide but maintain their differentiated functions. The new cells do not come from
stem cells, nor do they come from the dedifferentiation of the adult cells. Each cell
produces cells similar to itself; no mass of undifferentiated tissue forms. This type of
regeneration is characteristic of the mammalian liver.
Stem Cells
A stem cell can be defined as a relatively undifferentiated cell that when it divides
produces (1) one cell that retains its undifferentiated character; and (2) a second cell
that can undergo differentiation.

2. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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Examples of Stem Cell-Mediated Regeneration
Regular Regeneration: In some organs, such as the gut, epidermis, and bone marrow,
stem cells regularly divide to replace worn-out cells and repair damaged tissues.
Under special conditions: In other organs, such as the prostate and heart, stem cells
divide only under special physiological conditions, usually in response to stress or the
need to repair the organ.
Types of Stem Cells
There are two types of stem cells based on their origin:
 Embryonic stem cells are derived from the inner cell mass of mammalian blastocysts
(blastula) or fetal gamete progenitor (germ) cells. These cells are capable of
producing all the cells of the embryo (i.e., a complete organism).
 Adult stem cells are found in the tissues of organs after the organ has matured.
These stem cells are usually involved in replacing and repairing tissues of that
particular organ and can form only a subset of cell types (i.e., they cannot form a
complete organism).
Stem Cell Potency
The potency of a stem cell is its ability to generate numerous different types of
differentiated cells. Based on their potency, stem cells can be classified into the
following types:
 Totipotent cells are capable of forming every cell in the embryo and, also, the
trophoblast cells of the placenta. The only totipotent cells are the zygote and
(probably) the first 4-8 blastomeres.
 Pluripotent stem cells can become all the cell types of the embryo except
trophoblast. Usually, these embryonic stem cells are derived from the inner cell
mass of the mammalian blastocyst (blastula).

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 Multipotent stem cells are stem cells that can only form a relatively small subset of
all the possible cells of the body. These are usually adult stem cells, i.e. the
hematopoietic (blood cell forming) stem cells can form the granulocytes (WBCs),
platelets, and red blood cells.
 Unipotent stem cells are found in particular tissues and are involved in regenerating
a particular type of cell. Spermatogonia, for example, are stem cells that give rise
only to sperm.
The multipotent and unipotent stem cells are often called committed stem cells since
they have the potential to become relatively few cell types.
Epimorphosis (Regeneration of Salamander Limbs)
In epimorphosis, adult structures undergo de-differentiation to form an undifferentiated
mass of cells that then re-differentiate (differentiate again) to form the new structure.
When an adult salamander limb is amputated (cut), the remaining limb cells can
reconstruct a complete new limb. This is an example of epimorphosis. This epimorphic
regeneration is accomplished by cell de-differentiation to form a regeneration blastema
(an aggregation of relatively de-differentiated cells derived from the originally
differentiated tissue) which then proliferates (rapid reproduction of cells) and re-
differentiates into the new limb parts. Bone, dermis, and cartilage just beneath the site
of amputation contribute to the regeneration blastema, as do satellite cells from nearby
muscles.
1. Formation of Apical Ectodermal Cap (AEC) & Regeneration Blastema
When a salamander limb is amputated, a plasma clot forms; within 6-12 hours,
epidermal cells from the remaining stump migrate to cover the wound surface, forming
the wound epidermis. The nerves near the area of amputation (cut) degenerate.

4. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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During the next 4 days, the extracellular matrix of the tissues beneath the wound
epidermis is degraded by proteases, releasing single cells that undergo
dedifferentiation: bone cells, cartilage cells, fibroblasts, and myocytes all lose their
differentiated characteristics. Genes that are expressed in differentiated tissues are
downregulated (their expression is inhibited), while the genes that are associated with
the embryonic limb (undifferentiated) are expressed, i.e. “msx 1 gene is expressed
during regeneration of salamander limb, it is associated with the proliferating progress
zone mesenchyme of the embryonic limb”. This cell mass is the regeneration blastema,
and these are the cells that will continue to proliferate (rapid cell division), and which will
eventually re-differentiate to form the new structures of the limb. Moreover, during this
time, the wound epidermis thickens to form the apical epidermal cap (AEC).
2. Proliferation of the Blastema Cells
The growth of the regeneration blastema depends on the presence of both the apical
ectodermal cap (AEC) and nerves. The apical ectodermal cap stimulates the growth of
the blastema by secreting Fgf8 (Fibroblast growth factor 8 – a type of growth factor), but
the effect of the AEC is only possible if nerves are present. The neurons are also
believed to release factors necessary for the proliferation of the blastema cells, for
example: newt anterior gradient protein (nAG). This protein permits normal regeneration
in denervated limbs (where nerves have been removed/cut/degenerated).
The creation of the amphibian regeneration blastema also depends on the maintenance
of ion currents driven through the stump (the remainder area after the cut), if this electric
field is suppressed, the regeneration blastema fails to form. For example, when the tail
of Xenopus (a species of Frog) tadpole (Frog larva) is cut, the V-ATPase proton pump is
activated within 6 hours after tail amputation, changing the membrane voltage and
establishing flow of protons through the blastema. If this proton pump is inactivated then
there is no regeneration.

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Figure: Steps of epimorphic regeneration of salamander limb.
Morphallaxis (Regeneration in Hydra)
In morphallaxis, regeneration occurs through the repatterning of existing tissues, and
there is little new growth. Such regeneration is seen in Hydra (a cnidarian).
Hydra is a genus of freshwater cnidarians. Most hydras are tiny—about 0.5 cm long. A
hydra has a tubular body, with a "head" at its distal end and a "foot" at its proximal end.
The "foot," or basal disc, enables the animal to stick to rocks or the undersides of pond
plants. The "head" consists of a conical hypostome region (containing the mouth) and a
ring of tentacles.
If a hydra's body column is cut into several pieces, each piece will regenerate a head at
its original apical end and a foot at its original basal end. No cell division is required for
this to happen, and the result is a small hydra.
1. Head Activation Gradient
Every portion of the Hydra body column along the apical-basal (longitudinal) axis is
potentially able to form both a head and a foot. However, a series of morphogenetic
gradients (different concentrations of morphogenetic factors in different body parts)
allow the head to form only at one place and the basal disc to form only at another.

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This means that there exists a head activation gradient (highest at the hypostome/head
region) and a foot activation gradient (highest at the basal disc). The head activation
factor is concentrated in the head and decreases toward the basal disc.
2. Head Inhibition Gradient
There is a gradient of head inhibitor as well,
this is proven by the fact that:
(A) Subhypostomal tissue (tissue beneath
the hypostome that contains a high
amount of head activators) does not
generate a new head when placed close
to an existing host head.
(B) Subhypostomal tissue generates a head
if the existing host head is removed.
(C) Subhypostomal tissue generates a new
head when placed far away from an
existing host head.
3. The Hypostome as an Organizer
The hypostome acts as an "organizer" of Hydra, because:
 When transplanted, the hypostome can induce host tissue to form a second body
axis.
 The hypostome is the only "self-differentiating" region of the hydra.
 The hypostome produces both the head activation and head inhibition signals.
 The head inhibition signal is a signal to inhibit the formation of new organizing
centers.

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4. Basal Disc Activation & Inhibition Gradients
The basal disc is the source of both a foot inhibition and a foot activation gradient.
Several small peptides have been found to activate foot formation; several inhibition
factors are also present near the basal region. These inhibition and activation gradients
inform the hydra "which end is up" and specify positional values along the apical-basal
axis (where to have the head and where to have the foot).
Compensatory Regeneration (Mammalian Liver)
In compensatory regeneration, differentiated cells divide to recover the structure and
function of an injured organ. It has been demonstrated in the mammalian liver and the
zebrafish heart.
The human liver regenerates by the proliferation (rapid cell growth) of existing tissue.
The regenerating liver cells do not fully dedifferentiate when they re-enter the cell cycle.
No regeneration blastema is formed. Rather, the five types of liver cells (hepatocytes,
duct cells, fat-storing cells, endothelial cells, and Kupffer macrophages) all begin
dividing to produce more of themselves. Each cell type retains its cellular identity, and
the liver retains its ability to synthesize the liver-specific enzymes necessary for glucose
regulation, toxin degradation, bile synthesis, albumin production, and other hepatic
functions.
The removal or injury of the liver is sensed through the bloodstream, as some liver-
specific factors are lost while others (such as bile acids and gut lipopolysaccharides)
increase. These lipopolysaccharides activate two of the non-hepatocyte cells to secrete
paracrine factors (chemical messengers that affect neighboring cells) that allow the
hepatocytes to re-enter the cell cycle.
Because human livers have the power to regenerate, a patient's diseased liver can be
replaced by compatible liver tissue from a living donor (usually a relative). Although the

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removed lobe (of donor) does not grow back, the remaining lobes enlarge to
compensate for the loss of the missing tissue.
If the hepatocytes are unable to regenerate the liver sufficiently within a certain amount
of time, the oval cells divide to form new hepatocytes. Oval cells are a small progenitor
cell population that can produce hepatocytes and bile duct cells. They appear to be kept
in reserve and to be used only after the hepatocytes have attempted (and failed) to heal
the liver.