PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

Coexistence of perinatal and adult forms of a glial progenitor cell during development of the rat optic nerve

We have studied the developmental appearance of the O-2A(adult) progenitor cell, a specific type of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell that we have identified previously in cultures prepared from the optic nerves of adult rats. O-2A(adult) progenitors differ from their counterparts in perinatal animals (O-2A perinatal progenitor cells) in antigenic phenotype, morphology, cell cycle time, rate of migration, time course of differentiation into oligodendrocytes or type-2 astrocytes and sensitivity to the lytic effects of complement in vitro. In the present study, we have found that O-2A(adult) progenitor-like cells first appear in the developing optic nerve approximately 7 days after birth and that by 1 month after birth these cells appear to be the dominant progenitor population in the nerve. However, the perinatal-to-adult transition in progenitor populations is a gradual one and O-2A(adult) and O-2A perinatal progenitors coexist in the optic nerve for 3 weeks or more. In addition, cells derived from optic nerves of P21 rats express characteristic features of O-2adult and O-2A perinatal progenitors for extended periods of growth in the same tissue culture dish. Our results thus indicate that the properties that distinguish these two types of O-2A progenitors from each other are expressed in apparently identical environments. Thus, these cells must either respond to different signals present in the environment, or must respond with markedly different behaviours to the binding of identical signalling molecules.     

The relationship between cell division and melanocyte differentiation in epidermal cultures from mouse embryos

Cultures of 14-day embryonic mouse epidermis that include melanoblasts initiate melanin synthesis 30 hr after plating, a schedule that is 2.5 days earlier than in vivo. In order to determine if the accelerated differentiation of melanoblasts is related to a cessation of cell proliferation in the cultures, a study of [³H]thymidine incorporation by melanoblasts and melanocytes was made. Autoradiograms of 14-day epidermal cultures grown for 48 hr in medium containing [³H]thymidine revealed that melanoblasts continue to proliferate during this time period. A second population of melanoblasts that did not incorporate [³H]thymidine was also present in these cultures. The relative numbers of dividing and nondividing melanoblasts change with the age of the epidermis cultured. Ninety-one percent of the melanoblasts in 13-day epidermis take up [³H]thymidine, 63% incorporate [³H]thymidine in 14-day cultures, and only 29% take up label in cultures of 15-day epidermis. It appears from these results that melanoblasts during their migration from the neural crest are proliferative cells and that during the early invasion of the epidermis a nonproliferative population of melanoblasts is established. Both populations coexist in the epidermis and subsequently undergo differentiation on the same time schedule.     

The relationship between nerves and smooth muscle cells in the rat iris

Summary The dilatator muscle cells form short projections into the stroma of the iris. Close to these projections run several nerve bundles. The unmyelinated axons often show enlargements (varicosities) containing mitochondria and vesicles. Several of the varicosities are partly denuded of the Schwann cell and are covered only by a basement membrane. The varicosities are then separated from the muscle cells only by basement membranes and a 0.1–1 stromal space. The ultrastructure of the iris dilatator muscle thus also fits the view that the autonomic ground plexus with its varicosities forms the real innervation apparatus.The smallest space between axon and muscle has a width of 700–900 Å and is cemented with basement membrane material. It is suggested that the main function of these contact sites is not to transmit a nerve impulse but to anchor the nerves to their effector organ.This study has been supported by grants from the Swedish State Research Council (U 267) and the United States Public Health Service (N B 2854-04).     

Heterotypic and homotypic cellular interactions influencing the growth and differentiation of bipotential oligodendrocyte-type-2 astrocyte progenitors in culture

Cell populations highly enriched in oligodendrocyte-type-2 astrocyte (O-2A) progenitors (so defined by their ability to bind the monoclonal antibodies LB1 and O4, and by the lack of expression of the differentiated glial markers galactocerebroside and glial fibrillary acidic protein (GFAP) were obtained from rat mixed cortical glial cultures. The O-2A progenitors were grown at low density (2 X 10(4) cells/cm2) in BME + 10% fetal calf serum (FCS) on a poly-L-lysine (PLL) substrate (controls) or on a substrate of purified type-1 astrocytes (AS) killed by air drying (K-AS), in order to analyze the effects of the interaction between the two cell types on the growth and differentiation of the immature O-2A cells, independently of the mitogenic soluble factors (e.g., platelet-derived growth factor; see Raff, 1989, Science 243, 1450-1455) secreted by type-1 AS. While on PLL most of the progenitors differentiated into GFAP+ type-2 AS within 1 week, on K-AS they largely differentiated into GalC+ oligodendrocytes (OL). On the latter substrate, however, the precursors achieved a higher density, due to higher proliferative activity. The additional observation, that when immature O-2A cells were seeded at high density (greater than 5 X 10(4) cells/cm2) on PLL their differentiation into OL was much more pronounced than in cultures of lower density, indicates that there is a close correlation between the density of immature O-2A cells and lineage decision, and that the increased OL differentiation of the immature O-2A cells on K-AS is at least partly related to the higher density achieved by the cells on this substrate. The enhanced proliferation of immature O-2A cells on K-AS did not appear to be related to platelet-derived growth factor or fibroblast growth factor remaining attached to the substrate, nor to known components of the extracellular matrix (ECM), such as heparan sulfate, chondroitin sulfate, laminin, or fibronectin, but was probably due to other components of a polypeptide nature present in the ECM produced by type-1 AS. A cell-free ECM was in fact almost as mitogenic as the K-AS substrate, and the mitogenic activities of both K-AS and AS-ECM were similarly inhibited by a set of enzymatic (pronase, trypsin) and physicochemical (heat, pH) treatments.     

The relationship between nerves and smooth muscle cells in the rat iris

Summary The sphincter muscle in the rat iris forms irregular strands in the stroma. Bundles of unmyelinated axons run among the muscle cells. After sympathetic denervation some axons degenerate. This should indicate that sympathetic and parasympathetic nerves are present in the same nerve net. The parasympathetic axons possess varicosities, that is, enlargements containing mitochondria and synaptic vesicles. These varicosities show a similar structural relationship to the muscle cells as do the varicosities of sympathetic nerves. No obvious ultrastructural difference is observed between the sympathetic and parasympathetic varicosities.This study has been supported by research grants (U267 and Y247) from the Swedish Medical Research Council and by a Public Health Service Research Grant (NB05236-01) from the National Institute of Neurological Diseases and Blindness.     

Infection by coronavirus JHM of rat neurons and oligodendrocyte-type-2 astrocyte lineage cells during distinct developmental stages.

Primary telencephalic cultures derived from neonatal Wistar Furth rats were able to support the growth of coronavirus JHM if a viable neuronal population was maintained. This occurred under serum-free defined, but not serum-supplemented, growth conditions. The importance of neurons in establishing infections in mixed cultures was confirmed by immunocytochemical and electron microscopic studies. Glia, although more abundant than neurons in these cultures, were less frequently infected during the initial 48 h postinoculation. The two glial lineages present in mixed telencephalic cultures were separated into type-1 astrocytes and oligodendrocyte-type-2 astrocyte (O-2A) lineage cells and individually assessed for their ability to support virus growth. Infection could not be established in type-1 astrocytes regardless of the culture conditions employed, consistent with our previous study (S. Beushausen and S. Dales, Virology 141:89-101, 1985). In contrast, infections could be initiated in selected O-2A lineage cells grown in serum-free medium. Virus multiplication was however significantly reduced by preconditioning the medium with mixed telencephalic or enriched type-1 astrocyte cultures, suggesting that intercellular interactions mediated by soluble factor(s) can influence the infectious process in O-2A lineage cells. This presumption was supported by eliciting similar effects with basic fibroblast growth factor and platelet-derived growth factor, two central nervous system cytokines known to control O-2A differentiation. The presence of these cytokines, which synergistically block O-2A cells from differentiating into oligodendrocytes was correlated with specific and reversible resistance to JHM virus (JHMV) infection. These data, combined with our finding that accelerated terminal differentiation of the oligodendrocyte phenotype confers resistance to JHMV (Beushausen and Dales, Virology, 1985), suggest that the permissiveness of O-2A cells for JHMV is restricted to a discrete developmental stage.     

Proliferation, multipotency and neuronal differentiation of cryopreserved neural progenitor cells derived from the olfactory neuroepithelium of the adult rat

The use of olfactory neuroepithelium neural progenitor cells for transplantation has attracted attention in the treatment of many neurological disorders, which require efficient recovery methods and cryopreservation procedures. The purpose of this study was to evaluate different cryopreservation techniques for neural progenitor cells derived from the olfactory neuroepithelium (ONe NPCs) in adult rats. Initially, we compared the survival rates of cryopreserved ONe NPCs treated with six different cryoprotectants: dimethylsulfoxide (DMSO), ethylene glycol (EG) and glycerol, each with or without 10% FBS and with two different storage periods in liquid nitrogen (-196 degrees C), specifically 3 days short-term storage and 3 months long-term storage. We assessed the recovery efficiency of ONe NPCs after freezing and thawing by viability testing and colony-forming assay as well as immunocytochemistry under different conditions. No significant difference in the survival rate was observed among these different cryoprotectants. With these protocols, ONe NPCs retained their multipotency and differentiated into glial (GFAP-positive), neuronal (NeuN-positive) and oligodendroglia (Galc-positive) cells. Collectively, our results imply that, under optimal conditions, ONe NPCs might be cryopreserved for periods of >3 months without losing their proliferative and multipotency activities.     

The relationship between yeast cell size and cell division in Candida albicans

The mean size and percentage of budded and unbudded cells of Candida albicans grown in batch culture over a wide range of doubling times have been measured. Cell volume decreased with increased doubling time and a nonlinear approach to an asymptotic minimum was observed. When cells were separated by age according to bud scars, each age showed a similar decrease. During each cell division cycle, size increased slowly during both budded and unbudded periods so that each generation was significantly larger than the preceding. There was no difference in size between the parent portion of budded cells and unbudded cells of the same age. Time-lapse photomicroscopy of cells growing on solid medium showed that cells divide asymmetrically with larger parents having a shorter subsequent cycle time than the smaller daughter, although the time utilized for bud formation was similar. When cells were shifted from a medium supporting a low growth rate and small size to a medium supporting a faster growth rate and larger size, both budded and unbudded cells increased significantly in size. As the doubling time increased, both the budded and unbudded portions of parental and daughter cycles increased.     

The relationship between cell size and cell division in the yeast Saccharomyces cerevisiae.

Cell numbers in synchronous cultures of yeast cultured at fast growth rates increase from N to 2N after the first division and from 2N to 4N after the second division. At these fast growth rates, there are equal numbers of parents and daughters. In contrast, at slow growth rates the cell number increases from N to 2N after one division and from 2N to 3N rather than 4N after the second division. Moreover, the percentage of daughters increases with decreasing growth rate. Thus, slowly growing cultures actually consist of two sub-populations having different cell cycle transit times. These observations are predicted if a yeast cell requires a critical size before a particular cell cycle event can be completed and that after completion of this event cell division occurs following a period of time independent of growth rate.     

Repopulation of O-2A progenitor cells after irradiation of the adult rat optic nerve analyzed by an in vitro clonogenic assay.

In the central nervous system (CNS) O-2A (Oligodendrocyte type 2 Astrocyte) progenitor cells have been proposed as potential target cells, and their depletion by irradiation will cause demyelination. The extent and time course of repopulation of these glial stem cells were studied in the adult rat optic nerve after irradiation in vivo. The number of O-2A progenitor cells was measured quantitatively by an in vitro clonogenic assay. Although the CNS is typically a late-responding tissue, repopulation was initiated almost immediately after irradiation and after several weeks a plateau was reached that lasted up to 6 months. Single doses of 4-12 Gy of X rays caused a permanent reduction in the number of O-2A progenitor cells. An analysis of the colony size of O-2A progenitor cells showed a sustained reduction in the number of offspring of cells surviving a dose of 12 Gy. In addition, the colony size of unirradiated progenitors diminished with increasing age of the animals.     

The differentiation of oligodendrocytes in the rat optic nerve

The time of appearance and the rate of accumulation of specific myelin lipids and proteins were measured in the rat optic nerve during the period from birth to 18 days. The appearance of the activities of several enzymes involved in the synthesis of these lipids was also monitored. Correlation of these biochemical data with previously known morphological findings indicated that the “active” oligodendrocytes (detected between 5 and 15 days after birth) displayed maximal levels of synthesis of the components of myelin, and that these cells appeared to be responsible for the initial synthesis of myelin. Both young and mature oligodendrocytes showed limited capacity to synthesize these compounds. Furthermore, induction of the enzymes involved in the synthesis of myelin components appeared to take place in a simultaneous, rather than a sequential manner.     

The relationship between cell proliferation and differentiation and mapping of putative dental pulp stem/progenitor cells during mouse molar development by chasing BrdU-labeling

Human dental pulp contains adult stem cells. Our recent study demonstrated the localization of putative dental pulp stem/progenitor cells in the rat developing molar by chasing 5-bromo-2’-deoxyuridine (BrdU)-labeling. However, there are no available data on the localization of putative dental pulp stem/progenitor cells in the mouse molar. This study focuses on the mapping of putative dental pulp stem/progenitor cells in addition to the relationship between cell proliferation and differentiation in the developing molar using BrdU-labeling. Numerous proliferating cells appeared in the tooth germ and the most active cell proliferation in the mesenchymal cells occurred in the prenatal stages, especially on embryonic Day 15 (E15). Cell proliferation in the pulp tissue dramatically decreased in number by postnatal Day 3 (P3) when nestin-positive odontoblasts were arranged in the cusped areas and disappeared after postnatal Week 1 (P1W). Root dental papilla included numerous proliferating cells during P5 to P2W. Three to four intraperitoneal injections of BrdU were given to pregnant ICR mice and revealed slow-cycling long-term label-retaining cells (LRCs) in the mature tissues of postnatal animals. Numerous dense LRCs postnatally decreased in number and reached a plateau after P1W when they mainly resided in the center of the dental pulp, associating with blood vessels. Furthermore, numerous dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 and CD146. Thus, dense LRCs in mature pulp tissues were believed to be dental pulp stem/progenitor cells harboring in the perivascular niche surrounding the endothelium.     

Relationship between tracheary element differentiation and the cell cycle in single cells isolated from the mesophyll of Zinnia elegans

A relationship between tracheary element differentiation and the cell cycle was studied in single cells isolated from the mesophyll of Zinnia elegans L. cv. Canary bird. Almost all nuclei of isolated mesophyll cells were at the 2 C level of DNA, indicating that almost all cells were initially in the G₁ phase and that somatic polyploidy was absent. Cultured cells underwent partially synchronous DNA replication at 42 h and mitosis at 54 h of culture, and the first cell cycle time was approximately 58 h.
The occurrence and timing of DNA replication and mitosis during cytodifferentiation to tracheary elements were investigated using microspectrophotometry, microfluorometry, tritiated thymidine autoradiography, and serial observation. More than 55% of the nuclei of the immature tracheary elements were at the 2 C level of DNA and were not labeled by continuous feeding with tritiated thymidine, providing clear evidence that these cells differentiated without interventing DNA replication. Some tracheary elements (approximately 30%) were formed after one round of the cell cycle, and others (less than 5%) were formed after passing through the S phase, but without intervening mitosis. All types of tracheary elements appeared simultaneously after 58 h of culture, and their patterns of increase in number were similar. From the results, we propose a hypothesis concerning the relationship between cytodifferentiation and the cell cycle.