Modification of nanodiamonds for fluorescence bioimaging.

Non-invasive bioimaging is essential in enhancing pre-clinical diagnosis and therapy. Developing efficient imaging probes with high stability, low toxicity, and the potential of offering high resolution images is a very important aspect of developing non-invasive bioimaging techniques. Fluorescent nanodiamonds, which are produced by high energy beam irradiation and high temperature/pressure treatment, have been extensively investigated. In this study, we report the chemical modification of common nanodiamonds (prepared by detonation and high-pressure high-temperature milling) using a stable fluorophore (perylene diimide derivative) via carbodiimide coupling. The resulting nanodiamonds show good biocompatibility, cellular uptake and fluorescent imaging potential with mesenchymal stromal cells. This method provides an efficient alternative approach to the preparation and the use of fluorescent nanodiamonds for bioimaging, with the potential benefit of chemically adjusting the structure of perylene diimide for optimized emission/absorbance wavelength.

Describing Self-Advocacy in Underrepresented Women With Advanced Cancer.

PURPOSE: To describe the self-advocacy experiences of women from underrepresented groups who have advanced breast or gynecologic cancer. PARTICIPANTS & SETTING: To be eligible for the study, participants had to self-identify as vulnerable, which was defined as a member of a group considered at risk for poor cancer outcomes and underrepresented in clinical research. METHODOLOGIC APPROACH: This descriptive, longitudinal, qualitative study consisted of one-on-one interviews of women within three months of an advanced breast or gynecologic cancer diagnosis. FINDINGS: 10 participants completed 25 interviews. The average age of participants was 60.2 years (range = 38-75 years). Three major themes emerged: (a) speaking up and speaking out, (b) interacting with the healthcare team, and (c) relying on support from others. IMPLICATIONS FOR NURSING: Women with advanced cancer who are from underrepresented groups self-advocated in unique ways, learning over time the importance of how to communicate their needs and manage their healthcare team. Future research should incorporate these findings into tailored self-advocacy interventions.

Umbilical cord mesenchymal stromal cell-derived extracellular vesicles lack the potency to immunomodulate human monocyte-derived macrophages in vitro.

Mesenchymal stromal cells (MSCs) have been reported to display efficacy in a variety of preclinical models, but without long-term engraftment, suggesting a role for secreted factors, such as MSC-derived extracellular vesicles (EVs). MSCs are known to elicit immunomodulatory effects, an important aspect of which is their ability to affect macrophage phenotype. However, it is not clear if these effects are mediated by MSC-derived EVs, or other factors secreted by the MSCs. Here, we use flow cytometry to assess the effects of human umbilical cord (hUC) MSC-derived EVs on the expression of pro-inflammatory (CD80) and anti-inflammatory (CD163) surface markers in human monocyte-derived macrophages (hMDMs). hUC-MSC-derived EVs did not change the surface marker expression of the hMDMs. In contrast, when hMDMs were co-incubated with hUC-MSCs in indirect co-cultures, changes were observed in the expression of CD14, CD80 and CD163, particularly in M1 macrophages, suggesting that soluble factors are necessary to elicit a shift in phenotype. However, even though EVs did not alter the surface marker expression of macrophages, they promoted angiogenesis and phagocytic capacity increased proportionally to increases in EV concentration. Taken together, these results suggest that hUC-MSC-derived EVs are not sufficient to alter macrophage phenotype and that additional MSC-derived factors are needed.

Near infrared conjugated polymer nanoparticles (CPN™) for tracking cells using fluorescence and optoacoustic imaging.

Tracking the biodistribution of cell therapies is crucial for understanding their safety and efficacy. Optical imaging techniques are particularly useful for tracking cells due to their clinical translatability and potential for intra-operative use to validate cell delivery. However, there is a lack of appropriate optical probes for cell tracking. The only FDA-approved material for clinical use is indocyanine green (ICG). ICG can be used for both fluorescence and photoacoustic imaging, but is prone to photodegradation, and at higher concentrations, undergoes quenching and can adversely affect cell health. We have developed novel near-infrared imaging probes comprising conjugated polymer nanoparticles (CPNs™) that can be fine-tuned to absorb and emit light at specific wavelengths. To compare the performance of the CPNs™ with ICG for in vivo cell tracking, labelled mesenchymal stromal cells (MSCs) were injected subcutaneously in mice and detected using fluorescence imaging (FI) and a form of photoacoustic imaging called multispectral optoacoustic tomography (MSOT). MSCs labelled with either ICG or CPN™ 770 could be detected with FI, but only CPN™ 770-labelled MSCs could be detected with MSOT. These results show that CPNs™ show great promise for tracking cells in vivo using optical imaging techniques, and for some applications, out-perform ICG.

Intravenous Administration of Human Umbilical Cord Mesenchymal Stromal Cells Leads to an Inflammatory Response in the Lung.

Mesenchymal stromal cells (MSCs) administered intravenously (IV) have shown efficacy in preclinical models of various diseases. This is despite the cells not reaching the site of injury due to entrapment in the lungs. The immunomodulatory properties of MSCs are thought to underlie their therapeutic effects, irrespective of whether they are sourced from bone marrow, adipose tissue, or umbilical cord. To better understand how MSCs affect innate immune cell populations in the lung, we evaluated the distribution and phenotype of neutrophils, monocytes, and macrophages by flow cytometry and histological analyses after delivering human umbilical cord-derived MSCs (hUC-MSCs) IV into immunocompetent mice. After 2 hr, we observed a significant increase in neutrophils, and proinflammatory monocytes and macrophages. Moreover, these immune cells localized in close proximity to the MSCs, suggesting an active role in their clearance. By 24 hr, we detected an increase in anti-inflammatory monocytes and macrophages. These results suggest that the IV injection of hUC-MSCs leads to an initial inflammatory phase in the lung shortly after injection, followed by a resolution phase 24 hr later.

Quantifying acute kidney injury in an Ischaemia-Reperfusion Injury mouse model using deep-learning-based semantic segmentation in histology.

This study focuses on ischaemia-reperfusion injury (IRI) in kidneys, a cause of acute kidney injury (AKI) and end-stage kidney disease (ESKD). Traditional kidney damage assessment methods are semi-quantitative and subjective. This study aims to use a convolutional neural network (CNN) to segment murine kidney structures after IRI, quantify damage via CNN-generated pathological measurements, and compare this to conventional scoring. The CNN was able to accurately segment the different pathological classes, such as Intratubular casts and Tubular necrosis, with an F1 score of over 0.75. Some classes, such as Glomeruli and Proximal tubules, had even higher statistical values with F1 scores over 0.90. The scoring generated based on the segmentation approach statistically correlated with the semiquantitative assessment (Spearman's rank correlation coefficient=0.94). The heatmap approach localised the intratubular necrosis mainly in the outer stripe of the outer medulla, while the tubular casts were also present in more superficial or deeper portions of the cortex and medullary areas. This study presents a CNN model capable of segmenting multiple classes of interest, including acute IRI-specific pathological changes, in a whole mouse kidney section and can provide insights into the distribution of pathological classes within the whole mouse kidney section.

Multispectral optoacoustic tomography is more sensitive than micro-computed tomography for tracking gold nanorod labelled mesenchymal stromal cells.

Tracking the fate of therapeutic cell types is important for assessing their safety and efficacy. Bioluminescence imaging (BLI) is an effective cell tracking technique, but poor spatial resolution means it has limited ability to precisely map cells in vivo in 3D. This can be overcome by using a bimodal imaging approach that combines BLI with a technique capable of generating high-resolution images. Here we compared the effectiveness of combining either multispectral optoacoustic tomography (MSOT) or micro-computed tomography (micro-CT) with BLI for tracking the fate of luciferase+ human mesenchymal stromal cells (MSCs) labelled with gold nanorods. Following subcutaneous administration in mice, the MSCs could be readily detected with MSOT but not with micro-CT. We conclude that MSOT is more sensitive than micro-CT for tracking gold nanorod-labelled cells in vivo and depending on the route of administration, can be used effectively with BLI to track MSC fate in mice.

Harmonised culture procedures minimise but do not eliminate mesenchymal stromal cell donor and tissue variability in a decentralised multicentre manufacturing approach.

BACKGROUND: Mesenchymal stromal cells (MSCs), commonly sourced from adipose tissue, bone marrow and umbilical cord, have been widely used in many medical conditions due to their therapeutic potential. Yet, the still limited understanding of the underlying mechanisms of action hampers clinical translation. Clinical potency can vary considerably depending on tissue source, donor attributes, but importantly, also culture conditions. Lack of standard procedures hinders inter-study comparability and delays the progression of the field. The aim of this study was A- to assess the impact on MSC characteristics when different laboratories, performed analysis on the same MSC material using harmonised culture conditions and B- to understand source-specific differences. METHODS: Three independent institutions performed a head-to-head comparison of human-derived adipose (A-), bone marrow (BM-), and umbilical cord (UC-) MSCs using harmonised culture conditions. In each centre, cells from one specific tissue source were isolated and later distributed across the network to assess their biological properties, including cell expansion, immune phenotype, and tri-lineage differentiation (part A). To assess tissue-specific function, angiogenic and immunomodulatory properties and the in vivo biodistribution were compared in one expert lab (part B). RESULTS: By implementing a harmonised manufacturing workflow, we obtained largely reproducible results across three independent laboratories in part A of our study. Unique growth patterns and differentiation potential were observed for each tissue source, with similar trends observed between centres. Immune phenotyping verified expression of typical MSC surface markers and absence of contaminating surface markers. Depending on the established protocols in the different laboratories, quantitative data varied slightly. Functional experiments in part B concluded that conditioned media from BM-MSCs significantly enhanced tubulogenesis and endothelial migration in vitro. In contrast, immunomodulatory studies reported superior immunosuppressive abilities for A-MSCs. Biodistribution studies in healthy mice showed lung entrapment after administration of all three types of MSCs, with a significantly faster clearance of BM-MSCs. CONCLUSION: These results show the heterogeneous behaviour and regenerative properties of MSCs as a reflection of intrinsic tissue-origin properties while providing evidence that the use of harmonised culture procedures can reduce but do not eliminate inter-lab and operator differences.

Fate of intravenously administered umbilical cord mesenchymal stromal cells and interactions with the host's immune system.

Mesenchymal stromal cells (MSCs) are multipotent cells showing promise in pre-clinical studies and currently used in many clinical trials. The regenerative potential of MSCs is mediated, at least in part, by direct and indirect immunomodulatory processes. However, the mechanism of action is not fully understood yet, and there are still concerns about possible undesired negative effects associated with the administration of living cells. In this study, we (i) compare the long-term fate and safety of umbilical cord (UC-)MSCs administered to immunocompetent and immunocompromised (severe combined immunodeficient (SCID) and non-obese diabetic (NOD)/SCID) animals, and (ii) investigate the immunological response of the host to the administered cells. Intravenous administration of firefly luciferase expressing UC-MSCs revealed that the cells get trapped in the lungs of both immunocompetent and immunocompromised animals, with > 95% of the cells disappearing within 72 h after administration. In 27% of the SCID and 45% of the NOD/SCID, a small fraction of the cells lived up to day 14 but in most cases they all disappeared earlier. One NOD/SCID mouse showed a weak signal up to day 31. Immunocompetent mice displayed elevated percentages of neutrophils in the lungs, the blood, and the spleen 2 h after the administration of the cells. The concentration of neutrophil chemoattractants (MCP1, CCL7, Gro-α and IP-10) were also increased in the plasma of the animals 2 h after the administration of the MSCs. Our results suggest that although the UC-MSCs are short-lived in mice, they still result in an immunological response that might contribute to a therapeutic effect.

DEAE-Dextran Enhances the Lentiviral Transduction of Primary Human Mesenchymal Stromal Cells from All Major Tissue Sources Without Affecting Their Proliferation and Phenotype.

Genetic engineering of mesenchymal stromal cells (MSCs) is a tool widely used to explore MSC properties in vitro and in vivo. Lentiviral infection with the use of polycations as an adjuvant is a method that is commonly used to generate stably transduced cells. However, it is known that some polycations can negatively affect primary MSCs and to date, no study has explored the effect of different polycations on the transduction efficiency and properties of all main types of MSCs, namely those derived from umbilical cord, bone marrow and adipose tissue. Here we explore a range of polycations, using transduction protocols with and without spinoculation, to produce stably transduced MSCs from these three tissue sources. We identified that an overnight incubation with diethylaminoethyl-dextran (DEAE-Dextran) is the protocol associated with the best transduction efficiency without compromising the viability of the cells, and which worked consistently with lentiviral particles encoding for different transgenes. Transduced and sorted MSC populations revealed no significant changes in proliferation, morphology and expression of MSC markers compared to naïve MSCs. Following this study, we conclude that DEAE-Dextran is a polycation that can be successfully used to enhance the transduction of MSCs from all major tissue sources.

Extracellular Vesicles from Human Umbilical Cord-Derived MSCs Affect Vessel Formation In Vitro and Promote VEGFR2-Mediated Cell Survival.

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have emerged as novel tools in regenerative medicine. Angiogenesis modulation is widely studied for the treatment of ischaemic diseases, wound healing, and tissue regeneration. Here, we have shown that EVs from human umbilical cord-derived MSCs can affect VEGFR2 signalling, a master regulator of angiogenesis homeostasis, via altering the phosphorylation of AKT. This translates into an inhibition of apoptosis, promoting exclusively cell survival, but not proliferation, in human microvascular endothelial cells. Interestingly, when comparing EVs from normoxic cells to those obtained from hypoxia (1% O2) preconditioned cells, hypoxia-derived EVs appear to have a slightly enhanced effect. Furthermore, when studied in a longer term endothelial-fibroblast co-culture angiogenesis model in vitro, both EV populations demonstrated a positive effect on vessel formation, evidenced by increased vessel networks with tubes of significantly larger diameters. Our data reveals that EVs selectively target components of the angiogenic pathway, promoting VEGFR2-mediated cell survival via enhancement of AKT activation. Our data show that EVs are able to enhance specific components of the VEGF signalling pathway and may have therapeutic potential to support endothelial cell survival.

Evaluation of Cytotoxicity and Bioimaging of Nitrogen-Vacancy Nanodiamonds.

Nanodiamonds, due to their chemical inertness and biocompatibility, have found extensive uses in drug delivery and biomedical applications. Fluorescent nanodiamonds with fluorescent properties generated by nitrogen-vacancy defects have been intensively investigated for bioimaging, due to their high quantum yield and high photobleaching stability. In addition, the surface properties and particle size of nanodiamonds have significant impacts on cellular uptake and imaging quality. In this study, nitrogen-vacancy nanodiamonds with different particle sizes (40 nm and 90 nm) have been physicochemically characterised and investigated for their cytotoxicity and potential in fluorescence imaging. The nanodiamonds (with concentrations up to 100 µg/mL) showed cell viability >70% with mesenchymal stromal cells. The number of nanodiamonds was observed to have a larger impact on cell viability than the mass of nanodiamonds. Larger nanodiamonds (90 nm) exhibited a lower level of cytotoxicity, higher cellular uptake and fluorescence intensity. The results indicate the potential of using fluorescent nanodiamonds as a nanoprobe for effective bioimaging and cell tracking.

Optical Tissue Clearing to Study the Intra-Pulmonary Biodistribution of Intravenously Delivered Mesenchymal Stromal Cells and Their Interactions with Host Lung Cells.

Mesenchymal stromal cells (MSCs) injected intravenously are trapped in the capillaries of the lungs and die within the first 24 h. Studying the biodistribution and fate of labelled therapeutic cells in the 3D pulmonary context is important to understand their function in this organ and gain insights into their mechanisms of action. Optical tissue clearing enables volumetric cell tracking at single-cell resolution. Thus, we compared three optical tissue-clearing protocols (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC), modified stabilised 3D imaging of solvent-cleared organs (s-DISCO) and ethyl cinnamate (ECi)) to evaluate their potential to track the biodistribution of human umbilical cord MSCs expressing the tdTomato fluorescence reporter and investigate how they interact with host cells in the mouse lung. The results showed that although CUBIC clearing is the only method that enables direct imaging of fluorescently labelled MSCs, combining s-DISCO or ECi with immunofluorescence or dye labelling allows the interaction of MSCs with endothelial and immune cells to be studied. Overall, this comparative study offers guidance on selecting an optical tissue-clearing method for cell tracking applications.

Murine models of renal ischemia reperfusion injury: An opportunity for refinement using noninvasive monitoring methods.

BACKGROUND: Renal ischemia reperfusion injury (R-IRI) can cause acute kidney injury (AKI) and chronic kidney disease (CKD), resulting in significant morbidity and mortality. To understand the underlying mechanisms, reproducible small-animal models of AKI and CKD are needed. We describe how innovative technologies for measuring kidney function noninvasively in small rodents allow successful refinement of the R-IRI models, and offer the unique opportunity to monitor longitudinally in individual animals the transition from AKI to CKD. METHODS: Male BALB/c mice underwent bilateral renal pedicle clamping (AKI) or unilateral renal pedicle clamping with delayed contralateral nephrectomy (CKD) under isoflurane anesthetic. Transdermal GFR monitoring and multispectral optoacoustic tomography (MSOT) in combination with statistical analysis were used to identify and standardize variables within these models. RESULTS: Pre-clamping anesthetic time was one of the most important predictors of AKI severity after R-IRI. Standardizing pre-clamping time resulted in a more predictably severe AKI model. In the CKD model, MSOT demonstrated initial improvement in renal function, followed by significant progressive reduction in function between weeks 2 and 4. Performing contralateral nephrectomy on day 14 enabled the development of CKD with minimal mortality. CONCLUSIONS: Noninvasive monitoring of global and individual renal function after R-IRI is feasible and reproducible. These techniques can facilitate refinement of kidney injury models and enable the degree of injury seen in preclinical models to be translated to those seen in the clinical setting. Thus, future therapies can be tested in a clinically relevant, noninvasive manner.

Hypoxia-induced HIF1α activation regulates small extracellular vesicle release in human embryonic kidney cells.

Extracellular vesicles (EVs) are membrane enclosures released by eukaryotic cells that carry bioactive molecules and serve to modulate biological responses in recipient cells. Both increased EV release and altered EV composition are associated with the development and progression of many pathologies including cancer. Hypoxia, a feature of rapidly growing solid tumours, increases the release of EVs. However, the molecular mechanisms remain unknown. The hypoxia inducible factors (HIFs) are transcription factors that act as major regulators of the cellular adaptations to hypoxia. Here, we investigated the requirement of HIF pathway activation for EV release in Human Embryonic Kidney Cells (HEK293). Time course experiments showed that EV release increased concomitantly with sustained HIF1α and HIF2α activation following the onset of hypoxia. shRNA mediated knock-down of HIF1α but not HIF2α abrogated the effect of hypoxia on EV release, suggesting HIF1α is involved in this process. However, stabilization of HIF proteins in normoxic conditions through: (i) heterologous expression of oxygen insensitive HIF1α or HIF2α mutants in normoxic cells or (ii) chemical inhibition of the prolyl hydroxylase 2 (PHD2) repressor protein, did not increase EV release, suggesting HIF activation alone is not sufficient for this process. Our findings suggest HIF1α plays an important role in the regulation of EV release during hypoxia in HEK293 cells, however other hypoxia triggered mechanisms likely contribute as stabilization of HIF1α alone in normoxia is not sufficient for EV release.

Firefly luciferase offers superior performance to AkaLuc for tracking the fate of administered cell therapies.

INTRODUCTION: A novel, red-shifted bioluminescence imaging (BLI) system called AkaBLI has been recently developed for cell tracking in preclinical models and to date, limited data is available on how it performs in relation to existing systems. PURPOSE: To systematically compare the performance of AkaBLI and the standard Firefly luciferase (FLuc) systems to monitor the biodistribution and fate of cell therapies in rodents. METHODS: Umbilical cord mesenchymal stromal cells (MSCs) were transduced to produce two genetically engineered populations, expressing either AkaLuc or the engineered FLuc luc2. The bioluminescence of AkaLuc+ and FLuc+ cells was assessed both in vitro (emission spectra, saturation kinetics and light emission per cell) and in vivo (substrate kinetics following intraperitoneal and subcutaneous administration and biodistribution of the cells up to day 7). RESULTS: Introduction of the reporter genes has no effect on MSC phenotype. For BLI, the FLuc system is superior to AkaBLI in terms of (i) light output, producing a stronger signal after subcutaneous substrate delivery and more consistent signal kinetics when delivered intraperitoneally; (ii) absence of hepatic background; and (iii) safety, where the AkaLuc substrate was associated with a reaction in the skin of the mice in vivo. CONCLUSION: We conclude that there is no advantage in using the AkaBLI system to track the biodistribution of systemically administered cell-based regenerative medicine therapies in vivo.

Mesenchymal stromal cells: what have we learned so far about their therapeutic potential and mechanisms of action?

Mesenchymal stromal cells (MSCs) have been found to be safe and effective in a wide range of animal models of human disease. MSCs have been tested in thousands of clinical trials, but results show that while these cells appear to be safe, they tend to lack efficacy. This has raised questions about whether animal models are useful for predicting efficacy in patients. However, a problem with animal studies is that there is a lack of standardisation in the models and MSC therapy regimes used; there appears to be publication bias towards studies reporting positive outcomes; and the reproducibility of results from animal experiments tends not to be confirmed prior to clinical translation. A further problem is that while some progress has been made towards investigating the mechanisms of action (MoA) of MSCs, we still fail to understand how they work. To make progress, it is important to ensure that prior to clinical translation, the beneficial effects of MSCs in animal studies are real and can be repeated by independent research groups. We also need to understand the MoA of MSCs to assess whether their effects are likely to be beneficial across different species. In this review, we give an overview of the current clinical picture of MSC therapies and discuss what we have learned from animal studies. We also give a comprehensive update of what we know about the MoA of MSCs, particularly in relation to their role in immunomodulation.