Bone marrow niches for hematopoietic stem cells: life span dynamics and adaptation to acute stress.

ABSTRACT: Hematopoietic stem cells (HSCs) are instrumental for organismal survival because they are responsible for lifelong production of mature blood lineages in homeostasis and response to external stress. To fulfill their function, HSCs rely on reciprocal interactions with specialized tissue microenvironments, termed HSC niches. From embryonic development to advanced aging, HSCs transition through several hematopoietic organs in which they are supported by distinct extrinsic cues. Here, we describe recent discoveries on how HSC niches collectively adapt to ensure robust hematopoietic function during biological aging and after exposure to acute stress. We also discuss the latest strategies leveraging niche-derived signals to revert aging-associated phenotypes and enhance hematopoietic recovery after myeloablation.

Variation in mesenchymal KITL/SCF and IGF1 expression in middle age underlies steady-state hematopoietic stem cell aging.

ABSTRACT: Intrinsic molecular programs and extrinsic factors including proinflammatory molecules are understood to regulate hematopoietic aging. This is based on foundational studies using genetic perturbation to evaluate causality. However, individual organisms exhibit natural variation in the hematopoietic aging phenotypes and the molecular basis of this heterogeneity is poorly understood. Here, we generated individual single-cell transcriptomic profiles of hematopoietic and nonhematopoietic cell types in 5 young adult and 9 middle-aged C57BL/6J female mice, providing a web-accessible transcriptomic resource for the field. Among all assessed cell types, hematopoietic stem cells (HSCs) exhibited the greatest phenotypic variation in expansion among individual middle-aged mice. We computationally pooled samples to define modules representing the molecular signatures of middle-aged HSCs and interrogated, which extrinsic regulatory cell types and factors would predict the variance in these signatures between individual middle-aged mice. Decline in signaling mediated by adiponectin, kit ligand (KITL) and insulin-like growth factor 1 (IGF1) from mesenchymal stromal cells (MSCs) was predicted to have the greatest transcriptional impact on middle-aged HSCs, as opposed to signaling mediated by endothelial cells or mature hematopoietic cell types. In individual middle-aged mice, lower expression of Kitl and Igf1 in MSCs was highly correlated with reduced lymphoid lineage commitment of HSCs and increased signatures of differentiation-inactive HSCs. These signatures were independent of expression of aging-associated proinflammatory cytokines including interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor α and RANTES. In sum, we find that Kitl and Igf1 expression are coregulated and variable between individual mice at the middle age and expression of these factors is predictive of HSC activation and lymphoid commitment independently of inflammation.

Investigating retinal toxicity of a lutein-based dye in a model of isolated and perfused bovine retina.

PURPOSE: Retidyne™ is a new lutein-based dye for internal limiting membrane staining. It uses the intrinsic staining characteristics of lutein which is already known to act as an antioxidant and blue-light filter in the human retina. We investigated retinal tolerance to different staining times measured by the electroretinogram (ERG) of an isolated and perfused retina whole mount. METHODS: For functionality, testing bovine retinas were prepared and perfused with an oxygen saturated standard solution and the ERG was recorded until stable b-wave amplitudes were reached. Then the perfusion was stopped and Retidyne™ was applied directly onto the retinal surface for exposure times of 60 or 120 s. After restarting the perfusion with standard solution, the ERG amplitudes were monitored for 75 min. To investigate the effects on photoreceptor function alone, 1 mM asparate was added to block b-waves. RESULTS: For an exposure time of 60 s amplitudes of a- and b-waves remained stable throughout the experiment. Exposure times of 120 s caused an initial drop of amplitudes that reached statistical significance only for a-waves (a, - 21%, p = 0.047; b, - 14%, p = 0.052). This effect was only seen during the first minutes of the washout and the ERG recovered completely. CONCLUSIONS: In the model of isolated and perfused bovine retina, Retidyne™ showed a good safety profile for common intraoperatively used staining times. An initial toxic effect regarding the transient drop of amplitudes cannot be ruled out but the effect might also be explained by the partial blockage of the flashlight due to a more intense staining effect at the beginning of the washout.

Comprehensive Molecular Characterization of a Cisplatin-Specific Monoclonal Antibody.

Despite their immense and rapidly increasing importance as analytical tools or therapeutic drugs, the detailed structural features of particular monoclonal antibodies are widely unknown. Here, an antibody already in use for diagnostic purposes and for molecular dosimetry studies in cancer therapy with very high affinity and specificity for cisplatin-induced DNA modifications was studied extensively. The molecular structure and modifications as well as the antigen specificity were investigated mainly by mass spectrometry. Using nano electrospray ionization mass spectrometry, it was possible to characterize the antibody in its native state. Tandem-MS experiments not only revealed specific fragments but also gave information on the molecular structure. The detailed primary structure was further elucidated by proteolytic treatment with a selection of enzymes and high resolution tandem-MS. The data were validated by comparison with known antibody sequences. Then, the complex glycan structures bound to the antibody were characterized in all detail. The Fc-bound oligosaccharides were released enzymatically and studied by matrix-assisted laser desorption/ionization mass spectrometry. Overall 16 different major glycan structures were identified. The binding specificity of the antibody was investigated by applying synthetic single and double stranded DNA oligomers harboring distinct Pt adducts. The antibody-antigen complexes were analyzed by mass spectrometry under native conditions. The stability of the complex with double stranded DNA was also investigated.

Identification of Lewis and Blood Group Carbohydrate Epitopes by Ion Mobility-Tandem-Mass Spectrometry Fingerprinting.

Glycans have several elements that contribute to their structural complexity, involving a range of monosaccharide building blocks, configuration of linkages between residues and various degrees of branching on a given structure. Their analysis remains challenging and resolving minor isomeric variants can be difficult, in particular terminal fucosylated Lewis and blood group antigens present on N- and O-glycans. Accurately characterizing these isomeric structures by current techniques is not straightforward and typically requires a combination of methods and/or sample derivatization. Yet the ability to monitor the occurrence of these epitopes is important as structural changes are associated with several human diseases. The use of ion mobility-mass spectrometry (IM-MS), which separates ions in the gas phase based on their size, charge and shape, offers a new potential tool for glycan analysis and recent reports have demonstrated its potential for glycomics. Here we show that Lewis and blood group isomers, which have identical fragmentation spectra, exhibit very distinctive IM drift times and collision cross sections (CCS). We show that IM-MS/MS analysis can rapidly and accurately differentiate epitopes from parotid gland N-glycans and milk oligosaccharides based on fucosylated fragment ions with characteristic CCSs.

Cytotoxic Effect of Voriconazole on Human Corneal Epithelial Cells.

BACKGROUND: To assess the cytotoxic properties of voriconazole and sulfobutylether-ß-cyclodextrin (SBECD) on cultured primary human corneal epithelial cells. METHODS: Human corneal epithelial cells were cultured and exposed to various concentrations of SBECD (0.016-32 mg/ml) and voriconazole (0.001-2 mg/ml). Cellular cytotoxicity of SBECD and voriconazole on human corneal epithelial cells was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide test and the LIVE/DEAD Viability/Cytotoxicity Assay with fluorescence microscopy analysis. Cell damage was assessed with phase-contrast microscopy after 24 h of exposure to SBECD and voriconazole. RESULTS: The cytotoxicity tests and the morphological characteristic demonstrated the dose-dependent toxic effect of SBECD and voriconazole on human corneal epithelial cells. No corneal epithelial cytotoxicity was observed below the concentration of 0.08 and 0.025 mg/ml after 24-hour exposure to SBECD and voriconazole, respectively. CONCLUSIONS: The results of the study reveal the dose-dependent cytotoxic effect of SBECD and voriconazole on cultured human corneal epithelial cells. Therefore, voriconazole eye drops should be used cautiously in the treatment of fungal corneal ulcers.

Cytotoxic properties of sunitinib and sorafenib on human corneal epithelial cells.

PURPOSE: To generate toxicology profiles of individual drugs on human corneal epithelial cells (HCEC) and compare their in vitro cytotoxicity. METHODS: Monolayer cultures of HCEC were harvested from two human donor eyes. Sunitinib (0.3-10 µg/mL) and Sorafenib (0.3-100 µg/mL), diluted in culture medium (CnT-BM.1, CELLnTEC Advanced Cell Systems AG, Bern, Switzerland), 1% Penicillin and 1% Streptomycin were added to cells that were being grown in cell culture dishes. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed 24 hours, three days and five days after incubation. Live/dead viability/cytotoxicity assay (Live/dead assay) was performed and analyzed using fluorescence microscopy after 24 hours of incubation. The expression of p63, ABCG2 and PDGFRß was evaluated by immunocytochemistry prior to exposure. Cell morphology was assessed with a phase contrast microscope after 24 hours of exposure. RESULTS: Significant toxicity of Sunitinib was seen at concentrations of >3.3 µg/mL and of Sorafenib at concentrations of >1.0 µg/mL after 24 hours of incubation. Both drugs exhibited increasing toxicities over time. HCEC stained positively for p63, ABCG2 and PDGFRß. In comparison, the IC50 (inhibitory concentration 50) of Sorafenib was 2.26 times the IC50 of Sunitinib using Live/dead assay after 24 hours and 2.39, 1.29 and 0.78 times the IC50 of Sunitinib using the MTT test after 24 hours, three days and five days, respectively. CONCLUSIONS: These in vitro experimental findings support the safety of Sunitinib and Sorafenib on HCEC when used at a concentration of <3.3 µg/mL and <1.0 µg/mL, respectively, after 24 hours of exposure. The in vitro cytotoxicity of Sorafenib on HCEC was higher than Sunitinib.

Comparative toxicity and proliferation testing of aflibercept, bevacizumab and ranibizumab on different ocular cells.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is a key factor in the pathogenesis of neovascular retinal diseases including age-related macular degeneration. VEGF inhibitors including ranibizumab, pegaptanib or bevacizumab improve retinal morphology and vision in many patients. The recently approved drug aflibercept (VEGF Trap-Eye/Eyelea, Regeneron, Tarrytown, New York, USA) offers a new therapy modality. We therefore tested for toxic and anti-proliferating effects of aflibercept. METHODS: The effects of aflibercept (0.125, 0.5, 2 mg), ranibizumab (0.125 mg) and bevacizumab (0.3125 mg) after 1, 24, 48 and 72 h on cell morphology via phase contrast pictures, cell viability via MTS assay, total cell amount via crystal violet staining, apoptosis induction via caspase 3/7 assay and proliferation via BrdU assay were investigated. Three ocular cell lines were chosen for toxicology testing: ARPE19 cells, RGC-5 cells and 661W cells. RESULTS: Aflibercept did not cause changes in cell morphology, induce apoptosis or cause permanent decrease in cell viability, cell density or proliferation in any cell line or concentration investigated. In general, aflibercept had fewer effects (upregulation or downregulation) compared with controls than bevacizumab or ranibizumab. CONCLUSIONS: In our experiments, aflibercept did not lead to any negative effects on retinal cell lines and might therefore be used safely in clinical applications.

Decellularized bovine corneal posterior lamellae as carrier matrix for cultivated human corneal endothelial cells.

PURPOSE: To evaluate the potential of decellularized bovine corneas (DBCs) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs). METHODS: Posterior lamellae of ten bovine corneas were decellularized using ethylene diamin tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/mL) and 0.3% sodium dodecyl sulphate (SDS). Hematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining was done to confirm the absence of bovine cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using a DNA Purification Kit. HCECs were harvested from human donor eyes and seeded on the Descemet's membrane of the DBCs. Cell morphology was assessed after 6 h of incubation, and at days 1, 4, 7, 10 and 14. Expression of zonula occludens-1 (ZO-1), connexin-43 (CX-43), Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase), natrium hydrogen carboanhydrase (Na(+)/HCO(3)(-)), collagen type VIII, collagen type IV and cytokeratin-3 (AE5) were analyzed by immunohistochemistry. RESULTS: HE staining and DAPI staining showed that bovine cells were substantially removed from the stroma and Descemet's membrane. A significant DNA reduction (mean before decelluraziation 365.3 ± 88.6 ng/mg, mean after decelluarization 23.2 ± 7.9 ng/mg, p < 0.001) was observed. HCECs formed a continuous, viable, predominantly polygonal monolayer with a mean cell density of 2380 ± 179 cells/mm(2) on DBCs. Immunohistochemistry analysis demonstrated positive staining for AE5, collagen type VIII, ZO-1, CX-43, Na(+)/HCO(3)(-), and Na(+)/K(+)-ATPase. CONCLUSIONS: Phenotypical properties of HCECs on DBCs imply that the HCEC sheets are capable of maintaining an intact barrier and ionic pump function in vitro. DBCs might, therefore, be a promising scaffold for ex vivo expansion of HCECs. This xenogeneic substrate might be used for therapy of isolated corneal endothelial diseases.

Reconstruction of corneal stroma with decellularized porcine xenografts in a rabbit model.

PURPOSE: To evaluate the potential use of decellularized porcine stromal matrix (PSM) for reconstruction of corneal stroma in a rabbit model. METHODS: Ten chinchilla bastard rabbit corneas were exposed to a circular half-thickness keratotomy with a 3.0 mm diameter at the central cornea. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra-acetic acid (EDTA, 0.1%), aprotinin (10 K IU/ml) and 0.3% sodium dodecyl sulphate (SDS). The 3.0 mm in diameter decellularized corneal stromal xenograft was inserted into the pocket, and the incision was closed with four 10.0 nylon sutures. Clinical photographs were taken at day 1, day 7, day 30 and on a monthly basis for up to 6 months after transplantation. Six months after surgery, the rabbits were killed and eyes were enucleated. Haematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm the complete removal of the corneal cells after decellularization of porcine corneas and repopulation with rabbit cells. Alcian blue staining was performed for analysing the structure of the extracellular matrix (ECM). RESULTS: Efficient elimination of porcine cells was achieved by our decellularization protocol and confirmed via HE and DAPI stainings. Moreover, the major histoarchitectural ECM structure had been maintained as visualized by the alcian blue stain. Finally, the PSM was biocompatible with the host's epithelium evidenced as a regrowth covering the exposed xenograft. CONCLUSIONS: This novel technique of tissue engineering may provide one of many solutions to addressing anterior corneal pathological conditions in the face of a shortage of human corneal material.

Methyl blue and aniline blue versus patent blue and trypan blue as vital dyes in cataract surgery: capsule staining properties and cytotoxicity to human cultured corneal endothelial cells.

PURPOSE: To evaluate capsule-staining properties and biocompatibility of the triarylmethane dyes methyl blue and aniline blue compared with patent blue and trypan blue on cultured human corneal endothelial cells. SETTING: Centre for Ophthalmology, University of Tuebingen, Tuebingen, Germany. DESIGN: Experimental study. METHODS: Human corneal endothelial cell cultures were harvested from human donor cells and exposed to various concentrations (0.025 to 5.0 mg/mL) of methyl blue, aniline blue, patent blue, and trypan blue. Cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test after 24 hours of incubation. Calcein live cell staining was performed at the same time point. The dyes were also used to stain pig lens capsules in vitro by incubating the lenses for 1 minute with 3 concentrations (0.5, 1.5, and 2.5 mg/mL) of dye, after which the staining properties were evaluated. RESULTS: No significant cytotoxicity was detected for patent blue and methyl blue at any tested concentration. However, aniline blue exerted significant cytotoxicity at concentrations of 1.5 mg/mL or higher and trypan blue at 2.5 mg/mL or higher. Capsule staining of the tested triarylmethane dyes was suitable for performing capsulorhexis, but only at higher concentrations than with trypan blue. CONCLUSIONS: High concentrations and long incubation times of trypan blue and aniline blue showed significant cytotoxicity to human cultured endothelial cells in contrast to patent blue and methyl blue. All tested dyes were able to stain lens capsules sufficiently for capsulorhexis creation. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.