Evidence of In Vitro Preservation of Human Nephrogenesis at the Single-Cell Level

Abstract We investigated the effects of testosterone on FSH secretion from male rat anterior pituitary cells in culture at the single cell level. Anterior pituitary cells cultured with or without 10 ng/ml testosterone for 72 h were mono-dispersed and subjected to cell immunoblot assays for FSH. Cell blots specific for FSH were quantified by means of a microscopic image analyzer. The number of FSH-secreting cells detected as immunoreactive cell blots on the transfer membrane represented 4·1% of total pituitary cells applied on the membrane. The amount of FSH secreted by single cells varied from <20 to >8 000 fg/cell/h. The number of FSH-secreting cells was not changed by the addition of 10 ng/ml testosterone into the culture medium. Testosterone administration increased the mean FSH secretion by 64% after 3 h incubation, resulting in a shift to the right in the frequency distribution of FSH secretion from single cells. The total amount of FSH, namely the sum of FSH secreted by each FSH-secreting cell, was increased by 92% by the addition of testosterone. However, mean amounts of FSH secretion by the top ten cells of the largest secretor subgroup (>5 pg/cell/3 h) were not different between control and testosterone-treated groups. The present study analyzed, for the first time, FSH secretion from rat anterior pituitary cells at the single cell level. The results suggest that stimulation by testosterone of FSH secretion in vitro is not due to an increase in the number of FSH-secreting cells but to an increase in FSH secretion from each cell. Journal of Endocrinology (1996) 148, 427–433

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Luteinizing Hormone (LH) Drives Diverse Intracellular Calcium Second Messenger Signals in Isolated Porcine Ovarian Thecal Cells: Preferential Recruitment of Intracellular Ca2+ Oscillatory Cells by Higher Concentrations of LH*

Endocrinology ◽

10.1210/endo.141.6.7501 ◽

2000 ◽

Vol 141 (6) ◽

pp. 2220-2228 ◽

Cited By ~ 6

Author(s):

C. Aguirre ◽

F. C. L. Jayes ◽

J. D. Veldhuis

Keyword(s):

Single Cell ◽

Phospholipase C ◽

Pertussis Toxin ◽

Second Messenger ◽

High Amplitude ◽

Single Cell Level ◽

Cell Level ◽

Thecal Cells ◽

Second Messenger Signaling

Abstract The present study examines Ca2+ second messenger signaling driven by LH in isolated porcine thecal cells. To this end, we implemented semiquantitative fluorescent (fura-2) videomicroscopic imaging of single thecal cells in vitro. Stimulation of 388 cells with LH (5 μg/ml) elicited an intracellular Ca2+ ([Ca2+]i) signal in 85± 5.3% of individual thecal cells (n = 11 experiments). Among 337 LH-responsive cells, we identified four predominant temporal modes of [Ca2+]i signaling: 1)[ Ca2+]i oscillations with periodicities of 0.5 to 4.5 min−1 (63 ± 4.5%), 2) a[ Ca2+]i spike followed by a sustained plateau (17 ± 2.6%), 3) a [Ca2+]i spike only (5.8 ± 2.6%); and 4) a [Ca2+]i plateau only (3.8 ± 1.5%). The prevalence, but not the amplitude or frequency, of LH-induced [Ca2+]i oscillations in thecal cells was dependent on the agonist concentration. Reduced availability of extracellular Ca2+ induced by treatment with EGTA or cobaltous chloride did not block the initiation, but reversibly abolished ongoing [Ca2+]i oscillations (72% of cells) or increased the mean[ Ca2+]i interspike periodicity from 1.09± 0.16 to 0.59 ± 0.07 min−1 (P< 0.05). Putative phospholipase C inhibition with U-73122 (10μ m) also abolished or frequency-damped LH-driven[ Ca2+]i oscillations in 95 ± 4.7% of cells. [Ca2+]i oscillations in thecal cells were not abrogated by overnight pretreatment with pertussis toxin. We conclude that 1) thecal cells (unlike earlier findings in granulosa cells) manifest a diverse array of [Ca2+]i signaling responses to LH at the single cell level; 2) LH can dose dependently recruit an increasing number of individually[ Ca2+]i oscillating thecal cells; 3) extracellular Ca2+ is required for LH to sustain (but not initiate) frequent and high amplitude [Ca2+] oscillations in thecal cells; and 4) these signaling actions of LH are mediated via phospholipase C, but not a pertussis-toxin sensitive mechanism. Accordingly, the present data extend the apparent complexity of LH-induced [Ca2+]i second messenger signaling and identify at the single cell level LH’s dose-responsive drive of[ Ca2+]i oscillations in gonadal cells.

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A Lymphoid Progenitor Propagates AML in a Mouse Model of CALM/AF10 Positive Leukemia.

Blood ◽

10.1182/blood.v106.11.101.101 ◽

2005 ◽

Vol 106 (11) ◽

pp. 101-101

Author(s):

Aniruddha Deshpande ◽

Monica Cusan ◽

Viajy P.S. Rawat ◽

Hendrik Reuter ◽

Leticia Quintanilla-Martinez ◽

...

Keyword(s):

Single Cell ◽

Fusion Gene ◽

Growth Potential ◽

Single Cell Level ◽

Cell Level ◽

Major Step ◽

Post Transplantation ◽

Myeloid Lineage ◽

Igh Locus

Abstract The identification of cancer stem cells is a major step towards the understanding of the pathogenesis of solid and hematological neoplasias and might have direct implications for the development of innovative therapeutic strategies aiming at the eradication of the tumor propagating cell. Here we describe that acute myeloid leukemia (AML), induced by the CALM/AF10 fusion gene, is propagated by a transformed lymphoid progenitor in a murine bone marrow (BM) transplantation model of t(10;11)(p13;q14) positive AML. When mice were transplanted with BM cells retrovirally engineered to express the C/A fusion, all animals (n=13) died from AML showing DJ rearrangement of the heavy chain of the IgH locus after a median of 110 days post transplantation. Diseased mice showed an accumulation of myeloid Gr1+/Mac1+ cells in the peripheral blood and spleen and a multi-organ infiltration by myeloperoxidase and chloracetate esterase positive cells in immunohistochemical sections. In the leukemic mice only a minor population counting for 6.7 % (± 2.1) cells in the BM displayed the B220 lymphoid antigen and lacked myeloid markers (on average 9.4 % ± 3). The majority of cells expressed myeloid markers (on average 82.9 % (± 8.6) Mac1+ cells, 86.4 % (± 3.7) Gr-1+ cells). Additionally, in the leukemic mice an average of 26.0 % (± 8.6) and 32.5 % (± 13.2) of these cells co-expressed B220 and Mac1 or B220 and Gr1, respectively, compared to 2.1 % (± 0.7) and 1.3 % (± 0.3), respectively, in GFP controls. Importantly, in vitro only the B220+/Mac− cell population had growth potential at the single cell level (seeding efficiency 29 %) compared to the B220+/Mac+ (1%) and B220−/Mac+ cells (1%). When the frequency of leukemia propagating cells (LPC) of the three different populations isolated from primary leukemic mice was determined by limiting dilution transplantation and Poisson statistics the frequency of the LPC was more than 380 fold higher in the ‘B220+/Mac1−’ population (1 in 36 cells) than in the ‘Mac1+/B220−’ bulk population (1 in 13906 cells) and more than 12fold increased compared to the B220+/Mac+ cells (1 in 437 cells). In vitro a single B220+/Mac1− cell isolated from a leukemic mouse was able to give rise to the B220+/Mac1+ as well as the Mac1+/ B220− population, both populations showing the identical genomic DJ rearrangement at the IgH locus as the initial B220+/Mac1− cell, demonstrating its capacity to differentiate into the myeloid lineage at the single cell level. The B220+/Mac1− population displayed a CD43+/AA4.1+/HSA+/CD19−/IL-7R− phenotype, was promiscuous in its transcription profile with positivity for EBF, but also MPO and lacked Pax5. Taken together, this murine leukemia model indicates that AML can be propagated from an early transformed lymphoid progenitor cell. The transformation of an early lymphoid cell, which is re-directed into the myeloid lineage by appropriate oncogenes, could explain recurrent observations of immunoglobulin rearrangements in patients with AML and provide a rationale for therapies, aiming at the elimination of the leukemia propagating cell with lymphoid characteristics, but sparing normal HSCs.

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Accumulation of Amphotericin B in Human Macrophages Enhances Activity against Aspergillus fumigatus Conidia: Quantification of Conidial Kill at the Single-Cell Level

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.10.2569 ◽

1998 ◽

Vol 42 (10) ◽

pp. 2569-2575 ◽

Cited By ~ 21

Author(s):

Bernhard Jahn ◽

Albert Rampp ◽

Christian Dick ◽

Andreas Jahn ◽

Michael Palmer ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Single Cell ◽

Amphotericin B ◽

Mammalian Cells ◽

Single Cell Level ◽

Cell Level ◽

Streptolysin O ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony

ABSTRACT A cytofluorometric assay that allowed assessment of damage to phagocytosed Aspergillus fumigatus conidia at the single-cell level was developed. After ingestion by monocyte-derived macrophages (MDMs), conidia were reisolated by treatment of the cells with streptolysin O, a pore-forming toxin with lytic properties on mammalian cells but not on fungi. The counts obtained by staining of damaged conidia with propidium iodide and quantification by cytofluorometry correlated with colony counts. By the use of this method, we demonstrate that MDMs differentiated in vitro by low-dose granulocyte-macrophage colony-stimulating factor and gamma interferon have only a limited capacity to damageAspergillus conidia in vitro. The killing rate 12 h after phagocytosis was found to be only 10 to 15%. However, intracellular loading of the phagocytes with amphotericin B (AmB) dose dependently enhanced the anticonidial activity. Preincubation of macrophages with only 1 μg of AmB per ml resulted in an uptake of 18 fg of AmB/cell, leading to killing rates of 50 to 60%. The experimental protocol provides a new tool for the rapid quantification of anticonidial activity against A. fumigatus in vitro. Intracellular accumulation of AmB may represent an important factor underlying the efficacy of this antifungal drug in the prophylaxis and treatment ofAspergillus infections.

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Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo

Haematologica ◽

10.3324/haematol.2021.278896 ◽

2021 ◽

Author(s):

Leo Nicolai ◽

Rainer Kaiser ◽

Raphael Escaig ◽

Marie-Louise Hoffknecht ◽

Afra Anjum ◽

...

Keyword(s):

Single Cell ◽

Platelet Function ◽

Cell Tracking ◽

Mouse Strains ◽

Single Cell Level ◽

Cell Level ◽

Reporter Mouse ◽

Reporter Mice

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Credriven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of megakaryocytes in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to WT controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and megakaryocyte biology on a single cell level.

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Therapeutic Effect of a Traditional Chinese Medicine Compound Mixture Compound Kushen Injection in Treating Multiple Myeloma

Blood ◽

10.1182/blood-2020-141929 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-32

Author(s):

Hongqiang Jiang ◽

Honglei Tu ◽

Yanxia Jin ◽

Xianjin Wu ◽

Ziyi Luo ◽

...

Keyword(s):

Multiple Myeloma ◽

Single Cell ◽

Conflicts Of Interest ◽

Dependent Manner ◽

Single Cell Level ◽

Cell Level ◽

Rpmi 8226 ◽

Nsg Mouse ◽

Compound Kushen Injection

Abstract Background: Multiple myeloma (MM) is a hematology malignant disease originated from B-cell line and still incurable. Compound Kushen Injection (CKI) as a Traditional Chinese Medicines are promising agents in our previous research for treating cancer. The effect of CKI on multiple myeloma was still unknown. Methods: In vitro experiment, flow cytometry was used to evaluate effect of CKI on multiple myeloma cells. Optofluidic chip was applied to detect effect at single-cell level. And in vivo RPMI-8226 GFP+ B-NSG mouse model was built to assess the role of CKI in multiple myeloma treatment. Results: CKI inhibited MM cells proliferation of and increased its apoptosis rate. And the cell cycle of MM cells was also arrested by CKI treatment. In contrast, CKI has few toxic effects on mesenchymal stem cells (MSCs) and MC3T3 cells. At the single-cell level, MM cells was died in time and dose dependent manner. Transcriptome find that the expression of MYC and TERT in CKI-treated RPMI-8226 cells was significantly down-regulated and confirmed by qRT-PCR and Western blot. Overexpression of TERT can partly reverse the inhibition effect of CKI on RPMI-8226 cells. B-NSG mouse was injected with GFP+ RPMI-8226 cells through caudal vein, and the disease was partially alleviated by decreased tumor burden in the CKI-treated group. Furthermore, it is surprising that in animal models with myeloma bone disease, the bone mass was higher in CKI treatment group than control. Conclusions: CKI inhibits MM cells through the MYC/TERT signaling pathway and improve the quality of life of MM mouse. Our findings provide preclinical evidence to show that CKI could be a promising candidate in human MM therapy. Disclosures No relevant conflicts of interest to declare.

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A Microphysiological Cell-Culturing System for Pharmacokinetic Drug Exposure and High-Resolution Imaging of Arrays of 3D Microtissues

Frontiers in Pharmacology ◽

10.3389/fphar.2021.785851 ◽

2021 ◽

Vol 12 ◽

Author(s):

Christian Lohasz ◽

Jacqueline Loretan ◽

Dario Sterker ◽

Ekkehard Görlach ◽

Kasper Renggli ◽

...

Keyword(s):

Single Cell ◽

Drug Exposure ◽

Dependent Manner ◽

Single Cell Level ◽

Cell Level ◽

Treatment Regimens ◽

Cell Culturing ◽

Drug Concentrations

Understanding the pharmacokinetic/pharmacodynamic (PK/PD)-relationship of a drug candidate is key to determine effective, yet safe treatment regimens for patients. However, current testing strategies are inefficient in characterizing in vivo responses to fluctuating drug concentrations during multi-day treatment cycles. Methods based on animal models are resource-intensive and require time, while traditional in vitro cell-culturing methods usually do not provide temporally-resolved information on the effects of in vivo–like drug exposure scenarios. To address this issue, we developed a microfluidic system to 1) culture arrays of three-dimensional spheroids in vitro, to 2) apply specific dynamic drug exposure profiles, and to 3) in-situ analyze spheroid growth and the invoked drug effects in 3D by means of 2-photon microscopy at tissue and single-cell level. Spheroids of fluorescently-labeled T-47D breast cancer cells were monitored under perfusion-culture conditions at short time intervals over three days and exposed to either three 24 h-PK-cycles or a dose-matched constant concentration of the phosphatidylinositol 3-kinase inhibitor BYL719. While the overall efficacy of the two treatment regimens was similar, spheroids exposed to the PK profile displayed cycle-dependent oscillations between regression and regrowth. Spheroids treated with a constant BYL719 concentration regressed at a steady, albeit slower rate. At a single-cell level, the cell density in BYL719-treated spheroids oscillated in a concentration-dependent manner. Our system represents a versatile tool for in-depth preclinical characterization of PK/PD parameters, as it enables an evaluation of drug efficacy and/or toxicity under realistic exposure conditions.

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