Cell proliferation in vivo and in vitro in visceral organs of the adult newt, Triturus cristatus carnifex

The mitotic and labelling incidence of intestine, liver, spleen and pancreas cells of Triturus cristatus carnifex adults kept at 15°C, 20°C, 25°C and 30°C were examined. Intestine mitotic and labelling incidences were highest at 25°C and lowest at 30°C. There was no significant difference between 15°C and 20°C. No such relationship could be shown for liver, spleen or pancreas, which had very much lower mitotic and labelling incidences. In culture, intestine mitotic and labelling incidences fell significantly within the first four hours, and maintained these low levels for the next five days. In contrast, liver mitotic and labelling incidences rose for 9–11 days, and then began to fall, while pancreas mitotic and labelling incidences reached peak values at day 5, and were kept in good condition for up to 14 days.     

Prospective isolation of adult neural stem cells from the mouse subependymal zone

Neural stem cells (NSCs) have the remarkable capacity to self-renew and the lifelong ability to generate neurons in the adult mammalian brain. However, the molecular and cellular mechanisms contributing to these behaviors are still not understood. Now that prospective isolation of the NSCs has become feasible, these mechanisms can be studied. Here we describe a protocol for the efficient isolation of adult NSCs, by the application of a dual-labeling strategy on the basis of their glial identity and ciliated nature. The cells are isolated from the lateral ventricular subependymal zone (SEZ) of adult hGFAP-eGFP (human glial fibrillary acidic protein-enhanced green fluorescent protein) transgenic mice by fluorescence-activated cell sorting. Staining against prominin1 (CD133) allows the isolation of the NSCs (hGFAP-eGFP(+)/prominin1(+)), which can be further subdivided by labeling with the fluorescent epidermal growth factor. This protocol, which can be completed in 7 h, allows the assessment of quantitative changes in SEZ NSCs and the examination of their molecular and functional characteristics.     

Ependymal/subependymal zone cells of postnatal and adult songbird brain generate both neurons and nonneuronal siblings in vitro and in vivo

The songbird forebrain continues to generate neurons in adulthood, from precursor cells located in the ependymal/subependymal zone (SZ) over the mediocaudal neostriatum. Precursor mitosis is followed by migration of neuronal daughter cells into the underlying forebrain, along radial fibers derived from the SZ. To define the ontogeny of both the new neurons and their radial guide cells, we employed retroviral insertion of the lacZ gene into neostriatal SZ precursor cells derived from postnatal and adult songbirds. We found that single SZ cells generate both neurons and substrate glia in vitro, and in an analogous fashion, both neurons and radial cells in vivo. This suggests that newly generated neurons and radial cells of the adult avian brain derive from a common pluripotential progenitor. © 1996 John Wiley & Sons, Inc.     

Effects of progesterone derivatives, dihydroprogesterone and tetrahydroprogesterone, on the subependymal layer of the adult rat

Indirect evidence suggests that in the subependymal layer (SEL) steroid hormones could be partially involved in the modulation of neurogenesis, but little or nothing is known about a direct effect of these molecules on this cellular system. The possible effect of progesterone (P) and/or its neuroactive metabolites, dihydroprogesterone (DHP) and tetrahydroprogesterone (THP), on the two cellular components of the SEL (i.e., proliferating/migrating neuroblasts and protoplasmic astrocytes) has been analyzed in adult male rat. P, DHP, and THP were administered by intraventricular injections and after 2 days the SEL was analyzed by immunohistochemistry by using anti-glial fibrillary acidic protein (GFAP) and anti-vimentin antibodies, to label the glial compartment, anti-polysialylated form of the neural cell adhesion molecule (PSA-NCAM), anti-Stathmin, and anti-beta III Tubulin antibodies to label the migrating neuroblasts. Furthermore, the newly formed cells were identified by using intraventricular injections of 5-bromo-2'-deoxyuridine (BrdU) detected immunohistochemically. Our results demonstrate that DHP and THP treatments drastically decrease the number of BrdU-labeled cells within the SEL. THP, DHP, and to a lesser extent P, administrations also induce molecular and structural modifications of the SEL glial compartment. On the whole, the present results indicate that neuroactive derivatives of P (i.e., DHP and THP) exert direct effects on adult neurogenesis, strongly affecting both neuroblasts and astrocytes of the SEL.     

Comparison of growth and cell proliferation kinetics during mouse molar odontogenesis in vivo and in vitro

Abstract. The growth of embryonic first lower mouse molars in vitro was slow and reduced in comparison with in vivo development: the volume of teeth removed on day 15 and 16 of gestation and cultured for 6 days did not exceed the volume reached at day 18 in vivo . The volume of teeth removed on day 17 and 18 and cultured for 6 days either remained constant or decreased. The appearance of post-mitotic odontoblasts and ameloblasts was delayed in vitro . This behaviour might be correlated with a lengthening of the cell cycle ( T _c). In vitro , the average durations of T _c (established by the percentage labelled mitoses technique) were 17.4–20.2 hr and 19.1–19.4 hr for pre-odontoblasts and pre-ameloblasts respectively. In vivo , the corresponding T _c values were 13.9 hr and 13.5 hr. The coordination of mitotic activities of pre-odontoblasts and pre-ameloblasts existing in vivo was maintained in vitro , and therefore seemed to require intra-dental control mechanisms. Non-specific extra-dental serum factors may affect the duration of T _c.     

Comparison of growth and cell proliferation kinetics during mouse molar odontogenesis in vivo and in vitro

The growth of embryonic first lower mouse molars in vitro was slow and reduced in comparison with in vivo development: the volume of teeth removed on day 15 and 16 of gestation and cultured for 6 days did not exceed the volume reached at day 18 in vivo. The volume of teeth removed on day 17 and 18 and cultured for 6 days either remained constant or decreased. The appearance of post-mitotic odontoblasts and ameloblasts was delayed in vitro. This behaviour might be correlated with a lengthening of the cell cycle (Tc). In vitro, the average durations of Tc (established by the percentage labelled mitoses technique) were 17.4-20.2 hr and 19.1-19.4 hr for pre-odontoblasts and pre-ameloblasts respectively. In vivo, the corresponding Tc values were 13.9 hr and 13.5 hr. The coordination of mitotic activities of pre-odontoblasts and pre-ameloblasts existing in vivo was maintained in vitro, and therefore seemed to require intra-dental control mechanisms. Non-specific extra-dental serum factors may affect the duration of Tc.     

Cell proliferation in the germinal layer of the lateral brain ventricles in normal adult mice and under some experimental conditions.

Histological and autoradiographic studies revealed mitotic activity in the subependymal germinal layer in the brains of adult mice. The number of mitoses observed was higher in animals subjected to administration of haematoporphyrin and selenourea.     

Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro

Although possible biological functions of whey acidic protein (WAP) have been suggested, few studies have focused on investigating the function of WAP. This paper describes evidence for WAP function in lobulo-alveolar development in mammary glands in vivo and in the cell cycle progression of mammary epithelial cells in vitro. Ubiquitous overexpression of WAP transgene impaired only lobulo-alveolar development in the mammary glands of transgenic female mice but not other physiological functions, indicating that the inhibitory function of WAP is specific to mammary alveolar cells. The forced expression of WAP significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 cells and EpH4/K6 cells), whereas it did not affect that of NIH3T3 cells. Co-culturing of WAP-clonal cells and control cells using a transwell insert demonstrated that WAP inhibited the proliferation of HC11 cells through a paracrine action but not that of NIH3T3 cells, and that WAP was able to bind to HC11 cells but not to NIH3T3 cells. Apoptosis was not enhanced in the HC11 cells with stable WAP expression (WAP-clonal HC11 cells). BrdU incorporation and FACScan analyses revealed that cell cycle progression from the G0/G1 to the S phase was inhibited in the WAP-clonal HC11 cells. Among G1 cyclins, the expression of cyclin D1 and D3 was significantly decreased in the WAP-clonal HC11 cells. The present results provide the first documented evidence that WAP plays a negative regulatory role in the cell cycle progression of mammary epithelial cells through an autocrine or paracrine mechanism in vivo.     

Cell lineage in the cerebral cortex of the mouse studied in vivo and in vitro with a recombinant retrovirus

To analyze cell lineage in the murine cerebral cortex, we infected progenitor cells with a recombinant retrovirus, then used the retroviral gene product to identify the descendants of infected cells. Cortices were infected on E12-E14 either in vivo or following dissociation and culture. In both cases, nearly all clones contained either neurons or glia, but not both. Thus, neuronal and glial lineages appear to diverge early in cortical development. To analyze the distribution of clonally related cells in vivo, clonal boundaries were reconstructed from serial sections. Perinatally (E18-PN0), clonally related cells were radially arrayed as they migrated to the cortical plate. Thus, clonal cohorts traverse a similar radial path. Following migration (PN7-PN23), neuronal clones generally remained radially arrayed, while glial clones were variable in orientation, suggesting that these two cell types accumulate in different ways. Neuronal clones sometimes spanned the full thickness of the cortex. Thus, a single progenitor can contribute neurons to several laminae.     

Viability of mouse half-embryos in vitro and in vivo

Mouse embryos at the 2-, 4-, 8-cell, and morula stage were divided in half by using microsurgical procedures and were either grown in vitro up to the blastocyst stage or transferred at the late morula stage into the uteri of pseudopregnant recipients. A relatively high percentage of the half embryos from 2-cell (70%), 4-cell (75%), 8-cell (93%), or morula stage embryos (75%) developed into blastocysts in vitro. However, the overall development in vivo of half embryos was low, as 3%, 13%, 8%, and 1% of half embryos from the 2-cell, 4-cell, 8-cell, and morula stages, respectively, developed into live fetuses. Embryos which were divided in half at different stages developed at different rates in vitro. This determined the stage of embryonic development at the time of transfer, which might have interacted with the stage of pseudopregnancy of the recipients to influence embryo survival in vivo.     

BAG-1 is preferentially expressed in neuronal precursor cells of the adult mouse brain and regulates their proliferation in vitro

BAG-1 protein has been well characterized as necessary for proper neuronal development. However, little is known about the function of BAG-1 in the adult brain. In this work, the expression and localization of BAG-1 in the mature mouse brain was studied. The levels of both BAG-1 isoforms decrease significantly in the brain during development. BAG-1 was found preferentially expressed in Neuronal Precursor Cells (NPCs) in the two major niches of neurogenesis. Lentiviral mediated overexpression of BAG-1 increased the proliferation rate of cultured NPCs. In addition, depletion of BAG-1 from NPCs induced a decrease in NPCs proliferation in the presence of a stress hormone, corticosterone. These data suggest a role for BAG-1 in mechanisms of neurogenesis in the adult mouse brain.     

In vitro proliferation of adult human beta-cells

A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1) analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched ("beta-cells") population were plated on extracellular matrix from rat (804G) and human bladder carcinoma cells (HTB9) or bovine corneal endothelial ECM (BCEC). Cells were maintained in culture+/-liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted) or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted), independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05).These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.     

Aging of mouse eggs in vivo and in vitro

Morphological and cytochemical (acid phosphatase) changes associated with mouse ova and cumulus cells aged within the oviducts (in vivo) or in culture (in vitro; 1–24 hours postovulation) have been investigated. Structural alterations of cumulus cells were apparent immediately after ovulation and included nuclear pycnosis and cytoplasmic vacuolization. Nevertheless, approximately 30% of the cumulus masses examined contained cells that plated out when cultured and remained viable for up t o three days in vitro. From 12 t o 24 hours postovulation almost all cumulus cells of specimens aged in vivo showed signs of degeneration. Disruption of the meiotic spindle and an increase in acid phosphatase positive organelles were characteristic of in vivo and in vitro aging ova. The percentage of fragmented eggs obtained from super-ovulated (5 IU PMS followed by 5 IU HCG) mice approximately one and 24 hours postovulation was not significantly different. Eggs obtained from superovulated animals and aged in vitro for 24 hours yielded significantly more fragmented ova. Fragmented eggs were not obtained from cycling females on the morning of estrus. When such eggs were cultured in vitro for 24 hours the percent fragmentation was significantly lower than that for aged eggs obtained from super-ovulated mice. These results indicate that 1) similar morphological alterations occur among cumulus cells and eggs aged either in vitro or in vivo, 2) ova from superovulated mice do not constitute a homogeneous population and 3) the method of superovulation employed in this study induces the ovulation of a relatively large group of eggs that are susceptible to fragmentation when cultured in vitro.     

A cell-survival factor (N-acetyl-L-cysteine) alters the in vivo fate of constitutively proliferating subependymal cells in the adult forebrain

The adult mouse brain contains a population of constitutively proliferating subependymal cells that surround the lateral ventricle and are the direct progeny of the neural stem cell. Constitutively proliferating cells divide rapidly; 6 days after labeling, 60% of their progeny undergo cell death, 25% migrate to the olfactory bulbs, and 15% continue to proliferate within the subependyma. We have intraventricularly infused a cell survival factor N-acetyl-L-cysteine (NAC), which is known to have survival effects without concomitant proliferative effects on cells in vitro, and examined the resulting fate of cells spared from the normally occurring cell death. NAC infusion for 5 days results in a five-fold increase in the number of retrovirally labeled subependymal cells compared to saline-infused controls. The increase in the number of subependymal cells is directly proportional to the amount of time during which NAC is present and is not due to increased proliferation. While NAC is able to keep all the normally dying progeny alive, the cells spared from death remain confined to the subependyma lining the lateral ventricles and do not migrate to the olfactory bulbs (one normal fate of constitutively proliferating progeny) or into the surrounding brain parenchyma. When animals survive for an additional 6 days following NAC infusion, the number of retrovirally labeled subependymal cells returns to control values, indicating that the continued presence of NAC is necessary for cell survival. These data suggest that preventing cell death is not sufficient to keep all of the progeny of these cells in a proliferative mode.     

Cell proliferation in mouse tissues after thymectomy and the administration of T-activin

The epithelium of mouse cornea and lymph nodes was examined for DNA-synthetic and mitotic activity at different times after thymectomy and administration of T-activin, an active factor of the thymus. Thymectomy entails retardation of the rate of corneal epithelium regeneration, diminution in both tissues under study of the amplitude of oscillations in cell proliferation throughout the day. Administration to the animals of the immunoactive thymic factor T-activin makes the circadian rhythm of cell proliferation return to normal. It is assumed that T-activin raises the capacity of lymphocytes to interact with epithelial cells, which manifests itself in the enhancement of their mitotic activity.     

Parvalbumin in mouse muscle in vivo and in vitro

Parvalbumin is a cytosolic calcium-binding protein found in adult fast-twitch mammalian muscle. Using an antibody to paravalbumin, we have shown that its distribution in adult mouse muscles is associated with certain fibre types. It is absent from slow-twitch type 1 fibres, is absent or at low levels in fast-twitch type 2A fibres, but is present at moderate or high levels in fast-twitch type 2B fibres. When adult mouse muscle is cultured with embryonic mouse spinal cord, the regenerated fibres become innervated, express the adult fast isoform of myosin heavy chain and appear histochemically as fast-twitch fibres. We therefore investigated whether these apparently mature fibres also contained parvalbumin. Parvalbumin was not found in any fibres of twenty mature cultures, suggesting that neurotrophic activity in the absence of specific adult nerve activity patterns was insufficient to cause the expression of parvalbumin in the cultures.     

Cell proliferation and differentiation in the fetal and early postnatal mouse thymus

The relationships between cell proliferation and cell differentiation during thymus ontogeny were studied by labeling DNA-synthesizing thymocytes with bromodeoxyuridine and staining with antibodies against CD4, CD8, J11d, phagocytic glycoprotein 1, TCR V beta 8 chain, Thy-1, and IL-2R surface proteins. The development of the thymus was discontinuous, with two well defined growth periods from 13 days to 18 days of fetal life and from 3 days to 6 days after birth, and more progressive growth from day 8 to 2 wk. Cell proliferation started on fetal day 12, 1 day after the arrival of hemopoietic stem cells in the third branchial pouch. These cells were phagocytic glycoprotein 1-positive but IL-2R and Thy-1 negative. Thus, cell proliferation preceded IL-2R expression. Until day 15, CD4-8- thymocytes expanded without differentiation. Then CD4-8+ and CD4+8+ cells appeared; this induction was proliferation dependent and occurred on cells which had already lost IL-2R, but just after maximum expression of this receptor. During several days, the thymus remained of constant size (around 10(7) cells) and behaved like the steady state thymus. On day 3 after birth, expansion started again and was correlated with an increase in CD4-8- proliferation index and IL-2R expression. At the same time, the thymic subset capable of expansion without differentiation was again, transiently, detectable. These results suggest that the inflow of precursor cells into the thymus is permanent but transiently increased at several times during ontogeny. Moreover, the behavior of fetal CD4-8- cells does not appear radically different from that of adult precursors, but the actual difference resides in the variation of the relative proportion of CD4-8- cells at different maturation stages, as revealed by striking variations of IL-2R expression by cycling cells.     

Temperature-induced cell proliferation in mouse ear epidermis in vivo

Increasing environmental temperature of the animal room from 22 to 35 °C for 5 h induces cell proliferation in mouse ear epidermis in vivo. High temperature acts specifically during the G2 period of the cell cycle in initiating mitosis in mouse ear epidermal cells. Both cycling and noncycling G2 blocked ear epidermal cells are released from the G2 period into mitosis during the course of heat treatment.