Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse

Development ◽  
1987 ◽  
Vol 101 (3) ◽  
pp. 627-652 ◽  
Author(s):  
K.A. Lawson ◽  
R.A. Pedersen

The fate of the embryonic endoderm (generally called visceral embryonic endoderm) of prestreak and early primitive streak stages of the mouse embryo was studied in vitro by microinjecting horseradish peroxidase into single axial endoderm cells of 6.7-day-old embryos and tracing the labelled descendants either through gastrulation (1 day of culture) or to early somite stages (2 days of culture). Descendants of endoderm cells from the anterior half of the axis were found at the extreme cranial end of the embryo after 1 day and in the visceral yolk sac endoderm after 2 days, i.e. they were displaced anteriorly and anterolaterally. Descendants of cells originating over and near the anterior end of the early primitive streak, i.e. posterior to the distal tip of the egg cylinder, were found after 1 day over the entire embryonic axis and after 2 days in the embryonic endoderm at the anterior intestinal portal, in the foregut, along the trunk and postnodally, as well as anteriorly and posteriorly in the visceral yolk sac. Endoderm covering the posterior half of the early primitive streak contributed to postnodal endoderm after 1 day (at the late streak stage) and mainly to posterior visceral yolk sac endoderm after 2 days. Clonal descendants of axial endoderm were located after 2 days either over the embryo or in the yolk sac; the few exceptions spanned the caudal end of the embryo and the posterior yolk sac. The clonal analysis also showed that the endoderm layer along the posterior half of the axis of prestreak- and early-streak-stage embryos is heterogeneous in its germ layer fate. Whereas the germ layer location of descendants from anterior sites did not differ after 1 day from that expected from the initial controls (approx. 90% exclusively in endoderm), only 62% of the successfully injected posterior sites resulted in labelled cells exclusively in endoderm; the remainder contributed partially or entirely to ectoderm and mesoderm. This loss from the endoderm layer was compensated by posterior-derived cells that remained in endoderm having more surviving descendants (8.4 h population doubling time) than did anterior-derived cells (10.5 h population doubling time). There was no indication of cell death at the prestreak and early streak stages; at least 93% of the cells were proliferating and more than half of the total axial population were in, or had completed, a third cell cycle after 22 h culture.(ABSTRACT TRUNCATED AT 400 WORDS)

1990 ◽  
Vol 258 (2) ◽  
pp. E368-E376 ◽  
Author(s):  
G. Shillabeer ◽  
J. M. Forden ◽  
J. C. Russell ◽  
D. C. Lau

We have investigated the in vitro rate of replication and differentiation of preadipocytes derived from lean (+/+) and obese (cp/cp) male JCR:LA-corpulent (cp) rats in an attempt to identify mechanisms that regulate adipose tissue growth. Cp/cp rats were twofold heavier than age-matched lean rats by 9-10 mo. Cp/cp-derived preadipocytes demonstrated an inherently slower rate of replication than +/+ preadipocytes (population doubling time: cp/cp 52.3 +/- 9.6 h vs. +/+ 19.7 +/- 1.6 h), although the preadipocyte pool in the cp/cp was significantly greater. Cp/cp preadipocytes were resistant to hormonally induced differentiation (19.9 +/- 9.4% of cells accumulated lipid) but differentiated when cocultured with mature adipocytes to the same extent as preadipocytes derived from Sprague-Dawley (SD) rats (cp/cp 48.4 +/- 15.2% vs. SD 52.2 +/- 11.9%). In contrast, SD preadipocytes did not differentiate in response to mature adipocytes from +/+ rats (13.8 +/- 5.2%). Our observations suggest that preadipocyte replication and maturation may not be controlled in a coordinated manner.


1995 ◽  
Vol 37 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Jeffrey I. Zwicker ◽  
Robert T. Proffitt ◽  
C. Patrick Reynolds

2017 ◽  
Vol 5 (1) ◽  
pp. 62-66
Author(s):  
O. Kalmukova ◽  
A. Ustymenko ◽  
T. Lutsenko ◽  
P. Klymenko ◽  
V. Kyryk

Nail unit is a complex anatomical structure that is capable of rapid growth and regeneration throughout the life. Such significant reparative potential is associated with the presence different types of stem and progenitor cells, whose biology remains one of the fundamental issues today. Taking into account the active search for new stem cell sources for cell therapy, the view of the nail unit as a potential site for the localization of undifferentiated cells with stem potency is topical problem.Purpose. The study was conducted with an objective to establish the morphological, morphometric and proliferative characteristics of cultured cells isolated from the mouse nail unit.Materials and methods. Primary cultures of cells were obtained from tissue sampling, which included areas of the proximal nail fold, nail matrix and onychodermis of the FVB mouse nail organ. Cells were cultured in DMEM:F12 medium with 15 % fetal bovine serum during 6 passages. We determined the colony-forming activity, the population growth rate and doubling time, measured the area of cells, nuclei, and calculated the nuclear-cytoplasmic ratio. For cell morphology analysis, we used staining with Bemer’s hematoxylin and eosin, Heidenhain’s iron hematoxylin and May-Grünwald stain.Results. According to the morphological analysis in vitro the cells from mouse nail unit are heterogeneous with high synthetic activity and a low nuclear-cytoplasmic ratio – the features characteristic of the low-differentiated cells. The population doubling time of the culture was 80 ± 6.5 hours on average, the fastest growing cells were at the 4th passage (63 ± 7 hours). The specific growth rate for cell culture is low (0.01 ± 0.0007).The colony forming efficiency at the 5th passage was only 4 %. A significant number of colonies was small with large poorly proliferative cells, which may indicate a production of large numbers of transitional progenitor cells.Conclusion. The obtained cell culture from the mouse nail unit according to the analysis of their morphology, morphometry and proliferative potential is heterogeneous and requires the further development of pure culture technologies for the detailed characterization of separate subpopulations of cells.


2011 ◽  
Vol 1 (2) ◽  
pp. 1-9
Author(s):  
Min Rahminiwati ◽  
Ita Juwita ◽  
Ani Murtisari ◽  
Latifah K Darusman

Kurkumin yang terdapat dalam rimpang temulawak, selain dapat menginduksi terjadinya proliferasi sel progenitor pada otak tikus dewasa juga dapat menghambat kerja enzim tirosinkinaseyang berperan penting dalam mengatur pertumbuhan dan diferensiasi sel. Meskipun demikianrespon sel saraf terhadap ekstrak temulawak pada masa pertumbuhan perlu kajian lebih lanjut. Efekekstrak temulawak terhadap proliferasi dan diferensiasi sel otak besar atau serebrum pada masapertumbuhan anak diteliti pada sel otak anak tikus Spague Dawley berumur tiga hari yangditumbuhkan dalam media DMEM (Dulbeccos Modified Eagles Medium). Perlakuan dikelompokkan dalam kelompok kontrol positif (mDMEM+30 g/mL asiaticoside (AC), kontrolnegatif (mDMEM), kelompok yang memperoleh ekstrak temulawak (CZ) 100 ppm (mDMEM+100ppm CZ), CZ 200 ppm (mDMEM+200 ppm CZ), dan CZ 400 ppm (mDMEM+400 ppm CZ). Kultur diinkubasi pada suhu 37 o C dalam inkubator CO 5 % selama 6 hari. Parameter yang diamatiadalah population doubling time, komposisi sel saraf dan sel glia, panjang akson dan dendrite yangdiukur masing masing menggunakan hemositometer, pewarnaan Hematoxyilin Eosin (HE) danmikrometer. Hasil penelitian menunjukkan ekstrak temulawak pada konsentrasi 100 ppmmemperlambat prolfperasi, pada konsentrasi 400 ppm meningkatkan diferensiasi sel yangditunjukkan dengan meningkatnya ratio sel glia terhadap sel saraf dan mempengaruhi panjangakson dan dendrite.Kata kunci : Curcuma xanthorrhiza Roxb., neuron, sel glia, dendrite


Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4535-4535
Author(s):  
Xiao Chen Chen ◽  
De Pei Wu ◽  
Feng Chen ◽  
Wei Rong Chang

Abstract Objective To isolate and culture bone marrow mesenchymal stem cells (MSC) from chronic myelogenous leukemia (CML) patients and examine their functional characteristics; To investigate their biological characteristics like proliferation, apoptosis and tumorigenicity;To observe their effects on proliferation and apoptosis of K562 cells. Methods Bone marrow was extracted from the anterior superior iliac spines of 20 patients with CML. MSC were isolated and cultured in vitro and subcultured for three to five generations. Cell morphology was observed under microscope. The ultrastructure was observed with electron microscope. The immunophenotype was detected by flow cytometry (FCM). Different agents were used to induce the MSC to differentiate into osteocyte and adipocyte, and Von Kossa staining, oil-red staining was used to examine the ability of differentiation. The growth curve, cell cycle and apoptosis were investigated. MSC were cultured in soft agar for 2 weeks to observe the clone growth. BALB/C nude mice were inoculated with MSC to observe the tumorigenicity. MSC and K562 cells were cocultured in vitro and coinjected into subcutaneous of BALB/C nude mice, to observe the effects of MSC on proliferation and apoptosis of K562 cells. Results Fibroblast-like, positive in CD44, CD73 and CD90,and negative in CD34, CD45 and HLA-DR, the CML derived MSC could differentiate into osteocyte and adipocyte. The MSC showed normal ultrastructure. After 2 weeks’ culture, no clone was formed from the MSC. Four weeks after, no tumor was seen in the mice inoculated with MSC. The population doubling time of P3 MSC is (32.61±1.54)h. The population doubling time of P4 MSC is (32.59±1.23)h, The population doubling time of P5 MSC is (32.41±0.75)h. To investigate the cell cycle of the third passage MSC by FCM, there were(93.67%±1.66%)cells in phase G0/G1,(6.33%±1.66%)in phase S+G2+M. There weren’t any apoptosis observed. K562 cells adhensively cultivated with MSC were accelerated and the population doubling time decreased([29.59±0.46]h vs [37.49±2.19]h, P<0.05),cells in G0/G1 decreased ([16.43%±1.67%] vs [32.23%±3.35%], P<0.01),cells in phase S increased ([69.63%±3.09%] vs [59.37%±4.40%], P<0.05),and those in G2/M increased([13.93%±1.45%] vs [8.40%±1.05%], P<0.01),compared with that cultivated in suspension. The apoptosis in both conditions were not observed. Coinjected with MSC, K562 cells developed tumors in 100% of the mice by (12.00±0.82)d compared to (15.50±0.58)d when implanted alone, indicating a statistically earlier onset of tumor growth in presence of MSC (P<0.01). The tumor volume and tumor weight were also increased when K562 cells coinjected with MSC compared with implanted alone (K562+MSC vs K562):tumor volume([75.70±7.30]mm2 vs [37.38±2.39]mm2, P<0.01),tumor weight([0.64±0.08]g vs [0.32±0.06]g, P<0.01). Conclusion MSC from the bone marrow of CML patients can be isolated and cultured;MSC derived from CML seem to have the abilities of powerful proliferation in vitro and multiple differentiation and have no tumorigenicity. The MSC can accelerate the proliferation of K562 cells both in vitro and vivo.


2008 ◽  
Vol 18 (2) ◽  
pp. 339-344 ◽  
Author(s):  
H.-J. Schulten ◽  
J. Wolf-Salgó ◽  
C. Gründker ◽  
B. Gunawan ◽  
L. FÜZESI

We describe the newly established cell line CS-99 derived from a uterine carcinosarcoma retaining features of the sarcomatous phenotype in vitro. CS-99 cells exhibit a mesenchymal morphology with predominantly spindle-shaped cells at nonconfluence turning to pleomorphic appearance at confluence. The mesenchymal phenotype was evidenced immunohistochemically by strong vimentin and moderate SM-actin, which was similar to the sarcomatous component of the primary tumor. P53 was overexpressed in a subset of CS-99 cells. Epithelial membrane antigen was moderately expressed whereas other markers including pan CK, CK 5/6, CK 34, epidermal growth factor receptor, desmin, carcinoembryonic antigen, S100, KIT, ERBB2, and the hormone receptors, estrogen receptor and progesterone receptor revealed either weak or no specific staining in CS-99 cells. High self-renewal capacity corresponded to the population doubling time of 23 h in high passage. CS-99 cells were able to develop three-dimensional tumor spheroids in vitro. Cytogenetic analysis and multicolor fluorescence in situ hybridization of CS-99 demonstrated an almost stable karyotype including numerical changes +8, +18, and +20 and translocations, amongst others der(1)t(1;2), der(1)t(1;7), der(2)t(2;19), der(5)t(5;8), and der(5)t(5;14). Taken together, the cell line CS-99 exhibits strong growths dynamics and a complex but stable karyotype in higher passages, and can be further a useful in vitro model system for studying tumor biology of carcinosarcomas.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
A. Gericke ◽  
K. Suminska-Jasińska ◽  
A. Bręborowicz

AbstractChronic exposure of retinal endothelium cells to hyperglycemia is the leading cause of diabetic retinopathy. We evaluated the effect of high glucose concentration on senescence in human retinal endothelial cells (HREC) and modulation of that effect by Sulodexide. Experiments were performed on HREC undergoing in vitro replicative senescence in standard medium or medium supplemented with glucose 20 mmol/L (GLU) or mannitol 20 mnol/L (MAN). Effect of Sulodexide 0.5 LRU/mL (SUL) on the process of HREC senescence was studied. Glucose 20 mmol/L accelerates senescence of HREC: population doubling time (+ 58%, p < 0.001) β-galactosidase activity (+ 60%, p < 0.002) intracellular oxidative stress (+ 65%, p < 0.01), expression of p53 gene (+ 118%, p < 0.001). Senescent HREC had also reduced transendothelial electrical resistance (TEER) (− 30%, p < 0.001). Mannitol 20 mmol/L used in the same scenario as glucose did not induce HREC senescence. In HREC exposed to GLU and SUL, the senescent changes were smaller. HREC, which became senescent in the presence of GLU, demonstrated higher expression of genes regulating the synthesis of Il6 and VEGF-A, which was reflected by increased secretion of these cytokines (IL6 + 125%, p < 0.001 vs control and VEGF-A + 124% p < 0.001 vs control). These effects were smaller in the presence of SUL, and additionally, an increase of TEER in the senescent HREC was observed. Chronic exposure of HREC to high glucose concentration in medium accelerates their senescence, and that process is reduced when the cells are simultaneously exposed to Sulodexide. Additionally, Sulodexide decreases the secretion of IL6 and VEGF-A from senescent HREC and increases their TEER.


Development ◽  
1972 ◽  
Vol 27 (3) ◽  
pp. 543-553
Author(s):  
D. A. T. New ◽  
R. L. Brent

Rat embryos, explanted with their embryonic membranes during the early stages of organogenesis ( days gestation), were grown in culture in roller tubes. Yolk-sac antibody (sheep anti rat yolk-sac gamma globulin), known to be teratogenic when injected into pregnant rats, was added to the culture medium. At concentrations of 0·1 mg/ml or more the antibody caused gross retardation of growth and differentiation. Injection of antibody into the amniotic cavity so that it had direct contact with the embryo, or between the amnion and yolk sac so that it was in contact with the mesodermal surface of the yolk sac, had little or no effect on development of the embryo or its membranes. These in vitro experiments indicate that yolk-sac antibody has an effect on development independent of any immunological reaction of the mother, and the primary action is probably on the visceral yolk-sac endoderm.


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