The Resurrection of Mabrokan: Production of Multiple Cloned Offspring from Decade-Old Vitrified Tissue Collected from a Deceased Champion Show Camel

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to reproduce animals with superior genetics. Viable cell lines are usually established from tissues collected by biopsy from living animals in the SCNT program. In the present study, tissues were collected and preserved from a suddenly deceased champion camel. We established cell lines from these decade-old tissues and used them as nuclear donors. After 42 h of in vitro maturation, 68.00 ± 2.40% of oocytes reached the metaphase II (M II) stage while 87.31 ± 2.57% in vivo collected oocytes were matured at collection (p < 0.05). We observed a higher blastocyst formation rate when in vivo matured oocytes (43.45 ± 2.07%) were used compared to in vitro matured oocytes (21.52 ± 1.74%). The live birth rate was 6.45% vs. 16.67% for in vitro and in vivo matured oocytes, respectively. Microsatellite analysis of 13 camel loci revealed that all the SCNT-derived offspring were identical to each other and with their somatic cell donor. The present study succeeded in the resurrection of 11 healthy offspring from the decade-old vitrified tissues of a single somatic cell donor individual using both in vitro and in vivo matured oocytes.

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Constitutive secretion of cystatin C (gamma-trace) by monocytes and macrophages and its downregulation after stimulation.

Journal of Experimental Medicine ◽

10.1084/jem.166.6.1912 ◽

1987 ◽

Vol 166 (6) ◽

pp. 1912-1917 ◽

Cited By ~ 89

Author(s):

A H Warfel ◽

D Zucker-Franklin ◽

B Frangione ◽

J Ghiso

Keyword(s):

Cell Lines ◽

Cystatin C ◽

Peritoneal Macrophages ◽

Inflammatory Conditions ◽

Monocytes And Macrophages ◽

Constitutive Secretion ◽

Established Cell ◽

Mouse Peritoneal Macrophages

Cystatin C (gamma-trace) was found to be a constitutively secreted protein of isolated human monocytes and mouse peritoneal macrophages, as well as the histiocytic lymphoma cell lines U937, P388D.1, and J774. This proteinase inhibitor is not uniquely secreted by monocytes/macrophages, but was also identified in the conditioned media from several primary cells, including brain cells, and diverse established cell lines. In vitro treatment of resident mouse peritoneal macrophages with either LPS or IFN-gamma caused a downregulation in cystatin C secretion. Elaboration of this protein was also diminished by macrophages that had been stimulated by thioglycollate in vivo, and treatment of these cells with LPS led to further decline. It is suggested that, under some inflammatory conditions, downregulation of cystatin C may contribute to tissue pathology.

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Full-term development of enucleated mouse oocytes fused with embryonic stem cells from different cell lines

Reproduction ◽

10.1530/rep.0.1210729 ◽

2001 ◽

pp. 729-733 ◽

Cited By ~ 25

Author(s):

T Amano ◽

Y Kato ◽

Y Tsunoda

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Lines ◽

Formation Rate ◽

Embryonic Stem ◽

Clear Correlation ◽

Developmental Potential ◽

Mouse Oocytes

The developmental potential of enucleated mouse oocytes receiving embryonic stem cells from ten lines with either the same or different genetic backgrounds using the cell fusion method was examined in vitro and in vivo. The development of nuclear-transferred oocytes into blastocysts was high (34-88%). However, there was no clear correlation between development into blastocysts after nuclear transfer and the chimaera formation rate of embryonic stem cells. The development into live young was low (1-3%) in all cell lines and 14 of 19 young died shortly after birth. Most of the live young had morphological abnormalities. Of the five remaining mice, two died at days 23 and 30 after birth, but the other three mice are still active at days 359 (mouse 1) and 338 (mice 4 and 5) after birth, with normal fertility. However, the reasons for the abnormalities and postnatal death of embryonic stem cell-derived mice are unknown.

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An Experimentally Defined Hypoxia Gene Signature in Glioblastoma and Its Modulation by Metformin

Biology ◽

10.3390/biology9090264 ◽

2020 ◽

Vol 9 (9) ◽

pp. 264

Author(s):

Marta Calvo Tardón ◽

Eliana Marinari ◽

Denis Migliorini ◽

Viviane Bes ◽

Stoyan Tankov ◽

...

Keyword(s):

Cell Lines ◽

In Silico ◽

Chemotherapy Resistance ◽

Viable Cell ◽

Gene Signature ◽

Cell Number ◽

Viable Cell Number ◽

In Silico Analyses

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, characterized by a high degree of intertumoral heterogeneity. However, a common feature of the GBM microenvironment is hypoxia, which can promote radio- and chemotherapy resistance, immunosuppression, angiogenesis, and stemness. We experimentally defined common GBM adaptations to physiologically relevant oxygen gradients, and we assessed their modulation by the metabolic drug metformin. We directly exposed human GBM cell lines to hypoxia (1% O2) and to physioxia (5% O2). We then performed transcriptional profiling and compared our in vitro findings to predicted hypoxic areas in vivo using in silico analyses. We observed a heterogenous hypoxia response, but also a common gene signature that was induced by a physiologically relevant change in oxygenation from 5% O2 to 1% O2. In silico analyses showed that this hypoxia signature was highly correlated with a perinecrotic localization in GBM tumors, expression of certain glycolytic and immune-related genes, and poor prognosis of GBM patients. Metformin treatment of GBM cell lines under hypoxia and physioxia reduced viable cell number, oxygen consumption rate, and partially reversed the hypoxia gene signature, supporting further exploration of targeting tumor metabolism as a treatment component for hypoxic GBM.

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Adapter Chimeric Antigen Receptor (AdCAR)-Engineered NK-92 Cells for the Multiplex Targeting of Bone Metastases

Cancers ◽

10.3390/cancers13051124 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1124

Author(s):

Stefan Grote ◽

Frank Traub ◽

Joerg Mittelstaet ◽

Christian Seitz ◽

Andrew Kaiser ◽

...

Keyword(s):

Bone Metastases ◽

Cell Lines ◽

Tumor Antigens ◽

Fatal Outcome ◽

In Vivo Models ◽

Established Cell ◽

Metastatic Spreading ◽

Pro Inflammatory Cytokines

Background: Since metastatic spreading of solid tumor cells often leads to a fatal outcome for most cancer patients, new approaches for patient-individualized, targeted immunotherapy are urgently needed. Methods: Here, we established cell lines from four bone metastases of different tumor entities. We assessed AdCAR NK-92-mediated cytotoxicity in vitro in standard cytotoxicity assays as well as 3D spheroid models Results: AdCAR-engineered NK-92 cells successfully demonstrated distinct and specific cytotoxic potential targeting different tumor antigens expressed on cell lines established from bone metastases of mammary, renal cell and colorectal carcinoma as well as melanomas. In that process AdCAR NK-92 cells produced a multitude of NK effector molecules as well as pro inflammatory cytokines. Furthermore, AdCAR NK-92 showed increased cytotoxicity in 3D spheroid models which can recapitulate in vivo architecture, thereby bridging the gap between in vitro and in vivo models. Conclusions: AdCAR NK-92 cells may provide an interesting and promising “off-the-shelf” cellular product for the targeted therapy of cancers metastasizing to the bone, while utilization of clinically approved, therapeutic antibodies, as exchangeable adapter molecules can facilitate quick clinical translation.

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Transfer of Cleavage-Stage Embryos May Benefit Normal Responders With Low Rate of Morphologically Good Embryos Formation On Day 3: A Comparison Study

10.21203/rs.3.rs-860928/v1 ◽

2021 ◽

Author(s):

Xiuliang Dai ◽

Xiyang Xia ◽

Tingting Gao ◽

Chunmei Yu ◽

Fang Cao ◽

...

Keyword(s):

Formation Rate ◽

Implantation Rate ◽

Live Birth Rate ◽

Developmental Potential ◽

Basic Characteristics ◽

Day 3 Embryos ◽

Low Rate ◽

Blastulation Rate

Abstract Background Do morphologically good (MG) embryos from patients with high and low rate of MG embryos on day 3 (RMD3) show similar developmental potential (DP)? Methods This respective study finally included a total of 916 fresh cycles and related 1074 FET cycles from Jan 2017 to May 2020 in our reproductive center. Cycles with high RMD3 were defined as the H group, while cycles with low RMD3 were defined as the L group. The basic characteristics of patients and fresh cycles, blastulation rate, and clinical outcomes were compared between the H and L groups in either ET cycles with MG day 3 cleavage embryos (ETC group) or ET cycles with MG blastocysts (ETB group). Results The overall characteristics of patients and cycles were grossly comparable between the H and L groups either in ETC or ETB groups. In ETB group, useable blastocysts formation rate, implantation rate and live birth rate was significantly reduced in the L group, compared to the H group;In ETC group, useable blastocysts formation rate was significantly reduced in the L group. However, implantation rate and livebirth rate was similar between the L and H groups. Conclusion The in vitro DP of MG day 3 embryos and in vivo DP of MG blastocysts were reduced significantly, while a similar in vivo DP of MG day 3 embryos was observed in patients with low RMD3 as compared to patients with high RMD3. It seems that direct transfer of day 3 MG embryos instead of extended culture may benefit patients with RMD3.

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Elevated expression of c-myc in lymphoblastoid cells does not support an Epstein–Barr virus latency III-to-I switch

Journal of General Virology ◽

10.1099/0022-1317-82-12-3051 ◽

2001 ◽

Vol 82 (12) ◽

pp. 3051-3055 ◽

Cited By ~ 13

Author(s):

Alexander Pajic ◽

Axel Polack ◽

Martin S. Staege ◽

Dimitry Spitkovsky ◽

Barbara Baier ◽

...

Keyword(s):

Cell Lines ◽

Epstein Barr Virus ◽

Type I ◽

Barr Virus ◽

Virus Latency ◽

Elevated Expression ◽

Established Cell ◽

Epstein Barr

Epstein–Barr virus (EBV) transforms primary B cells in vitro. Established cell lines adopt a lymphoblastoid phenotype (LCL). In contrast, EBV-positive Burkitt’s lymphoma (BL) cells, in which the proto-oncogene c-myc is constitutively activated, do not express a lymphoblastoid phenotype in vivo. The two different phenotypes are paralleled by two distinct programmes of EBV latent gene expression termed latency type I in BL cells and type III in LCL. Human B cell lines were established from a conditional LCL (EREB2-5) by overexpression of c-myc and inactivation of EBV nuclear protein 2 (EBNA2). These cells (A1 and P493-6) adopted a BL phenotype in the absence of EBNA2. However, the EBV latency I promoter Qp was not activated. Instead, the latency III promoter Cp remained active. These data suggest that the induction of a BL phenotype by overexpression of c-myc in an LCL is not necessarily paralleled by an EBV latency III-to-I switch.

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Characterization and banding of mitotic chromosomes from two established cell lines of the German cockroach Blattella germanica

Canadian Journal of Genetics and Cytology ◽

10.1139/g83-007 ◽

1983 ◽

Vol 25 (1) ◽

pp. 40-43 ◽

Cited By ~ 1

Author(s):

Michael S. Krawczun ◽

Asit B. Mukherjee

Keyword(s):

Cell Lines ◽

Banding Pattern ◽

Blattella Germanica ◽

German Cockroach ◽

Mitotic Chromosomes ◽

Established Cell Lines ◽

Established Cell ◽

Cockroach Blattella Germanica

A detailed analysis of karyology, chromosome measurement, and chromosome constitution in different cell populations from two established cell lines of the German cockroach Blattella germanica is provided. All 12 pairs of chromosomes in somatic cells are clearly identifiable. All chromosomes except No. 5 display distinct C- bands. The G- and Q-banding patterns are not consistent, but many cross bands were observed. The N-banding pattern is reproducible and quite similar to the C-banding pattern. The cells in vitro as compared with cells in vivo are found to be extremely suitable for analysis of chromosomes of this species.

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SIRPαFc, a CD47-Blocking Cancer Immunotherapeutic, Sensitizes Malignant B Cells to Macrophage-Mediated Destruction

Blood ◽

10.1182/blood.v126.23.2191.2191 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2191-2191

Author(s):

Natasja Nielsen Viller ◽

Saman Maleki Vareki ◽

Karen Dodge ◽

Hui Chen ◽

Vivian Lee ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Tumor Cells ◽

Cell Lines ◽

Research Funding ◽

B Lymphoma ◽

Established Cell Lines ◽

Established Cell

Abstract Macrophages commonly infiltrate tumor microenvironments and can phagocytose and destroy malignant cells. Cancer cells, however, can inhibit the tumoricidal activity of macrophages by expressing CD47 on their surface. CD47 delivers an anti-phagocytic ("do not eat") signal by binding signal-regulatory protein α (SIRPα) on the surface of macrophages. There is strong evidence that many liquid and solid tumors exploit the CD47-SIRPα pathway to escape macrophage-mediated destruction. Blockade of this inhibitory axis using a soluble SIRPα-Fc fusion protein (SIRPαFc) has emerged as a promising strategy to neutralize the suppressive effects of CD47 and promote the eradication of tumor cells. Here we have examined the effect of SIRPαFc on malignant human B cells in vitro and in vivo. We first assessed the binding of SIRPαFc to a panel of established cell lines and primary cells from patients with diffuse large B cell lymphoma, Burkitt's lymphoma, multiple myeloma and acute lymphoblastic leukemia. SIRPαFc exhibited strong, dose-dependent binding to all tumor cells, with an average effective half-maximal concentration of approximately 150 nM. Next, the ability of SIRPαFc to promote macrophage-mediated phagocytosis of human tumor cells was examined using confocal microscopy. In cultures left untreated or treated with a control Fc fragment, macrophages exhibited a low level of phagocytosis, consistent with CD47-mediated suppression. Blockade of CD47 on the target cells using SIRPαFc dramatically increased macrophage phagocytosis of tumor cells. The majority of established cell lines and all primary human tumors were sensitized to macrophage-mediated destruction, including both peripheral blood- and bone marrow-derived primary tumor samples. Finally, we assessed the in vivo activity of SIRPαFc in CD20hi (Raji) and CD20low (Namalwa) B lymphoma xenograft models. SIRPαFc treatment significantly reduced Raji growth and increased host mouse survival (time to euthanasia), and completely ablated the growth of Namalwa tumors, the latter being insensitive to rituximab therapy. In conclusion, SIRPαFc demonstrated in vitro activity against a broad range of human B cell tumors and was highly effective at controlling the growth of aggressive B lymphoma xenografts in mice, including a CD20low tumor that was non-responsive to rituximab. These data support the evaluation of SIRPαFc in patients with B cell malignancies. Disclosures Nielsen Viller: Trillium Therapeutics Inc.: Employment. Vareki:Trillium Therapeutics Inc.: Research Funding. Dodge:Trillium Therapeutics Inc.: Employment. Chen:Trillium Therapeutics Inc.: Employment. Lee:Trillium Therapeutics Inc.: Employment. Chai:Trillium Therapeutics Inc.: Employment. Pang:Trillium Therapeutics Inc.: Employment. Wong:Trillium Therapeutics Inc.: Employment. Trudel:Novartis: Honoraria; Oncoethix: Research Funding; BMS: Honoraria; Trillium Therapeutics Inc.: Research Funding; Celgene: Equity Ownership, Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Figueredo:Trillium Therapeutics Inc.: Research Funding. Pampillo:Trillium Therapeutics Inc.: Research Funding. Koropatnick:Trillium Therapeutics Inc.: Research Funding. Petrova:Trillium Therapeutics Inc.: Employment. Uger:Trillium Therapeutics Inc.: Employment.

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36 IMPROVING THE APPLICATION OF NUCLEAR TRANSFER FOR PRODUCING NON-DOMESTIC FELIDS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab36 ◽

2005 ◽

Vol 17 (2) ◽

pp. 168 ◽

Cited By ~ 3

Author(s):

M.C. Gomez ◽

C.E. Pope ◽

L. Lyons ◽

A. Cole ◽

M. Lopez ◽

...

Keyword(s):

Somatic Cell ◽

Cell Lines ◽

Nuclear Transfer ◽

Implantation Rate ◽

Domestic Cat ◽

Developmental Competence ◽

In Vitro Production ◽

Fetal Survival

One of the most remarkable aspects of somatic cell nuclear transfer (NT) is the possibility of avoiding extinction when there are few remaining animals of a specific felid population. Previously, we produced live male African Wildcat (AWC; Felis lybica) cloned kittens using inter-species nuclear transfer (Gomez et al. 2004 Cloning and Stem Cells 6, 217–228). The production of females is a primary objective of most breeding programs. Therefore, the purpose of the present study was to determine (1) if we could produce live female AWC cloned kittens at a proportion similar to that previously demonstrated with males, and (2) if our inter-species NT technique used to produce AWC is applicable to in vitro production of another non-domestic felid species. Specifically, we evaluated the in vivo developmental competence of NT embryos derived by fusion of Black footed cat (BFC, Felis nigripes) and AWC fibroblasts with domestic cat (DSH, Felis catus) cytoplasts, after transfer into domestic cat recipients. Fibroblast cell lines were established from skin biopsies of BFC (6-year-old), and AWC (12-year-old) adult females. After at least three passages, cells were serum-starved for 5 days and injected into the perivitelline space of enucleated domestic cat oocytes. Fusion of cell-cytoplast couplets was induced by applying a 3-s AC pre-pulse of 20 V, 1 MHz, followed by two 30-μs DC pulses of 240 V/mm. Fused couplets were activated 2 to 3 h after fusion by exposure to two 60 μsec DC pulses of 120 V/mm, followed by 4 h incubation with 10 μg/mL cycloheximide and 5 μg/mL cytochalasin B. Reconstructed BFC (n = 16) and AWC (n = 536) NT Day 1 embryos were transferred by laparoscopy into the oviducts of 1 and 12 gonadotrophin-treated DSH recipients, respectively, on Day 1 after induced ovulation. Pregnancy was assessed by ultrasonography on Day 22. One cat (100%) receiving BFC NT embryos and 5 (41.6%) cats receiving AWC NT embryos became pregnant. Twenty-three AWC cloned embryos implanted and 11 kittens were born. Three BFC NT embryos implanted and the pregnancy is currently ongoing. AWC cloned kittens were phenotypically and genetically identical to their somatic cell donor. Their clonal identity was assessed by multiplex PCR amplification of 20 microsatellite markers, including seven markers that are known to be on the X chromosome. In summary, these results indicate that female AWC cloned kittens can be produced and BFC pregnancy can be established in domestic cat recipients. The embryo implantation rate and viability of AWC female cloned embryos was higher than that observed after the transfer of AWC male cloned embryos. The difference may be due to improvements in the NT procedure, rather than to differences in the sex of the cell lines. Table 1. Implantation rate and fetal survival to term of AWC and BFC NT embryos in pregnant domestic cat recipients

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LPTO-06. A NOVEL BRAIN-PERMEANT CHEMOTHERAPEUTIC AGENT FOR THE TREATMENT OF BREAST CANCER LEPTOMENINGEAL METASTASIS

Neuro-Oncology Advances ◽

10.1093/noajnl/vdz014.029 ◽

2019 ◽

Vol 1 (Supplement_1) ◽

pp. i7-i7

Author(s):

Jiaojiao Deng ◽

Sophia Chernikova ◽

Wolf-Nicolas Fischer ◽

Kerry Koller ◽

Bernd Jandeleit ◽

...

Keyword(s):

Breast Cancer ◽

Brain Tumors ◽

Cell Lines ◽

Chemotherapeutic Agent ◽

Leptomeningeal Metastasis ◽

Breast Cancer Cell Lines ◽

Normal Brain ◽

Breast Cancers

Abstract Leptomeningeal metastasis (LM), a spread of cancer to the cerebrospinal fluid and meninges, is universally and rapidly fatal due to poor detection and no effective treatment. Breast cancers account for a majority of LMs from solid tumors, with triple-negative breast cancers (TNBCs) having the highest propensity to metastasize to LM. The treatment of LM is challenged by poor drug penetration into CNS and high neurotoxicity. Therefore, there is an urgent need for new modalities and targeted therapies able to overcome the limitations of current treatment options. Quadriga has discovered a novel, brain-permeant chemotherapeutic agent that is currently in development as a potential treatment for glioblastoma (GBM). The compound is active in suppressing the growth of GBM tumor cell lines implanted into the brain. Radiolabel distribution studies have shown significant tumor accumulation in intracranial brain tumors while sparing the adjacent normal brain tissue. Recently, we have demonstrated dose-dependent in vitro and in vivo anti-tumor activity with various breast cancer cell lines including the human TNBC cell line MDA-MB-231. To evaluate the in vivo antitumor activity of the compound on LM, we used the mouse model of LM based on the internal carotid injection of luciferase-expressing MDA-MB-231-BR3 cells. Once the bioluminescence signal intensity from the metastatic spread reached (0.2 - 0.5) x 106 photons/sec, mice were dosed i.p. twice a week with either 4 or 8 mg/kg for nine weeks. Tumor growth was monitored by bioluminescence. The compound was well tolerated and caused a significant delay in metastatic growth resulting in significant extension of survival. Tumors regressed completely in ~ 28 % of treated animals. Given that current treatments for LM are palliative with only few studies reporting a survival benefit, Quadriga’s new agent could be effective as a therapeutic for both primary and metastatic brain tumors such as LM. REF: https://onlinelibrary.wiley.com/doi/full/10.1002/pro6.43

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