Effect of donor variation on osteogenesis and vasculogenesis in hydrogel cocultures

Abstract Abstract 844 Background: RBC transfusion has clear efficacy in treating various types of anemia. However, in recent decades, there is a renewed focus on the potential for negative clinical sequelae from transfusing stored RBCs. Whether or not older, stored RBC units are associated with adverse outcomes remains controversial. However, there are clear cellular and biochemical data that RBC units accumulate particles and molecules over time with known toxicity when administered to animals and/or humans. Among the most potent of these are prostaglandins and leukotrienes (jointly known as eicosanoids), which are potent mediators of inflammation and vascular pathology. Indeed, arachidonic acid (AA), and 5-, 12-, and 15-hydroxyeicsotetranoic acid (HETE) accumulate during storage of human RBCs and are biologically active in priming neutrophils (Silliman et al., Transfusion 2011 51(12):2549-54). It is well known that there is substantial donor-to-donor variation in how well RBCs store from the standpoint of post-transfusion RBC recovery; however, it is unclear whether the accumulation of eicosanoids varies substantially among donors. If differences exist, then it may be useful to screen RBC units prior to transfusion into patients with illnesses likely to be affected by eicosanoid exposure. Using a well characterized mouse model of RBC storage, and different strains of donor mice, we tested the hypothesis that there are genetic determinants affecting eicosanoid levels in stored RBCs. Methods: RBCs from C57BL/6 (B6) and FVB mice were collected in CPDA-1, filter leukoreduced, and stored under conditions previously shown to model human RBC storage. Samples collected on days 0, 5, 9, and 14 were analyzed by small molecule mass spectrometry. The study was repeated 3 times and combined data were analyzed. Results: AA accumulated over storage time in both B6 and FVB RBC units to a similar level. In contrast, although essentially no accumulation of eicosanoids was observed in B6 RBC units, substantial time-dependent increases (compared to day of collection) were observed in FVB RBC units for 5-HETE (4-fold) and 15-HETE (12-fold). In addition, a greater than 10-fold increase was observed for prostaglandin E2 in FVB RBC units with no detectable prostaglandin E2 in B6 RBC units [ findings were consistent in all 3 experiments and all differences had p values of <.05]. As oxidized phospholipids are better substrates for phospholipases that generate AA, we hypothesized that oxidative damage would be increased in FVB RBCs. Additional metabolomic analysis demonstrated that B6 RBCs had decreased levels of oxidative damage compared to FVB RBCs, including substantially lower levels of 9,10-epoxystearate, an oxidized membrane lipid. Consumption of glucose and production of lactate were similar in both strains; however, B6 RBCs had increased total glutathione (GSH), oxidized GSH, and cysteine-GSH disulfides. No difference in flux through the pentose-phosphate shunt was observed (including similar NADPH levels); this suggests that the increased GSH in C57BL/6 RBCs was due to de novo synthesis and not to regeneration by GSH reductase. C57BL/6 RBCs also had increased levels of natural anti-oxidants, including ergothioneine and alpha-tocopherol. [All the above differences achieved a p value of <.05]. Conclusion: The current findings take advantage of a mouse model of RBC storage. Given that two inbred, genetically distinct strains demonstrate significant differences in the rate and magnitude of eicosanoid generation, these findings strongly support both the existence of donor variability in eicosanoid accumulation during RBC storage and a genetic basis thereof (in the context of a mouse system). In addition to providing a tractable platform to dissect the genetics of donor-specific RBC storage biology, these findings suggest the hypothesis that similar differences in eicosanoid generation occur with human RBC storage. Subsequent analysis of human specimens will need to be performed to test whether these findings extend to stored human RBC units. Disclosures: No relevant conflicts of interest to declare.

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High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells By AAV6 Vectors: strategies for Overcoming Donor-Variation and Implications in Genome Editing

Blood ◽

10.1182/blood.v128.22.5889.5889 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5889-5889

Author(s):

Chen Ling ◽

Kanit Bhukhai ◽

Zifei Yin ◽

Mengqun Tan ◽

Mervin Yoder ◽

...

Keyword(s):

Progenitor Cells ◽

Genome Editing ◽

High Efficiency ◽

Ex Vivo ◽

Transduction Efficiency ◽

Triple Mutant ◽

Hematopoietic Stem ◽

Combined Use ◽

Donor Variation ◽

Viral Capsid Proteins

Abstract We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs). However, the transduction efficiency of the wild-type (WT) AAV6 vector varies greatly in HSPCs from different donors. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705+Y731F+T492V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, 'cross-transduction'. The combined use of these strategies resulted in transduction efficiency exceeding ~90% in HSPCs. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs. Disclosures Leboulch: bluebird bio: Patents & Royalties. Payen:bluebird bio: Patents & Royalties. Srivastava:AGTC; Genzyme: Patents & Royalties.

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Application of hepatocyte-like cells to enhance hepatic safety risk assessment in drug discovery

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2017.0228 ◽

2018 ◽

Vol 373 (1750) ◽

pp. 20170228 ◽

Cited By ~ 8

Author(s):

Dominic P. Williams

Keyword(s):

Risk Assessment ◽

Drug Discovery ◽

High Throughput Screening ◽

Three Dimensional ◽

Induced Pluripotent Stem Cell ◽

Genetic Characteristics ◽

Short Period ◽

Donor Variation ◽

Discovery Pipeline

Hepatic stress and injury from drugs continues to be a major concern within the pharmaceutical industry, leading to preclinical and clinical attrition precautionary warnings and post-market withdrawal of drugs. There is a requirement for more predictive and mechanistically accurate models to aid risk assessment. Primary human hepatocytes, subject to isolation stress, cryopreservation, donor-to-donor variation and a relatively short period of functional capability in two-dimensional cultures, are not suitable for high-throughput screening procedures. There are two areas within the drug discovery pipeline that the generation of a stable, metabolically functional hepatocyte-like cell with unlimited supply would have major impact. First, in routine, cell health risk-assessment assays where hepatic cell lines are typically deployed. Second, at later stages of the drug discovery pipeline approaching candidate nomination where bespoke/investigational studies refining and understanding the risk to patients use patient-derived induced pluripotent stem cell (iPSC) hepatocytes retaining characteristics from the patient, e.g. HLA susceptibility alleles, iPSC hepatocytes with defined disease phenotypes or genetic characteristics that have the potential to make the hepatocyte more sensitive to a particular stress mechanism. Functionality of patient-centric hepatocyte-like cells is likely to be enhanced when coupled with emerging culture systems, such as three-dimensional spheroids or microphysiological systems. Ultimately, the aspiration to confidently use human-relevant in vitro models to predict human-specific hepatic toxicity depends on the integration of promising emerging technologies. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

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Donor Variation In Biological Mediators During Storage Of Packed Red Blood Cells

Blood ◽

10.1182/blood.v122.21.3655.3655 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3655-3655 ◽

Cited By ~ 3

Author(s):

Melinda M Dean ◽

Luke D Samson ◽

Kelly Rooks ◽

Jesse Fryk ◽

Shoma Baidya ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Immune Modulation ◽

Time Course ◽

Conflicts Of Interest ◽

Biological Response ◽

Storage Lesion ◽

Packed Red Blood Cells ◽

Donor Variation ◽

Time Point

Abstract Introduction During routine storage, packed red blood cells (PRBC) undergo numerous biochemical and biophysical changes collectively referred to as the “RBC storage lesion”. A number of factors reported to accumulate during the routine storage of PRBCs are hypothesized to mediate inflammatory cell responses and contribute to poor patient outcomes following transfusion. In addition, donor variability in red blood cell (RBC) characteristics and onset of the storage lesion has been reported. We investigated changes in levels of potential biological response modifies in the supernatant (SN) of PRBC relevant to storage, and, variance between donations. Methods Cytometric bead array was utilised to quantify a panel of 32 potential biological response modifiers (BRMs) in the SN of PRBC during storage. Potential BRMS were analysed in the SN of 8 leukodepleted PRBC units at weekly intervals (D2, D7, D14, D21, D28, D35, D42). The CBA panel was comprised of soluble(s) CD40 Ligand, sCD62E, sCD62L, sCD14, sCD54 (ICAM-1), sCD106 (VCAM-1), CXCL9, VEGF, Fractalkine (CX3CL1), IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF-α, MIP-1α, MIP-1β, IP-10, RANTES, sCD62P, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, IFN-α, IFN-γ, angiogenin, MCP-1. Storage related changes were analysed using ANOVA (95% CI). Donor variance was indicated by fold difference and range. “High” sub population of donations compared to remaining donations at each time point using Mann-Whitney (95% CI). Results Of the 32 potential BRMs studied, angiogenin, sCD14, sCD106 (VCAM-1), sCD62L, sCD62P, ICAM-1, IL-1α, IP-10, RANTES and IL-9 were consistently detected in all units throughout the time course. There was no evidence of a storage related increase in these biological mediators during storage of the PRBC, although angiogenin levels significantly declined during storage (P<0.001, ANOVA). Of particular interest, the concentrations of these nine biological mediators varied greatly between the individual PRBC units. ICAM-1, VCAM-1 and IL-1α concentrations each varied 10 fold between units (range 1000 – 10 000 pg/mL for each), sCD14 varied 5 fold (range 20 000 - 100 000 pg/mL), sCD62L varied 4.4 fold (range 9000 – 40 000 pg/mL), and sCD62P varied 6.5 fold (range 200 -1300 pg/ml). In addition, it was apparent that a sub population (3/8) of the units assessed consistently had the highest levels of ICAM-1, sCD106 (VCAM-1), sCD14, sCD62L, IL-1α, sCD62P and angiogenin. For sCD62P, in particular, this “high” sub population had significantly different levels of sCD62P at each time point compared to the other five units (P<0.05 at each time point). The remaining BRMs studied were at the limits of detection (<20 pg/mL) for every unit at each time point, and no storage related changes were evident. Conclusions There was minimal change in the BRMs studied relevant to storage duration of the PRBC units. The most notable differences in the levels of biological mediators present in PRBC SN were due to donor-to-donor variation. These data suggest high levels of BRMs and potential immune modulation in transfusion recipients may be the result of donor-associated differences rather than storage-associated differences in blood components. Disclosures: No relevant conflicts of interest to declare.

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