by
Stanley Shostak[3]
University of Pittsburgh
Pittsburgh, PA
USA
Prolongemmena
Introduction
Success and Problems
The Proposal
A (Very) Brief History of Histologys Nomenclature
Historys Legacy
The Missing Parameters: Embryology and Evolution
Reclassifying Adult Tissues
The Ambiguous Stem Cell
Potency as a criterion for stemness
The self-renewing stem cells
Identifying Types of Proliferative Cells
ASCs and TACs
Cache Cells
Reserve Cells
Amending Histologys Traditional Nomenclature
Indigenous Tissues
Epithelia
Traditional nomenclature
New nomenclature
Proto-epithelia: general cell division balanced by cell death
Meta-epithelia: clones of TACs arise from ASCs
Muscle
Traditional nomenclature
New nomenclature
Cellular muscle
Syncytial/reserve-cell muscle
Nerve
Problems from the past
Traditional nomenclature
New nomenclature: Germinal zones
Exogenous Tissues
Connective Tissue (CT)
Traditional nomenclature
New nomenclature
Fixed CT
Dynamic CT
Vascular Tissue (Blood and Lymphatic Tissue)
Traditional nomenclature
New nomenclature: Clonal Hierarchies
Germ Tissue
Traditional nomenclature
New nomenclature
Speculation on the Evolution of Tissues
The question provoking this paper has been in the literature since 1997: Why had [m]ultiple myeloma remained an orphan disease in terms of understanding its biology and of improving therapeutic modalities for almost 3 decades after the discovery of principles that led to cure acute leukemia?[1] I wondered if part of the blame rested with language—with ambiguous usage and inadequate definitions. Does language create problems in the descriptions of normal and disease organisms, organs, tissues, and cells that translate to failures in making connections?
Consequently, I recommended changes in the definition of stem cell,[2] and now I recommend redefining histologys tissues in order to bring clarity to otherwise clouded nomenclature. Specifically, my intention is to incorporate tissue dynamics into histologys language and reclassify dividing cells (stem cells and their cognates) in precise categories based on evolutionary conjectures.
The proposed changes also lead to operative consequence: (1) The transplantation of stem cells will not work therapeutically for damaged or diseased tissues in which stem cells or their cognates are intrinsic elements and not normally recruited from remote sites. (2) Therapeutic approaches in those tissues should be based on mobilizing stem cells within the intrinsic population. (3) The transplantation of stem cells should have therapeutic efficacy for tissues composed of exogenous cells recruited from remote sources. (4) Research should be directed at discovering the sources normally tapped by those tissues and maximizing their contribution to inoculates and grafts.
Adult mammalian epithelia are reclassified as meta-epithelia and proto-epithelia. In meta-epithelia, ASCs give rise to transient clones of TACs, and these cells, in turn, to TDCs, whereas in proto-epithelia differentiated cache cells undergo widespread (if cryptic) cell division. Mutations in cache cells and TACs may transform them into the self-renewing Adult stem-like cancer stem cells (CSCs) of carcinomas.
Mammalian muscle is reassigned to categories of cellular muscle (smooth and cardiac muscle) and syncytial/reserve-cell muscle (skeletal muscle). Cellular muscle resembles epithelium exhibiting either (or both) cache-like or ASC–TAC-like dynamics following injury or stress. Syncytial/reserve-muscle resembles meta-epithelium to the extent that reserve (satellite) cells resemble mitotically arrested ASCs that divide asymmetrically when stress or traumatized produce TAC-like cells that fuse and differentiate as muscle.
Nervous tissue in the central nervous system (CNS) is largely static in adults but germinal zones (e.g., the subventricular zones) containing Adult stem-like cells capable of generating TAC-like cells that movend differentiate as glial or neuronal cells. Cells in the peripheral nervous system (PNS) are established through the movement and invasive properties cells may be mobilized locally in the wake of trauma.
Mobilization of cells from local sources and recruitment from remote sites are employed in mechanisms of maintenance and repair in exogenous tissues. Least intrusive, embryonic fibroblasts (mesenchymal cells) invade epithelial, muscle, and nerve tissue up to their basal (external) lamina. Adult connective tissue (ct) falls into two categories: fixed and dynamic ct. Fixed ct (virtually all connective tissues except bone and adipose tissue) is static except where injury or disease induce mobilization and recruitment. Dynamic ct (bone and adipose tissue) is routinely remodeled by mobilizing cells locally and by recruitment. Excessive recruitment may be a source of benign fibromas whereas malignant transformations may require a transformation to a self-renewing CSC.
Vascular tissue is a far more exogenous tissue, colonizing and replacing other tissues during development. Indeed, vascular tissue invading fetal cartilage drives its replacement with cancellous bone. The circulating and fixed (arrested) blood cells and lymphocytes of adult vascular tissue arise from hierarchically determined clones of post-invasion precursors. So-called hematopoietic stem cells (HSCs) are not ASCs, since they do not undergo asymmetric division (i.e., are not self-renewing), although excess cell division may leave behind memory cells. Matire HSCs produce clones of determined cells. The proliferation of HSCs may be suspended, however, until provoked by specific growth factors or cytokines. Mutations turning HSCs into genuine self-renewing Adult stem-like cells may trigger transformations into self-renewing CSCs.
Germ-line tissue also invades and colonizes. Its target is the embryonic germinal ridge where Adult stem-like germ stem cells (GSCs) becoe primordial germ cells (PGCs) become oogonia and spermatogonia after embedding in epithelia in the developing ovary or testis. Reserve oocytes may (or may not) be recruited ectopically or mobilized from reserves in the germinal epithelium in adult mammalian ovaries, but in mammalian testes, self-renewing spermatogonia (i.e., resembling ASCs) are present throughout adult life. Cell division is constrained in some spermatogonia to the point of turning them into reserve cells.
Speculation about the evolution of tissues provides a rationalization for identifying two fundamental types of tissues (indigenous and exogenous) and for introducing tissue dynamics into histologys nomenclature. Extant mammalian tissues may have evolved from the sorting out and amalgamation of ur-tissue features from the pre-Ediacara (Vendobionta?). Pro-epithelium consisting of cache cells may have evolved into mata-epithelium when constraints on cell division reduced dividing differentiating cells into ASCs and TACS; muscle may have arisen from epithelium by specializing in the contractile properties of actin/myosin found in epithelium, and nerve may have arisen from muscle by specializing in its excitability. Vascular and germ tissue may have evolved from exogenous connective tissue.
Since the 17th century, if not before, the language employed by histologists to identify and describe tissues has shaped thinking about the development, maintenance, and aging of organisms, as well as the effects of disease and the causes of cancers. The success of this language does not preclude the necessity to improve it in order to keep up with other fields and technology.
During the last quarter of the 19th century and the greater part of the 20th, descriptions of tissues relied on magnified images of stained histological sections (uniformly thin slices) of organs viewed in the microscope. Subsequently, the language that evolved for description, analysis, and testing hypotheses helped to modernize medicine.
The synergism of laboratory and clinic was so successful, that histologist minimized (ignored?) the fact that stained sections and other standard preparations had been intentionally distorted to produce heterogeneity and enhance contrast. Furthermore, converting microscopic images to histologic descriptions depended on translating from the two dimensions of sections to the three dimensions of organs. Difficulties were monumental:
It [wa]s like trying to reconstruct a car [automobile] from a slice through 1 to 10 percent of its thickness--not only without prior knowledge to adjust what the object is, but without any knowledge that such a thing as a car with a particular structure exists.[3]
Today, the problem faced by histologists extends to four-dimensions and is vastly more complicated. To paraphrase, it is like trying to decipher the origin and destination of a car in route over the course of time in addition to reconstructing the car itself. Of course, contemporary microscopy allows one to see more than mere slices of organs, but the struggle is still to find the language for description, for posing research and clinical problems, for proposing hypotheses and experiments, for monitoring and evaluating therapies, for teaching histology to students of biology and medicine, and for elucidating issues of health and disease for the taxpaying public supporting medical research and practice.
Histologists have had their successes. For example, around the clock sampling of tissue has driven chronotherapeutics (the synchronization of therapy with circadian rhythms) that has not only boosted the bio-effectiveness of chemotherapeutic drugs[4] but has led to predictable changes in tolerability and unprecedented long-term survival.[5]
Furthermore, the adage that the fewer tumor cells are present at the time of chemotherapy the greater the chance for complete eradication is a tacit acknowledgment of the therapeutic value of research performed with histologys methods. And years of effort by histologists working experimentally on rabbits, monkeys, mice, and dogs, demonstrated in 1959 that the more isologous [i.e., close] the relationship is between host and donor the less marrow material is necessary to provide protection or repopulation.[6] Consequently, spectacular successes have been achieved in human beings by treating some blood cancers with autologous (self) bone marrow transplantation.
But histology also has its problems. Specifically, histologys contemporary language does not incorporate the technological and theoretical progress made in tissue dynamics. In particular, vacillation around definitions of stem cells have not been resolved by consensus, and consequent chaos about the constitution of tissues has not ended after 30 years of debate.[7] Instead of agreement, [v]arious definitions for a stem cell have been adopted by different authors.[8] Moreover, some criteria are impossible to meet: Defining a population of cells in vitro as stem cells presents inherent problems, including, most importantly, the demonstration that the cells retain the capacity to fully develop into all of the mature fates of the cells for which the putative stem cell is supposed to be a precursor.[9]
The fundamental issue of the relationship of embryonic stem cells (ESCs) to adult stem cells (ASCs) is yet to be resolved. Do ESCs mature into ASCs or are ASCs reinvented stem cells of the adult?
[ASCs] may not be the first cells that are present embryonically in a specific tissue to create that tissue, but rather appear later in development where they can replenish adult tissue populations
The shift from a large number of more restricted progenitors capable of tissue formation to a later-emerging population of multipotent lifetime self-renewing stem cells participating in repopulation suggests that these stem cells may be differentiated for a specific adult task necessary for the organisms survival.[10]
This issue of cellular equivalence (do ESCs = ASCs?) also arises around the relationship of ASCs to cancer stem cells (CSCs) (do ASCs = CSC?). These rare CSC with indefinite proliferative potential drive the formation and growth of tumors, but do transforming mutations create Adult stem-like CSCs? Are CSCs present among ordinary members of cell populations? Do CSCs gain ascendance stochastically or through a cascade of mutations?[11] Indeed, molecular similarities between ASCs and CSCs suggest a genealogical link. For example, Notch, Sonic hedgehog (Shh), and Wnt signaling pathways operating in oncogenesis also operate in the regulation of normal self-renewal in ASC.[12] The question is, how far do these similarities go, and do they lead to opportunities to prevent the appearance ofCSCs or to derail this progress?
Adding to the confusion over the definition of stem cells are results in vivo on the alleged transmutation of HSCs to nerve and other nonhematopoietic tissues in contrast to the much narrower transformation of stromal cells from marrow into osteoblasts, chondrocytes, adipocytes and possibly myoblasts.[13] The plasticity or potency of stem cells may have been exaggerated as consequences of cell fusion.[14] At stake in this confusison is popular enthusiasm for stem-cell research. Public support must not be allowed to falter in the wake of contradictory results, of failure (if not fraud),[15] but the confusion looks set to continue.[16]
The present modest proposal will fill the dearth of well-defined terms with a precise language that integrates tissue dynamics into histologys lexicon. Fortunately, the proposed changes and remedies for vacillation and confusion in histologys language do not impose theoretical hurdles and their usage will not present practical obstacles.
Precision in language will result from introducing a minimum of new terms, adding prefixes to some old terms, redefining some existing terms, narrowing some over-broad definitions, and limiting usage—in other words, by making amendments to existing language rather than inventing new language.
In amending histologys nomenclature, care is taken to qualify the fixity and flexibility of a tissues cell dynamics: Are they (a) a one-way street with flow-through dynamics (i.e., as in some surface epithelia and germ tissue)? Do they play (b) hide-and-seek (i.e., cryptic) dynamics of division in which differentiated cells reproduce themselves surreptitiously as in some glandular epithelia? Or are they (c) the stop and go or reactive dynamics that follow mobilization of dormant cells in the wake of trauma or stress as in skeletal muscle and germ-line tissue? In addition, attention should be drawn to (1) cell durability in potentially dividing cells as opposed to stasis in non-dividing cells; (2) to cellular mobilization of local cells in contrast to recruitment via circulation (of blood cells or blood-borne mesenchymal cells); and (3) whether mobilized and recruited cells contribute to maintenance and regeneration or only wound healing and repair.[17], [18].
More attention should also be paid to the language of cell loss. In adult mammalian tissues, cell loss either follows differentiation (e.g., sloughing of epidermis and intestinal villus epithelium) or occurs as part of differentiation demonstrated by nuclear falling off, known as apoptosis, and phagocytosis (i.e., engulfment) by circulating monocytes and mobile tissue macrophages.[19] previously dismissed as pycnosis (the thickening of nuclei; Gk. thick or dense), apoptosis is now routinely equated with programmed cell death, although an important part of the so-called program is the recruitment of monocytes and macrophages by chemokines (as innocuous as free nucleotides[20]) to the vicinity of the apoptotic cell and its identification by complement and other elements of the immune system.
One of the great ironies of modern tissue dynamics is the discovery of the role of tumor suppressor genes in apoptosis. In fact, tumour progression is often accompanied by a reduced propensity to undergo cell death.[21] Indeed, cell death is retarded in transgenic hematopoietic stem cells (HSCs) by over-expression of the cancer inducing oncogene bcl-2 in the presence of serum containing the KIT ligand (KL also known as the steel factor cytokine or stem cell factor [SCF]) that also stimulates cell division and the onset of differentiation in the same HSCs.[22], [23] The abrogation of the normal cell death program coupled to the self-renewal and persistent cell division may lie at the heart of adenocarcinoma dynamics as well.
Thus, the objective here is to make systematic and coherent amendments to existing terms thereby helping language catch up with other fields of biology and technology. Hopefully, extending and revamping usage in histologys nomenclature to include tissue dynamics will advance knowledge of growth, differentiation, and aging, and a new, robust language will aid the transfer of technology from the laboratory to the clinic, and from hypothetical explanations to solutions of lifes amenable problems.
Histologys nomenclature has not been stagnant. Indeed, it has changed over the centuries to incorporate new understanding, especially advances driven by technology and biologys concepts.
Histology (Gk., histos mast or beam of a loom or web + ology theory or doctrine of) entered the English language in 1848 according to the editors of the Oxford English Dictionary.[24] Histology was then the study of the weave, or structure of tissues (membranes and tunics) and the functions arising from their composition. Tissue, derived from the French tissu (woven: Lat., texere to weave) had already appeared in English in 1831 apropos of the texture of organs.
Today, histology is inextricable from the microscopic the anatomy of tissues, but this has not always been the case. Indeed, with the exception of Robert Hookes (1635–1703) discovery of cells (or, more precisely, the walls surrounding minute holes in cork) and his suggestion that tissues were made of cells, the classic 17th century microscopists[25] contributed little to histology. Their failure to solidify a discipline of histology or galvanize a study of tissue does not rest with the microscope, which was available commercially, but with the technical problem of preparing solid tissue for microscopic examination.
Instead of microscopy, histologys roots are planted in morbid anatomy and the look-and-feel observations made by physicians on cadaveric organs. Indeed, some of their terms (e.g., parenchyma) are still with us, and their conception of organs as the products of combined tissues continues to be the foundation of special histology (the study of tissues in organs).
The Greek physician and Erasistratus of Ceos (born 3 BCE) used (coined?) the word parenchyma (parenkhuma; Gk., para beside + enkhuma infusion, hence something poured in beside) for the soft parts of organs in the belief that blood poured into organs coagulated there (The term was later adapted in botany for soft, succulent parts of plants and fruit poured in by plant vessels in the pith, xylem, phloem, and bark).
Parenchyma became the general term for the major or distinctive material composing a tissue or comprising the bulk of an organ and was used in this context for the substance of lungs in 1578. Robert Hooke also used parenchyma to designate the mucous jelly of a sponge in 1665, and in the 18th century, parenchyma appeared frequently in volumes of the Philosophical Transactions of the Royal Society to designate the normal or inflamed juicy or fatty parts of vertebrate organs (e.g., skin, liver, and spleen).
Well into the 19th centry, morbid anatomists used methods of chemistry for the study of organs. Parts of previously healthy or diseased cadavers were allowed to putrefy or dry. Decayed and dessicated tissues were placed in crucibles and boiled in acids, alkalis, and salts in order to discover their chemical properties.
Thus, Marie Francois Xavier Bichat (1771–1802) earned the title of histologys first parent[26] (without so much as touching a microscope for the study of tissues). He left his mark on special histology by introducing the concept of collaborative membranes of organic economy[27] and coining the terms mucus, serous, and fibrous membranes in use today:
We may distribute the simple membranes into three general classes; the first comprises the mucous membranes, so named from the fluid which habitually moistens their unconnected surface, and which line all the hollow organs which communicate exteriorly by different openings through the skin In the second class are found the serous membranes, also characterized by the lymphatic fluid, which incessantly lubricates them
The third and last class comprehends the fibrous membranes; these, not moistened by any fluid, are thus named from their texture, composed of a white fibre
Each of the preceding simple membranes concurs, in different parts, to form the compound membranes.[28]
But toward the mid-19th century, histology turned away from morbid anatomy and toward microscopic anatomy. Several events conspired in the transition. In 1827 Giovanni Battista Amici (1786–1863) and Ernst Abb (1840–1905[8?]) began to produce apochromatic lenses and immersion objectives with optimal optical resolution. Robert Remak (1815–1865) then began the systematic study of tissue fixation (i.e., hardening in preparation of sectioning), while Karl Wilhelm von Ngeli (1817–1891) advanced the use of stains to better visualize cell parts, and Wilhelm His (1831–1904) invented a microtome for routinely cutting thin sections (slices) of tissue suitable for microscopic observation. European investigators[29] soon left their indelible mark on histologys imminent nomenclature.[30], [31], [32].
Parenchyma became equated with the most conspicuous or major type of cell comprising an organ while the other parts of organs (i.e., not washed away when parenchyma was broken down and removed) became known as stroma (Gk., bed covering). Stroma first appeared in the English literature in 1835 when Richard Owen (1804–1892) used it to designate the fine fibrous tissue embedding eggs in the ovary, and, later, stroma became associated with pathology through benign fibrous tumors and malignant cancers (Today, stroma is equated with connective tissue, from its dense, irregular variety (capsules, trabeculae) to loose connective tissue embedding parenchyma [fibrillar and reticular]).
Most dramatically, the new microscopic technology led to explanations for phenomena only partially appreciated previously. Earlier, Casper Friedrich Wolff (1738–1794) described granules in the membranes (i.e., germ layers) that created the chick embryo within an egg, and Lorenz Oken (born Okenfuss, 1779–1851) suggested that cells built organisms. In 1839, Theodor Schwann (1810–1882), building on the work of Matthias Jakob Schleiden (1804–1881) epitomized this idea in the cell theory, namely, that the growth (Wachsthum) of plants and animals depended on the same elementary parts (i.e., all living things were made by cells):
Wir haben geshen, dass alle Organismen aus wesentlich gleichen Theilen, nmlich aus Zell zusammengesetzt sind.[33]
(We have seen, that all organisms are assembled by essentially the same parts, namely by cells.)
Understandably, the new technology also led to confusion. but notions of how cell populations grew. Indeed, cell division was mired in mystification arising from a combination of 19th century materialist ideas of lifes chemistry and vitalist notions of spontaneous generation.
The rudiments of a correction were not provided in 1852 when Remak described cleavage in amphibian embryos, and, in the 1870s, an essentially correct microscopic description of mitosis (the nuclear events accompanying cell division) was thrashed outt hrough the efforts of several investigators,[34] and histology was ready for Rudolf Virchow (1821–1902), histologys second parent.
Virchows view of organismic growth is epitomized by his adage Where a cell arises, there a cell must have previously existed (omnis cellular e cellula)[35] or No developed tissues can be traced back either to any large or small simple element, unless it be unto a cell.[36] And thus Virchow left his mark on general histology. Henceforth, histology was synonymous with the microscopic study of tissues:
We either have tissues which consist exclusively of cells, where cell lies close to cell—in fact, cellular tissue in the modern sense of the word—or we find tissues, in which one cell is regularly separated from the other by a certain amount of intermediate matter (intercellular substance), and, therefore, a kind of uniting medium exists, which, while it visibly connects the individual elements, yet holds them separate. To this class belong the tissues which are now-a-days generally comprehended under the name of connective tissues Finally, there is a third group of tissues, in which the cells have attained specific, higher forms of development To this class belong the nervous and muscular systems, the vessels and the blood.[37]
Virchows four tissues—epithelia, connective, muscle, and nerve—were his legacy for general histology. His vascular tissue is easily redefined as blood cells and lymphocytes without stretching too far, and germ tissue—egg and sperm and their antecedents—can be added with the reach provided by August Weismanns (1834–1914) distinction between germ and soma.[38]
Beyond fashioning histologys tissues and their identifying characteristics, Virchow launched histology on its mission: understanding the role of cells in the anatomy, physiology, and pathology of tissues. And today, histology is firmly grounded in the microscopic anatomy of tissues and the analysis of cells many roles. Similarities among cells are reflected in criteria for identifying tissues; differences among cells and their products provide the criteria for classification within tissues; and deviations in tissues from the norm are analyzed as causes and consequences of disease and trauma.
But Virchow did not come to grips with the dynamics implied by his adage, omnis cellular e cellula. On the contrary, his interest in pathology did not spill over into interest in how cellular dynamics maintain tissues. He was not even concerned with the interface of evolution, development, and the upkeep of adult tissues. Indeed, for most of his mature years, the histologist Virchow was not even on speaking terms with his erstwhile student, the evolutionist Ernst Haeckel,[39] and Karl Ernst von Baer, the parent of embryology, is not even mentioned in Virchows Magnum opus, Cellular Pathology.[40] Regrettably, embryology, evolution and histology failed to become bedfellows in the academy.
Thus, tissue dynamics were not originally included among criteria for identifying tissues, their varieties, and pathological alterations. Two major reasons have prevailed since then for excluding tissue-dynamics from histologys nomenclature: (1) in practice, dividing cells (i.e., mitotic figures) were elusive in histological sections,[41] and (2), in theory, stem cell were ambiguously defined. Consequently, confusion reigned (and reigns) about the dynamic qualities of turnover, repair, and regeneration in tissues, and even about the identity of new cells generally and stem cells in particular.
Virchow cast histology on a oourse bereft of a comparative tradition and, thus, lacking an evolutionary branch. What is more, histology lacked strong ties with embryology. Although the embryonic sources of mammalian tissue are fairly well known descriptively, histology is not grounded in developmental biology, neither its evo nor devo facets.
On the plus side, histology has been spared teleological speculation over recapitulation that frequently accompanies evolutionary and embryologic inquiry, but, on the other side, histology has failed to incorporate the dynamics that complements evolutionary and embryologic research. In effect, without a commitment to developmental biology, histology became a study of stasis. Development, aging, healing, and regeneration were outliers instead of fundamental features of histology. Tissue turnover was of secondary interest in histology, and, most unfortunately, stem cells were simply components to tissues playing a quotidian role in tissue maintenance and not central players in the game of determination or changes in potency.
Embryologists divided the Eumetazoans, or multicellular animals into the Radiata at the tissue grade and having embryos with only two germ layers, and the Bilateria at the organ grade and having embryos with three germ layers. The radiates sometimes include the Poriphera (more often considered Mesozoa beneath the tissue grade), Placozoa, Cnidaria, and their sister group, Ctenophora. The Bilateria now consist of two branches of protostomes, the Lophotrochozoa and Ecdysozoa, and the deuterostomes. In practice, embryologists study the growth, morphogenesis, and differentiation of organisms in these separate groups, but theory in embryology is dominated with a unifying concern: how are morphogenesis and differentiation integrated by growth.[42] Determination either takes place before growth (determinate development) or after (indeterminate or regulative development).
Haeckel placed the radiates at the base of his gastrea theory and at the root of animal evolution.[43] Fully appreciating that many adult sponges and cnidarians had great powers of growth and regeneration, and demonstrating experimentally that a siphonophor, Crystallodes (a Cnidarian), was indeterminate during cleavage, Haeckel viewed regulative development as ancestral.
The opposite conclusion was reached by embryologists studying the lophotrochozoans epitomized by Caenorhabdites elegans the model roundworm. But regulative development is embodied, however, by the fruit fly Drosophila melanogaster representing Ecdysozoa and by vertebrates representing deuterostomes.
Determination relys on a few cells sequestered early in development that acquire fixed fates while producing larval structures. In regulative development, determination is held in abeyance until many cells are produced. These cells receive a variety of environmental cues that shunt them into different fates in adult development.
The difference between determinism and regulation are stark, but both sorts of development can go on simultaneously in different parts of the same organism, and the two modes of development can overlap. For example, in extreme indirect development, a small number of determined cells produce a larva while a distinctly different adult is produced by set-aside cells within the larva.[44] In the ecdysozoan ectopterygote (hemimetabolic) insects the set-aside cells are regulative as demonstrated experimentally by shifting their relative position in the larvas body,[45] while in echinoderms, the earliest cells cleaved out of the egg are regulative.
A dramatic example of overlap occurs in endopterygote (holometabolic) insects, such as Drosophila, where set-aside cells comprise imaginal disks. These disks are largely dedicated to the development of specific adult structures (such as antennae, wings, etc.) but can be shifted to the development of other structures under the influence of mutant homeotic genes[46] or when grown excessively via transplantation.[47]
The obvious question raised by set-aside cells is, where do stem cells fit in the spectrum of determinant and indetminant development? The anser is not intuitively obvious.
For example, if the development of amniotes (reptiles,
birds, mammals) is compared to extreme indirect development, then
extraembryonic membranes are the amniotes equivalent of the larva and the
embryonic stem cells in the geminal disk are the amniotes set aside cells
destined to produce the adult. In that case, the production of adult tisssues follows metamorphosis and the
production of adult stem cells and their cognates is independent of embryonic
stem cells.
If, on the other hand, the development of amniotes represents the conversion of regulative embryonic stem cells to determined adult tissues then reversal of adult stem cells to embryonic stem cells would seem to be a realistic possibility and one might hopfully expect to coax adult stem cells into becoming induced pluripotential stem cells (iPSCs).
Histologys tissues are reclassified here by incorporating insights from embryology and evolution. Adult tissues are assigned one of two classes: (1) tissues with cells bound by or resting on basal or external laminas or (2) tissues with cells lacking laminas although they may produce components of the laminas of other tissues. Cells in contact with laminas are inevitably polarized in some plane or dimension, whereas the default setting of cells without laminas is nonpolar. Furthermore, cell populations with laminas tend to come into contact with each other, while cells without laminas have extracellular matrix between them and may not have contact with other cells (osteocytes making contact through canaliculli being an exception). The movement of cells on laminas would be restricted to areas covered by the lamina, while cells lacking laminas could move more freely.
In adult vertebrates, epithelia, muscle and nerve tissues form the parenchyma of cells in contact with lamina. Laminas costrain growth, maintenance, healing and regeneration of their cell populations. Hence, the parenchyma of these tissue are give the name indigenous tissues and their cells indigenous cells.
In contrast, cells constituting various types of connective tissue, blood cells, and lymphocytes lack lamina. Cells giving rise to these tissues tend to move in the embryo and fetus and contact other tissues. In vertebrates, connective tissue cells (fibroblasts) make intimate contact with epithelia, muscle, and nerve (e.g., form the reticularis, endomytium, and endoneurium). Blood forming cells invade and colonize embryonic endothelium (in the yolk sac and para-aortic splanchnopleura, gonad, and mesonephros) and bone marrow mesenchyme,[48] while lymphocytes invade and colonize embryonic epithelium (famously the endoderm of the thymus) and endothelium (e.g., in the spleen, lymph nodes, and aggregated lymph nodules of the ileum). Germ tissue (egg, sperm, and their antecedents) is also nonlaminar during early development, albeit germ cells take on characteristics of laminar tissue at maturity.[49] Primordial germ cells (PGCs) invade and colonize the germinal ridge. Hence, connective tissue, blood cells, lymphocytes, and germ tissue are given the name exogenous tissues and their cells exogenous cells.
These classes of tissues and cells must be considered in terms of overriding qualities rather than strict adherence to one or another scenario. Indeed, over the course of development the division between cells with lamina and those without may change. In vertebrate embryos and fetuses, tissues undergo transitions between cells with and without lamina: de-epithelialization breaks cells contact with a lamina and sets them loose (e.g., the mobilization of myoblasts from the dermatome), while re-epithelialization binds cells to a lamina and secures them in place (e.g., the sequestration of myoblasts in limb buds).[50] Most dramatically, the neural crest de-epithelializes and gives rise to a host of cells with properties ranging from nerve and muscle to connective tissue.
Germ cells are inevitably the most enigmatic—in a class of their own—since they ultimately combine the qualities of both indigenous and exogenous cells. Developmentally, early mammalian germ cells have no lamina, and some of them will wander to and invade sites destine to become gonads (ovary or testis). Following successful invasion, however, the germ cells become wrapped in epithelia. Within the testis the epithelia comprise the supportive (sustentacular) cells of seminiferous tubules, while in the ovary, the epithelia comprise ovarian follicles. Moreover, during their development, oocytes produce their own lamina, while differentiating spermatids remain in intimate, epithelial-like contact with supporting epithelial cells.
Stem cell had antecedents in botany where the meristem or growing parts of stems and roots contain small dividing stem cells. Stem-cell theory might have advanced beyond the present impass if botanists conception of stem cells had been adopted for animals, but, unfortunately, botanical usage was not aadopted by zoologists.
Stem cells entered zoology in 1892 when Valentin Hacker used the term for the germ cells of a crustacean embryo.[51] The term became entrenched in this context after E. B. Wilson used it in his historic The Cell[52], for fertilized eggs of the crustacean Cyclops, the round worm Ascaris, several dipterans and higher invertebrates. Thereafter, stem cells were equated to germ cells capable of giving rise to the entire organism following fertilization,[53] and this germ-cell usage carried over from regulative embryonic cells[54] to the celebrated embryonic stem cell (ESC) of tissue culture fame. Today, pluripotency has been reinvented for so-called induced pluipotent stem cells (iPSCs)[55], [56] with their glowing promise to transform regenerative medicine.[57]
In current usage, the stem cell floats between two extremes: development revolving around potency and homeostasis centering on self-renewal. What is missing is a conception of stem cell related to their place in a regulative/determinant dialect. (from induction to feed back). The popular expectation that pluripotent stem cells can operate theraptuetically has dominated the discussion of stem cells, but the implications of stem cells for the control of cell population size may be closer to adult stem cells operative potential. Regrettably, the possibility of seeing adult stem cells as determined elements of indigenous cell populations, playing roles in tissue homeostasis seems to be ignored in too many wish lists proposals stem-cell therapy.
Potency as a
criterion for stemness
ASCs have lost their multilineage differentiation potential[58] and have only limited potency. Indeed, even the well-established regulator of pluripotency in ESCs, the Oct4 transcription factor is dispensable in ASCs.[59]
In contrast to ESCs, ASCs give rise to clones of monopotent, oligopotent, or, at best, multipotent TACs capable of differentiating within a range of related cell types. For example, oligopotent epidermal ASCs in the bulge of the outer root sheath of hair follicles give rise to the keratinocytes of soft keratin squames, of hard keratin hoofs, nails, and hair, and sebum secreting cells of sebaceous glands.[60] Indeed, claims for great potency in ASCs[61] are probably exaggerated and certainly controversial.[62]
Other problems with tying notions of potency to stem cells surfaced by the late 1970s when Christopher Potten admitted that stem cells cannot be reliably morphologically identified and their study is restricted to various functional tests.[63] Refining the problem, Marcus Loeffler joined Potten to proclaim the stem cell uncertainty principle according to which answer[ing] the question whether a cell is a stem cell alter[s] its circumstances and in doing so inevitably lo[ses] the original cell.[64] Indeed, thirty years ago, What fraction of the proliferative pool of cells in epithelial tissues functions is stem cells [was] uncertain.[65] This fraction remains uncertain today when hundreds of different human cell lines from embryonic, fetal and adult sources have been called stem cells, even though they range from pluripotent cells to adult stem cell lines[66],[67]
The self-renewing stem cells
The place of stem cells in tissue dynamics began to emerge in the 1920s when hematopoiesis and the cell lineage leading to erythrocytes (red blood cells) were brought under the rubric of stem cells. Then, in the post-World War II years, concern over the hazards of radiation along with enthusiasm for its therapeutic potential motivated research on dividing cells. Until then, undifferentiated dividing cells were thought to provide the precursors of differentiated cells, but studies on tissue dynamics led to the discovery of dividing differentiated cells.
Spleens were found to harbor a class of cells capable of giving rise to macroscopic colonies in the spleens of irradiated mice.[68] Theoretically, this class comprised a unique stem cell compartment of colony forming units (CFUs) consisting of a small population of cells (approximately one per ten thousand nucleated cells) capable, on average, of producing one similar cell while another cell joined the differentiating population. Similar CFUs in bone marrow were concentrated in a light fraction by velocity sedimentation.[69]
Stem cells, thus, were conceived of as the fountain showering tissues with replacement cells that maintained the tissue through turnover and were also capable of responding to contingency by accelerated proliferation and differentiation.[70] Whether stem cells were a deus ex machina or a genuine entity remained contentious, however, and provoked considerable research during the early post-war years of tracers, pulse/chase experiments, and autoradiography.
Distinguishing between types of dividing cells will ultimately depend on biochemical criteria. Particular types of cells should sustain gene expression within predictable ranges (e.g., c-Myc), produce particular gene products (e.g., b1 integrins in epidermis), generate specific cocktails of factors regulating cell fates and transitions between fates (including microRNAs targeting messenger RNAs and translation[71]), and display precise epigenetic conditions such as the configuration of the methylome.[72], [73] Today, biomarkers, immunofluorescence, confocal microscopy, and fluorescent flow cytometry hold promise for providing these kinds of a exact and systematic criteria for characterizing stem cells in tissue and plotting the course of their dynamics.[74]
And, indeed, the rigorous application of modern techniques to stem cells has produced vast amounts of data. The more than two hundred genes of transcription factors, stage-specific antigens, histotypic cell surface antigens, and expressed sequence tags (ESTs) of unknown provenance shared by hematopoietic, neural, and embryonic stem cells reinforce the belief that core stem cell properties (stemness) underlie self-renewal and the ability to generate differentiated progeny [although] most if not all of the [stem cell-] SC-enriched genes are not expressed exclusively in SCs.[75] Furthermore, these genes and their cognates are present in mice and humans,[76] and many of them provide valuable and useful cues for enrichment protocols.[77] The goal of reaching consensus on a list of stem cell genes has proven elusive, however,[78] and a contemporary list of markers does not constitute a reliable molecular signature for stem-cells generally.[79]
It is rare indeed to be able to identify stem cells within tissues using histological methods. Stem cells are supposed to have inherent properties, such as DNA label retention, but specific molecular markers of stem cells have not been found in many tissues.[80]
How then is one to define stem cells beyond the embryo, tissue culture ESCs, and iPSCs? Can ASCs be distinguished from TACs, cache, and reserve cells in adult tissues? Might they all be defined by specific behaviors?
ASCs versus TACs
Merely distinguishing between ASCs and the broader proliferative population poses difficulties. Probably most investigators share the notion that ASCs divide rarely, but cell markers do not distinguish between rarely dividing ASCs and TACs in steady-state tissues. Morphologically, criteria would seem unreliable, although actively cycling cells can sometimes be distinguished from non-proliferative cells by a preponderance of heterochromatin (deeply staining masses) in the nuclei of quiescent cells in contrast to dividing cells.
Researchers resort to a variety of criteria to confront the problem of identifying stem cells in situ (often without being overly scrupulous). First of all, in searching for ASCs, researchers assume they are looking for a small fraction of a larger proliferative population. Thus, as a result of radiation dose-survival studies, 2–7% of basal-layer cells in the mouse epidermis are identified as ASCs.[81]
Slow growth is also a requirement for ASCs. Slow growth is typically demonstrated in prolonged pulse chase experiments. After labeling cells in rapidly growing, postnatal and prepubertal animals with a nucleotide precursor or DNA analogue (bromodeoxyuridine [BrdU]), the fraction of label-retaining cells (LRCs) found following a prolonged chase (i.e., in the absence of label) identifies cells dividing rarely. (This LRC test for ASCs ties into another criterion for ASCs, namely, the notion that ASCs retain the old strand of DNA during replication and thus tend to maintain label DNA [see below]).
Other tests rely on the efflux or exclusion (with the help of a transporter) of dyes such as Hoechst 33342 (or Rhodamine 123). Cells isolated by fluorescent-activated cell sorting that have lost much of their Hoechst 33342 dye constitute a side population (SP) frequently containing a high proportion of ASCs.[82] The SP fraction of low Hoechst 33342 cells in viable murine bone marrow cells comprises 0.05–0.10%.[83] and carry bona-fide stem cell markers such as Sox2, Sox9, and Oct4 antigens, and (less reliably) the glycolipid markers of embryonic stem cells, SSEA4, Nanog, Sox4, Isl-1, and Pax6. In addition, immunofluorescent antibodies reveal the presence of specific markers related to the tissue of origin, such as pituitary specific factor (Prop1) and even cocktails of markers.[84]
But the gold standard for ASCs is their morphological niche, a unique site populated exclusively by ASCs, sometimes en masse. Ideally, anatomically distinctive niches, such as the bulge of the outer root sheath of hair follicles have definable microanatomies (microenvironments) that concentrate ASCs, sequester or induce them, and nurture them specifically. At a minimum, qualified niches would provide some microenvironmental details such as traces of antigens oriented with a specific polarity[85] that could be involved in the maintenance and/or control of ASC behavior.[86]
This gold standard is not especially useful, however. Historically, a degree of circularity crept into the definition of niche. Instead of a niche defining the stem cell, the stem cell defined the niche. Indeed, some so-called niches are nothing more than sites occupied by cells bearing a putative progenitor or stem cell marker.
Histologists should not relax lexical constraints when identifying niches. For example, the basal-layer of the epidermis (stratum basal) is not an ASC niche, since it supports TACs as well as ASCs (if not cache cells as well, see below) and, therefore, does not nurture ASCs exclusively. Likewise, LRCs alleged to be ASCs lie in regions that are not niches in the tongue, palatal papillae and the epidermis of the mouse ear.[87] At best, cells expressing stem cell markers may be located in sites qualifying as pool/niches as opposed to pure niches.
Language will have to be tightened up when it comes to niches. Extracellular matrix as such should not be considered a niche without demonstrating that it regulates stem-cell behavior. This is not to say that components of extracellular matrix may not exercise control over ASCs. In human epidermis, for example, cellular localization is constrained by the differential expression of b-1 integrins and binding to matrix,[88] but location alone does not dictate expression or, therefore, define a niche. Histologists will be well served were they to recognize that the simple location of stem cells is not sufficient to define a niche. The niche must have both anatomic and functional dimensions, specifically enabling stem cells to reproduce or self-renew.[89]
But the most rigorous criterion for ASCs relies on a specific definition of self-renewal: the segregation of new and old strands of DNA during cell division. The fresh ASC retains the bulk of the old-strand DNA (i.e., the template strand upon which the new-strand DNA is modeled), while a transit amplifying cell (TAC) receives the new strand of DNA.[90], [91] Now known as asymmetric division, this sort of division is associated exclusively with the self-renewal of ASCs. In theory, differential division giving rise to an ASC with old-strand DNA and a TAC with new-strand DNA protects the DNA in the ASC from damage if only because its DNA is potentially exposed to less frequent breaks and errors inherent in DNA synthesis.[92], [93], [94]
The transformation from ASC to TAC is not trivial. In the case of spermatogonia, an RNA-binding protein maintains the self-renewing germ-line population of ASCs in mice while preventing differentiation.[95]
In contrast to ASCs, TACs (and cache cells; see below) undergo symmetric division, producing two identical TACs or precursor cells without differentially sorting out old and new DNA strands. Rapid, multiple cell division gives rise to a clone of identical precursor cells that become terminally differentiating cells (TDCs) and have ceased proliferating.
Eventually, a consensus definition or, at least a good sense compromise of criteria based on asymmetric division will lead to a workable definition of ASCs. One caveat should be added, however: reversibility. Indeed, by exchanging a cyclin for a cell cycle inhibitor, the cell-cycle exit program of proliferating progenitor cells (i.e., a transient population of noncycling precursors) may go into reverse and become a cell-cycle reentry program for differentiated cells.[96] Presumably, reversals of this sort could backup beyond TACs all the way to ASCs, potentially turning TACs into ASCs or possibly cancer stem cells (CSCs).
Cache Cells
Cache cells comprise the differentiated parenchyma of many glandular organs and violate the classic axiom that differentiated cells are quiescent while undifferentiated cells divide actively. Although their kinetics are not well known and mitotic figures (i.e., dense chromosomes appearing during cell division) are as hard to find as are pycnotic figures (i.e., dense, misshapen nuclei appearing in dying cells), cache cells are presumably cyclically proliferative. Their population size, therefore, would seem to be regulated, at least in part, by cell death in equal and opposite amounts to cell division. Presumably, balance involves negative feedbacks from population pressure.
Parenchymal cells have long been thought to be non-proliferative. Hepatocytes, for example, were thought to be mitotically quiescent despite the frequent appearance of binuclear centrolobular cells. But since labeled nucleotide precursors became available, the perception of quiescence has been eclipsed by the acceptance of cell division. Indeed, hepatocytes restore as much as two-thirds of a liver (i.e., following partial hepatectomy) in massive rounds of local cell divisions[97], [98] (macrophages [hepatic dendritic cells] are thought to be recruited from bone marrow as well).
Curiously, unlike connective tissue fibroblasts in culture (see below), cache cell division does not seem to be limited by the number of times a cell has divided. Indeed, new cells appear mitotically equivalent that is, each gave rise to approximately the same number of descendants.[99]
Since, all parenchymal cells seemed to retain, if not exercise, the ability to divide, parenchymal cell populations were dubbed expanding.[100] The connotation of expansion seems misapplied, however, since cell-population size is regulated and governed, at least in part, by cell death via feedback. Cell division attributed to cache cells is not burdened by the connotation of expansion.
Cache cells are not ASCs. Cache cells do not occupy niches, although dividing cache cells may be concentrated in areas where they are more likely to divide than others (e.g., peribilliary or periportal hepatocytes compared to centrolobular hepatocytes). Moreover, cache cells do not divide asymmetrically or sort out the old and new DNA strands differentially at division. Cache cells are not self-renewing, therefore, although they are self-maintaining in the sense of supporting the maintenance of their population counter to the negative effects of cell death.
Finally, some tissues may contain mixed populations of cache cell and ASCs (and/or reserve cells). In mouse tail epidermis, for example, homeostasis results from the regulation of cells undergoing both symmetric and asymmetric division.[101] Indeed, more than one mechanism may govern cell population size in the same tissue. Liver maintenance, for example, may normally be performed by cache cells, whereas a liver with its regenerative capacity exhausted by severe or chronic liver disease may yet regenerate as a function of small Adult stem-like oval cells originating from the intrahepatic bile ductules and (possibly) through the action of extra-hepatic stem cells recruited from bone marrow.[102]
The pancreas too may be a mixed bag: pancreatic acini would seem to be the home of ASCs (and reserve cells),[103] while B islet cells are cache cells,[104] although, developmentally, both pancreatic parenhymal cells come out of the same basket.[105] Either the pancreass parenchymal proliferative population consists of asymmetrically dividing cells mixed with symmetrically dividing cells or another, variable enters the mix and proliferative cells have the ability to be either Adult stem-like or cache-like. Of course, were such ambivalent cells to exist, discovering the factors determining how they decide to divide one way or the other would be of considerable interest for controlling tissue maintenance and regeneration.
Reserve Cells
Like ASCs, reserve cells divide asymmetrically.[106] The renewed reserve cell retains the old DNA strand, while the new TCA cell receives the new DNA strand. But unlike ASCs that spend most of their time in G1 (the gap between mitosis and DNA synthesis) while gradually moving through the cell cycle, reserve cells are typically under mitotic arrest and spend their time in G0 (in suspension following mitosis but without entering the period of DNA synthesis). Reserve cells are released from custody, or mobilized, by trauma or stress and subsequently divide. Indeed, they seem primed for reentering the cell cycle and dividing when the occasion arises.
Reserve cells tend to be distributed individually, but they may occupy specific locations within a tissue. These locations may be compared to niches, since they are morphologically distinct, but reserve cells do not congregate, and their locales do not resemble the bulge occupied by epidermal stem cells such as the hair-follicle bulge. For example, the reserve cells of skeletal muscle known as satellite cells (also known as quiescent myoblasts), are distributed individually within or beneath the external lamina of muscle fibers (sublaminal space or zone between the lamina and the sarcolemma of the muscle fiber),[107] while the reserve cells of germ tissue in male mammals, or dormant spermatogonia, are distributed individually in the basal zone of seminiferous tubules. The sites occupied by reserve cells seem to ensure a regenerative capacity for tissue distinctly beyond the normal self-renewing function of ASCs or the maintenance function of cache cells.
Hematopoietic Stem Cells
A completely different type of cell supports the blood cell/lymphocyte system (vascular tissue). It is the hematopoietic stem cell (HSC) and it provides all the cells normally found in blood and lymph.
Like ASCs, HSCs are rare, proliferative cells that spawn clones of cells destined to differentiate in specific (although not necessarily terminal) pathways pathways. And like ASCs, HSCs congregate in niches (i.e., in bone marrow, thymus, spleen, lymph nodes, and lymph nodules, etc.).
But, unlike ASCs, HSCs do not retain nuclear label (they are not LRCs); they exhibit symmetrical division (i.e., division is not asymmetrical); and their old and new DNA strands are not segregated on chromosomes during self-renewing cell division.[108] Moreover, like TACs, HSCs respond to growth factors and cytokines.[109] Hence, HSCs are not ASCs![110]
HSCs (aka mammals common myeloid progenitors [CMPs] and similar cells in Drosophila[111]) give rise to so many types of of blood cells and lymphocytes that HSCs qualify as multipotent. HSCs may also be long-lived dormant cells, and clones descended from HSCs include memory cells that resume dormancy. Memory cells are not products of self-renewal (i.e., 1:1 asymmetric division) as much as of excess cell division. They lay in wait for the specific stimuli that provoke cell division and differentiation.
The drop-off of immunity accompanying aging may be the consequence of loss of memory cells. On the other hand, the induction of leukemic stem cells may be the result of the transformation of HSC cells into self-renewing adult stem-like cells with its prospects for the endless production of new clones of cells.[112]
Introducing tissue dynamics into histologys nomenclature results in systematic changes rather than interfering with traditional usage. Since indigenous tissues support cell division within their cell populations, while exogenous tissues rely on mobilizing new cells from unrelated sources and/or recruiting additional cells from remote sites, changes in nomenclature fall into predictable categories. Some overlap in sources of new cells is also said to occur: otherwise indigenous tissues may exhibit mobilization and recruitment (e.g., smooth muscle), while exogenous tissues may exhibit indigenous growth (e.g., HSCs). The validity of these claims remains to be established.
Traditional nomenclature
Epithelia take their name from modern Latin, epi- above and from Greek thele teat referring to the simple [concave] cellular tissues we meet upon the external surface of the body, and in the cylindrical and scaly mucous and serous membranes.[113] Traditionally, epithelia cover surfaces (externally: epidermis; and internally: intestinal epithelia) or form tubular structures (i.e., glands, acini, ducts). In both cases, epithelia hate a free edge and form surfaces without borders by enclosing or lining structures while being broadly in contact with each other and resting on a submicroscopic basal lamina or microscopic basement membrane.
Types of epithelia are distinguished by the number of cell layers comprising the tissue: simple epithelia have one cell layer; stratified epithelia have more than one layer, and pseudostratified are actually simple but have the appearance of stratified epithelia due to the differential apical and basal distribution of nuclei. Finer distinctions are drawn from the shape of cells in the outermost layer: squamous, cuboidal, columnar, and transitional (swollen).
In addition, epithelia are classified as keratinized, when any type of keratin is synthesized within the cell, or nonkeratinized, when keratins are not synthesized; ciliated, when cilia are present at the cells apical surface, or nonciliated when they are not. Epithelia have a brush border when microvilli are abundant on the cells apical surface, and stereocilia (actually long microvilli) decorate the apical surface of epithelia in the epididymis. An epithelium is striated when mitochondria near the base and oriented perpendicularly between deep folds in the plasma membrane give the appearance of basal striations.
Two simple squamous epithelia are identified by their location: endothelium lines blood and lymphatic vessels and the heart (endothelium is also distinguished by overlapping rather than abutting tight junctions and in some vessels by holes [fenestrae] penetrating the cytoplasm); mesothelium is the thin pavement epithelium lining the peritoneal, plural, and pericardial cavities. The stratified epithelium lining the cornea is also called endothelium, but this is an idiosyncratic usage and should be abandoned.
Further distinctions among epithelia are grounded in cell polarity: hemidesmosomes are present basally; intercellular junctions (tight or occluding, gap or communicating, adhering or anchoring, zonal or macular [desmosomal] attachments, folded membranes, paracellular barriers, and apical complexes) are present laterally; and accessory structures (cilia, microvilli, stereocilia) functioning in absorption, secretion, propulsion, filtering, and the reception of external signals are present apically.
In addition, cellular guests are frequently present within epithelia: microphages (transient neutrophils), macrophages (fixed [histiocytes] and transient monocytes]), microglia (dendritic cells), lymphocytes, melanocytes and neural crest derivatives with neuro-sensory activity. Indeed, lymphocytes may completely replace epithelia in tonsils and at the luminal end of lymphoid nodules in the wall of the ileum. Moreover, functionally differentiated cells carrying specific antigens (fluorescently identified), may be regionally distributed or preferentially localized within epithelia.
New nomenclature
The presence of asymmetrically dividing ASCs versus symmetrically dividing cache cells provides a criterion for placing epithelia in two classes. Typically, ASCs plus their derived clones of TACs maintain simple and stratified epithelia covering surfaces (epidermis, intestinal epithelium [including intestinal glands known as crypts]) as well as some epidermal-derivatives (pilo-sebaceous systems, mammary glands, and parts of sex ducts), as well as some carcinomas. Cache cells maintain glandular epithelia (liver, pancreas, salivary glands, pituitary, urinary system, accessory sex glands and their ducts).
An evolutionary argument (see Speculation on the Evolution of Tissues below) suggests that the ancestral form of cell division is the cache type: widespread, unlimited, symmetrical proliferation or differentiated parenchyma. The evolved form of cell division, thus, is the self-renewing cell division by ASCs: occurring in small numbers, asymmetrical, distributing old and new DNA strands differentially, and giving rise to TACs. Hence, the new classes of epithelia are named proto-epithelia, exhibiting cache dynamics and meta-epithelia. exhibiting ASC dynamics.
In proto-epithelia, parenchymal cells are cache cells, each of which has the capacity to divide. Cache cells are differentiated and their division contributes to the differentiated cell population. Cell division is not self-renewing and asymmetrical: Neither one cell nor the other cell produced by a division retains the preponderance of old (template) or new (replicated) DNA strands.
Cell cycling is presumably controlled by cyclins, cyclin-dependent kinases, and breaking proteins. Hence, cache cells suffer from multiple points of vulnerability to malignant transformation. Curiously, cancer stem cells (CSCs) of glandular carcinomas exhibit Adult stem-like self renewal[114] suggesting a mismatch between the sources of new cells and the differentiated cell type. Malignancy would seem to be the consequences of such a mismatch.
Mitotic figures and dividing cells are not typically reported in histological preparations (i.e., paraffin sections stained with hematoxylin and eosin) of proto-epithelia, hence the name cache. The mitotic figures may also be unevenly distributed (e.g., concentrated in peribilliary or periportal regions of liver lobules), and cell division may exhibit a circadian rhythm (e.g., rat kidney,[115] rat uterine luminal epithelium,[116] mouse liver,[117] and Ehrlich ascites carcinoma[118]). Rarely, reserve cells are said to occur among cache cells (e.g., pancreatic acini[119]).
Cell death would seem to balance cell division in proto-epithelia. The control of cell death is obscure, but death may be mediated by feedback constraints exercised by position or access to resources (e.g., in liver lobules).
Meta-epithelia contain two different types of dividing parenchymal cells—ASCs and TACs— and nondividing TDCs. ASCs are self-renewing (i.e., exhibit asymmetric division) and the size of ASC populations may not change with age,[120], although ASCs competences may decline.[121] ASC behavior is presumably influenced by signal transduction pathways originating in niches.[122] ASCs also exhibit circadian rhythms (e.g., in mouse cornea,[123] hairless mouse dorsum,[124] mouse tongue keratinocytes[125]),
TACs produce a clone through a limited number of symmetric divisions before the appearance of TDCs. Multiple pathways of differentiation would seem available to TACs (for example, in intestinal glands[126]) as a function of local conditions[127], [128] The passage from TAC to TDC is fundamental to the normal turnover of cells in tissues,[129] but it may also be reversible.[130]
CSCs may also be products of ASCs.[131], [132] ASCs would seem to be vulnerable to malignant cancerous transformations via Wnt-pathway-activating mutations in genes: adenomatous polyposis coli [APC]). TACs, on the other hand, may undergo malignant transforation no further than benign tumors.[133] A distinction betwen ASCs and TACs, therefore, may be useful for distinguishing between malignant cancers and benign tumors (e.g., of the prostate and vagina).
Traditional nomenclature
Muscle is traditionally classed as smooth (aka involuntary), cardiac, and skeletal muscle (aka voluntary): cardiac and skeletal muscles are striated; smooth and cardiac muscle are cellular. Skeletal muscle consists of syncytial muscle fibers (aka myotubules). Smooth muscle cells can divide (e.g., during the expansion of the gravid uterus), and cardiac muscle cells may contribute to regeneration of a damaged or diseased heart, but skeletal muscle fibers do not support cell division.[134] Uniquely, in skeletal muscle, so-called satellite cells embedded in the external lamina of fibers or in the sublaminal space may add fibers to the muscle as a consequence of exercise (known as load-induced or compensatory hypertrophy) or during regeneration following trauma. Cells produced by satellite cell division fuse with each other and existing fibers thereby add to the muscle mass.[135]
New nomenclature
Muscle shares many characteristics with epithelium. Indeed, muscles motor protein, myosin resembles the nonmuscle myosin of epithelia. Remarkably,
vertebrate smooth muscle myosin is more similar to nonmuscle [epithelial] myosin than to striated muscle myosin, both in sequence and in biochemical characteristics [Amino acid sequences indicate that] smooth muscle and striated muscle myosins branch independently from nonmuscle myosin. [I]n fact, the two types of muscle tissue may also be independently derived from nonmuscle tissue. [136]
Other epithelial qualities present in muscle include the presence of an external lamina resembling the basal lamina of epithelia; dense bodies in smooth muscle resembling desmosomes (anchoring points of intermediate filaments); nexus or gap junctions (between the sarcolemmas of smooth muscle cells and cardiomyocytes); fascia adherens (adhering junctions) in cardiac muscle resembling zona adherens in epithelia.
The cells comprising smooth and cardiac muscle, moreover, exhibit cache-like dynamics (dividing differentiated cells) when provoked by stress or trauma. Skeletal muscle, moreover, exhibits adult stem-like dynamics when satellite cells are provoked to divide. Division is asymmetrical, producing TAC-like cells that fuse and differentiate into TDC-like muscle fibers.
While rejecting the ct route to dynamics (see below), muscle seems to have inherited the epithelial routes and taken them further in the case of skeletal muscle. Hence, two classes of muscle are distinguished: cellular muscle (cardiac and smooth) exhibiting cache-like dynamics under stress or trauma, and syncytial/reserve-cell muscle (skeletal) exhibiting ASC -like dynamics under stress or trauma.
The heart is not a bag of muscle. Rather it is a system of muscle, blood vessels, ct, and its own intrinsic conductive system derived from muscle, all originating in multipotent stem cells: the heart is built from a pool of multipotent cells that persists and differentiates as the heart grows.[137] In mice and human beings, this cellular pool has multiple origins: mesoderm forms cardiovascular progenitor (aka colony forming) cells, endothelial cells lining cardiac vasculature, and vascular smooth muscle;[138] and embryonic proepicardial cells de-epithelialize (become mesenchymal), invade the developing heart, transiently express their T-box transcription factor (i.e., become Tbx18-expressing cardiac progenitors), and give rise to cardiomyocytes, interstitial cardiac fibroblasts, and coronary (vascular) smooth muscle cells.
Hence, one is not surprised when fetal proepicardial cells explanted in vitro prove to be oligopotent stem-like cells differentiating as cardiomyocyte and cardiac smooth muscle fates.
No instances were observed where derivatives of a single clone formed only cardiomyocytes and not smooth muscle cells. This demonstrated that a large proportion of proepicardial cells are pluripotent and can adopt either cardiomyocyte or smooth muscle cell fates.[139]
Furthermore, adult epicardial cells adopt angiogenic cell fates in vitro differentiating as fibroblasts, smooth muscle cells, and endothelium.[140] And aging myopathy in the heart is retarded in mice transgenic for insulin-like growth factor-1 (IGF-1) otherwise known to promote stem cell growth and survival.[141]
In human beings specifically, fetal cardiac transcription factor ISL1+ (islet 1 positive) cardiovascular progenitors display multipotency, giving rise to cardiomyocytes, smooth muscle, and endothelial cell lineages:
In contrast to murine cardiogenesis, large numbers of multipotent ISLI+ progenitors persist during late stages of human fetal cardiogenesis, suggesting a stem cell paradigm for the exponential growth of the human fetal heart and outflow tract over many weeks.[142]
The possibility that adult hearts are maintained and restored by local cardiac stem-like cells (and possibly epicardial stem-like cells) would be supported were one to identifying proliferating and subsequently differentiating members of a side population (SP) among isolated heart cells. Cardiac tissue would then have to be reclassified as ASC-like. Were this possibility to prove correct, a great deal will have to be learned about cardiomyocyte kinetics in adults, but the effort will undoubtedly be rewarding by way of opeining possibilities for cardiac regeneration. But the large numbers of cells expressing markers of cardiac tissue, cardiac myocytes, smooth muscle, and endothelium in adult tissue suggest that regeneration following infarction is a function of cache-like cells, not adult stem-like cells.[143]
The alternative to regeneration by native, multipotent cardiac Adult stem-like or cache-like myocytes is recruitment of cells from circulation.[144] Indeed, the migration of cells to sites of myocardial infarction is a tantalizing hypothesis for proponents of embryonic stem cell (ESC) therapies for injured hearts. And myocardium formed following direct injection into peri-infarcted left ventricles of lineage-negative (Lin-) bone marrow cells obtained from transgenic mice by fluorescence-activated cell sorting for c-kitPOS stem cells.[145] Furthermore, transplanted autologous skeletal myoblasts experience high rates of early cell death and limited success relieving infarcted myocardium.[146] The transplantation of foreign cells to repair adult cardiac muscle has not, therefore, been demonstrated in this proof of concept experiment.
Hints of cache-like dynamics are tantalizing if not definitive for smooth muscle. An epithelial-like pattern of division was first attributed to smooth muscle in 1987 in a review on atherosclerosis:
Benditt's observation of monoclonality also implies some intrinsic mechanism allowing cells to grow in a focal manner. It is intriguing to consider the possibility that this commitment process could require the release of cells from the intrinsic inhibitory effects of heparan sulfate located around the cells or the synthesis of growth factors secreted by the smooth muscle cells themselves. If we add the hypothesis that only some cells are capable of such a response, we would expect the sort of oligodense phenomenon demonstrated by Benditt. Proof of such a hypothesis, however, will have to await development of methods to explore these mechanisms directly in the vessel wall responding to injury.[147]
Earl Benditts original article in 1977 concerned atherosclerotic lesions containing variant (disorganized) smooth muscle (i.e., tunica media) cells in plaques beneath endothelium. Seventy-five percent or more of these plaques were monotypic (i.e., made up of cells exhibiting only one isozyme pattern). Similarly, samples of nonartherosclerotic aortic wall showed clustering [that] may be a manifestation of confined growth of a group of cells recruited during early embryonic development from the mesenchyme to form the arterial media[148]
Benditt concluded that a significant proportion of the plaques were monoclonal. He speculated on causes of plaque formation, but did not extend his conclusion to normal smooth muscle. Twenty years later, Benditt and others concluded that, smooth muscle cells from normal arteries can show monoclonal characteristics,[149] i.e., normal smooth muscle would seem to exhibit cache-like dynamics.
However, label-retaining cells (LRCs) resembling ASCs are found in smooth muscle in the endometrium following pulse-chase experiments with labeled DNA analogues. A model system for the regeneration of mouse myometrium showed that after stimulation by human chorionic gonadotropin, LRCs produced clones of cells in the uterine stroma followed by growth in the myometrium. Since the clonogenic division of the LRCs was consistent with asymmetric division in which an ASC retains the old strand DNA and a TAC obtains the new strand DNA, and while acknowledging that additional studies are required to make definitive conclusions, the authors propose that the lineage differentiation of uterine myometrial cells may represent a continuum, with LRCs being the most primitive [i.e., ASCs], followed by transient amplifying cells [i.e., TACs], and finally terminal differentiation into myometrial cells [i.e., TDCs]. [150] Nevertheless, if only because the source of the dividing cells would seem to be differentiated smooth muscle cells, cache-like dynamics remains the hypothesis of choice.
Sublaminal satellite cells are a distinguishing characteristic of mammalian skeletal muscle. While typically considered undifferentiated or primitive, satellite cells are reserve cells, specialized for their roles in hypertrophy and regeneration.
Skeletal muscle is typically long-lived but satellite cells may sustain growth and replacement.[151] Division in satellite cells resembles that of ASCs by way of being self-renewing and asymmetric, returning the old DNA strand to the satellite cell and shunting the new DNA strand to a TSC-like precursor[152] that divides and forms a (small) clone of myoblasts that eventually fuses differentiate into fibers (myotubules). Indeed, while the typical satellite cell is Pax7+/Myf5-, its division contributes a Pax7+/Myf5- satellite cell to the reservoir of reserve cells and a Pax7+/Myf5+ muscle precursor to the supply of differentiating skeletal myoblasts.[153] Uncertainty remains, however over the number of times a satellite cells can divide and contribute to muscle regeneration.[154]
Despite early reports to the contrary,[155], injured striated muscle in adults would not seem capable of successfully recruiting cells from remote sites. Indeed, the notions that skeletal muscle can arise from non-satellite cells associated with blood vessels or interstitial tissue are challenged by the finding that virtually all satellite cells in regenerated muscle were marked as being derived from Pax7 ablated progenitors[156] i.e., satellite cells. Rather than trying to transplant stem cells as an aid to regeneration, efforts might be directed toward mobilizing native reserve cells already present in muscle.
Neurons are cells that convey electro/chemical stimuli (both excitatory and inhibitory) through their bodies and processes to other neurons or effectors such as muscle and glands. Neuroglial cells develop and function in the support of neurons and their processes. Neuroglial cells are the astorcytes, oligodendroglia, and ependymal cells of the central nervous system (CNS), while neurolemmocytes and capsule (aka satellite) cells surrounding aggregates of neurons (known as ganglia) are present in the peripheral nervous system (PNS). Microglia, or dendritic cells, present in the brain and spinal cord are not conductive or supportive in origin or function. Instead, dendritic cells are part of the macrophage/monocyte system of vascular tissue. Indeed, in a dramatic demonstration of cell-based gene therapy, transduced (allogenic) hematopoietic cell transplantation (HCT) had clinical benefits for two X-linked adrenoleukodystropy (ALD) patients via the macrophages/microglia compartment.[157]
Adult nerve tissue has long been considered static. Indeed, the view is largely justified: retrospective birth dating demonstrates that occipital neurons of the human cortex are as old as the individual.[158] Some parts of the nervous system are not quite as static as other parts, however. For example, the subventricular zone (SVZ) of the adult mammalian [mouse] brain retains the potential to generate new neurons.[159] Moreover, adult human neural progenitors (AHNPs) extracted from many areas of the adult forebrain and expanded in tissue culture subsequently differentiated into glial and neuronal cell types both in vivo and in vitro.[160] Similar stem-like cells are obtained from various parts of mammalian brains whether plated as monolayers in tissue culture or reared as floating aggregates called neurospheres.[161]
Problems from the past
Today, nerve tissue is synonymous with the organization and distribution of neurons (i.e., nerve cells), their processes (axons and dendrites), and associated glial cells. Nerve tissue was only this clearly defined in the 20th century. Indeed, nerve tissue was the last holdout against the cell theory.
Confusion began with the spinal cord and the structures called nerves running through the body to muscles. These had been placed in the same class of bodily cords as tendons and sinews (Latin nervus sinew akin to Greek neuron string). And confusion lingered over the presence of individual cells in the nervous system versus networks of fused cells. Ironically, Santiago Ramn y Cajal (1852–1934) demonstrated the boundaries of neurons within the nervous system and demolished the notion of networks with his own adaptations of the silver impregnating techniques devised by Camillo Golgi (1843–1926) to demonstrate the existence of networks. (The two neurologists shared the Nobel Prize in Physiology or Medicine in 1906 without reconciling their differences.) Ramn y Cajal also contributed to the current notion that nerve-cell axons transmitted excitation impulses to nerve-cell dendrites and cell bodies via synaptic junctions as well as to effectors such as muscle via synapses with motor endplates.
Confusion over cellularity in the nervous system is understandable: in ordinary histological sections, the cytoplasm of neurons seem to merge with surrounding material in the so-called gray matter of the brain and spinal cord. Historically, the immediate surroundings of neuronal nuclei in gray matter were called perikaryon (i.e., about or around the nucleus), while the felt work surrounding the perikaryon (consisting of axons, dentrites, glial cells and their processes) was called the neuropil (neural felt).
Today, the brain and spinal cord are recognized as highly complex networks of neurons, their processes, and central glial cells covered by layers of connective tissue (the meninges), and the peripheral nerves connecting the brain and spinal cord to effectors (muscles and glands) are recognized as bundles of individual neuronal fibers, each either surrounded by a myelin sheath or tucked into a fold in the plasma membrane of a peripheral glial cell (a neurolemmocyte) sealed beneath an external lamina and wrapped in layers of connective tissue (endoneurium, perineurium, and epineurium).
Neuronal fibers are either dendrites (drawn out extensions of neurons) generally carrying stimuli to neurons (afferent impulses) or axons (specialized fibers arising in axon hillocks) generally carrying stimuli away from neurons (efferent impulses). Afferent sensory fibers originate in general or special peripheral structures, and efferent fibers of motor neurons end in terminal boutons that synapse with dendritic spines of ganglionic neurons, motor endplates of muscle, or other specialized sites in effectors. Between sensory and motor neurons in the CNS are association neurons having multiple synapses on their own dendritic spines and synaptic junctions with other neurons.
Non-conductive nere-tissue cells in the CNS are central glial cells and those in the PNS are peripheral glial cells. Central glial cells fill the neuropil. They are astrocytes whose foot processes surrounded blood vessels, oligodendrocytes that invested axons in myelin sheaths, and ependymal cells lining the cavities of the brain and spinal cord. Radial glial cells (radial astrocytes) appear transiently during early development but may persist as neuro/glial stem cells in parts of the adult brain.
Traditional nomenclature
Anatomically, the nervous system is divided into the CNS consisting of brain and spinal cord, and the PNS consisting of most other conductive elements, including the enteric nervous sytem (ENS), with the exception of the hearts intrinsict conductive system (sinoatrial and atrioventricular nodes, connecting fibers and branches to ventricular muscles). The CNS and most of the PNS are, by and large, bilaterally symmetrical, having right and left halves which may seem identical although frequently specialized in different functions. Both systems are differentiated dorso/ventrally with sensory functions dominating dorsal portions and motor functions dominating ventral portions and anaterior/posteriorly with sensory functions dominating anterior portions.
In the PNS, sensory neurons reside in spinal ganglia and send fibers (axons) into the spinal cord through a dorsal root. Within the CNS, sensory fibers synapse wtih association neurons and these, in turn, to motor neurons completing reflexive and other sensory/motor pathways including cognitive pathways. Motor fibers (axons) arising from motor neurons leave the brain through cranial nerves and the spinal cord through the ventral root. Voluntary motor nerves pass uninterrupted to effectors, while axons of involuntary motor neurons (pre-ganglionic fibers) synapse in ganglia with neurons that send (post-ganglionic) fibers to effectors.
The CNS is invested in ct sheaths (meninges) and a richly vascular pia mater, but, beyond the vicinity of blood vessels, ct does not penetrated nerve tissue in the CNS, and a blood brain barrier keeps most blood cells and lymphocytes as well as blood-borne proteins otherwise in plasma out of cerebral-spinal fluid. Nervous tissue in the CNS proper is divided into gray matter containing neurons and white matter containing axons, dendrites, central glial cells, the fibers of astrocytes, and conspicuously the myelin sheaths laid down by oligodentrocytes. In cortical regions of the brain (cerebral hemispheres and cerebellum), gray matter is organized in distinct (or not so distinct) layers, while in non-cortical regions gray matter is concentrated in (unfortunately named) nuclei.
The brains subdivisions are defined by cavities persisting from embryonic stages of development. The anterior cavities of the forebrain are lateral ventricles (I and II) and the rostral portion of the third ventricle. Forebrain (telencephalon) surrounding these cavities consists of the cerebral cortex, corpora striata, and rhinencephalon. The remainder of the third ventricle lies in the caudal portion (diencephalon) of the forebrain, enclosed by the epithalamus, thalamus (including the metathalmus), and hypothalamus. The mid-brain, containing the narrow cerebral aqueduct, gives rise to the colliculi, tegmentum and crura cerebri. Finally, the hindbrains fourth ventricle lies between the cerebellum and pons, runs into the medulla oblongata, and merges with the central canal of the spinal cord.
Developmentally, the CNS is formed entirely by cells derived from the embryos neural tube, whereas all neural and glial cells residing in the PNS are derived from neural crest formed at the edges of neural folds and not included in the neural tube. In addition to sensory and post-ganglionic cells, the neural crest provides several other cells to the nervous system: the neurolemmocytes of nerves and capsular (satellite) cells surrounding ganglia, sensory cells (e.g., heat and pain receptors), and cells composing sensory structures such as spindle stretch receptors in striated muscle and structural cells comprising deep and superficial touch and pressure receptors in skin, lips, and internal organs (e.g., pancreas).
With the exception of dendritic cells (from the macrophage/monocytes system), nerve tissue is especially successful at keeping the vascular system at bay. Walls formed by the podocysts of astrocytes create the blood-brain barrier. Responsibility for keeping lymphocytes out of the CNS in adults would seem to be exercised by nerve tissue, since T-cell acute lymphoblastic leukemia cells infiltrate brain vessels when mutations in the Notch1 oncogene trigger expression of the Ccr7 gene. The CCR7 chemokine receptor is an essential adhesion entry signal employed by invading leukemic cells.[162]
New nomenclature: Germinal zones
[I]n the past few years, scientists have found that certain kinds of neurons can grow in adult brains, including those of humans. Not only has this turned scientific dogma on its head, but it has also provided the first glimmer of hope for those suffering from degenerative brain disease or paralyzing spinal cord injuries.[163]
The concept of shifting neuronal germinal zones containing Adult stem-like neuronal stem cells (NSCs) has replaced the notion of a strictly static nervous system. The first germinal zone is the embryonic neuro-epithelium where mitotic activity takes place in the matrix near the outer surface producing neuroblasts and then glioblasts.[164], [165] The matrix is also called the ventricular zone (VZ), since it will later line the ventricle and neural canal formed when the neural plate folds into neural ridges that fuse dorsally into the neural tube.
Before the neural canal is completely enclosed, post-mitotic neuroblasts move out of the matrix and disperse tangentially, parallel to the surface (along the forming axis) and radially, climbing elongated extensions of radial glial cells into peripheral parts of the forming CNS (e.g., to cortical and subcortical regions of the future brain). Migrating glioblasts follow the neuroblasts until the only cells left in the ventricular lining of the neural canal are ependymal cells.
Radial glial cells play a major morphogenic role in the radial migration of neuroblast. These glial cells form long processes stretching across the entire neuroepithelium from its basal membrane (the embryos pial membrane) to the ventricular border (facing the cavity of the neural canal). The stretched processes undergo fasciculation (lateral binding into fascicles) becoming a framework guiding migrating neuroblasts to their ultimate destinations in the CNS. In cortical regions, late neuroblasts move past and displace early neuroblasts (i.e., cortical layers form centrifugally from the inside out). Consequently, in cortical regions, late migrating neuroblasts make sequential contacts with already settled early neuroblasts thus building a chronologically integrated network. In contrast, in non-cortical regions, early neuroblasts become entangled in fascicles and are not displaced by late-migrating neuroblasts (i.e., non-cortical regions form centripetally from the outside in). Thus the arrangement of cells and the structure of networks differs in the cortical and non-cortical regions.[166]
At the same time that the neural tube is closing, mitotic activity moves peripherally into the second germinal zone, the subventricular zone (SVZ) where mitotic activity will remain in portions of the adult CNS. The (blast) cells that divide in the adult SVZ have the potential to differentiate into neurons and central glial cells,[167] and some carry specific earmarks qualifying them as neural precursors. Specifically, some cells stain positively for MAP-2 (microtubule-associated protein 2), NF (neurofilament), and NSE (neuron-specific enolase) with specific neuronal antibodies but not GFAP [glial fibrillary acidic protein]), indicating that the cells are not simply dividing glial cells. SVZ cells also retain labeled DNA (i.e., they are LRCs), and exhibit self-renewal asymmetric cell division.[168] Furthermore, the post-natal SVZ germinal zone also qualifies as a niche: the extra-cellular matrix molecule tenascin C (highly expressed in the SVZ) promotes the accumulation of the ectodermal growth factor receptors (EGFRs) and consequently the proliferation of multipotent NSCs.[169]
Adult NSCs are especially reactive to growth factors. For example, intraventricular administration of mitogens such as epidermal growth factor (EGF) induces proliferation of subependymal cells, their migration away from lateral ventricle walls, and differentiation into astrocytes and neurons.[170] What is more, in combination, EFG and basic fibroblast growth factor (bFGF) in vitro induce proliferation in cells obtained from the ependymal layer of mouse thoracic and lumbar/sacral segments of the spinal cord and fourth ventricle of the brain.[171]
Localized (circumscribed) germinal zones in the adult SVZ are found in the lateral ventricle of the brain, hippocampus,[172],, [173] and the subependymal regions of the spinal cord in mammals from mice to cows.[174] NSCs present in these regions produced TAC-like cells that differentiate preferentially as glial cell as opposed to neurons, but, in rodents, cells differentiating as neurons (i.e., neuroblasts) move chain-like in a well-defined rostal migratory stream (RMS) to the olfactory bulb where they become integrated granular interneurons.[175]
Intriguingly, human neural precursor cells (hNPCs) obtained from fetal cortices and cultured in vitro formed neurospheres (balls of neural-like cells) and grew rapidly in the presence of EGF and FGF-2. These neurospheres shifted toward less growth and showed increased rates of cell death after withdrawal of the growth factors. Moreover, the number of undifferentiated hNPCs decreased, and the number of cells expressing the neuron specific marker, b-tubulin III increased when the neurospheres were allowed to settle on coverslips. Then hNPCs differentiated into neuron-like cells, extending thick, radially oriented dendritic processes.[176] No oligodendrocyte-like cells differentiated, possibly due to the depression of cell division.
Of course, the discovery of germinative zones in the CNS has raised expectations for the treatment of injuries to the nervous system and neuropathies. These expectations must not be allowed to run ahead of cautious optimism, however.
The problem is that the NSCs produced in adult germinal zones exhibit chronological or staged losses of morphogenetic plasticity.
Timing seems to be encoded in progenitor cells so that besides positional information they have temporal information, which is seen as stage-dependent changes.[177]
In other words, stem cells obtained from a given region of the embryonic brain are different from those derived from the structure in the adult brain that the embryonic region gave rise to.[178] Thus, the promise held out by potential NSCs is undermined by their loss of plasticity. Similar changes (e.g., perinatal restrictions in neuronal subtype potential, failure to generate serotonergic neurons, and a decline with age in ability to generate noradrenergic neurons) are found in precursors derived from the neural crest in the enteric nervous system.[179] In other words, history has its consequences.
Traditional nomenclature
Fibroblasts (or fibrocytes) would seem to be the one consistent element found in connective tissues. Virchow characterized these cells in embryos as spindle-shaped cells [that] remain the same [throughout a lifetime], although they are often not easy to see.[180] Virchows assumption seems to have been that fibroblasts produce all the different sorts of extracellular material composing the bulk of bone and cartilage, loose webs of fibers, and fluid filled cavities he lumped together in connective tissue. He added adipose tissue to the category if only because as fat comes and goes the adipose tissue is once more reduced to the state of simple, gelatinous connective, or mucous tissue[181] Moreover, apropos of his prevailing interest in tissue pathology, Virchow found corpuscles of the connective tissues [when] fatty tumour arises in fatty tissue, or a connective tissue (fibrous) tumour in connective tissue.[182]
Connective tissues are divided morphologically into loose and dense ct mainly on the basis of extracellular material: proteoglycan, collagen (of different types), structural glycoproteins (fibrillin and fibronectin), and glycosaminoglycans (GAGs). Loose ct (including embryonic mesenchyme and fetal umbilical cord jelly) comprises reticular ct (typically the stroma of lymphatic tissue: nodes, spleen, and thymus), and the ubiquitous areolar ct (loose [loosely speaking]) that fills extracellular spaces within organs and connects blood vessels to other tissues (via adventitia [meaning foreign or coming from outside]).
Dense ct is either irregular (dermis of the skin, perichondrium, periosteum, elastic and fibrous membranes [tunics, theca, submucosa, lamina, capsules], trabeculae and septa) or regular (ligaments, tendons, fascia). Cartilage comes in several varieties (hyaline, articular, elastic, and fibro-cartilage), and adult bone comes in two major types: cancellous (spongy: filling marrow cavities and diploe) and compact (membranous bones of the skull and cortical bones of the appendicular and axial skeletons). Both types of adult bone are lamellellar, and both are constantly subject to remodeling, although cancellous bone is more active than compact bone. Adipose tissue is found in two types: white (unilocular) and brown (multilocular). Both are dynamic and highly active metabolically.
New nomenclature
Connective tissue is divided into two broad classes: fixed and dynamic. Fixed ct (loose, dense regular, and cartilage with the exception of fibro-cartilage) is virtually static in adults in the absence of trauma or disease. The major cell types of fixed ct are the mitotically quiescent fibrocyte (also called a fibroblast) of loose and dense ct, perichondrium, and most chondrocyte of cartilage. Indeed, cartilage undergoing healing, presumably by local mobilization of perichondrocytes is frequently replaced with fibrocartilage rather than restored to its original condition, and bony spurs that replace degenerated cartilage in joints are the baine of those with osteorarthritis.
Dynamic ct (bone and adipose tissue) contains mitotically active osteoblasts. Additional cells may be mobilized localled from osteocytes previously embedded in bony matrix or from periosteum, or new osteoblasts may be recruited from circulation.
The most conspicuous feature of fixed ct is dormancy in situ. Retrospective birth dating demonstrates that turnover in the connective tissue of the jejunum is extremely sluggish, taking fifteen to sixteen years.[183] Even chondrocytes that contribute to interstitial growth (forming isogenous groups within their cartilaginous matrix) quickly cease dividing.
Dormancy is lifted, however, under a variety of conditions indicating that constraints on cell division in ct are imposed extrinsically and not through any intrinsic failure of fibroblasts. For example, the endometrial stroma of ovariectomized mice undergoes massive cell division when exposed to exogenous estrogen.
Dividing stromal cells in developing ct should not to be confused with ASCs. Thus, when endometrial stroma, cells are labeled postnatally and prepubertally with the DNA analogue bromodeoxyuridine (BrdU), the rubric for ASCs dictates that the number of label-retaining cells (LRCs) stabilizes. Data show, however, that the number of label-bearing ct cells declines during the chase period and, indeed, only [o]ccasional stromal LRCs were present after a 12 week chase.[184] Furthermore, BrdU LRCs did not coexpress with Sca-1, a known stem cell marker.[185] But ambiguity remains, since stromal LRCs were CD45- [ and, therefore,] not CD45+ leukocytes of hematopoietic origin[186] recruited from circulation.
Dormancy is famously lifted when fibrocytes are explanted to tissue culture. After a brief lag, the fibrocytes become fibroblasts and divide rapidly. Indeed, not only do fibroblasts divide in vitro, but an underlying feeder layer of irradiated, non-multiplying fibroblasts is commonly employed to condition tissue culture media thereby promoting cell division and aiding the establishment and upkeep of other fragile cells (e.g., epithelial and cancer cells).[187]
Division of explanted fibroblasts in tissue culture has attracted a great deal of attention especially because of one peculiarity: division stops after a fixed number as a function of the age of the explant. The cells then enter a period of mitotic quiescence[188] that may last months but is eventually followed by death. Although many tissue culturists knew that fresh (aka primary) cultures of fibroblasts ultimately died, their failure to achieve cellular immortality was commonly attributed to culture conditions and not to any inherent property of cells. The phenomenon of a timetable for cell mortality measured in cell divisions was only recognized after Leonard Hayflick showed that mitotic quiescence followed a predictable number of divisions now known as the Hayflick limit. Indeed, cells exceeding their Hayflick limit have cancer cell properties including higher rates of telomerase activity compared to normal cells.[189]
Of course, the possibility that organisms age and die when their fibroblasts reach their Hayflick limit is an alluring hypothesis[190] (and a contentious one[191]), but even fibroblasts from the elderly seem to have some unspent mitotic activity left when explanted to tissue culture. The mortality of organisms, therefore, would not seem to be a simple function of the mortality of fibroblasts.
Anyone suffering from osteoporosis, periodontal disease, arthritis, and osteolysis induced by tumors has firsthand knowledge of consequences arising from dysfunctional dynamic ct, while those with healed fractures of bone will hold cts functional dynamics in high regard. What is rarely appreciated is the massive amount of remodeling going on unbidden in normal dynamic ct:
Bone remodeling occurs in small packets of cells called basic multiellular units (BMUs), which turn bone over in multiple bone surfaces [A]t any one time, ~20% of the cancellous bone surface is undergoing remodelling.[192]
Bone is a spectacularly dynamic tissue, and bone remodeling is a symphony of cellular counterpoint: myf-5 and myoD genes are inactivated in osteocytes, and a signal pathway utilizing prostaglandins is activated; osteocytes secrete insulin-like polypeptide growth factor (IGF-I) mobilizing local osteoclasts and recruiting precursors of hematopoietic origin (monocytes?); osteoclasts create resorption lacunae (aka compartments) and lay down a substrate of osteoblast-attachent matrix; parathyroid hormone (PtH), 1,25-hydroxyvitamin D, and cytokines keep osteoclasts active for their brief life span; a reversal phase is initiated, possibly under the influence of high calcium levels, and osteoclasts disappear; transforming growth factor (TGF-b) blocks osteoclast resorption of bone; mitotically active mesenchymal osteo-progenitor (pre-osteoblasts) and recycled osteocytes from resorbed bone mature as osteoblasts, probably under the influence of left-over type I collagen fragments, osteoblast-derived TGF-b, IGF-I and II, fibroblast growth factors (FGFs) as well as platelet-derived growth factor (PDGF) and plasminogen activators; mature osteoblasts commence the complex cascade of bone formation: osteoid is laid down in a thick layer, matrix is formed, and osteocytes become embedded in new matrix until the lacuna is filled with new bone and osteoblast activity ceases (probably under the influence of negative feedback.)[193]
Clearly, the process of bone remodeling couples local dynamics with a reach far beyond the local. 1,25-dihyroxyvitamin D3 augments proliferation of bone-marrow stromal cells in vitro and increases the percentage of osteoblast-forming colonies (known as colony forming units or CFUs) formed by bone marrow cells.[194] Once mobilized, osteoblast-precursors in vivo attach to heparin-binding growth-associated molecule (HB-GAM) and commence matrix formation.
Inevitably, the same factors implicated in normal local and systemic outreach have their pathogenic side. For example, for persons with multiple myeloma, painful bone resorption (osteolysis) is attributable to large amounts of interleukin 6 (IL-6). This very same cytokine critical for the recruitment of of osteoblasts and osteoclasts during bone remodeling[195] is also a potent myeloma cell growth factor.[196]
Finally, adipose tissue is placed in the category of dynamic ct because adipocytes are mitotically active. However, the high metabolic activity of adipocytes suggests that they may not be ct cells in the first place. In addition, adipose/smooth muscle metaplasia in uterine fatty tumors (UFT: lipomas and mixed lipoma/leiomyoma = lipoleiomyoma)[197] suggests that adipocytes are more closely related to active smooth muscle cells than lethargic fibrocytes. Were adipocytes a variety of smooth muscle, intrinsic mititoic activity would reasonably be attributed to cache-like cells.
Vascular tissue, which is to say cells suspended in plasma and lymph, is hardly restricted to the contents of blood and lymphatic vessels. Indeed, most of the same cells circulating in the organism through vessels are ubiquitous in the loose ct of every organ (with the exception of brain and spinal cord).
Traditional nomenclature
Peripheral blood and aspirated bone marrow are traditionally studied in smears (films) spread on slides and stained with one or another brew: Romanowsky stain (of Marshall), May-Grnwald Giemsa stain, Wrights, or Leishman stain. Each stain has its fans, but the results are very nearly interchangeable.[198]
Blood cells fall into two main categories: white blood cells (leucocytes) and red blood cells (RBCs or erythrocytes).[199] Leucocytes are further divided into granulocytes (aka myeloid [bone marrow] cells) and agranulocytes or monocytes. Nuclear morphology and staining properties separate the granulocytes into neutrophils, eosinophils, and basophils. Mammalian RBCs lack nuclei and cell organelles (mitochondria) and are not, therefore, complete cells. In addition, mammalian platelets (thrombocytes) are blood-borne cytoplasmic fragments of medullary megakaryocytes.
Lymphocytes were once placed in the agranulocyte class with monocytes with no greater rationalization than their having single, round nuclei and a dardkly staining (basophilic) rim of cytoplasm. Today, lymphocytes comprise a class of their own, but the three recognized types of lymphocytes, B-, T-, and natural killer (NK) cells, are only lumped together for want of microscopic criteria for identifying them separately.
Ironically, the same leucocytes seen in blood and bone-marrow smears are abundantly present in loose ct seen in sections (and whole mounts of mesenteries) but the intimate relationship of circulating and fixed cells was not appreciated. Indeed, more than half the cells seen in loose ct are monocytes (macrophages [histiocytes]). In fact, blood vessels are just the tracks for transporting monocytes and granulocytes from their sites of origin in bone marrow to loose ct where they perform most of their functions.
In contrast, although their functions were obscure, lymphocytes have long been observed in abundance in the reticular ct of thymus, the lymph nodes, tonsils, aggregated nodules in the ileum, and in the spleen. Although they are indistinguishable in the light microscope, B lymphocytes are concentrated in the cortex of lymph nodes and the crown of nodules. T lymphocytes are concentrated in the subcortical region and more nearly at the germinal center of lymph nodules, in the peri-arteriolar region of the spleen, and throughout the cortex of the thymus.
New nomenclature: Clonal Hierarchies
Two engines drive contemporary wisdom on vascular dynamics: hematology and immunology. No other part of histology is as indebted to pathology. Indeed, from acute leukemia to zoonosis and from diagnosis to discharge, a patients blood is examined microscopically, cells examined, counted, and results compared to those for blood in normal individuals. Consequently, the hematologist, immunologist, and pathologist have contributed enormously to understanding blood cell and lymphocyte dynamics in health and disease.
Relying on stained bone marrow films, researchers have pieced together the lineage of blood cells and lymphocytes from the size of cells, shapes of nuclei, and the color and density of cytoplasmic granules. In the language that evolved for the myeloid (granulocyte) line, early dividing cells had -blast attached to their name (e.g., myeloblast). Early post-mitotic cells with few granules were called promyelocytes and later cells with specific granules were called metamyelocytes. Finally, stab or band cells developed as the granulocytes nucleus underwent various contortions and the cytoplasm filled with specific granules.
Dividing cells in the monocyte lineage were promonocytes and simply monocytes when they became post-mitotic and entered circulation. Likewise, but without leaving the bone marrow, megakaryoblasts became megakaryocytes that budded off or fractured into platelets.
Dividing lymphocytes were lymphoblasts while they had a nucleolus and prolymphocytes when they had no nucleolus. They entered circulation at this stage as mitotic large lymphocytes and (probably) non-dividing medium and small lymphocytes. All these cells became the T, B, and NK lymphocytes proliferating and dying in the thymus, spleen, lymph nodes, tonsils, aggregated lymph nodules, and everywhere else where lymphocytes congregate.
Proerythroblasts represent the mitotically active, erythropoietin (aka erythropoietic stimulating factor [ESF]) sensitive stage[200] that ends after some three to five divisions. Names employed for cells on later echelons along the erythrocyte ladder of differentiation are poorly chosen. Consistency would have demanded the removal of the -blast ending since later cells are post-mitotic, but -blast stuck, unfortunately, to post-mitotic normoblasts (aka erythroblasts). These cells past into early normoblasts (or basophilic erythroblasts), followed by intermediate normoblasts (or polychromatic erythroblasts), late normoblasts (or orthochromatic erythroblasts), and definitive normoblasts (or acidophilic erythroblasts). At this ultimate stage, the mammalian normoblast loses its nucleus on its way to circulation, becoming a transient reticulocyte, and finally an erythrocyte.
The source(s) of blood cells and lymphocytes has been hotly pursued. Although cells were initially thought to originate in separate spleen and lymphocyte cell lines, one multipotent stem cell known as the hematopoietic stem cell (HSC) is now widely assumed to be the universal blood/lymphocyte precursor.[201] Over the years, notions of hematopoiesis have been reconfigured around clones arising from HSCs in a hierarchy of increasingly limited potentency.[202]
Initially, the clones exhibit multipotency: Repeated plating reveals a hierarchy among the cell types differentiating, with primitive hemopoietic progenitors leading to spleen colony-forming units and granulocyte-erythrocyte-megakaryocyte-macrophage colony-forming units (CFU-GEMM). Granulocytes break out in three steps. Eosinophils and basophils arise from granulocyte precursors, while a neutrophil-monocyte line gives rise to neutrophil and monocyte (macrophage) clones. Further up the hierarchy, bipotent megakaryocyte-erythrocyte progenitor gives rise to monopotent proerythroblasts and megakaryotcyte-committed progenitors (MKP) that differentiate as megakaryotcytes.[203]
The unraveling of hierarchial clones stemming from HSCs begins with (Frank) Macfarlane Burnets (1899–1985) clonal selection theory of immunity based on colony forming units (cells produced from a single progenitor) borrowed from microbiology.[204] Burnet postulated that in adults all antibodies were encoded in individual quiescent lymphocytes. A specific lymphocyte wore a specific antibody (or part thereof) on its sleeve (so to speak), that is, its external membrane. The cell released stored reservoirs of the same specific antibody in the presence of the corresponding antigen thereby becoming an effector cell. At the same time, leucocytes (and lymphocytes) produced factors that promoted cell division in the effector cell and hence the growth of a clone of identical antibody-producing cells. Some of these cells, known as memory cells returned to a quiescent pool of specific antibody-coated cells that would become active in the anamnestic (memory) or secondary response characteristic of immunity. Thus, in addition to effector cells long-lived memory [cells] persisted but their functional activity only became apparent after secondary antigenic stimulation.[205]
Burnets idea was quickly extended from lymphopoiesis to hematopoiesis generally, and Mako Ogawa and colleagues used a soft tissue culture medium (containing methylcellulose and conditioned by human lyphoblasts) to raise clones of suspended cells (from adult bone marrow, spleen umbilical cord and elsewhere).[206], [207] Serial plating of cells from these clones revealed that colony forming units (i.e., CFSs or dividing cells) were rare (e.g., occurring one to two per 106 primary culture bone marrow cell) but could divide endlessly (unlike fibroblasts). These cells were vulnerable, however, to directed differentiation. Donald Metcalf discovered that interleukin 5 and stem cell factor (SCF, aka c-kit ligand or steel factor) increased the frequency of eosinophil-committed progenitor cells in multicentric colonies, and thrombopoietin with SCF increased the frequency of megakaryoctye-committed progenitor cells.[208]
A consensus has yet to form around a modern language of blood cell and immunocyte dynamics. Such a language should insert a degree of rationality into the present hodgepodge of hemato/lymphopoietic terms. Lines should connect the dots of cell production and cells at comparable levels of proliferation should be given comparable names. Some of todays terms should be retained: common lymphoid progenitors (giving rise to Pro-B, Pro-T, and Pro-NK [natural killer] lymphocytes) and myeloid progenitors (giving rise to macrophages, granuolocytes, and dendritic cells) are useful terms;[209] but other terms, such as blast forming units should be abandoned along with some of the conveniences frequently used to shorten expression such as long term and short term stem cells. And, if reason prevailed, some definitions would be used consistently: the term progenitor refers to a cell with a more restricted potential than a stem cell. Precursor is a less stringent term that refers to any cell that is earlier in a developmental pathway than another. [210] Histologists, hematologists, and immunologists will have a far better idea of what theyre talking about when that these changes are incorporated into the canon.
Traditional nomenclature
Germ tissue is singular, although, surrounded by somatic epithelium in the ovary and testis, germ tissue has some epithelial features. In the testis, sustentacular (aka supportive) cells comprise the epithelium of seminiferous tubules. Tight junctions between the sustentacular cells divide the seminiferous tubulules into two compartments: a basal compartment containing spermatogonia and an adluminal compartment containing spermatocytes undergoing meiosis and spermatids differentiating into spermatozoa. The lumen of the tubule accommodates released spermatozoa that mature and become decapacitated in the epididymis.
In the ovary, follicular epithelium surrounds each oocyte. In a fecund ovary, abundant primordial follicles lie under the ovarian mesothelium. These follicles consist of a single layer of flattened follicle cells surrounding a primary oocyte. Moving inward in the ovary, primordial follicles become transformed into primary follicles as the layer of follicle cells becomes cuboidal and multilayered. At the same time, the oocyte grows and secretes its zona pellucida (basement membrane). In the secondary follicle, islands of follicular fluid appear among follicle cells, and in the tertiary follicle these islands coalesces into an antrum surrounded by follicular epithelium now renamed the granulosum (or zona granulosum).[211] The oocyte, which has grown to its mature size, is embedded on a cumulus oophorus of radially oriented cumulus cells. As the antrum swells and the granulosum thins, the mature follicle moves toward the ovarys surface where a stigma bulges prior to ovulation. In most mammals (canids being an exception), the oocyte completes its first meiotic division to become a secondary oocyte, which, following ovulation, is surrounded by a thin corona radiata of cumulus cells (bovines being an exception).
The major difference between testis and ovary (aside from the morphology of the cellular products) is that the testis supports ASCs in the form of spermatogonia whereas the ovary has no comparable cell. Until recently, the adult mammalian ovary was thought to contain nothing resembling an oogonium. Presently, prospects have risen for oogonia in the mammalian ovary (see below).
New nomenclature
The line drawn between somatic and germ tissues by August Weismann[212] has persisted despite efforts to erase it.[213], [214] If new evidence for the transformation of somatic epithelium into oocytes is borne out, the line between somatic and germ tissues will all but disappear.
Mammalian females have long been thought to be born with a fixed number of oocytes in follicles and no mitotically competent oogonia whatsoever (prosimians and rabbits being exceptions). Recently, however, estimates of the number of oocytes at birth, suggest that ovaries simply do not contain enough oocytes enclosed within follicles to support all the oocytes released at ovulation and the far greater number that die in atretic (unperforated) follicles during the femals reproductive lifetime.[215]
Estimates of early follicle number leave room for doubt,[216] and far greater doubt surrounds the possible sourc(s) of additional oocytes were they, indeed, mobilized. One possible source would be the mesothelium surrounding the ovary, or ovarian surface epithelium (OSE),[217] once called the germinal epithelium, and renewal of oocytes has been demonstrated experimentally.[218] Markers indicate, moreover, that the production of new oocytes is not due to circulating germ cells.[219]
All is not lost for the notion of separate germ and somatic lines, however. Instead of new oocytes emerging from somatic epithelium, possibly, oogonia descended from primordial germ cells are buried among the mesothelial cells and rise under under the influence of specific cytokines to replace lost oocytes.
Thus, adult female mammals, like their male counterpart may have germinal stem cells (GSCs). GSCs in both sexes would have passed through an establishment phase during development and reside in a maintenance or self-renewing phase in adults. The establishment phase would seem to be governed by extracellular signals from hormones and local interactions and the maintenance phase by somatic (epithelial?) tissue and not germ stem cells.[220], [221] Indeed, in mouse testes lacking spermatogenesis because of a mutation in the c-kit gene or following treatment with busulfan (that denudes the testis of spermatogonia), infusions of spermatogonia acquired from neonates and from cryptorchid adult testes have similar chances of establishing themselves, but the immature pup testis is superior to the adult testis as a site for colonization and repopulation.[222]
In adult male mammals, a one-way spermatogenic wave begins the terminal DNA synthesis of GSCs[223] and ends with the completion of meiosis and differentiation of spermatids. The spermatogenic wave of rat testes is set off simultaneously at as many as fourteen sites dispersed along the length of seminiferous tubules. In human beings, multiple onsets are not set off simultaneous and the wave is, therefore, not synchronized.
However. in
mammalian testes, various hypotheses have been proposed to describe the exact identity of GSCs and their pattern of division, most of which point to a subset of spermatogonia (Type A or a subtype of Type A) as GSCs Despite this, it has not been unequivocally shown whether GSCs in the mammalian testis divide asymmetrically[224]
One possibility casts pale (Ap) or single (As) spermatogonia in the role of self-renewing spermatogenic stem cells (SSCs) that give rise to paired (Apr) and aligned (Aal) or intermediate spermatogonia (also known as A1–A4, intermediates, and B-type spermatogonia in rodents). In this case, the latter spermatogonia should be reclassified as TACs in the process of differentiating into spermatids and spermatocytes.[225]
Remarkably, the progeny of spermatogonia (and early oogonia from Drosophila[226], [227] to mammals[228]) remain attached to each other by intercellular bridges. In female vertebrates, the bridges break prior to oocyte differentiation and the commencement of meiosis. In males, bridges link cells throughout three to six synchronous mitotic divisions. The bridges become filled with midbodies or remnants of mitotic spindles and sealed by membranous septa in incipient spermatocytes, but, following meiotic divisions, the bridges open again as spermatids differentiate.[229], [230] Complete strings of attached cells (as many as 256) could occur, but cell death breakes the string along its length and nests of spermatids are smaller. The causes of these cell deaths are unknown.
In addition to active spermatogonia, dormant (?) dark spermatogonia (Ad aka A0) would seem to play the role of reserve stem cells. They are beyond the reach of normal spermatogenesis but may become active in the wake of trauma and repopulate the seminiferous tubule with active spermatogonia.[231]
Reserve germ cells may thus be present in both male and female adult mammals and in the event of trauma or disease may operate in regeneration or replacement. In females, these reserve cells may normally be an extra source of oocytes, while in males reserve spermatogonia are germ cells of last resort.
Typically, the origin and evolution of animals are portrayed as a progressive series of linear transitions. Thus, an ancestral animal consisting of a single-layered epithelium in the shape of a sphere (or compressed into a disk), is thought to have invaginated (folded inward at a point) forming a cup-shaped animal with inner and outer layers. The inner of these layers proceeded to fold or delaminate cells into the space between the two layers thereby forming a third, middle layer and evolving into a three-layered animal.
Then, in the course of time, animals with simple layered forms evolved more complex forms, changing progressively from spherical to radial symmetry to biradial and bilateral symmetry. Through adaptive advantages implicit in the division of labor, the outer layer specialized as an ectoderm, the inner layer an endoderm, and the layer in between, a mesoderm. And these layers gave rise to all the tissues present in extant metazoans. Today, all these transitions are recapitulated among the Bilateria in changes from fertilized egg to early embryonic forms, to fetal or larval forms, and, ultimately, to even more complex adult forms.
The main problem with this scenario is that the fossil record, development, and the welter of comparative genomic evidence are inconsistent with linearity. The fossil record jumps around from one period of mass extinction and explosive evolution to another; development is frequently heterochronic (simply out of step with linearity); and homologies among genes indicate that genes influencing development surface before their time.[232] Genes implicated in early development,[233], body axes formation, and transduction pathways stretch across phyla and back beyond the origins of their present hosts.[234], [235] In short, recapitulation may be an excellent metaphor and teaching device, but the fossil record of evolution, development, and the genome do not repeat one another. Indeed. even the relationship of genes in ESC to genes in ASCs supports neither linearity nor recapitulation:
Pax7 dependency [defines] the transition from muscle progenitor to adult stem-cell state [i.e., satellite cell], which ensures that muscles achieve regenerative capacity. Given the essential roles for Pax3 and Pax7 in embryonic and for Pax7 alone in perinatal myogenic progenitors, it was entirely unexpected that adult satellite cells require neither Pax7 nor Pax3 for muscle regeneration. We imagine that postnatal changes of muscle organization, mechanics and physiology demand stem cells to alter their transcriptional program as a means to adapt to these challenges. Changes in genetic requirement for muscle stem cells from embryonic to juvenile to adult stages elucidate the inadequacy of applying knowledge gained from developmental studies to adult stem-cell biology.
Alternatively, Donald Williamson has proposed a different, non-linear, non-recapitulative idea of how animals evolved. Williamson was drawn to evolutionary theory by contrasts in the development of many marine invertebrates with extreme indirect development (in which adult develops independently within the larva) and the many arthropods he studied throughout his career exhibiting only moderate indirect development (in which the larva develops into an adult through several molts).
Williamsons larval transfer hypothesis accounts for extreme indirect development and less extreme forms of development as well as alternation of generation seen in cnidarians [polyp followed by medusa] by proposing that the genes that specify them [stages of a life cycle] have been transferred from one hereditary animal lineage to another by cross-species, cross-genera and even cross-phyla fertilizations.[236] According to Williamson, moreover, serial chimeras produced all present-day animals and not through one hybridization but many.
Williamson has produced intriguing spinning top larvas as consequences of experimental cross-fertilizations.[237] The relevance to evolution of these hopeful monsters remains speculative, but they vindicate Williamsons larval transfer hypothesis as evolutionary conjecture.
Imagine a time before barriers to cross-fertilization were as high as they generally are today in animals. At that time, cells might merge occasionally, with adaptive results. The merger need not have been complete.[238] Indeed, if the frequency of symbiotic relationships in present marine invertebrates is any indication, many incomplete mergers were successful and have been perpetuated through geologic time.[239] But, however rarely, more profound mergers might also have taken place. For example, cells might have formed cellular slimemold-like creatures,[240] of fused more profoundly into plasmodia resembling the true slime mold (Physarum). Lateral or horizontal gene transfer might have resulted in the incorporation of genes into a dominant nucleus,[241], [242] or nuclear fusion might have produced one or more nuclei in a mature cell.[243] Cells might have conjugated to form heterokaryons, containing two nuclei, and these nuclei might ultimately have fused (nuclear copulation), or syncytia might have formed with many nuclei that came together into a single nucleus.
At the same time, other cells presumably discovered adaptive advantages in protecting their own genomes against fusion. These cells would have begun the process of throwing up barriers to conjugation and copulation (e.g., mating types and heterogameity) culminating in impediments to hybridization such as those represented by eggs and sperm. Ultimately the true origin of species began when impediments to illegitimate mating and blocks to fertilization became insurmountable—hence, true species.
The evolution of crossbreeding hermaphrodites took second place in competition with separate male and female adults, and subsequent sexual dimorphism triumphed in the drive toward raising species barriers. Certainly, coupled to gametic differentiation, the very existence of male and female types of organisms limits the possibilities of conjugation and copulation even within a species.
Barriers of different heights to chimera-formation would seem to be all but universal today. Remnants of a past more prone to merging may be with us still, however. Symbionts in cnidarians (e.g., Symbiodinium, the zooxanthellae dinoflagellate symbionts of reef-building corals), for example, are not passed through eggs but are taken up de novo by larvae,[244] and many protistan species have not bothered to evolve egg and sperm, although similar and, presumably, closely related species have. Morphologically identical isogametes occur in both protozoa (such as the familiar Tetrahymena) and algae (e.g., Chlorophyta [green algae], Class Chlorophyceae, virtually all Tetrasporales, Trentepohliales, and Siphonales, in many Zygnematales, Ulotrichales, Ulvales, Siphonocladales, Microsporales, Cladophorales, Chlorococcales, and Chaetophorales, in the Chrysophyta [yellow-green algae], in Ceratium among the dinoflagellates [Pyrrhophyta], and in some brown algae [Class Isogeneratae].[245]
The fossil record (albeit controversial) provides abundant time for evolution by hybridization. To begin with, eukaryotes molecular clock seems to have begun ticking as far back as 3500 million years ago (Ma).[246] Steranes, telltale biomarkers of eukaryotes (i.e., cells with nuclei), appear in northwestern Australias ancient sedimentary shales as old as 2700Ma.[247] Progress was not uniform, however, and lifes evolution seems to have been slowed by snowball Earth (global glaciation) somewhere about 2300 to 2200 Ma,[248] leaving the cenancester or last common ancestor of prokaryotes and eukaryotes to branch definitively in the vicinity of 2000 Ma.[249], [250], [251], [252], [253]
The rate of evolution seems to have stepped up thereafter. Ancestral eukaryotes that followed in the fossil record lacked a gullet or gut but were probably chock-full of photosynthetic or chemosynthetic symbionts; protistan (heterotrophic?) cells occurred about 1800–1300 MA; filamentous eukaryotes may date back to 1,700 and 1,600 Ma to the end of the Paleoproterozoic;[254] and the major eukaryotic clades (fungi, plants, animals) diverged 1300–720 Ma, while diversity of animals began at the Ediacaran-Cambrian radiation ( 600 Ma).[255]
Between the Varangian ice age (590 Ma) and the base of the Cambrian System (the Phanerozoic about 550 Ma), an odd assemblage of large, dieverse, but structurally rather simple, mainly wormlike and jellyfishlike creatures make up what is known as the Ediacaran Fauna.[256] A second group of Ediacarans called the Vendobionta, concentrated in strata about 560 Ma, were leaf-like organisms (colonies?), some mounted on disklike holdfasts consisted of a central axis from six inches to more than a foot long and two rows of thirty to fifty branches composed of interlocking tubes a few millimeters wide that may be fused and may bear flaps marked by parallel grooves on one face. Other vendobionts had three or more wings attached to a central axis forming a fan or cup. Were they cnidarian-like colonies that thrived in the Cambrian, or were they quiltlike organisms made up of cylindrical tubes filled with plamodial fluid rather than cellular tissue [that became] an extinct kingdom separate from the animals, an experiment in macroscopic multicellularity that blossomed for a geological moment but ultimately failed?[257]
Certainly, Ediacaran multicellular forms are problematic fossils. For example, some characteristics of Venustovermis may indicate a possible molluscan affinity These characters, however, are found separately within different molluscan groups [and] could represent an independently evolved animal lying outside of molluscs.[258] On the other hand, Williamson-like hybridization might explain the mixture of characteristics without making th assumptions inherent in a defense of covergence.
How then might tissues, specifically, have evolved in a Williamson-like scenario assuming that protistan-like organisms were present and conjugating before obstacles to hybridization were thrown up or thrown as high as they are today. Possibly, a first step consisted of conjugation among single-cell isogametic species resulting in the formation of ur-tissues: a chimeric ur-epithelium might have been an epithelial-like pseudo-plasmodial aggregate of cells or a syncytial-like plasmodium; an ur-connective tissue might have been merely ameba-like cells, although pseudoplasmodia or a true plasmordia are also possibilities. Subsequently, Williamson-like hybridizations might have brought these ur-tissues together. A paeloproterozoic biota might then have emerged of chimeric epithelial/cellular, epithelial/plasmodial, and epithelial/pseudoplasmodial organisms, and these may have evolved specialized and differentiated tissues, preserving some of the originary characteristics and mixing and merging others.
One would expect that some the ancient characteristics of thee chimeric organisms are preserved (if only in modified form) in contemporary animals.[259] In sponges, an ur-ameboid tissue may be represented today by archeocytes (amebocytes); an ur-epitheliim may be represented by collar cells (choanocytes) and pinacocytes; an ur-plasmodium may be represented by the syncytial exopinacoderm. Collar cells and pinacocytes do not quite qualify as epithelial, since they lack intercellular junctions (although septate junctions appear among sclerocytes).[260] Division in archeocytes and collar cells may be cache-like, since mitosis is widespread and abundant when food is plentiful and ceases when food is sparse.[261]
The disk-like placozoan, Trichoplax, hardly seems to consist of much more than an ur-epithelium and possibly some muscle and nerve elements derived from it. Trichoplax has a continuous epithelium differentiated into dorsal and ventral surfaces separated by an interspace containing a syncytium of mesenchymal fiber cells delaminated from the epithelia. Since the fiber cells are contractile and may also be involved in the coordination of movement, they seem to combine the functions of muscle and neurons on a primitive level.[262] All the epithelial cells are flagellated and joined apically by belt desmosomes while non-flagellated gland cells are interspersed among ventral epithelial cells. Division may be cache-like.
Cnidaria has been nominated for the post of the oldest eumetazoan phylum[263] based on complexity built into its simple appearance. Were cnidarians to have arisen from hybridization, the ur-epithelium might be represented by ectoderm and endoderm,[264], [265], [266] and the ur-connective tissue by interstitial cells (aka amoebocytes) normally tucked into the interstices of epithelial cells of polyps and thinly spread throughout the bell of medusas.
Cnidarian endoderm and ectoderm consist of classic epithelial cells linked by occluding septate junctions and communicating gap junctions. Cell division in cnidarian epithelial cells is of the cache style, widespread and copious when resources are available and ceasing when deprived of resources. Indeed, every epithelial cell in the body column (but not tentacles) would seem capable of division. When mitoses are abundant, excess cells are either utilized in intrinsic or extrinsic growth (i.e., increased body mass or asexual reproduction), but, when resources are scarce, tissues and the animals body ceases growing, asexual reproduction stops, followed by autophagy and shrinkage.[267]
Interstitial cells are a potpourri of adult stem-like cells, producing clones of determined cells that cease dividing and go on to differentiate into nematocytes, neurons, gland cells, and germ line cells. Remarkably suggestive of separate origins, 86 genes are expressed exclusively in various terminally differentiated derivatives of interstitial cells, while 25% of the selected genes turned out to be epithelial cell-specific when tested by in situ hybridization.[268]
The freshwater polyp, Hydra, became a well-known model organism since research on its regeneration began in the 18th century. Hydra consists entirely of the epithelia composed of cache-like cells, interstitial cells squeezed in between the bases of the ectodermal cells, and their terminally differentiated products. Muscle would seem to be derived from epithelia, since, in Hydra and other hydrozoan polyps, muscle-like fibers form the base of epithelial cells (i.e., the cells are epitheliomuscular or myoepithelial cells). Free muscle is present on the concave sides of medusas (jellyfish) and in thick muscular bands lining the pharyngeal septa of anemones.
The conversion of originally separate epithelial and cellular or plasmodial ur- components into an integrated whole would, presumably, also involve the mixing of some features. Epithelium and connective tissue, as such, seem to retain many originary characteristics. Muscle seems to be derived fairly straightforwardly from epithelium, and vascular tissue could be derived fairly straightforwardly from connective tissue. But nerve, while epithelial-like in many respects has some connective-tissue characteristics (e.g., neural crest migration), and germ tissue, while connective-tissue-like with respect to the migration of primordial germ cells in embryos has some epithelial characteristics (e.g., the presence of adult stem-like cells in germinative zones).
Ur-epithelial traits would seem to be the direct source of proto-epithelium (glandular epithelium if not glandular tumors[269]) exhibiting cache-like cell division: universal cell division in differentiated parenchymal cells. Rates of cell division are regulated at least in part by cell-position in an organ and, hence by access to resources. But population size is also regulated by cell loss under feedback constraints much like budding in Hydra.[270], [271]
The evolution of constraints on cell division may have begun with the adoption by proto-epithelia of connective tissue-like cell cell division. Meta-epithelia may then have evolved by exchanging adult stem-like cell division for cache-like cell division. Clones of TACs formed from the progeny of asymmetric division in ASCs would then have opened up vast areas for evolutionary exploitation by terminally differentiated cells. The size of a meta-epithelial tissue might then be regulated by the number of niches available and filled by ASCs coupled to the rate of differentiation and cell loss rather than feedback from dead tissue.
Connective tissue may have been derived from ameboid cells, a plasmodium, or pseudoplasmodium that retained the ability to invade other tissues as far as their basal (external) lamina. Loose connective tissue fibroblasts in situ are long-lived and their cell division highly constrained but hardly immortal. Indeed, in vitro the cells proliferate wildly, but even there, they are limited and, after a more or less precise number of cell divisions (Hayflick limit) they enter senescence and die in tissue culture.
The mixing of cell features might account for the transitions between epithelial-like and connective tissue-like cells during development. For example, embryonic dermo-myotome cells de-epithelialize into fibroblast-like cells prior to becoming muscle, mesenchyme, and bone formation, and neural crest cells de-epithelialize from the edges of the forming neural folds, wander as fibroblast-like cells throughout the organisms, and settle down as melanocytes, ganglion cells, and sensory cells of the peripheral nervous system.
Like proto-epithelium, cellular muscle (smooth and cardiac muscle) retains cellularity and exhibits universal cell division among differentiated cells resembling cache cells. Striated muscles in vertebrates constitutes a unique class of syncytial/reserve muscle in which undividing fibers are accompanied by reserve (satellite) cells resembling highly constraind ASCs.
Like epithelia and muscle, neurons have a basal lamina. A close relationship with muscle is suggested, moreover, by nerves excitability. But nerve has its own peculiar type of terminal junctions and both neuroblasts and their processes are migratory and, hence, more like connective tissue than sessile epithelia or differentiated muscle. Curiously, like connective tissue connecting blood vessels to epithelia, nerve tissues astrocytes connect neurons to blood vessels, but unlike fibroblasts, astrocytes form a blood-brain barrier excluding most elements in plasma from cerebrro-spinal fluid. The spinning of myelin sheaths by oligodendrocytes in the CNS and neurolemmocytes in the PNS have no counterpart in connective tissue but may resemble the intimate lateral contact of striated muscle fibers with each other.
Vascular cells (blood and lymphocytes) would also seem to be derived from ur-connective tissue, but instead of merely contacting, vascular tissue invades and colonizes tissues, epithelia in the case of thymus and tonsils and connective tissue in the case of bone marrow, spleen, and intestine. Ironically, so-called hematopoietic stem cells, an early model for adult stem cells, are not ASCs (defined narrowly), sinse they do not exhibit label retention or self-renewing asymmetric division. Rather, HSCs are connective tissue-like leaving excess, left-over memory cells from clones—cells given another chance to proliferate and release their specific product in response to stimulation.
Germ-line tissue would also seem to be derived from an ur-connective tissue, since germ tissue invades and colonizes epithelia of the embryonic germinal ridge, but the origin of germ cells is still an enigma. Asymmetric division in germ line stem cells (GSCs) is reminiscent of ASCs in epithelia and suggestive of an evolutionary relationship with epithelial tissues. Moreover, the Piwi gene, highly expressed and required for asymmetric division in the germ cells of diverse organisms,[272] is also expressed in epithelial cells where it may be required for asymmetric division. Indeed, Cniwi, the Piwi homologue in the marine cnidarian Podocoryne carnea, is also up-regulated during the transdifferentiation of mitotically inactive striated muscle into mitotically active and differentiating smooth muscle[273] suggesting that Piwis expression in GSCs may be an expression of reprogramming (the erasure or resetting of epigenetic patterns in the genome)[274] rather than purely a function of asymmetric division.
In most animals, the germ-line separates from somatic-lines relatively early in development. PGCs are isolated during cleavage in nematodes[275] and are present in the mesenteries and germinal ridges of five to nine-week old human embryo.[276] One does not ordinarily think of their early departure from embryonic epithelium as deepithelialization nor of the move back into epithelial containers, follicles and tubules in the developing gonad as reepithelizalization. Rather, PGCs leave the impression of invasive cells colonizing the germinal ridge. Both impressions are consistent with different possibilities raised by the hybridization hypothesis for tissues.
Were an ur-connective tissue of isolated protistan-like cells or a plasmodium to have invaded and parasitized an ur-epithelial animal, the tissue destined to become germ tissue might invade the host to the core, namely, to chromosomal genes, ultimately condensing and consolidating all the genes present in a single genome. Such a re-packaging would provide the added advantage of allowing segregation and recombination during meiosis and fertilization. Of course, the purity of the original ur-tissues genome would be lost in the process, and the original genomes would never again be free agents capable of independent life, but, if the evolution of sexual reproduction is any indication, the ur-tissues would have struck a good bargain.
Of course, Williamsons larval transfer hypothesis with multiple cross-fertilizations encounters multiple objections, but the hypothesis answers two questions that make it attractive as an explanation for the evolution of tissues. The first question is why do tissues utilize such diverse ways of integrating their growth and homeostasis as contolling cell loss (cache cells), limiting cell division (ASC kinetics), and becoming static (fixed tissues and reserve cells). The answer virtually popping out of Williamsont hypothesis is that those were the controls, limits, and capacities of ur-tissues that came with them and were available to evolve during the integration that followed hybridizations.
The second question, in Williamson terms is, how does it so happen that larval parts of the genome are separate from adult parts, and larvae use larval parts of the genome, while adults uses adult parts without the two parts becoming twisted? Williamsons answer is that the two (or more) parts work separately because they originated separately and remain separate having only come together in the same animal via hybridization. Williamsons hypothesis, thus, explains separated domains on chromosomes by the fusion of separate genomes. Hybridization also explains the clustering of related genes in domains without having to invoke mysterious forces fomenting local gene duplication without global gene duplication.
In the case of tissues, domains, clusters, and gene complexes on chromosomes are not only separate, but the order of genes and the pattern of gene expression in the organism are frequently correlated (known as colinearity). For example, the 20 Hox-related genes in the starlet sea aneone, Nematostella vectinsis occur in nested domains on chromosomes and are expressed in distinct regions along the primary body axis. Remarkably, the biradially symmetrical anemone has a pattern of Hox genes on chromosomes and expresses them in body regions in a pattern reminiscent of dorsoventral patterning in bilateral animals (i.e., supporting the existence of a pre-bilaterian Hox code[277]).
Indeed, The [eumetazoan] ancestor had blocks of linked genes that remain together in the modern human and anemone genomes—the oldest known conserved synteny outside of prokaryotic operons.[278] One can be forgiven for speculating that the bilaterian Hox code was inherited from a bilateral ancestor that happened to hybridize with a radial ancestor creating a bi-radial anemone in the process.
In effect, the two classes of ancestral tissues proposed here, indigenous and exogenous, might have been united by some mechanism analogous (or homologous) to Williams hypothesis of larval transfer. The tissues, distinguished by the attachment of cells to a basal or external lamina, might have been unified in a single organism by some mechanism akin to hybridization. Indigenous tissues mounted on or enclosed by a basal or external lamina would then have evolved into epithelia and their derivative muscle and nerve, while exogenous tissues lacking a lamina might have evolved into connective tissue, blood cells, lymphocytes, and germ cells.
Today, parenchymas of indigenous tissues are epithelia, muscle, and nerve tissue. In vertebrates, these tissues are in intimate contact through their basal or external lamina. The parenchymal cells are radially symmetrical along an axis: epithelial cells resting on a basal lamina are anatomically polarized along an apical-basal axis; neurons enclosed by an external lamina are functionally polarized by the direction of excitation; muscle, also enclosed by its external lamina is functionally polarized by dendritic spines and motor endplates and the like.
Exogenous tissues lack a lamina and form extracellular matrix that frequently separates cells (osteocytes in cancellous bone making contact through gap junctions are an exception). In vertebrates, connective tissue, blood cells and lymphocytes, and germ tissue are exogenous tissues. They all interact with other tissues: Connective tissue fuses with the basal or external lamina of indigenous tissues and forms their stroma; blood cells and lymphocytes invade and colonize epithelia and connective tissue; and primoridial germ cells invade and colonize the embryonic germinal ridge only to become encapsulated by epithelium.
Indigenous and exogenous tissues exhibit different methods of normal tissue maintenance and repair. Cell division occurs within the confines of indigenous tissue and is balanced by cell loss and death. Indigenous tissue rely on cell division among their own kind, whether differentiated cache and cache-like cells or self-renewing adult stem cells that give rise to dividing transit amplifying cells[279]; exogenous tissues rely on mobilizing unrelated cells locally and recruiting additional cells from remote sites via a combination of coordinated local and systemic stimuli.
Of course, the notion that genomes are combined, whether through cross-fertilization or some other mechanism, means that genomes can also cooperate, and indigenous and exogenous tissues display a degree of integration. For example, during bone remodeling, osteocytes (ostensibly members of an exogenous tissue) behave like cache cells (members of an indigenous tissue). But germ cells represent by far the greatest degree of integration, as indeed, they should inasmuch as they will ultimately reproduce the entire spectrum of indigenous and exogenous tissues. Embryonic cells and induced pluripotent stem cells are not pluripotent so much as they are sharing the fundamental qualities of an unencumbered, hybridized genome.
Ultimately, the value of language is communication, and the value of communication is provoking thought. If (Re)Defining Tissues is successful, it will provoke research in classifying tissues, in unraveling mechanisms of tissue dynamics (steady-state, statics, recruitment), and in the roles of different types of dividing cells in growth and homeostasis, normal and disease states, turnover and healing, aging and regeneration.
[1] The style and usage employed are consistent with guidelines in Nomina Histologica Third Edition (by International Anatomical Nomenclature Committee; 1977). Hence, the text contains no eponyms. In keeping with American spelling, however, diphthongs are reduced, and singular and plural endings are Anglicized.
[2] Note: It was the authors intention to cite only articles available freely on line. All other literature cited is from the aurhors library and reprint collection.
[3] 2307 Pittock Street, Pittsburgh PA 15217, e-mail: sshostak@pitt.edu
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[279] The traditional adage that dividing cells are not differentiated and differentiated cells do not divide may still apply, if loosely, to slowly dividing undifferentiated ASCs and rapidly dividing TACs. But the adage breaks down completely with cognate cells, since cache cells appear differentiated but divide actively, and reserve cells are morphologically undifferentiated yet do not ordinarily divide.