Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

REGENERATION IN ANIMAL MODELS

Dr. Péter Balogh and Dr. Péter EngelmannTransdifferentiation and regenerative medicine – Lecture 3

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

TÁMOP-4.1.2-08/1/A-2009-0011

Regeneration

Regeneration is the sequence of morphogenetic events that restores the normal structure of an organ after its partial or total loss/amputation. It exists at different levels in plants, invertebrate and vertebrate animals

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Physiological regeneration

Reparative regeneration

Hypertrophy

Morphallaxis

Types of regeneration in multicellular organisms

Tissue damage or loss

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• An ancient conglomerate of flagellate protists is developed.

• All cells at the surface shifted into subgroups of dividing cells and non-dividing cells. Those can be named as unipotent stem cells and non-stem cells.

• When multicellularity developed, there was a requirement for migrating stem cells to replace other cells inside the body.

Evolution of stem cells

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Regeneration in Porifera (sponges)• One cell population of sponges, the so called

archeocytes are active stem cells.• Archeocytes are able to differentiate into various

types of cells and repopulate themselves by self-renewal.

• Archeocytes give rise to choanocytes (participate in respiratory and digestive function), sclerocytes (important cells in innate immunity).

• Archeocytes also produce oocytes, while choanocytes produce sperm.

• In special occasions choanocytes transdifferentiate into archaeocytes.

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Regeneration in Hydra• Artificially dissected Hydra polyps can retain their

aggregation within 48 hours.• Individual animals do not increase their size since

growth is just balanced by loss of tissue in the form of buds in the lower gastric region and by sloughing of tissue at the ends of the tentacles and from the basal disk.

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Stem cell lineages in Hydra

• Epithelial cells in the Hydra body column continuously undergo mitotic divisions. Moreover, ectodermal and endodermal epithelial cells also exist.

• These two epithelial cell layers made up by stem cells. Hydra epithelial cells are capable, by successive divisions, both of indefinite self-renewal and of producing different types of specialized cells such as tentacle or foot specific epithelial cells.

• In addition, an interstitial stem cell layer is developed.

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Molecular factors of Hydra stem cells• Notch signaling• Wnt signaling• BMP molecules• JAK/STAT • Gene screen for stem cell related genes

(Sox2+, Nanog, Oct3/4??)

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Regeneration in planaria I• Planarians are bilaterally symmetrical animals found

in freshwater streams and ponds.• Planarians have the capacity to replace enormous

amount of missing regions through regeneration.• Planarian regeneration is referred to as morphallaxis.• Morphallaxis means cell proliferation / regeneration

events away from the wound tissue.

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Regeneration in planaria II Neoblasts• Planarian bidirectional regeneration is mediated by

neoblasts.• Approx. 30% of the total cells in the planaria are

neoblasts• Neoblasts can be found in the entire mesenchymal

region of the body with the exception of the pharyngeal region.

• Neoblasts divide by mitosis and can repopulate themselves. They are the only dividing cells in planaria.

• When a planaria is wounded, neoblasts migrate to the site and begin dividing.

• Neoblasts can become any cell type the planaria needs—nerve cells, reproductive cells, etc.

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Molecular pattern of neoblasts• Nanos RNA• Piwi RNA• Piwi subfamily - Argonaute proteins • miRNA• Wnt pathway• Shh pathway• FGF family

TÁMOP-4.1.2-08/1/A-2009-0011Regenerative capacity of axons inC. elegans• Axons are able to regrow in many animals after

injury except in mammals.• In C. elegans axonal regeneraton appears as early

as 4-5 hours following laser surgery, a growth cone-like structure is developed after 6-10 hours.

• DLK-1 pathway involved mainly in this regeneration process.

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Regeneration in annelids• It has been known for decades that annelids are

capable for efficient regeneration of their injured body parts.

• However the molecular aspects of this regeneration is more hidden.

• After 6-10 hours of the injury, neoblast cells are present and give rise to other tissue cells.

• Moreover, transdifferentiation of epithelial cells into nerve cells was observed.

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Regeneration in insects• Some insects are able to regenerate their legs and

other appendage organs.• Other insect species (flies) such as Drosophila do not

regenerate adult appendages, but from their imaginal discs they have a great regenerative behaviours during larval life.

• In this process several factors are participating such as decapentaplagic (dpp), wingless (wg) etc. molecules.

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Regeneration and colony fusion in protochordates• Protochordates share developmental history with

vertebrates at least through their early stages.• These colonial metazoans can give hints for

regeneration mediated by stem cell activity.• Colony development is dependent on self / non self

recognition mediated by a polymorphic gene family (Fu/HC) along with germ and somatic stem cell circulation.

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Regeneration of vertebratesThere are two types of regeneration:• Epimorphosis or epimorphic regeneration: This type

of regeneration involve the reconstruction of the missing parts by local proliferation from the blastema, or addition of parts to the remaining piece. For example: regeneration of tail, limbs and lens in amphibians and other vertebrates.

• Morphallaxis or morphallactic regeneration: This type of regeneration involving reorganization of the remaining part of the body of an animal. For example: Hydra, planaria and other invertebrates e.g. regeneration of the new individual from body pieces.

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Regeneration in fish IFin regeneration divided into four successive steps:1 Wound healing / closure within 3 hrs2 Blastema formation within 1 day3 Regenerative outgrowth concomitant to

differentiation within 2 days4 Patterning of blastema

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Regeneration in fish IIHeterogeneity of cell sourceDuring fin regeneration epidermis contains different compartments of blastema cells:• Distal• Proximal• Lateral

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Regeneration in fish IIImolecular patterns• Shh• Wnt• FGF• Activin b A• C-Jun, JunB

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Epimorphosis or epimorphic regenerationRegeneration of tail in amphibians and reptilia:Amphibia: The tail lacks vertebrae and has an unsegmented cartilaginous tube, which contains the regenerated spinal cord which form mainly of the ependymal lining of the central canal . At first very few cells accumulate under the wound epithelium . The ependyma and the various connective tissues dermis, muscle septa, adipose tissues and osteocytes of vertebrae are the sources of cells for the generate. The non-nervous elements proliferate behind the apex, forming both the muscle and cartilage tube ,then the ependyma proliferate and gradually extend dorsally.

Reptilia: For example lizard, the regenerated tail is a quite imperfect tail. It lacks vertebrae, and in their place, has an unsegmented cartilaginous tube. This tube contains the regenerated spinal cord, including the extension of the ependymal lining of the central canal of the spinal cord.

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Similarities in regeneration

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PD deletion PD duplication

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Imprinting in regenerationAmputation

MA11A13

MA11A13

MA11A13

InactivationOff

ActivationOnOn On

Never expressed

MemorizedRenewed

Gene expression

On Off

Off

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Regeneration of the limbRegeneration begins in 3 phases:1 Phase of wound healing or pre -blastema stage:

Blood clotting and migration of epidermal cells from the basal layer of epidermis toward the centre of the wound. The wound is covered with epithelium which is thicker than the epidermis of the limb.

2 Phase of blastema formation:Cells accumulate beneath the epithelial coverings and form the blastema. Mesenchymal cells accumulate beneath the cap. Mesenchymal – blastemal cells differentiate into myoblasts and muscle cells, early cartilage cells and cartilage. During the dedifferentiation phase hyaluronate (HA) increases in the distal stump to form blastema . As the blastema forms, the HA concentration will be decreased. The production of HA and break down of collagen represent the establishment of migration from stump tissues.

3 Phase of dedifferentiation and morphogenesis:The blastema begins to restore the part of which the limb was deprived. Specifically, if the fore arm is removed, the blastema differentiated directly into the muscle, bone, cartilage and skin of the fore arm.

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Regeneration of amphibian lens 1 After removal or injury of the lens the dorsal region of the iris thickens

and a cleft arises between inner and outer lamellae of the iris.2 Amoeboid cells move from the stroma into the cleft followed by

marked increase of RNA and DNA synthesis as well as of mitotic cell division.

3 The pigmented cells of the dorsal region is engulfed by invading amoeboid cells.

4 The formed non- pigmented cubical cells form hollow epithelial vesicle and extends with inner and outer lamellae.

5 The vesicle inner wall cells elongated into the lumen and form primary lens fibers.

6 The lens-specific crystalline proteins are formed.7 The primary lens fibers push to the front of vesicle to form a nucleus

behind the lens epithelium which form the secondary lens fibers.8 The nucleus of primary lens fibers is enclosed by secondary lens fibers.9 In the central lens fibers the nucleus degenerate ,primary and

secondary lens fibers are the components of the lens.

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Neural stem cells differentiation capacity

Differentiation

NogginLow-RA FGF-2 FGF-2

Passage (6 days)

in vitro

in vivo

Somatic cells

Neurogenesis

Neurogenesis

Early neurogenesisProjecting neuron

Cholinergic neuronDopaminergic neuron

Motor neuron

Neuron

Gliogenesis

Gliogenesis

Late neurogenesisInterneuronGABAergic

neuron

NeuronAstrocyte

Oligodendrocyte

Differentiation

iPS cellsES cells

Embryoidbody

Primaryneurosphere

Embryos

Secondaryneurosphere

Neonatals Adults

Blastocysts

Blastocysts

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Factors controlling regeneration in vertebrates• Nervous system• Animal size• Pituitary gland• Vitamin A and its derivatives• Insulin

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Summary• All organisms possess a certain level of regeneration

capacity after tissue injury.

• At early stage of evolution, animals are able to regenerate whole body, however this capacity is more restricted to specialized tissues and organs in course of evolution.

• Neoblasts, hemoblast, progenitor, stem cells are participating in this process.