A Comparison of Exogenous Labels for the Histological Identification of Transplanted Neural Stem Cells

The interpretation of cell transplantation experiments is often dependent on the presence of an exogenous label for the identification of implanted cells. The exogenous labels Hoechst 33342, 5-bromo-2′-deoxyuridine (BrdU), PKH26, and Qtracker were compared for their labeling efficiency, cellular effects, and reliability to identify a human neural stem cell (hNSC) line implanted intracerebrally into the rat brain. Hoechst 33342 (2 mg/ml) exhibited a delayed cytotoxicity that killed all cells within 7 days. This label was hence not progressed to in vivo studies. PKH26 (5 μM), Qtracker (15 nM), and BrdU (0.2 μM) labeled 100% of the cell population at day 1, although BrdU labeling declined by day 7. BrdU and Qtracker exerted effects on proliferation and differentiation. PKH26 reduced viability and proliferation at day 1, but this normalized by day 7. In an in vitro coculture assay, all labels transferred to unlabeled cells. After transplantation, the reliability of exogenous labels was assessed against the gold standard of a human-specific nuclear antigen (HNA) antibody. BrdU, PKH26, and Qtracker resulted in a very small proportion (<2%) of false positives, but a significant amount of false negatives (~30%), with little change between 1 and 7 days. Exogenous labels can therefore be reliable to identify transplanted cells without exerting major cellular effects, but validation is required. The interpretation of cell transplantation experiments should be presented in the context of the label's limitations.

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WAF1 retards S-phase progression primarily by inhibition of cyclin-dependent kinases.

Molecular and Cellular Biology ◽

10.1128/mcb.17.8.4877 ◽

1997 ◽

Vol 17 (8) ◽

pp. 4877-4882 ◽

Cited By ~ 131

Author(s):

V V Ogryzko ◽

P Wong ◽

B H Howard

Keyword(s):

Mammalian Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

S Phase ◽

In Vivo Studies ◽

Nuclear Antigen ◽

Phase Progression

The p21(WAF1/CIP1/sdi1) gene product (WAF1) inhibits DNA replication in vitro (J. Chen, P. Jackson, M. Kirschner, and A. Dutta, Nature 374:386-388, 1995; S. Waga, G. Hannon, D. Beach, and B. Stillman, Nature 369:574-578, 1994), but in vivo studies on the antiproliferative activity of WAF1 have not resolved G1-phase arrest from potential inhibition of S-phase progression. Here, we demonstrate that elevated WAF1 expression can retard replicative DNA synthesis in vivo. The WAF1-mediated inhibitory effect could be antagonized by cyclin A, cyclin E, or the simian virus 40 small-t antigen with no decrease in the levels of WAF1 protein in transfected cells. Proliferating-cell nuclear antigen (PCNA) overexpression was neither necessary nor sufficient to antagonize WAF1 action. Expression of the N-terminal domain of WAF1, responsible for cyclin-dependent kinase (CDK) interaction, had the same effect as full-length WAF1, while the PCNA binding C terminus exhibited modest activity. We conclude that S-phase progression in mammalian cells is dependent on continuing cyclin and CDK activity and that WAF1 affects S phase primarily through cyclin- and CDK-dependent pathways.

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Immunomodulatory Activities of Selected Essential Oils

Biomolecules ◽

10.3390/biom10081139 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1139 ◽

Cited By ~ 2

Author(s):

Georg Sandner ◽

Mara Heckmann ◽

Julian Weghuber

Keyword(s):

Essential Oils ◽

Study Data ◽

In Vivo Studies ◽

Immunomodulatory Effects ◽

Regulatory Pathways ◽

Cellular Effects ◽

Anti Inflammatory ◽

Toxic Potential

Recently, the application of herbal medicine for the prevention and treatment of diseases has gained increasing attention. Essential oils (EOs) are generally known to exert various pharmacological effects, such as antiallergic, anticancer, anti-inflammatory, and immunomodulatory effects. Current literature involving in vitro and in vivo studies indicates the potential of various herbal essential oils as suitable immunomodulators for the alternative treatment of infectious or immune diseases. This review highlights the cellular effects induced by EOs, as well as the molecular impacts of EOs on cytokines, immunoglobulins, or regulatory pathways. The results reviewed in this article revealed a significant reduction in relevant proinflammatory cytokines, as well as induction of anti-inflammatory markers. Remarkably, very little clinical study data involving the immunomodulatory effects of EOs are available. Furthermore, several studies led to contradictory results, emphasizing the need for a multiapproach system to better characterize EOs. While immunomodulatory effects were reported, the toxic potential of EOs must be clearly considered in order to secure future applications.

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RBP-J Deficiency Promoted The Proliferation and Differentiation of CD133-Positive Ependymal Cells In Vitro and In Vivo Studies

10.21203/rs.3.rs-663313/v1 ◽

2021 ◽

Author(s):

Xin Ye ◽

Mengyi Li ◽

Wei Bian ◽

Junwei Li ◽

Ting Zhang ◽

...

Keyword(s):

Notch Signaling ◽

Signaling Pathway ◽

Notch Signaling Pathway ◽

In Vivo Studies ◽

Ependymal Cells ◽

Ependymal Layer ◽

Proliferation And Differentiation ◽

Mature Neurons

Abstract Although the ependymal cells were reported to have the characteristics of neural stem cells (NSCs), the properties of CD133-ependymal cells have not been uncovered, in particular, it is largely unknown about the effect of Notch signaling pathway on the neurogenesis of CD133-positive ependymal cells. By using the transgenic mouse and primarily cultured ependymal cells, we found that the immunoreactivity for prominin-1/CD133 was exclusively localized in the subventricular zone (SVZ) and ependymal layer of ventricles, moreover, most CD133-positive ependymal cells were co-labeled with Nestin. In addition, RBP-J, a key nuclear effector of Notch signaling pathway, was highly active in CD133-positive ependymal cells. Our results demonstrated that CD133-positive ependymal cells can differentiate into the immature and mature neurons, in particular, the number of CD133-positive ependymal cells differentiating into the immature and mature neurons was significantly increased following the deficiency or interference of RBP-J in vivo or in vitro. By using real-time qPCR and Western blot, we found that RBP-J and Hes1 were down-regulated while Notch1 was up-regulated in the expression levels of mRNAs and proteins following the deficiency or interference of RBP-J in vivo or in vitro. These results demonstrated RBP-J deficiency promoted the proliferation and differentiation of CD133-positive ependymal cells. Therefore, we speculated that RBP-J could maintain CD133-positive ependymal cells in the characteristics of NSCs possibly by regulating Notch1/RBP-J/Hes1 pathway.

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The importance of N- and O-linked oligosaccharides for the biosynthesis and in vitro and in vivo biologic activities of erythropoietin

Blood ◽

10.1182/blood.v77.12.2624.bloodjournal77122624 ◽

1991 ◽

Vol 77 (12) ◽

pp. 2624-2632 ◽

Cited By ~ 2

Author(s):

LC Wasley ◽

G Timony ◽

P Murtha ◽

J Stoudemire ◽

AJ Dorner ◽

...

Keyword(s):

Chinese Hamster Ovary Cells ◽

Critical Role ◽

Chinese Hamster ◽

In Vivo Studies ◽

Erythroid Precursor ◽

Ovary Cells ◽

Biologic Activity ◽

Proliferation And Differentiation

Erythropoietin (EPO) plays a critical role in stimulating the proliferation and differentiation of erythroid precursor cells. EPO is heavily glycosylated with three asparagine (N)-linked tetraantennary oligosaccharides that may contain N-acetyl-lactosamine repeats and a single serine (O)-linked oligosaccharide. EPO expressed in Chinese hamster ovary cells exhibits biologic properties and amino acid and carbohydrate composition similar to natural urinary EPO. The importance of the complex N-linked and the O-linked carbohydrate was studied by expressing EPO in cells that are deficient in UDP-galactose/UDP-N- acetylgalactosamine 4-epimerase activity. In these cells, the ability to add galactose and N-acetylgalactosamine to glycoproteins can be controlled by the addition of these sugars to the culture medium. The results demonstrate that a block in O-linked glycosylation and/or the ability to process N-linked carbohydrate to completion does not alter EPO secretion. EPO produced without O-linked carbohydrate exhibits normal in vitro and in vivo biologic activity and in vivo clearance. However, EPO produced with incompletely processed N-linked oligosaccharides exhibits normal in vitro activity but is at least 500- fold less effective in stimulating erythropoiesis in vivo. Studies on the survival of bioactive EPO remaining in the circulation demonstrated that EPO with incomplete N-linked oligosaccharides exhibits a sevenfold increased rate of clearance. However, this increased clearance may not fully account for the 500-fold loss of in vivo activity. These results suggest a potentially important unique requirement for appropriate complex N-linked oligosaccharides for the intrinsic biologic activity of EPO in vivo.

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Prolonged underfeeding of sheep increases myostatin and myogenic regulatory factor Myf-5 in skeletal muscle while IGF-I and myogenin are repressed

Journal of Endocrinology ◽

10.1677/joe.0.1760425 ◽

2003 ◽

Vol 176 (3) ◽

pp. 425-437 ◽

Cited By ~ 56

Author(s):

F Jeanplong ◽

JJ Bass ◽

HK Smith ◽

SP Kirk ◽

R Kambadur ◽

...

Keyword(s):

Satellite Cells ◽

Muscle Growth ◽

Cell Activity ◽

Nuclear Antigen ◽

Myogenic Regulatory Factor ◽

Proliferation And Differentiation ◽

Igf I ◽

Muscle Necrosis

The IGF axis is nutritionally sensitive in vivo and IGFs stimulate myoblast proliferation and differentiation in vitro, while myostatin inhibits these processes in vitro. We hypothesised that underfeeding would reversibly inhibit the myogenic activity of satellite cells in vivo together with decreased IGF-I and increased myostatin in muscle. Satellite cell activity was measured indirectly from the expression of proliferating cell nuclear antigen (PCNA) and the myogenic regulatory factors (MRFs), MyoD, Myf-5 and myogenin. Young sheep were underfed (30% of maintenance) and some killed after 1, 4, 12, 17, 21 and 22 weeks. Remaining underfed animals were then re-fed a control ration of pellets and killed after 2 days, and 1, 6 and 30 weeks. Expression of PCNA and MRFs decreased during the first week of underfeeding. This coincided with reduced IGF-I and myostatin mRNA, and processed myostatin. Subsequently, Myf-5, MyoD, myostatin mRNA and processed myostatin increased, suggesting that satellite cells may have become progressively quiescent. Long-term underfeeding caused muscle necrosis in some animals and IGF-I and MRF expression was increased in these, indicating the activation of satellite cells for muscle repair. Re-feeding initiated rapid muscle growth and increased expression of PCNA, IGF-I and the MRFs concurrently with decreased myostatin proteins. In conclusion, these data indicate that IGF-I and myostatin may work in a coordinated manner to regulate the proliferation, differentiation and quiescence of satellite cells in vivo.

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Suppression of C6 Gliomas via Application of Rat Hyperplasia Gene

The International Journal of Biological Markers ◽

10.5301/jbm.5000114 ◽

2014 ◽

Vol 29 (4) ◽

pp. 411-422 ◽

Cited By ~ 1

Author(s):

Peng Gao ◽

Zhanwei Wang ◽

Bin Zhang ◽

Yourui Zou ◽

Hui Guo ◽

...

Keyword(s):

Cell Proliferation ◽

Recombinant Adenovirus ◽

Glioma Cells ◽

In Vivo Studies ◽

Nuclear Antigen ◽

Sprague Dawley ◽

Rat Glioma ◽

Suppression Mechanism

Background Among all neurological tumors, tumor incidence of the neuroepithelial tissue is the highest, where 50% are gliomas. Treatment for gliomas has traditionally included surgery and adjuvant therapy. With advancements in medicine, gene therapy has entered the clinical setting, in which control of tumor growth, tumor volume and decrease of supply of blood to the tumor have been observed. Rat hyperplasia suppressor gene (rHSG) has been proven to inhibit the injury-mediated proliferation of vascular smooth muscle cells. Methods A recombinant adenovirus, Adv-rHSG-GFP, was constructed and characterized by in vitro and in vivo studies. The function of rHSG on cell proliferation was determined in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) exclusion assay and plate clone formation, while a C6/Sprague Dawley rat glioma model was established to observe the effect of rHSG in vivo. Results Overexpression of rHSG displayed a strong effect on suppressing C6 cells proliferation in vitro and growth of glioma in vivo, which suggests the use of rHSG as a possible treatment strategy for glioma. p21Cip1, p27Kip1 and proliferating cell nuclear antigen were found to be involved in the tumor suppression mechanism of rHSG. Conclusions rHSG can markedly inhibit of the growth of rat glioma cells. The suppression mechanism of rHSG may be related to cell cycle regulation, which shows that rHSG is a potential therapeutic target of glioma tumor. This preclinical study supports a further in-depth study on the effect of rHSG on cell proliferation, migration and change in the extracellular matrix component of glioma cells.

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SKP-SCs transplantation alleviates 6-OHDA-induced dopaminergic neuronal injury by modulating autophagy

Cell Death and Disease ◽

10.1038/s41419-021-03967-3 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Chengxiao Ma ◽

Wen Zhang ◽

Wengcong Wang ◽

Jiabing Shen ◽

Kefu Cai ◽

...

Keyword(s):

Schwann Cells ◽

Cell Transplantation ◽

Dopaminergic Neurons ◽

Therapeutic Option ◽

In Vivo Studies ◽

And Function ◽

Disease Cell ◽

Neuronal Autophagy

AbstractParkinson’s disease is a common neurodegenerative disease. Cell transplantation is a promising therapeutic option for improving the survival and function of dopaminergic neurons, but the mechanisms underlying the interaction between the transplanted cells and the recipient neurons remain to be studied. In this study, we investigated the effects of skin precursor cell-derived Schwann cells (SKP-SCs) directly cocultured with 6-OHDA-injured dopaminergic neurons in vitro and of SKP-SCs transplanted into the brains of 6-OHDA-induced PD mice in vivo. In vitro and in vivo studies revealed that SKP-SCs could reduce the damage to dopaminergic neurons by enhancing self-autophagy and modulating neuronal autophagy. Thus, the present study provides the first evidence that cell transplantation mitigates 6-OHDA-induced damage to dopaminergic neurons by enhancing self-autophagy, suggesting that earlier transplantation of Schwann cells might help alleviate the loss of dopaminergic neurons.

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The importance of N- and O-linked oligosaccharides for the biosynthesis and in vitro and in vivo biologic activities of erythropoietin

Blood ◽

10.1182/blood.v77.12.2624.2624 ◽

1991 ◽

Vol 77 (12) ◽

pp. 2624-2632 ◽

Cited By ~ 108

Author(s):

LC Wasley ◽

G Timony ◽

P Murtha ◽

J Stoudemire ◽

AJ Dorner ◽

...

Keyword(s):

Chinese Hamster Ovary Cells ◽

Critical Role ◽

Chinese Hamster ◽

In Vivo Studies ◽

Erythroid Precursor ◽

Ovary Cells ◽

Biologic Activity ◽

Proliferation And Differentiation

Abstract Erythropoietin (EPO) plays a critical role in stimulating the proliferation and differentiation of erythroid precursor cells. EPO is heavily glycosylated with three asparagine (N)-linked tetraantennary oligosaccharides that may contain N-acetyl-lactosamine repeats and a single serine (O)-linked oligosaccharide. EPO expressed in Chinese hamster ovary cells exhibits biologic properties and amino acid and carbohydrate composition similar to natural urinary EPO. The importance of the complex N-linked and the O-linked carbohydrate was studied by expressing EPO in cells that are deficient in UDP-galactose/UDP-N- acetylgalactosamine 4-epimerase activity. In these cells, the ability to add galactose and N-acetylgalactosamine to glycoproteins can be controlled by the addition of these sugars to the culture medium. The results demonstrate that a block in O-linked glycosylation and/or the ability to process N-linked carbohydrate to completion does not alter EPO secretion. EPO produced without O-linked carbohydrate exhibits normal in vitro and in vivo biologic activity and in vivo clearance. However, EPO produced with incompletely processed N-linked oligosaccharides exhibits normal in vitro activity but is at least 500- fold less effective in stimulating erythropoiesis in vivo. Studies on the survival of bioactive EPO remaining in the circulation demonstrated that EPO with incomplete N-linked oligosaccharides exhibits a sevenfold increased rate of clearance. However, this increased clearance may not fully account for the 500-fold loss of in vivo activity. These results suggest a potentially important unique requirement for appropriate complex N-linked oligosaccharides for the intrinsic biologic activity of EPO in vivo.

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Chemopreventive Effect of Cratoxylum formosum (Jack) ssp. pruniflorum on Initial Stage Hepatocarcinogenesis in Rats

Molecules ◽

10.3390/molecules26144235 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4235

Author(s):

Piman Pocasap ◽

Natthida Weerapreeyakul ◽

Rawiwan Wongpoomchai

Keyword(s):

Hepatocellular Carcinoma ◽

Early Stage ◽

In Vivo Studies ◽

Nuclear Antigen ◽

Apoptotic Cells ◽

Glutathione S Transferase ◽

Initial Stage ◽

Indigenous Plant

Cratoxylum formosum ssp. pruniflorum (Kurz) Gogelein (CP) is an indigenous plant found mainly in southeast Asia. Several in vitro studies have confirmed its activity against hepatocellular carcinoma; however, in vivo studies of the effect of CP on liver cancer are needed. This study investigated the effect of CP on early-stage hepatocarcinogenesis in rat liver when using diethylnitrosamine (DEN) as a carcinogen. Immunohistochemistry was used to detect (a) upregulation of glutathione S-transferase placental (GST-P) positive foci, (b) the proliferating cell nuclear antigen PCNA, and (c) apoptotic cells in the liver as indicators of early-stage carcinogenesis. Immunohistochemical parameters were observed in rats given CP orally following DEN injection. Rats given DEN presented overexpression of GST-P positive foci, PCNA, and apoptotic cells, indicating the formation of cancerous tissues, and these effects were diminished by CP treatment. CP thus inhibited hepatocarcinogenic effects in an animal model. These results could help plan further in vivo studies and support the use of CP to prevent processes that promote the pathogenesis of hepatocellular carcinoma in humans.

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