Growth & Development

From FETAL PRECURSOR CELL TRANSPLANTATION (FPCT)
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As stated in the earlier chapter of the text, the early development of most vertebrate CNS is similar. The early mammalian neural tube is a straight structure. The most anterior part of the tube balloons into three primary vesicles: forebrain, midbrain, and hindbrain. By the time the posterior end of the neural tube closes, optic vesicles bulge laterally from each side of the developing forebrain.


The forebrain subdivides into the anterior telencephalon, and caudal diencephalon. Mesencephalon does not subdivide. Rhombencephalon subdivides into anterior metencephalon and posterior myelencephalon.


Neural tube closure in mammals is initiated at several places along the AP axis. Failure to close the posterior neuropore at day 27 results in spina bifida, failure to close anterior neural tube results in lethal anencephaly, where the forebrain remains in contact with amniotic fluid and inevitably degenerates.


The reader is advised to pay attention to the following explanation of the reason for intraventricular implantation of fetal precursor cell transplants.


The original neural tube is composed of a one-layer thick germinal neuroepithelium. It is a rapidly dividing cell population. DNA synthesis, i.e. S phase of mitosis, occurs while the nucleus is at the external side of the tube, and as mitosis proceeds the nucleus migrates luminally. Mitosis occurs on the luminal side of the cell layer.


During early mammalian development 100% of neural tube cells divide. Shortly thereafter certain cells stop participating in mitosis. These post-mitotic cells, neuronal and glial, differentiate on the external side of the neural tube, while those that continue to participate in mitosis, i.e. the germinal cells, are on the luminal side of the neural tube.


The neurons that stopped participating in mitosis always migrate away from the luminal side of the neural tube. Time when the neuron precursor divided last time is the neuron’s ‘birthday’. Different types of neurons and glial cells have different ‘birthdays’. Cells with the earliest birthdays migrate the shortest distances. Cells with later birthdays, i.e. younger, migrate through all layers to form the more superficial regions of the brain cortex. This forms an ‘inside-out gradient of development’. Subsequent differentiation of these precursor cells is dependent upon their position in terms of cell layer that these neurons occupy once outside the layer of dividing cells. A single stem cell in the ventricular layer can produce neurons (and glial cells) that end up in any of the cortical layers. Determination of laminar identity, i.e. which layer a cell migrates to, is made during the final cell division.


As cells on the luminal side of the neural tube, i.e. germinal layer, later on called ‘ependymal cells’, continue to divide, the migrating cells form a second layer, progressively thicker by a continuous addition of new cells from ependyma, the mantle zone -


In the mantle zone the migrating cells differentiate into neurons and glial cells. Neurons develop dendrites to make connections between themselves, and send forth axons away from the lumen, and axons create a cell-poor marginal zone. Eventually, glial cells cover many axons with myelin sheaths, thereby giving this part of the marginal zone a whitish appearance, ‘white matter of the brain’. The portion of the mantle zone with neuronal bodies is referred to as ‘gray matter of the brain’.


Cell migration, differential growth, and selective cell death, produce modifications of the three-zone pattern in the brain that is quite apparent when one compares the cortex of cerebrum and cerebellum.


Cerebrum is organized in two distinct ways:

1/ vertically into layers that interact with each other. Certain neuroblasts from the mantle zone migrate upon glia (see below) through the white matter to create the second zone of neurons. This newly created mantle zone is called ‘neopallial cortex’. It eventually stratifies into six layers of cell bodies. It takes up until the middle of childhood before these neurons attain adult forms. Each layer of the cerebral cortex differs from the other in its functional properties, the types of neurons found therein, and the type of neuronal connections that develop.

2/ horizontally into over 40 regions that regulate geographically distinct processes and functions.


Neither vertical nor horizontal organization is clonally specified. On the contrary, there is a lot of cell movement that mixes the progenies of various precursor cells.


The primary mechanism for positioning young neurons within the developing mammalian brain is ‘glial guidance’. Neurons ride the ‘glial monorail’ to their respective destinations. When the neuronal cell membrane first makes contact with the glial membrane, the glial cell stops proliferating and begins to differentiate and extends its glial process. This process is controlled by glial cells and not by neurons. Neuron binds to the glial cell and begins to express calcium ion channels. Influx of calcium ions is necessary for the motility of the leading process. The neuron wraps this long leading process around the glial fiber, and as it migrates along the process, it continues to signal the glial cells to retain its differentiated form. Neurons maintain its adhesion to the glial cell through a number of proteins, the most important being the adhesion protein astrotactin.


While 80% of the young neurons migrates radially, about 12% migrates laterally from one functional region of the cortex into another. Neural descendants of a single germinal cell are dispersed across the functional regions of the cortex. Specification of these cortical areas into specific functions occurs after neurogenesis.


Human brain continues to develop at fetal rates even after birth. At birth humans are prepared for independent life much less than primates, and continuous fast brain development is mandatory. For that reason some feel that during our first year of life we are essentially ‘extrauterine fetuses’. Much of human intelligence comes from the stimulation of the nervous system during the first year after birth.


Human cerebral cortex has no neuronal connections at 12 weeks gestation, and therefore cannot move in response to a thought, nor experience consciousness or fear. Measurable electrical activity characteristic of neural cells, i.e. EEG, is first seen at 7 months gestation.


Human brain consists of 100 billion neurons associated with 1 trillion of glial cells.

At birth cortical neurons have very few dendrites, but during the first year after birth each cortical neuron develops so many dendrites that it can accommodate 100,000 connections with other neurons.


Each neuron has one axon, which may extend for several feet. It is an extension of neuronal body. To prevent dispersion of the electrical signal and to facilitate its conduction , each axon in CNS is insulated by processes handled by oligodendrocytes. Oligodendrocyte wraps itself around the developing axon, and then produces a specialized cell membrane rich in myelin basic protein that spirals around the central axon. In the peripheral nervous system another glial cell called Schwann cell accomplishes the same.


Axon must be specialized for secreting a specific neurotransmitter across the synaptic clefts that separate axon of one neuron from the surface of its target cell.


Development of neural crest cells of most vertebrates is similar. Derived from ectoderm, neural crest cells originate at the dorsal-most region of the neural tube and migrate extensively to generate a large number of differentiated cells types:

1/ neurons and glial cells of sensory, sympathetic, and parasympathetic nervous systems,

2/ the epinephrine-producing medulla cells of adrenal gland,

3/ pigment containing cells of the epidermis,

4/ skeletal and connective tissue of the head.


Neural crest is divided into:

1/ cephalic neural crest, the cells of which migrate dorso-laterally to produce the craniofacial mesenchyme; these cells enter also pharyngeal arches to give rise to thymic cells, odontoblasts, cartilage of inner ear and mandible,

2/ trunk neural crest: those cells that migrate dorso-laterally become melanocytes, while those that migrate ventro-laterally form either dorsal root ganglia, or sympathetic ganglia, adrenal medulla and para-aortic bodies,

3/ vagal and sacral neural crest, the cells of which generate the parasympathetic (enteric) ganglia of the gut,

4/ cardiac neural crest, the cells of which develop into melanocytes, neurons, mesenchyme of the 3rd, 4th, 6th pharyngeal arches, and musculo-connective tissue wall of the large arteries as they arise from the heart, and septum that separates the pulmonary circulation from aorta.


The above is of great help in selecting the cell types for treatment of certain pathological conditions by fetal cell transplantation..


Neural crest cells are exceptionally strongly pluripotential.


Epidermis:


We lose and replace 1.5 gm of dried epidermal cells a day. The replacement is coming from the population of stem cells. In adult skin a cell born in the Malpighian layer takes approximately 8 weeks to reach stratum corneum, and remains there for about 2 weeks. In psoriasis characterized by exfoliation of enormous numbers of epidermal cells, the time cells spend in stratum corneum is only 2 days. This is due to the over-expression of transforming growth factor TGF-beta.


In male pattern baldness the scalp follicles revert back to producing under-pigmented and very fine vellus hair of newborn. There appears to be a pluripotent epidermal stem cell the progeny of which can become epidermis, sebaceous gland or hair follicle.


Mesoderm:


The formation of mesodermal and endodermal organs occurs synchronously with neural tube formation.


Mesoderm is divided into:

1/ chorda-mesoderm, that forms notochord without which the neural tube formation is not possible;

2/ somatic dorsal mesoderm , that becomes somites, blocks of mesodermal cells on both sides of neural tube that produce many kinds of connective tissue of the back;

3/ intermediate mesoderm that forms urinary system and genital ducts;

4/ lateral plate mesoderm that gives rise to the heart, blood vessels, blood cells of the circulatory system, lining of body cavities, all mesodermal components of the limbs except the muscles, and all extraembryonic membranes;

5/ head mesenchyme necessary for the development of the face.


The mechanism for creating mesodermal somites and body cavity linings has changed little throughout vertebrate evolution.


Somites are transient structures, any cells of which can become any of the somite derived structures.


Myogenesis cannot take place without cell-cell-recognition and cell fusion. But species specificity is not necessary, i.e. myoblasts fuse only with other myoblasts but those myoblasts need not be of the same species.


Osteogenesis:There are two major modes of bone formation, and both involve transformation of a pre-existing mesenchymal tissue into bone tissue.

1/ Intramembraneous ossification is a direct conversion of mesenchymal tissue into bone: some mesenchymal cells develop into capillaries, others into osteoblasts capable to secrete bone matrix. The secreted collagen-glycosaminoglycan matrix binds calcium salts brought in by capillaries, and thereby the matrix calcifies. External layer of mesenchymal cells creates periosteum, and the cells on the inner surface of periosteum become osteoblasts.

2/ Endochondral ossification, where mesenchymal cells differentiate into cartilage first, and cartilage is later replaced by bone. The cartilage tissue becomes a model for the bone that is created during the next step. This remarkable process coordinates chondrogenesis with osteogenesis.


Cardinal difference from intramembraneous ossification is that in the endochondral ossification ‘epiphyseal plates’ are created, the cartilagineous regions at the end of long bones, through which bone lengthens. The proliferation of epiphyseal plate is controlled by hormones: growth hormone and insulin-like growth factor. At the end of puberty, high levels of estrogen or testosterone cause the remaining epiphyseal plate cartilage to hypertrophy, leading eventually to the death of cartilage cells and their replacement by bone. Without cartilage cells, the growth of bone ceases.


As new bone tissue is added by the periosteal osteoblasts, there is a hollowing out of the internal region of bone to form the bone marrow cavity. This destruction of bone tissue is carried out by osteoclasts, multinucleated cells that enter the bone from capillaries and dissolve both the organic and inorganic portions of matrix. They are derived from the same precursor cells as granulocytes and macrophages.


Blood vessels import the blood-forming cells that eventually reside in the bone marrow for the duration of life.


Kidneys: Mammalian nephron contains over 10,000 cells and 12 different cells types, each cell type located in a particular place in relation to the other cell types along the length of nephron.


Circulatory system is the first functioning system in the developing fetus and the heart is the first functioning organ. The presumptive heart cells originate in the early primitive streak, just posterior to the Hensen’s node and then at the age of 20 days migrate anteriorly between ectoderm and endoderm. The direction of this migration is controlled by endoderm. The presumptive heart cells of birds and mammals form a double walled tube consisting of an inner endocardium and outer epimyocardium. Pulsations of the heart begins while the paired primordial chambers are still fusing, and the pacemaker function is assumed by the sinus venosus.


Haematopoietic system: We lose and replace 100 billion of red blood cells each day. The replacement is coming from the population of hematopoietic stem cells.


Red blood cells, white blood cells, i.e. granulocytes, lymphocytes, monocytes, macrophages, osteoclasts, platelets, share a common precursor: the pluripotential hematopoietic stem cell. Hematopietic stem cells die without the growth factors of the bone marrow, produced also by stromal cells, i.e. fibroblasts and other connective tissue elements of bone marrow, and other growth factors traveling through blood. In the spleen stem cells are committed predominantly to erythroid development, while in the bone marrow granulocyte development dominates.


In mammals the location of the sites of early hematopiesis has not been completely elucidated. In the fetus the main hematopoietic organ is liver, and it is believed that stem cells from liver migrate to the bone marrow. In the adult the major source of blood cells is bone marrow, although in mice also spleen participates.


Endoderm: We lose and replace 100 billion of intestinal cells each day and the replacement is coming from the population of stem cells in intestinal mucosa.


Oral plate ectoderm is in contact with brain ectoderm, and these two ectoderms interact: the roof of oral ectoderm forms the Rathke’s pouch that becomes anterior lobe of pituitary.


The second pharyngeal pouch (PP) gives rise to the walls of tonsils, the third PP to thymus and one pair of parathyroid bodies, and the second pair of parathyroids comes from 4th pharyngeal pouch. Small central diverticulum is formed between two 2nd pharyngeal pouches and this pocket of endoderm and mesenchyme will migrate down and become thyroid.


Between the 4th pharyngeal pouches the laryngotracheal groove extends ventrally.


Lungs are among the last of mammalian organs to fully differentiate. They must be able to draw in oxygen with the first baby’s breath. To accomplish this, alveolar cells secrete a surfactant into the fluid bathing the lungs. This surfactant, consisting of phospholipids such as sphingomyelin and lecithin, is secreted very late in gestation, and reaches useful level for full performance at 34th week of human gestation. It enables the alveolar cells to touch one another without sticking together. VI.BIBLIOGRAPHY [148]


Differentiation is the development of specialized cell types from the single fertilized egg. Such change in biochemistry and function of a cell is preceded by a process involving the covert commitment of a cell to a particular fate. After the commitment, the cell does not appear phenotypically different from its uncommitted state, but somehow its developmental fate has become restricted.


The commitment takes place in various ways:

1/ autonomous specification, where each cell becomes specified by the type of cytoplasm it acquires during mitosis, and cell fate is thereby determined without any relationship to neighboring cells;

2/ conditional specification, where the cells originally have the ability to follow more than one path of differentiation, but the interaction of these cells with other cells or tissues restricts the fates of one or both participants. Here the future of the cell depends upon the conditions in which it finds itself, i.e. this is a pattern of embryogenesis called regulative development, and that brings us to secondary inductions.


Organs are complex structures composed of numerous types of tissues. The precise arrangement of tissues cannot be disturbed without damaging its function. In the construction of organs there is a coordination between groups of cells whereby one group of cells changes the behavior of an adjacent set of cells, causing them to change their shape, mitotic rate, or differentiation. This action at close range, called secondary induction, or proximate interaction, enables one group of cells to respond to a second group of cells, and then via such change, to alter a third set of cells.


In ‘instructive interaction’ a signal from the inducing cell is necessary for initiating new gene expression in the responding cell. Without the inducing cell, the responding cell is not capable of differentiating in that particular way.


In ‘permissive interaction’ the responding tissue contains all the potentials to be expressed, but it requires an environment that allows the expression of these traits. At the same time the responding tissue must be ‘competent’ to respond, i.e. a cell must synthetize a receptor for the inducing molecule, or a cell must synthetize a molecule that allows the receptor to function, or a cell must repress the inhibitor.


Among interactions the epithelio-mesenchymal ones are the best known, where epithelial sheets from any germ layer interact with mesenchymal cells, usually neural crest cells or loose mesodermal cells. Ectodermal or endodermal epitheliums respond differently to different regionally specific mesenchymes, i.e. liver, intestine or lungs, but only as far as their genomes would permit. The instructions sent by mesenchymal tissue can sometimes cross species barriers, but are always organo-specific, they continue until an organ is formed with organ specific mesenchymal cells and organ specific epithelia. VI.BIBLIOGRAPHY [148]


Local induction occurs via cell-cell contact, cell-matrix contact, or a diffusion of soluble signals, i.e. paracrine factors. Distant induction is carried out by diffusible regulators of development that travel through the blood to cause changes in the differentiation and morphogenesis of other tissues, e.g. hormones. VI.BIBLIOGRAPHY [148]


Already in 1950 a report on embryonic transplantation via vascular route concluded that injected early embryonic tissue survived in the blood stream of the recipient, infiltrated into the tissues, reached their ‘normal’ sites, and multiplied and differentiated there in a typical fashion, i.e. description of ‘organospecificity’, and of ‘homing’ VI.BIBLIOGRAPHY [189]


Moscona showed in 1952 that “cells destined to give rise to cartilage or kidney, when reassembled after isolation and reared in tissue culture, continue in their erstwhile course of histogenesis, producing cartilage and nephric tubules, respectively... ..and if provided with proper stroma, a glandular blastema may give rise to.... ...morphogenesis”…


Andres observed that random scrambled embryonic cell suspension incorporated in the yolk sac of chick embryo can develop into organized, very complex bodies, containing many different and well-differentiated organ parts (brain, ganglia, skeleton with joints and muscles, skin with feathers, etc..).


Cells from chick embryonic kidneys, liver and skin were minced and then deposited on the chorio-allantoic membrane of 8-days old chick embryos. The same organs were recreated with a typical architectural pattern, including connective and vascular tissue, polarization, and evidence of functional activity. Since the transplanted tissue fragments accomplished on a neutral test site a second organogenesis, strictly corresponding to the organ from which they had been isolated, they must have achieved this by virtue of ‘information’ distinctive of the kind of organ which they had formed part of and must have been capable of translating that ‘information’ into a repeat performance. VI.BIBLIOGRAPHY [190]


Already in 1898 E.B. Wilson reported on his observation that in various genera (flatworms, mollusks, annelids) the same organs always originated from the same group of cells, i.e. organospecificity.


Only about 33 animal body plans (‘Bauplans’) are presently used on this planet. These constitute all animal phyla in existence today. Eukaryotic cells emerged some 1.4 billion years ago, and all known Metazoan phyla were formed in the Cambrian radiation which began 544 million years ago, and lasted some 10 million years, and no new morphological pattern (‘Bauplan’) has been added since then. A new bauplan could be created only by a modification of existing bauplan in the earliest stages of the development of an organism. VI.BIBLIOGRAPHY [148]


Developmental mechanisms:


During the evolution altogether 17 types of multicellular organisms emerged from the single-celled ones. But only three groups, those that generated fungi, plants and animals, evolved the ability to form multicellular aggregates that differentiate into particular cell types, i.e. embryo.


Only those that retained or produced non-ciliated cells learned how to divide cells . Those that differentiated cilia never learned how to divide cells, while those that had no cilia, i.e. lacked motility, learned to migrate inside of blastocoel, and create a federation of cells. Blastula arose as a means of joining autonomous cells into a federation, i.e. each cell gave up its autonomy in order to create a community of cells. While individual cells were totipotent, in the aggregate each cell restricted its potency, and that applied to all its neighboring cells. Once one inside population of blastocoel could interact with another, or with an outside population, induction events could occur that had given rise to new organs.


Metchnikoff wrote in 1891 that evolution consists of modifying embryonic organisms, not adult ones. Organisms evolved through changes in their embryonic development.

Re-arrangement of development during early embryonic stage brings about new types of organization that are one of the key mechanisms for establishing new phyla and classes.


Early developmental changes can be affected by changing the localization of cytoplasmatic determinants, changing the ratio of mitosis of one cell, or a group of cells, relative to the others, and changing the positions of the cells as they divide.


Von Baer stated that animals of different species but of the same genus diverge very late in their development. The more divergent are the species, the earlier can one distinguish (recognize) their embryos.


Vertebrates are thought to have arisen from invertebrates in several steps that involved the formation of and modification of new cell types. The ability of mesoderm to form a notochord, and its overlying ectoderm to become a neural tube, separated chordates from the lower invertebrates. The development of neural crest cells, and the epidermal placodes that give rise to the sensory nerves of the face, distinguish vertebrates from the protochordates. Once a vertebrate it is difficult to develop into anything else due to the constrains imposed upon evolution: physical, morphogenetic construction rules, historical restrictions based on genetics of the development of an organism. E.G. Conklin stated in 1915 that we are vertebrates, because our mothers were vertebrates, and produced eggs of the vertebrate pattern.


Bobshansky stated in 1937 that evolution means changes in gene frequencies in a population over time. When the interaction between genes is changed, new cellular phenotypes can arise. The creation of a new cell type is a rare event in Nature and often can change the entire phenotype of an organism. Mutations in regulatory genes can create large changes in morphology. Large morphological changes seen during evolutionary history could be explained by the accumulation of small genetic changes. But Van Valen in 1976 expressed an alternative opinion that evolution can be defined as the control of the development by ecology.