PTPmu-targeted nanoparticles label invasive pediatric and adult glioblastoma.

Poor prognosis for glioblastoma (GBM) is a consequence of the aggressive and infiltrative nature of gliomas where individual cells migrate away from the main tumor to distant sites, making complete surgical resection and treatment difficult. In this manuscript, we characterize an invasive pediatric glioma model and determine if nanoparticles linked to a peptide recognizing the GBM tumor biomarker PTPmu can specifically target both the main tumor and invasive cancer cells in adult and pediatric glioma models. Using both iron and lipid-based nanoparticles, we demonstrate by magnetic resonance imaging, optical imaging, histology, and iron quantification that PTPmu-targeted nanoparticles effectively label adult gliomas. Using PTPmu-targeted nanoparticles in a newly characterized orthotopic pediatric SJ-GBM2 model, we demonstrate individual tumor cell labeling both within the solid tumor margins and at invasive and dispersive sites.

Volumetric characterization of human coronary calcification by frequency-domain optical coherence tomography.

BACKGROUND: Coronary artery calcification (CAC) presents unique challenges for percutaneous coronary intervention. Calcium appears as a signal-poor region with well-defined borders by frequency-domain optical coherence tomography (FD-OCT). The objective of this study was to demonstrate the accuracy of intravascular FD-OCT to determine the distribution of CAC. METHODS AND RESULTS: Cadaveric coronary arteries were imaged using FD-OCT at 100-µm frame interval. Arteries were subsequently frozen, sectioned and imaged at 20-µm intervals using the Case Cryo-Imaging automated system(TM). Full volumetric co-registration between FD-OCT and cryo-imaging was performed. Calcium area, calcium-lumen distance (depth) and calcium angle were traced on every cross-section; volumetric quantification was performed offline. In total, 30 left anterior descending arteries were imaged: 13 vessels had a total of 55 plaques with calcification by cryo-imaging; FD-OCT identified 47 (85%) of these plaques. A total of 1,285 cryo-images were analyzed and compared with corresponding co-registered 257 FD-OCT images. Calcium distribution, represented by the mean depth and the mean calcium angle, was similar, with excellent correlation between FD-OCT and cryo-imaging respectively (mean depth: 0.25±0.09 vs. 0.26±0.12mm, P=0.742; R=0.90), (mean angle: 35.33±21.86° vs. 39.68±26.61°, P=0.207; R=0.90). Calcium volume was underestimated in large calcifications (3.11±2.14 vs. 4.58±3.39mm(3), P=0.001) in OCT vs. cryo respectively. CONCLUSIONS: Intravascular FD-OCT can accurately characterize CAC distribution. OCT can quantify absolute calcium volume, but may underestimate calcium burden in large plaques with poorly defined abluminal borders.

Optimal slice thickness for improved accuracy of quantitative analysis of fluorescent cell and microsphere distribution in cryo-images.

Cryo-imaging has been effectively used to study the biodistribution of fluorescent cells or microspheres in animal models. Sequential slice-by-slice fluorescent imaging enables detection of fluorescent cells or microspheres for corresponding quantification of their distribution in tissue. However, if slices are too thin, there will be data overload and excessive scan times. If slices are too thick, then cells can be missed. In this study, we developed a model for detection of fluorescent cells or microspheres to aid optimal slice thickness determination. Key factors include: section thickness (X), fluorescent cell intensity (Ifluo), effective tissue attenuation coefficient (µT), and a detection threshold (T). The model suggests an optimal slice thickness value that provides near-ideal sensitivity while minimizing scan time. The model also suggests a correction method to compensate for missed cells in the case that image data were acquired with overly large slice thickness. This approach allows cryo-imaging operators to use larger slice thickness to expedite the scan time without significant loss of cell count. We validated the model using real data from two independent studies: fluorescent microspheres in a pig heart and fluorescently labeled stem cells in a mouse model. Results show that slice thickness and detection sensitivity relationships from simulations and real data were well-matched with 99% correlation and 2% root-mean-square (RMS) error. We also discussed the detection characteristics in situations where key assumptions of the model were not met such as fluorescence intensity variation and spatial distribution. Finally, we show that with proper settings, cryo-imaging can provide accurate quantification of the fluorescent cell biodistribution with remarkably high recovery ratios (number of detections/delivery). As cryo-imaging technology has been used in many biological applications, our optimal slice thickness determination and data correction methods can play a crucial role in further advancing its usability and reliability.

Deep learning multi-organ segmentation for whole mouse cryo-images including a comparison of 2D and 3D deep networks.

Cryo-imaging provided 3D whole-mouse microscopic color anatomy and fluorescence images that enables biotechnology applications (e.g., stem cells and metastatic cancer). In this report, we compared three methods of organ segmentation: 2D U-Net with 2D-slices and 3D U-Net with either 3D-whole-mouse or 3D-patches. We evaluated the brain, thymus, lung, heart, liver, stomach, spleen, left and right kidney, and bladder. Training with 63 mice, 2D-slices had the best performance, with median Dice scores of > 0.9 and median Hausdorff distances of < 1.2 mm in eightfold cross validation for all organs, except bladder, which is a problem organ due to variable filling and poor contrast. Results were comparable to those for a second analyst on the same data. Regression analyses were performed to fit learning curves, which showed that 2D-slices can succeed with fewer samples. Review and editing of 2D-slices segmentation results reduced human operator time from ~ 2-h to ~ 25-min, with reduced inter-observer variability. As demonstrations, we used organ segmentation to evaluate size changes in liver disease and to quantify the distribution of therapeutic mesenchymal stem cells in organs. With a 48-GB GPU, we determined that extra GPU RAM improved the performance of 3D deep learning because we could train at a higher resolution.

Expression of membrane Hsp90 is a molecular signature of T cell activation.

Heat shock protein 90 (Hsp90) maintains cellular proteostasis during stress and has been under investigation as a therapeutic target in cancer for over two decades. We and others have identified a membrane expressed form of Hsp90 (mHsp90) that previously appeared to be restricted to rapidly proliferating cells exhibiting a metastatic phenotype. Here, we used HS-131, a fluor-tethered mHsp90 inhibitor, to quantify the effect of T cell activation on the expression of mHsp90 in human and mouse T cells. In cell-based assays, stimulation of human T cells induced a 20-fold increase in mHsp90 expression at the plasma membrane, suggesting trafficking of mHsp90 is regulated by TCR and inflammatory mediated signaling. Following injection of HS-131 in mouse models of human rheumatoid arthritis and inflammatory bowel disease, we detected localization of the probe at sites of active disease, consistent with immune cell invasion. Moreover, despite rapid hepatobiliary clearance, HS-131 demonstrated efficacy in reducing the mean clinical score in the CIA arthritis model. Our results suggest mHsp90 expression on T cells is a molecular marker of T cell activation and potentially a therapeutic target for chronic diseases such as rheumatoid arthritis.

Fast lipid and water levels by extraction with spatial smoothing (FLAWLESS): three-dimensional volume fat/water separation at 7 Tesla.

PURPOSE: To quickly and robustly separate fat/water components of 7T MR images in the presence of field inhomogeneity for the study of metabolic disorders in small animals. MATERIALS AND METHODS: Starting with a Markov random field (MRF) based formulation for the 3-point Dixon separation problem, we incorporated new implementation strategies, including stability tracking, multiresolution image pyramid, and improved initial value generation. We term the new method FLAWLESS (Fast Lipid And Water Levels by Extraction with Spatial Smoothing). RESULTS: Compared with non-MRF techniques, FLAWLESS decreased the fat-water swapping mistakes in all of the three-dimensional (3D) animal volumes that we tested. FLAWLESS converged in approximately 1/60th of the computation time of other MRF approaches. The initial value generation of FLAWLESS further improved robustness to field inhomogeneity in 3D volume data. CONCLUSION: We have developed a novel 3-point Dixon technique found to be useful for high field small animal imaging. It is being used to assess lipid depots and metabolic disorders as a function of genes, diet, age, and therapy.

Mouse and human phenotypes indicate a critical conserved role for ERK2 signaling in neural crest development.

Disrupted ERK1/2 (MAPK3/MAPK1) MAPK signaling has been associated with several developmental syndromes in humans; however, mutations in ERK1 or ERK2 have not been described. We demonstrate haplo-insufficient ERK2 expression in patients with a novel approximately 1 Mb micro-deletion in distal 22q11.2, a region that includes ERK2. These patients exhibit conotruncal and craniofacial anomalies that arise from perturbation of neural crest development and exhibit defects comparable to the DiGeorge syndrome spectrum. Remarkably, these defects are replicated in mice by conditional inactivation of ERK2 in the developing neural crest. Inactivation of upstream elements of the ERK cascade (B-Raf and C-Raf, MEK1 and MEK2) or a downstream effector, the transcription factor serum response factor resulted in analogous developmental defects. Our findings demonstrate that mammalian neural crest development is critically dependent on a RAF/MEK/ERK/serum response factor signaling pathway and suggest that the craniofacial and cardiac outflow tract defects observed in patients with a distal 22q11.2 micro-deletion are explained by deficiencies in neural crest autonomous ERK2 signaling.

Quantitative analysis of metastatic breast cancer in mice using deep learning on cryo-image data.

Cryo-imaging sections and images a whole mouse and provides ~ 120-GBytes of microscopic 3D color anatomy and fluorescence images, making fully manual analysis of metastases an onerous task. A convolutional neural network (CNN)-based metastases segmentation algorithm included three steps: candidate segmentation, candidate classification, and semi-automatic correction of the classification result. The candidate segmentation generated > 5000 candidates in each of the breast cancer-bearing mice. Random forest classifier with multi-scale CNN features and hand-crafted intensity and morphology features achieved 0.8645 ± 0.0858, 0.9738 ± 0.0074, and 0.9709 ± 0.0182 sensitivity, specificity, and area under the curve (AUC) of the receiver operating characteristic (ROC), with fourfold cross validation. Classification results guided manual correction by an expert with our in-house MATLAB software. Finally, 225, 148, 165, and 344 metastases were identified in the four cancer mice. With CNN-based segmentation, the human intervention time was reduced from > 12 to ~ 2 h. We demonstrated that 4T1 breast cancer metastases spread to the lung, liver, bone, and brain. Assessing the size and distribution of metastases proves the usefulness and robustness of cryo-imaging and our software for evaluating new cancer imaging and therapeutics technologies. Application of the method with only minor modification to a pancreatic metastatic cancer model demonstrated generalizability to other tumor models.

Altered hypoxia-inducible factor-1 alpha expression levels correlate with coronary vessel anomalies.

The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5-positive tissues were also specifically positive for nuclear-localized hypoxia inducible factor-1 alpha (HIF-1alpha), the oxygen-sensitive component of the hypoxia inducible factor-1 (HIF-1) heterodimer. This led to our hypothesis that there is a "template" of hypoxic tissue that determines the stereotyped pattern of the major coronary vessels. In this study, we disturbed this template by altering ambient oxygen levels (hypoxia 15%; hyperoxia 75-40%) during the early phases of avian coronary vessel development, in order to alter tissue hypoxia, HIF-1alpha protein expression, and its downstream target genes without high mortality. We also altered HIF-1alpha gene expression in the embryonic outflow tract cardiomyocytes by injecting an adenovirus containing a constitutively active form of HIF-1alpha (AdCA5). We assayed for coronary anomalies using anti-alpha-smooth muscle actin immunohistology. When incubated under abnormal oxygen levels or injected with a low titer of the AdCA5, coronary arteries displayed deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicated that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF-1 regulation of gene expression.

Cryo-imaging of Stem Cell Biodistribution in Mouse Model of Graft-Versus-Host-Disease.

We demonstrated the use of multispectral cryo-imaging and software to analyze human mesenchymal stromal cells (hMSCs) biodistribution in mouse models of graft-versus-host-disease (GVHD) following allogeneic bone marrow transplantation (BMT). We injected quantum dot labeled MSCs via tail vein to mice receiving BMT and analyzed hMSC biodistribution in major organs (e.g. lung, liver, spleen, kidneys and bone marrow). We compared the biodistribution of hMSCs in mice following allogeneic BMT recipients (with GVHD) to the biodistribution following syngeneic BMT (without GVHD). Cryo-imaging system revealed cellular biodistribution and redistribution patterns in the animal model. We initially found clusters of cells in the lung that eventually dissociated to single cells and redistributed to other organs within 72 h. The in vivo half-life of the exogenous MSCs was about 21 h. We found that the biodistribution of stromal cells was not related to blood flow, rather cells preferentially homed to specific organs. In conclusion, cryo-imaging was suitable for analyzing the cellular biodistribution. It could provide capabilities of visualizing cells anywhere in the mouse model with single cell sensitivity. By characterizing the biodistribution and anatomical specificity of a therapeutic cellular product, we believe that cryo-imaging can play an important role in the advancement of stem and stromal cell therapies and regenerative medicine.

Photodynamic Therapy Is an Effective Adjuvant Therapy for Image-Guided Surgery in Prostate Cancer.

Local and metastatic relapses of prostate cancer often occur following attempted curative resection of the primary tumor, and up to 66% of local recurrences are associated with positive margins. Therefore, technologies that can improve the visualization of tumor margins and adjuvant therapies to ablate remaining tumor tissues are needed during surgical resection of prostate adenocarcinoma. Photodynamic agents have the potential to combine both fluorescence for image-guided surgery (IGS) and photodynamic therapy (PDT) to resect and ablate cancer cells. The objective of this study was to determine the utility of a targeted PDT agent for IGS and adjuvant PDT. Using a previously developed prostate-specific membrane antigen (PSMA)-targeted PDT agent, PSMA-1-Pc413, we showed that PSMA-1-Pc413 selectively highlighted PSMA-expressing tumors, allowing IGS and more complete tumor resection compared with white light surgery. Subsequent PDT further reduced tumor recurrence and extended animal survival significantly. This approach also enabled identification of tumor cells in lymph nodes. In summary, this study presents a potential new treatment option for patients with prostate cancer undergoing surgery, which improves tumor visualization and discrimination during surgery, including identification of cancer in lymph nodes. SIGNIFICANCE: These findings present a photodynamic agent that can be used for both photodynamic therapy and image-guided surgery, allowing better visualization of tumor margins and elimination of residual tumor tissues.

High temporal resolution OCT using image-based retrospective gating.

High temporal resolution OCT imaging is very advantageous for analyzing cardiac mechanics in the developing embryonic heart of small animals. An image-based retrospective gating technique is presented to increase the effective temporal resolution of an OCT system and to allow visualization of systolic dynamics in 3D. The gating technique employs image similarity measures for rearranging asynchronously acquired input data consisting of a time series of 2D images at each z position along the heart volume, to produce a time sequence of 3D volumes of the beating heart. The study includes a novel robust validation technique, which quantitatively evaluates the accuracy of the gating technique, in addition to visual evaluations by 2D multiplanar reformatting (MPR) and 3D volume rendering. The retrospective gating and validation is demonstrated on a stage 14 embryonic quail heart data set. Using the validation scheme, it is shown that the gating is accurate within a standard deviation of 4.7 ms, which is an order of magnitude shorter than the time interval during which systolic contraction (approximately 50 ms) occurs in the developing embryo. This gating method has allowed, for the first time, clear visualization of systolic dynamics of the looping embryonic heart in 3D.

Denoising and 4D visualization of OCT images.

We are using Optical Coherence Tomography (OCT) to image structure and function of the developing embryonic heart in avian models. Fast OCT imaging produces very large 3D (2D + time) and 4D (3D volumes + time) data sets, which greatly challenge ones ability to visualize results. Noise in OCT images poses additional challenges. We created an algorithm with a quick, data set specific optimization for reduction of both shot and speckle noise and applied it to 3D visualization and image segmentation in OCT. When compared to baseline algorithms (median, Wiener, orthogonal wavelet, basic non-orthogonal wavelet), a panel of experts judged the new algorithm to give much improved volume renderings concerning both noise and 3D visualization. Specifically, the algorithm provided a better visualization of the myocardial and endocardial surfaces, and the interaction of the embryonic heart tube with surrounding tissue. Quantitative evaluation using an image quality figure of merit also indicated superiority of th new algorithm. Noise reduction aided semi-automatic 2D image segmentation, as quantitatively evaluated using a contour distance measure with respect to an expert segmented contour. In conclusion, the noise reduction algorithm should be quite useful for visualization and quantitative measurements (e.g., heart volume, stroke volume, contraction velocity, etc.) in OCT embryo images. With its semi-automatic, data set specific optimization, we believe that the algorithm can be applied to OCT images from other applications.

Cryo-Imaging and Software Platform for Analysis of Molecular MR Imaging of Micrometastases.

We created and evaluated a preclinical, multimodality imaging, and software platform to assess molecular imaging of small metastases. This included experimental methods (e.g., GFP-labeled tumor and high resolution multispectral cryo-imaging), nonrigid image registration, and interactive visualization of imaging agent targeting. We describe technological details earlier applied to GFP-labeled metastatic tumor targeting by molecular MR (CREKA-Gd) and red fluorescent (CREKA-Cy5) imaging agents. Optimized nonrigid cryo-MRI registration enabled nonambiguous association of MR signals to GFP tumors. Interactive visualization of out-of-RAM volumetric image data allowed one to zoom to a GFP-labeled micrometastasis, determine its anatomical location from color cryo-images, and establish the presence/absence of targeted CREKA-Gd and CREKA-Cy5. In a mouse with >160 GFP-labeled tumors, we determined that in the MR images every tumor in the lung >0.3 mm2 had visible signal and that some metastases as small as 0.1 mm2 were also visible. More tumors were visible in CREKA-Cy5 than in CREKA-Gd MRI. Tape transfer method and nonrigid registration allowed accurate (<11 µm error) registration of whole mouse histology to corresponding cryo-images. Histology showed inflammation and necrotic regions not labeled by imaging agents. This mouse-to-cells multiscale and multimodality platform should uniquely enable more informative and accurate studies of metastatic cancer imaging and therapy.

A Fluorescent Hsp90 Probe Demonstrates the Unique Association between Extracellular Hsp90 and Malignancy in Vivo.

Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is correlated with malignancy. Development of small molecule probes that can detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a cell impermeable far-red fluorophore-tagged Hsp90 inhibitor to target eHsp90 in vivo. High resolution confocal and lattice light sheet microscopy show that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is actively internalized. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by overexpressing p110HER2. Whole body cryoslicing, imaging, and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression and internalization is a phenomenon unique to tumor cells in vivo and may provide an "Achilles heel" for the early diagnosis of metastatic disease and targeted drug delivery.

Automatic Stem Cell Detection in Microscopic Whole Mouse Cryo-Imaging.

With its single cell sensitivity over volumes as large as or larger than a mouse, cryo-imaging enables imaging of stem cell biodistribution, homing, engraftment, and molecular mechanisms. We developed and evaluated a highly automated software tool to detect fluorescently labeled stem cells within very large ( âˆ¼ 200 GB) cryo-imaging datasets. Cell detection steps are: preprocess, remove immaterial regions, spatially filter to create features, identify candidate pixels, classify pixels using bagging decision trees, segment cell patches, and perform 3D labeling. There are options for analysis and visualization. To train the classifier, we created synthetic images by placing realistic digital cell models onto cryo-images of control mice devoid of cells. Very good cell detection results were (precision=98.49%, recall=99.97%) for synthetic cryo-images, (precision=97.81%, recall=97.71%) for manually evaluated, actual cryo-images, and false positives in control mice. An α-multiplier applied to features allows one to correct for experimental variations in cell brightness due to labeling. On dim cells (37% of standard brightness), with correction, we improved recall (49.26%→ 99.36%) without a significant drop in precision (99.99%→ 99.75%) . With tail vein injection, multipotent adult progenitor cells in a graft-versus-host-disease model in the first days post injection were predominantly found in lung, liver, spleen, and bone marrow. Distribution was not simply related to blood flow. The lung contained clusters of cells while other tissues contained single cells. Our methods provided stem cell distribution anywhere in mouse with single cell sensitivity. Methods should provide a rational means of evaluating dosing, delivery methods, cell enhancements, and mechanisms for therapeutic cells.

Processing to determine optical parameters of atherosclerotic disease from phantom and clinical intravascular optical coherence tomography three-dimensional pullbacks.

Analysis of intravascular optical coherence tomography (IVOCT) data has potential for real-time in vivo plaque classification. We developed a processing pipeline on a three-dimensional local region of support for estimation of optical properties of atherosclerotic plaques from coronary artery, IVOCT pullbacks. Using realistic coronary artery disease phantoms, we determined insignificant differences in mean and standard deviation estimates between our pullback analyses and more conventional processing of stationary acquisitions with frame averaging. There was no effect of tissue depth or oblique imaging on pullback parameter estimates. The method's performance was assessed in comparison with observer-defined standards using clinical pullback data. Values (calcium [Formula: see text], lipid [Formula: see text], and fibrous [Formula: see text]) were consistent with previous measurements obtained by other means. Using optical parameters ([Formula: see text], [Formula: see text], [Formula: see text]), we achieved feature space separation of plaque types and classification accuracy of [Formula: see text]. Despite the rapid [Formula: see text] motion and varying incidence angle in pullbacks, the proposed computational pipeline appears to work as well as a more standard "stationary" approach.

Three-dimensional registration of intravascular optical coherence tomography and cryo-image volumes for microscopic-resolution validation.

Evidence suggests high-resolution, high-contrast, [Formula: see text] intravascular optical coherence tomography (IVOCT) can distinguish plaque types, but further validation is needed, especially for automated plaque characterization. We developed experimental and three-dimensional (3-D) registration methods to provide validation of IVOCT pullback volumes using microscopic, color, and fluorescent cryo-image volumes with optional registered cryo-histology. A specialized registration method matched IVOCT pullback images acquired in the catheter reference frame to a true 3-D cryo-image volume. Briefly, an 11-parameter registration model including a polynomial virtual catheter was initialized within the cryo-image volume, and perpendicular images were extracted, mimicking IVOCT image acquisition. Virtual catheter parameters were optimized to maximize cryo and IVOCT lumen overlap. Multiple assessments suggested that the registration error was better than the [Formula: see text] spacing between IVOCT image frames. Tests on a digital synthetic phantom gave a registration error of only [Formula: see text] (signed distance). Visual assessment of randomly presented nearby frames suggested registration accuracy within 1 IVOCT frame interval ([Formula: see text]). This would eliminate potential misinterpretations confronted by the typical histological approaches to validation, with estimated 1-mm errors. The method can be used to create annotated datasets and automated plaque classification methods and can be extended to other intravascular imaging modalities.

3D registration of intravascular optical coherence tomography and cryo-image volumes for microscopic-resolution validation.

High resolution, 100 frames/sec intravascular optical coherence tomography (IVOCT) can distinguish plaque types, but further validation is needed, especially for automated plaque characterization. We developed experimental and 3D registration methods, to provide validation of IVOCT pullback volumes using microscopic, brightfield and fluorescent cryo-image volumes, with optional, exactly registered cryo-histology. The innovation was a method to match an IVOCT pull-back images, acquired in the catheter reference frame, to a true 3D cryo-image volume. Briefly, an 11-parameter, polynomial virtual catheter was initialized within the cryo-image volume, and perpendicular images were extracted, mimicking IVOCT image acquisition. Virtual catheter parameters were optimized to maximize cryo and IVOCT lumen overlap. Local minima were possible, but when we started within reasonable ranges, every one of 24 digital phantom cases converged to a good solution with a registration error of only +1.34±2.65µm (signed distance). Registration was applied to 10 ex-vivo cadaver coronary arteries (LADs), resulting in 10 registered cryo and IVOCT volumes yielding a total of 421 registered 2D-image pairs. Image overlays demonstrated high continuity between vascular and plaque features. Bland-Altman analysis comparing cryo and IVOCT lumen area, showed mean and standard deviation of differences as 0.01±0.43 mm2. DICE coefficients were 0.91±0.04. Finally, visual assessment on 20 representative cases with easily identifiable features suggested registration accuracy within one frame of IVOCT (±200µm), eliminating significant misinterpretations introduced by 1mm errors in the literature. The method will provide 3D data for training of IVOCT plaque algorithms and can be used for validation of other intravascular imaging modalities.

Inactivated Mesenchymal Stem Cells Maintain Immunomodulatory Capacity.

Mesenchymal stem cells (MSC) are studied as a cell therapeutic agent for treatment of various immune diseases. However, therapy with living culture-expanded cells comes with safety concerns. Furthermore, development of effective MSC immunotherapy is hampered by lack of knowledge of the mechanisms of action and the therapeutic components of MSC. Such knowledge allows better identification of diseases that are responsive to MSC treatment, optimization of the MSC product, and development of therapy based on functional components of MSC. To close in on the components that carry the therapeutic immunomodulatory activity of MSC, we generated MSC that were unable to respond to inflammatory signals or secrete immunomodulatory factors, but preserved their cellular integrity [heat-inactivated MSC (HI-MSC)]. Secretome-deficient HI-MSC and control MSC showed the same biodistribution and persistence after infusion in mice with ischemic kidney injury. Both control and HI-MSC induced mild inflammatory responses in healthy mice and dramatic increases in interleukin-10, and reductions in interferon gamma levels in sepsis mice. In vitro experiments showed that opposite to control MSC, HI-MSC lacked the capability to suppress T-cell proliferation or induce regulatory B-cell formation. However, both HI-MSC and control MSC modulated monocyte function in response to lipopolysaccharides. The results of this study demonstrate that, in particular disease models, the immunomodulatory effect of MSC does not depend on their secretome or active cross-talk with immune cells, but on recognition of MSC by monocytic cells. These findings provide a new view on MSC-induced immunomodulation and help identify key components of the therapeutic effects of MSC.