Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation

Abstract Spermatogonial stem cells (SSCs) have significant applications in both reproductive and regenerative medicine. However, primary human SSCs are very rare and a human SSC line has not yet been available. In this study, we have for the first time reported a stable human SSC line by stably expressing human SV40 large T antigen. RT-PCR, immunocytochemistry and Western blots revealed that this cell line was positive for a number of human spermatogonial and SSC hallmarks, including VASA, DAZL, MAGEA4, GFRA1, RET, UCHL1, GPR125, PLZF and THY1, suggesting that these cells are human SSCs phenotypically. Proliferation analysis showed that the cell line could be expanded with significant increases of cells for 1.5 years and high levels of PCNA, UCHL1 and SV40 were maintained for long-term culture. Transplantation assay indicated that human SSC line was able to colonize and proliferate in vivo in the recipient mice. Neither Y chromosome microdeletions of numerous genes nor tumor formation was observed in human SSC line although there was abnormal karyotype in this cell line. Collectively, we have established a human SSC line with unlimited proliferation potentials and no tumorgenesis, which could provide an abundant source of human SSCs for their mechanistic studies and translational medicine.

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Electrogenic sodium absorption and chloride secretion by an inner medullary collecting duct cell line (mIMCD-K2)

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.2.f347 ◽

1995 ◽

Vol 268 (2) ◽

pp. F347-F355 ◽

Cited By ~ 17

Author(s):

N. L. Kizer ◽

B. Lewis ◽

B. A. Stanton

Keyword(s):

Cell Line ◽

Collecting Duct ◽

T Antigen ◽

Sodium Absorption ◽

Sv40 Large T Antigen ◽

Permanent Cell Line ◽

Inner Medullary Collecting Duct ◽

Permanent Cell ◽

Medullary Collecting Duct

The initial segment of the inner medullary collecting duct (IMCDi) absorbs Na+ by an electrogenic mechanism and plays an important role in regulating the composition and volume of the urine. The purpose of the present study was to establish a permanent cell line derived from the IMCDi, which has the ion transport properties of the IMCDi in vivo. To this end, we isolated IMCD cells from the IMCDi of a mouse, Tg(SV40E) Bri 7, transgenic for the early region of SV40 (large T antigen) and established a permanent cell line, mIMCD-K2, by clonal dilution. mIMCD-K2 cells retain many differentiated characteristics of the IMCDi, including amiloride-sensitive electrogenic Na+ absorption stimulated by nanomolar concentrations of aldosterone. Aldosterone (1.5 x 10(-6) M) increased Na+ absorption from 0.2 +/- 0.1 to 4.6 +/- 1.7 microA/cm2. In addition, the cells secrete Cl- by an electrogenic mechanism at a rate of 0.5 +/- 0.1 microA/cm2. We propose that IMCDi cells either absorb or secrete NaCl depending on NaCl homeostasis. The mIMCD-K2 cell line should be useful for studying the cellular mechanisms responsible for electrogenic Na+ and Cl- transport in the IMCDi.

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Expression of Tyrosine Hydroxylase in an Immortalized Human Fetal Astrocyte Cell Line; in Vitro Characterization and Engraftment into the Rodent Striatum

Cell Transplantation ◽

10.1177/096368979600500206 ◽

1996 ◽

Vol 5 (2) ◽

pp. 145-163

Author(s):

Carlo Tornatore ◽

Belinda Baker-Cairns ◽

Gal Yadid ◽

Rebecca Hamilton ◽

Karen Meyers ◽

...

Keyword(s):

Tyrosine Hydroxylase ◽

Cell Line ◽

Ethical Issues ◽

Neuron Specific Enolase ◽

T Antigen ◽

Sv40 Large T Antigen ◽

Th Cells ◽

Xenograft Rejection

The use of primary human fetal tissue in the treatment of neurodegenerative disorders, while promising, faces several difficult technical and ethical issues. An alternative approach that would obviate these problems would be to use immortalized cell lines of human fetal central nervous system origin. An immortalized human fetal astrocyte cell line (SVG) has been established (45) and herein we describe the in vitro and in vivo characteristics of this cell line which suggest that it may be a useful vehicle for neural transplantation. The SVG cell line is vimentin, GFAP, Thy 1.1 and MHC class I positive, and negative for neurofilament and neuron specific enolase, consistent with its glial origin. To determine whether the cell line could be used as a drug delivery system, a cDNA expression vector for tyrosine hydroxylase was constructed (phTH/Neo) and stably expressed in the SVG cells for over 18 months as demonstrated by immunohistochemistry and Western blotting of the stable transfectants. HPLC analysis of the supernatant from these cells, termed SVG-TH, consistently found 4-6 pmol/ml/min of 1-dopa produced with the addition of BH4 to the media. Furthermore, in cocultivation experiments with hNT neurons, PC-12 cells and primary rat fetal mesencephalic tissue, both the SVG and SVG-TH cells demonstrated neurotrophic potential, suggesting that they constituitively express factors with neuroregenerative potential. To determine the viability of these cells in vivo, SVG-TH cells were grafted into the striatum of Sprague-Dawley rats and followed over time. A panel of antibodies was used to unequivocally differentiate the engrafted cells from the host parenchyma, including antibodies to: SV40 large T antigen (expressed in the SVG-TH cells), human and rat MHC class 1, vimentin, GFAP, and tyrosine hydroxylase. While the graft was easily identified with the first week, over the course of a four week period of time the engrafted cells decreased in number. Concomittantly, rat CD4 and CD8 expression in the vicinity of the graft increased, consistent with xenograft rejection. When the SVG-TH cells were grafted to the lesioned striatum of a 6-hydroxydopamine lesioned rats, rotational behavior of the rat decreased as much as 80% initially, then slowly returned to baseline over the next four weeks, parallelling graft rejection. Thus, the SVG-TH cells can induce a functional recovery in an animal model of Parkinson's disease, however as a xenograft, the SVG cells are recognized by the immune system.

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RABL6A promotes pancreatic neuroendocrine tumor angiogenesis and progression in vivo

10.1101/2021.03.17.435790 ◽

2021 ◽

Author(s):

Chandra Kumar Maharjan ◽

Courtney A. Kaemmer ◽

Viviane P. Muniz ◽

Casey Bauchle ◽

Sarah L. Mott ◽

...

Keyword(s):

Tumor Angiogenesis ◽

Pancreatic Neuroendocrine Tumor ◽

Antigen Expression ◽

T Antigen ◽

Tumor Formation ◽

Sv40 Large T Antigen ◽

Bona Fide ◽

New Biomarkers

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet β cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.

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RABL6A Promotes Pancreatic Neuroendocrine Tumor Angiogenesis and Progression In Vivo

Biomedicines ◽

10.3390/biomedicines9060633 ◽

2021 ◽

Vol 9 (6) ◽

pp. 633

Author(s):

Chandra K. Maharjan ◽

Shaikamjad Umesalma ◽

Courtney A. Kaemmer ◽

Viviane P. Muniz ◽

Casey Bauchle ◽

...

Keyword(s):

Tumor Angiogenesis ◽

Pancreatic Neuroendocrine Tumor ◽

Antigen Expression ◽

T Antigen ◽

Tumor Formation ◽

Sv40 Large T Antigen ◽

Bona Fide ◽

New Biomarkers

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Tumor Suppressive Role of miR-342-5p in Human Chondrosarcoma Cells and 3D Organoids

International Journal of Molecular Sciences ◽

10.3390/ijms22115590 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5590

Author(s):

Clément Veys ◽

Abderrahim Benmoussa ◽

Romain Contentin ◽

Amandine Duchemin ◽

Emilie Brotin ◽

...

Keyword(s):

Luciferase Reporter ◽

Functional Screening ◽

Western Blots ◽

Egfr Expression ◽

Reporter Assays ◽

Direct Target ◽

Induced Apoptosis ◽

First Time

Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.

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lncRNA CASC19 Contributes to Radioresistance of Nasopharyngeal Carcinoma by Promoting Autophagy via AMPK-mTOR Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22031407 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1407

Author(s):

Hongxia Liu ◽

Wang Zheng ◽

Qianping Chen ◽

Yuchuan Zhou ◽

Yan Pan ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Cell Line ◽

Malignant Tumors ◽

Underlying Mechanism ◽

Mtor Pathway ◽

Rna Seq ◽

Cellular Autophagy ◽

First Time

Nasopharyngeal carcinoma (NPC) is one of the most frequent head and neck malignant tumors and is majorly treated by radiotherapy. However, radiation resistance remains a serious obstacle to the successful treatment of NPC. The aim of this study was to discover the underlying mechanism of radioresistance and to elucidate novel genes that may play important roles in the regulation of NPC radiosensitivity. By using RNA-seq analysis of NPC cell line CNE2 and its radioresistant cell line CNE2R, lncRNA CASC19 was screened out as a candidate radioresistance marker. Both in vitro and in vivo data demonstrated that a high expression level of CASC19 was positively correlated with the radioresistance of NPC, and the radiosensitivity of NPC cells was considerably enhanced by knockdown of CASC19. The incidence of autophagy was enhanced in CNE2R in comparison with CNE2 and another NPC cell line HONE1, and silencing autophagy with LC3 siRNA (siLC3) sensitized NPC cells to irradiation. Furthermore, CASC19 siRNA (siCASC19) suppressed cellular autophagy by inhibiting the AMPK/mTOR pathway and promoted apoptosis through the PARP1 pathway. Our results revealed for the first time that lncRNA CASC19 contributed to the radioresistance of NPC by regulating autophagy. In significance, CASC19 might be a potential molecular biomarker and a new therapeutic target in NPC.

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Erratum: Corrigendum: Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation

Scientific Reports ◽

10.1038/srep43195 ◽

2017 ◽

Vol 7 (1) ◽

Author(s):

Jingmei Hou ◽

Minghui Niu ◽

Linhong Liu ◽

Zijue Zhu ◽

Xiaobo Wang ◽

...

Keyword(s):

Stem Cell ◽

Cell Line ◽

Tumor Formation ◽

Germline Stem Cell ◽

Stem Cell Line

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Growth Properties of Neural Cell Lines Immortalized with the Tsa58 Allele of Sv40 Large T Antigen

Cell Transplantation ◽

10.1177/096368979700600306 ◽

1997 ◽

Vol 6 (3) ◽

pp. 231-238 ◽

Cited By ~ 5

Author(s):

M.E. Truckenmiller ◽

Ora Dillon-Carter ◽

Carlo Tornatore ◽

Henrietta Kulaga ◽

Hidetoshi Takashima ◽

...

Keyword(s):

Amino Acid ◽

Cell Line ◽

Cell Lines ◽

T Antigen ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Large T Antigen ◽

Wild Type ◽

Sv40 Large T Antigen ◽

Growth Properties

In vitro growth properties of three CNS-derived cell lines were compared under a variety of culture conditions. The M213-20 and J30a cell lines were each derived from embryonic CNS culture with the temperature-sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cell line was immortalized using wild-type SV40 large T antigen. Cells immortalized with tsA58 SV40 large T proliferate at the permissive temperature, 33° C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5°C. Both the M213-20 and J30a cell lines were capable of proliferating at 39.5°C continuously for up to 6 mo. All three cell lines showed no appreciable differences in growth rates related to temperature over a 7-day period in either serum-containing or defined serum-free media. The percentage of cells in S-phase of the cell cycle did not decrease or was elevated at 39.5°C for all three cell lines. After 3 wk at 39.5°C, the three cell lines also showed positive immunostaining using two monoclonal antibodies reacting with different epitopes of SV40 large T antigen. Double strand DNA sequence analyses of a 300 base pair (bp) fragment of the large T gene from each cell line, which included the ts locus, revealed mutations in both the J30a and M213-20 cell lines. The J30a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were also found. While the ts mutation of the M213-20 cells was retained, an additional bp substitution with a predicted amino acid change was found. The A7 cell line sequence was identical to the reference wild-type sequence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained.

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Prostaglandin A1 inhibits the phosphorylation of tau via activating protein phosphotase 2A in a michael addition mechanism at the site of cysteine 377

10.21203/rs.3.rs-56068/v1 ◽

2020 ◽

Author(s):

Guo-Biao Xu ◽

Pei-Pei Guan ◽

Pu Wang

Keyword(s):

Cognitive Decline ◽

Michael Addition ◽

Dependent Manner ◽

Western Blots ◽

Michael Adduct ◽

Term Administration ◽

Prostaglandin A1

Abstract Background: Prostaglandin (PG) A1 is a metabolic product of cyclooxygenase 2 (COX-2), which potentially involved in regulating the development and progression of Alzheimer’s disease (AD). As a cyclopentenone (cy) PG, PGA1 is characterized by the presence of a chemically reactive α, β-unsaturated carbonyl. Although PGA1 is potentially involved in regulating multiple biological processes via michael addition, its specific roles in AD remained unclear.Methods: The tauP301S transgenic (Tg) mice were employed as in vivo AD models and neuroblastoma (N) 2a cells as in vitro neuronal models. By intracerebroventricular injected (i.c.v) with PGA1, the binding proteins to PGA1 are analyzed by HPLC-MS-MS. In addition, western blots are used to determine the phosphorylation of tau in PGA1 treated Tg mice in the absence or presence of okadaic acid (OA), an inhibitor of protein phosphotase (PP) 2A. Combining a synthesis of pull down assay, immunoprecipitation, western blots and HPLC-MS-MS, PP2A scaffold subunit A alpha (PPP2R1A) was identified to be activated by directly binding on PGA1 in cysteine 377-dependent manner. Via inhibiting the hyperphosphorylation of tau, morris maze test was employed to determine the inhibitory effects of PGA1 on cognitive decline of tauP301S Tg mice.Results: By incubation with neuroblastoma (n)2a cells and pull down assay, mass spectra (MS) analysis revealed that PGA1 binds with more than 1000 proteins, among which contains the proteins of AD, especially tau protein. Moreover, short-term administration of PGA1 to tauP301S Tg mice significantly decreased the phosphorylation of tau at the sites of Thr181, Ser202 and Ser404 in a dose-dependent manner. To the reason, it’s caused by activating PPP2R1A in tauP301S Tg mice. More importantly, PGA1 has the ability to form michael adduct with PPP2R1A via its cysteine 377 motif, which is critical for the enzymatic activity of PP2A. By activating PP2A, long-term application of PGA1 to tauP301S Tg mice significantly reduced the phosphorylation of tau, which results in improving the cognitive decline of tauP301S Tg mice.Conclusion: Our data provided the first insights needed to decipher the mechanisms underlying the ameliorating effects of PGA1 on cognitive decline of tauP301S Tg mice via activating PP2A in a PPP2R1AC377-dependent Michael adducting mechanisms.

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Dynamics of intravital concentration of amino acid metabolites in human brain in post-traumatic period

Доклады Академии наук ◽

10.31857/s0869-56524842238-242 ◽

2019 ◽

Vol 484 (2) ◽

pp. 238-242

Author(s):

N. A. Semenova ◽

P. E. Menshchikov ◽

A. V. Manzhurtsev ◽

M. V. Ublinskiy ◽

T. A. Akhadov ◽

...

Keyword(s):

Traumatic Brain Injury ◽

Amino Acid ◽

Human Brain ◽

Leading Role ◽

Acid Metabolites ◽

Post Traumatic ◽

First Time

Intracellular concentrations of N acetyaspartate (NAA), aspartate (Asp) and glutamate (Glu) were determined for the first time in human brain in vivo, and the effect of severe traumatic brain injury on NAA synthesis in acute and late post-traumatic period was investigated. In MRI‑negative frontal lobes one day after injury Asp and Glu levels were found to decrease by 45 and 35%, respectively, while NAA level decreased by only 16%. A negative correlation between NAA concentration and the ratio of Asp/Glu concentrations was found. In the long-term period, Glu level returned to normal, Asp level remained below normal by 60%, NAA level was reduced by 65% relative to normal, and Asp/Glu ratio significantly decreased. The obtained results revealed leading role of the neuronal aspartate-malate shuttle in violation of NAA synthesis.

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