Toward a new histology
Amazingly, histology has remained the same as the world has changed. Histologists are in thrall to Virchow but he is only histology's patron saint; he is not its deity. Let us break the bonds of epithelia, connective tissue, muscle, and nerve that bind histologists to an arcane nomenclature! Certainly, germ, blood, and lymphatic cells are entitled to recognition as tissues. Above all, different mechanisms of development should be incorporated into a new histological nomenclature, and, where possible, the evolution of tissues should be acknowledged among their identifying features. Genomics and informatics will provide data, but histologists must provide the impetus!
Please join me in creating a rational new histology where tissues are named, discussed, understood, and taught in ways that reflect what is known and not merely what was known.
11 comments:
I like the idea of hierarchical blood cells as colonizers in alternate
tissues but.. Gilbert Smith
Since histology emerged around a tool (microscope), I guess the first question is what tools should the 21st century histologist master?
Ola Histo master.
Couldn't stay retired? Go fishing?
Mall walking? Play chess in the park?
Ya had to stick your finger back into the bio pot and stir it up.
I think I have what might be an answer to Daniel's question.
UNM has a telescope. You just look into the big end of the scope. It would be the biggest microscope around.
I'm thinking of posting my entire (Re)Defining Tissues paper.
(Re)Defining Tissues ,
by
Stanley Shostak
University of Pittsburgh
Pittsburgh, PA
USA
Reclassifying Adult Tissues
Histology’s 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, 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. 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). 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.
(Re)defining tissues (continued 1)
The Ambiguous Stem Cell
“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. The term became entrenched in this context after E. B. Wilson used it in his historic The Cell , 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, and this germ-cell usage carried over from regulative embryonic cells to the celebrated embryonic stem cell (ESC) of tissue culture fame. Today, pluripotency has been reinvented for so-called induced pluipotent stem cells (iPSCs) , with their glowing promise “to transform regenerative medicine.”
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” and have only limited potency. Indeed, even the well-established regulator of pluripotency in ESCs, the Oct4 transcription factor is “dispensable” in ASCs.
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. Indeed, claims for great potency in ASCs are probably exaggerated and certainly controversial.
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.” 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.” Indeed, thirty years ago, “What fraction of the proliferative pool of cells in epithelial tissues functions is stem cells … [was] uncertain.’ 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” ,
(Re)defining tissues (continued 2)
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.” 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.
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. 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 Types of Proliferative Cells
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., β1 integrins in epidermis), generate specific cocktails of factors regulating cell fates and transitions between fates (including microRNAs targeting messenger RNAs and translation ), and display precise epigenetic conditions such as the configuration of the methylome. , 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.
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. Furthermore, these genes and their cognates are present in mice and humans, and many of them provide valuable and useful cues for enrichment protocols. The goal of reaching consensus on a list of stem cell genes has proven elusive, however, and a contemporary list of markers does not constitute a reliable “molecular signature” for stem-cells generally.
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.
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?
(Re)defining tissues (continued 3)
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.
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. The SP fraction of low Hoechst 33342 cells in viable murine bone marrow cells comprises 0.05–0.10%. 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.
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 that could be involved in the maintenance and/or control of ASC behavior.
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. At best, cells expressing stem cell markers may be located in sites qualifying as “pool/niches” as opposed to “pure” niches.
(Re)defining Tissues (continued 4)
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 β-1 integrins and binding to matrix, 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.”
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. , 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. , ,
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.
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. 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 organ’s 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 , (macrophages [hepatic dendritic cells] are thought to be recruited from bone marrow as well).
(Re)defining tissues (Continued 5)
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 β-1 integrins and binding to matrix, 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.”
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. , 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. , ,
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.
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. 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 organ’s 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 , (macrophages [hepatic dendritic cells] are thought to be recruited from bone marrow as well).
(Re)defining tissues (Continued 6)
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. Moreover, like TACs, HSCs respond to growth factors and cytokines. Hence, HSCs are not ASCs!
HSCs (aka mammal’s common myeloid progenitors [CMPs] and similar cells in Drosophila ) 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.
(and there's much more: if you're interested get in touch with me at sshostak@pitt.edu)
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