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Autologous bone marrow mononuclear cells (BMMNCs) infused after severe traumatic brain injury have shown promise for treating the injury. We evaluated their impact in children, particularly their hypothesized ability to preserve the blood-brain barrier and diminish neuroinflammation, leading to structural CNS preservation with improved outcomes. We performed a randomized, double-blind, placebo-sham-controlled Bayesian dose-escalation clinical trial at two children's hospitals in Houston, TX and Phoenix, AZ, USA (NCT01851083). Patients 5-17 years of age with severe traumatic brain injury (Glasgow Coma Scale score ≤ 8) were randomized to BMMNC or placebo (3:2). Bone marrow harvest, cell isolation and infusion were completed by 48 h post-injury. A Bayesian continuous reassessment method was used with cohorts of size 3 in the BMMNC group to choose the safest between two doses. Primary end points were quantitative brain volumes using MRI and microstructural integrity of the corpus callosum (diffusivity and oedema measurements) at 6 months and 12 months. Long-term functional outcomes and ventilator days, intracranial pressure monitoring days, intensive care unit days and therapeutic intensity measures were compared between groups. Forty-seven patients were randomized, with 37 completing 1-year follow-up (23 BMMNC, 14 placebo). BMMNC treatment was associated with an almost 3-day (23%) reduction in ventilator days, 1-day (16%) reduction in intracranial pressure monitoring days and 3-day (14%) reduction in intensive care unit (ICU) days. White matter volume at 1 year in the BMMNC group was significantly preserved compared to placebo [decrease of 19 891 versus 40 491, respectively; mean difference of -20 600, 95% confidence interval (CI): -35 868 to -5332; P = 0.01], and the number of corpus callosum streamlines was reduced more in placebo than BMMNC, supporting evidence of preserved corpus callosum connectivity in the treated groups (-431 streamlines placebo versus -37 streamlines BMMNC; mean difference of -394, 95% CI: -803 to 15; P = 0.055), but this did not reach statistical significance due to high variability. We conclude that autologous BMMNC infusion in children within 48 h after severe traumatic brain injury is safe and feasible. Our data show that BMMNC infusion led to: (i) shorter intensive care duration and decreased ICU intensity; (ii) white matter structural preservation; and (iii) enhanced corpus callosum connectivity and improved microstructural metrics., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Introduction: Traumatic brain injury (TBI) is a leading cause of death and morbidity in the trauma population. Microglia drive the secondary neuroinflammatory response after TBI. We sought to determine if the microglial response to neurologic injury was exacerbated by a second stimulus after exposure to neurologic injury., Methods: Sprague-Dawley rats (age 2-3 wk) were divided into injured and noninjured groups. Injured rats underwent a controlled cortical impact injury; noninjured rats remained naïve to any injury and served as the control group. Primary rat microglia were isolated and applied to in vitro cultures. After incubation for 24 h, the microglia were stimulated with lipopolysaccharide (LPS) or norepinephrine. Twenty-four hours after stimulation, cell culture supernatant was collected. Tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) production were measured by standard enzyme-linked immunosorbent assays. GraphPad Prism was used for statistical analysis., Results: When compared to noninjured microglia, LPS induced a significantly greater production of TNF-α in microglia isolated from the injured ipsilateral (versus noninjured = 938.8 ± 155.1, P < 0.0001) and injured contralateral hemispheres (versus noninjured = 426.6 ± 155.1, P < 0.0001). When compared to microglia from noninjured cerebral tissue, IL-6 production was significantly greater after LPS stimulation in the injured ipsilateral hemisphere (mean difference versus noninjured = 9540 ± 3016, P = 0.0101) and the contralateral hemisphere (16,700 ± 3016, P < 0.0001). Norepinephrine did not have a significant effect on IL-6 or TNF-α production., Conclusions: LPS stimulation may amplify the release of proinflammatory cytokines from postinjury microglia. These data suggest that post-TBI complications, like sepsis, may propagate neuroinflammation by augmenting the proinflammatory response of microglia., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background Aims: Mesenchymal stromal cells (MSCs) are multipotent adult cells that can be isolated from tissues including bone marrow [MSC(BM)], adipose [MSC(AT)] and umbilical cord [MSC(CT)]. Previous studies have linked expression of tissue factor (TF) on MSC surfaces to a procoagulant effect. Venous thromboembolism (VTE), immediate blood-mediated inflammatory reaction (IBMIR) and microvascular thrombosis remain a risk with intravascular MSC therapy. We examined the effect of low molecular weight heparin (LMWH) on clinical-grade MSCs using calibrated automated thrombography (CAT)., Methods: Clinical grade MSC(BM)s, MSC(AT)s and MSC(CT)s harvested at passage 4 were added to normal pooled plasma (NPP) to a final concentration of either 400 000 or 50 000 cells/mL. LMWH was added to plasma in increments of 0.1 U/mL. Thrombin generation (TG) was measured using CAT. Flow cytometry was conducted on the cells to measure MSC phenotype and TF load., Results: Presence of MSCs decreased lag time and increased peak TG. All cell lines demonstrated a dose response to LMWH, with MSC(AT) demonstrating the least thrombogenicity and most sensitivity to LMWH. TG was significantly reduced in all cell lines at doses of 0.2 U/mL LMWH and higher., Discussion: All MSC types and concentrations had a decrease in peak thrombin and TG with increasing amounts of LMWH. While this in vitro study cannot determine optimal dosing, it suggests that LMWH can be effectively used to lower the risk of VTE associated with intravascular administration of MSCs. Future in vivo work can be done to determine optimal dosing and effect on IBMIR and VTE., Competing Interests: Declaration of Competing Interest/Funding CSC has the following relationships to disclose: he has existing or previous sponsored research agreements with CBR Systems, Inc. and has served on CBR’s Scientific & Medical Advisory Board, as well as on Biostage, Inc./Harvard Apparatus Regenerative Technology, Inc.’s Scientific Advisory Board. BSG has the following relationships to disclose: Coagulex, Inc.: Sponsored Research Funding and Equity/Royalty (interest via UTHealth). UTHealth has an institutional COI for holding equity in Coagulex. JCC has funding from the Department of Defense, Aniara, and has received speaker honoraria and research funding from Grifols. SDO is supported by NINDS R21NS116302. FT and SDO have existing or previous sponsored research agreements with Biostage, Inc./Harvard Apparatus Regenerative Technology, Inc., and CBR Systems, Inc. JBS was supported by a T32 fellowship from the National Institute of General Medical Sciences of the National Institutes of Health under award number 2T32GM008792. Manufacturing of MSC(AT)s and corresponding validation was funded by FT’s and SDO’s Sponsored Research Agreements with Biostage, Inc. The manufacturing and validation of MSC(CT)s were funded by a Sponsored Research Agreements with CBR Systems, Inc. in which CSC is the primary investigator and FT and SDO are coinvestigators., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Cardiac co-morbidities and in-hospital cardiac complications significantly contribute to COVID-19 mortality. However, their influence on mortality between 2021 and 2020 may differ due to the availability of vaccines, different viral strains, and therapeutic advancements., Methods: We performed a retrospective chart review and individual patient analysis of all COVID-19 associated in-patient deaths in 2020 ( n  = 346) and 2021( n  = 527) in a large Minneapolis health system. Cause of death was adjudicated by at least two health care providers, including one cardiologist., Results: Patients who died in 2021 were younger, of similar race/ethnicity, and body mass index compared to 2020. In 2021, 24 % of the cohort was full or partially vaccinated, while none were vaccinated in 2020. Patients who died in 2021 had significantly fewer cardiovascular co-morbidities and major adverse cardiovascular events prior to COVID-19 infection, resulting in significantly fewer in-hospital cardiac adverse events compared to patients who died in 2020, including myocardial infarction, stroke, and atrial fibrillation. In contrast, patients in 2021 had significantly higher rates of venous thromboembolic events., Conclusion: Patients who died from COVID-19 in 2021 had significantly fewer cardiovascular co-morbidities and in-hospital cardiovascular complications compared to patients who died in 2020. Sixteen percent of patients stipulated as dying from COVID-19 actually die from other causes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Introduction: Citrate-phosphate-dextrose (CPD) is the most common anticoagulant for blood product storage in the United States. It was developed to prolong shelf life, though there is little research regarding its impact on function following transfusion. We used flow cytometry (FC), thromboelastography (TEG), and a clot contraction assay called the zFlex platform to measure platelet activation and global clot formation in blood samples anticoagulated with either CPD or in a standard blue top citrate (BTC) tube., Methods: Samples were obtained through venipuncture of the antecubital fossa from healthy donors who had not recently taken antiplatelet medication. Samples for FC analysis were spun to obtain platelet-rich plasma, while TEG and zFlex utilized recalcified whole blood., Results: Mean fluorescence intensity for CD62p (P-selectin, marker of platelet activation) in baseline samples was equal, while mean fluorescence intensity in samples activated with thrombin receptor activating peptide was higher in CPD than BTC (65,814 ± 4445 versus 52,483 ± 5435, P = 0.007). TEG results demonstrated similar maximum amplitude for CPD (62.7 ± 1.8 mm versus 61 ± 1 mm) (P = 0.33), though reaction time and kinetics time were significantly longer in CPD versus BTC. CPD R-time: 7.9 ± 0.4 min versus BTC: 3.8 ± 0.4 (P < 0.001). CPD K-time: 2.2 ± 0.2 min versus BTC: 1.6 ± 0.1 min (P < 0.001). Clot contraction strength was not different between the two groups on zFlex: CPD 4353 ± 6 = 517 μN versus BTC 4901 ± 390 μN (P = 0.39)., Conclusions: Our findings suggest that CPD does not affect platelet function (minimal difference on FC and no difference in ultimate clot strength, which is ∼80% due to platelet function) but may alter clot dynamics by attenuating thrombin generation., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The Blood-Brain Barrier (BBB) is a highly-selective physiologic barrier responsible for maintaining cerebral homeostasis. Innovative in vitro models of the BBB are needed to provide useful insights into BBB function with CNS disorders like traumatic brain injury (TBI). TBI is a multidimensional and highly complex pathophysiological condition that requires intrinsic models to elucidate its mechanisms. Current models either lack fluidic shear stress, or neglect hemodynamic parameters important in recapitulating the human in vivo BBB phenotype. To address these limitations in the field, we developed a fluid dynamic novel platform which closely mimics these parameters. To validate our platform, Matrigel-coated Transwells were seeded with brain microvascular endothelial cells, both with and without co-cultured primary human astrocytes and bone-marrow mesenchymal stem cells. In this article we characterized BBB functional properties such as TEER and paracellular permeability. Our platform demonstrated physiologic relevant decreases in TEER in response to an ischemic environment, while directly measuring barrier fluid fluctuation. These recordings were followed with recovery, implying stability of the model. We also demonstrate that our dynamic platform is responsive to inflammatory and metabolic cues with resultant permeability coefficients. These results indicate that this novel dynamic platform will be a valuable tool for evaluating the recapitulating BBB function in vitro, screening potential novel therapeutics, and establishing a relevant paradigm to evaluate the pathophysiology of TBI., (© 2023. Springer Nature Limited.)

Background: Inflammation and white matter injury are consequences of neonatal intraventricular hemorrhage (IVH). Both white matter and the neuroimmune system are developing during the time which IVH occurs and its consequences develop. IVH has been studied in many different animal models; however, the effects of IVH occurring at different developmental time points in the same model have not been examined. Understanding how the timing of IVH affects outcome may provide important insights into both IVH pathophysiology and innate immune development., Methods: We used intraventricular injection of lysed whole blood to model neonatal IVH in postnatal day (P)2 and P5 rats. Flow cytometry was used to detect innate immune activation. MRI was used to screen animals for the development of increased ventricular size. Immunohistochemistry for myelin basic protein was used to quantify white matter and corpus callosum thickness., Results: P5 animals exhibited significant increases in several measures of classically pro-inflammatory innate immune activation that P2 animals did not. Animals with IVH induced at P5 also developed ventricular enlargement visible on MRI whereas animals with IVH induced at P2 did not. On histological analysis, there were no significant effects of IVH in P2 animals, but IVH in P5 animals reduced white matter labeling and corpus callosum thickness., Conclusions: IVH induces a strong innate inflammatory response in P5 as well as changes in ventricular size and reduction of white matter. P2 animals do not exhibit significant changes in innate immune activation or white matter structure after IVH. This suggests that white matter pathology from IVH is due in part to innate immune activation; and that the developmental stage of the innate immune system is a key determinant of IVH pathology., Competing Interests: Declaration of Competing Interest Not applicable., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Microglia are a primary mediator of the neuroinflammatory response to neurologic injury, such as that in traumatic brain injury. Their response includes changes to their cytokine expression, metabolic profile, and immunophenotype. Dexmedetomidine (DEX) is an α 2 adrenergic agonist used as a sedative in critically ill patients, such as those with traumatic brain injury. Given its pharmacologic properties, DEX may alter the phenotype of inflammatory microglia., Methods: Primary microglia were isolated from Sprague-Dawley rats and cultured. Microglia were activated using multiple mediators: lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly I:C), and traumatic brain injury damage-associated molecular patterns (DAMP) from a rat that sustained a prior controlled cortical impact injury. After activation, cultures were treated with DEX. At the 24-h interval, the cell supernatant and cells were collected for the following studies: cytokine expression (tumor necrosis factor-α [TNFα], interleukin-10 [IL-10]) via enzyme-linked immunosorbent assay, 6-phosphofructokinase enzyme activity assay, and immunophenotype profiling with flow cytometry. Cytokine expression and metabolic enzyme activity data were analyzed using two-way analysis of variance. Cell surface marker expression was analyzed using FlowJo software., Results: In LPS-treated cultures, DEX treatment decreased the expression of TNFα from microglia (mean difference = 121.5 ± 15.96 pg/mL; p < 0.0001). Overall, DEX-treated cultures had a lower expression of IL-10 than nontreated cultures (mean difference = 39.33 ± 14.50 pg/mL, p < 0.0001). DEX decreased IL-10 expression in LPS-stimulated microglia (mean difference = 74.93 ± 12.50 pg/mL, p = 0.0039) and Poly I:C-stimulated microglia (mean difference = 23.27 ± 6.405 pg/mL, p = 0.0221). In DAMP-stimulated microglia, DEX decreased the activity of 6-phosphofructokinase (mean difference = 18.79 ± 6.508 units/mL; p = 0.0421). The microglial immunophenotype was altered to varying degrees with different inflammatory stimuli and DEX treatment., Conclusions: DEX may alter the neuroinflammatory response of microglia. By altering the microglial profile, DEX may affect the progression of neurologic injury., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature and Neurocritical Care Society.)

This narrative review article seeks to highlight the effects of citrate on physiology during massive transfusion of the bleeding patient., Data Sources: A limited library of curated articles was created using search terms including "citrate intoxication," "citrate massive transfusion," "citrate pharmacokinetics," "hypocalcemia of trauma," "citrate phosphate dextrose," and "hypocalcemia in massive transfusion." Review articles, as well as prospective and retrospective studies were selected based on their relevance for inclusion in this review., Study Selection: Given the limited number of relevant studies, studies were reviewed and included if they were written in English. This is not a systematic review nor a meta-analysis., Data Extraction and Synthesis: As this is not a meta-analysis, new statistical analyses were not performed. Relevant data were summarized in the body of the text., Conclusions: The physiologic effects of citrate independent of hypocalcemia are poorly understood. While a healthy individual can rapidly clear the citrate in a unit of blood (either through the citric acid cycle or direct excretion in urine), the physiology of hemorrhagic shock can lead to decreased clearance and prolonged circulation of citrate. The so-called "Diamond of Death" of bleeding-coagulopathy, acidemia, hypothermia, and hypocalcemia-has a dynamic interaction with citrate that can lead to a death spiral. Hypothermia and acidemia both decrease citrate clearance while circulating citrate decreases thrombin generation and platelet function, leading to ionized hypocalcemia, coagulopathy, and need for further transfusion resulting in a new citrate load. Whole blood transfusion typically requires lower volumes of transfused product than component therapy alone, resulting in a lower citrate burden. Efforts should be made to limit the amount of citrate infused into a patient in hemorrhagic shock while simultaneously addressing the induced hypocalcemia., Competing Interests: Drs. Cox and Gill received sponsored research funding from Coagulex and Equity/Royalty (interest via UTHealth). UTHealth has an institutional conflicts of interest for holding equity in Coagulex (to Dr. Gill). Dr. Olson is supported by National Institute of Neurological Disorders and Stroke R21NS116302. Dr. Schriner was supported by a T32 fellowship from the National Institute of General Medical Sciences of the National Institutes of Health under award number 2T32GM008792. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

During cell division, the microtubule nucleating and organizing organelle, known as the centrosome, is a critical component of the mitotic spindle. In cells with two centrosomes, each centrosome functions as an anchor point for microtubules, leading to the formation of a bipolar spindle and progression through a bipolar cell division. When extra centrosomes are present, multipolar spindles form and the parent cell may divide into more than two daughter cells. Cells that are born from multipolar divisions are not viable, and hence clustering of extra centrosomes and progression to a bipolar division are critical determinants of viability in cells with extra centrosomes. We combine experimental approaches with computational modeling to define a role for cortical dynein in centrosome clustering. We show that centrosome clustering fails and multipolar spindles dominate when cortical dynein distribution or activity is experimentally perturbed. Our simulations further reveal that centrosome clustering is sensitive to the distribution of dynein on the cortex. Together, these results indicate that dynein's cortical localization alone is insufficient for effective centrosome clustering and, instead, dynamic relocalization of dynein from one side of the cell to the other throughout mitosis promotes timely clustering and bipolar cell division in cells with extra centrosomes.

Background: Prepandemic hospital guidelines were unable to support an acute influx of patients with respiratory deterioration. New processes for general care practice were needed to facilitate patient care., Purpose: To develop and evaluate guidelines to safely treat patients with COVID-19 respiratory deterioration in the general care setting., Methods: A quality improvement project with 2 PDSA (Plan-Do-Study-Act) cycles was used to develop guidelines for high-flow oxygen and prone positioning, along with frequent monitoring and collaboration with virtual critical care support., Results: Over 6 months, 126 patients with COVID-19 were cared for on general care units. Zero intubations occurred on the general care units, with 211 patient hospital days spent in general care that previously would have required an intensive care unit bed., Conclusions: Patients in the general care setting with respiratory decline can safely be managed with appropriate monitoring criteria, oxygen device settings, and nursing support unitizing technology., Competing Interests: There are no conflicts of interest with any of the authors to disclose. There has been no funding associated with this work to report. This was not part of a clinical trial., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Increased microvascular permeability at the level of the blood-brain barrier (BBB) often leads to vasogenic brain edema following traumatic brain injury (TBI). These pathologic conditions compromise the integrity of the neurovascular unit resulting in severe brain dysfunction. To quantify this permeability and assess ionic equillibrium, preclinical researchers have relied on the use of various molecular weight permeable dyes such as Evans Blue that normally cannot enter the brain parenchyma under homeostatic conditions. Evans Blue, the most cited of the molecular weight dyes, has reported reproducibility issues because of harsh extraction processes, suboptimal detection via absorbance, and wide excitation fluorescence spectra associated with the dye. Our laboratory group transitioned to Alexa Fluor 680, a far-red dye with improved sensitivity compared to Evans Blue and thus improved reproducibility to alleviate this issue. To evaluate our reproducibility and increase the rigor of our experimental design, we retrospectively analyzed our controlled cortical impact (CCI) experiments over the past 10 years to evaluate effect size with larger samples and potential sources of variability. All of our BBB permeability experiments were performed with Male, Sprague Dawley rats weighing between 225 and 300 g. Historically, Sprague Dawleys were randomly divided into treatment groups: SHAM, CCI, and a stem cell-based treatment from years 2007-2020. The assessment of microvascular hyperpermeability were evaluated by comparing the mean at minimum threshold, area at 1 k-2 k, and intensity density obtained from Alexa Fluor 680 permeability data. Studies utilizing Evans Blue were further compared by tip depth, diameter size, and the hemisphere of injury. Statistical evaluation utilizing the G Power software analysis did not yield a significant difference in sample size comparing experimental groups for Evans Blue and Alexa Fluor 680 analyzed brain tissue. Our analysis also demonstrated a trend in that recent studies (years 2018-2020) have yielded more compact sample sizes between experimental groups in Alexa Fluor 680 analyzed rats. This retrospective study further revealed that Alexa Fluor 680 image analysis provides greater sensitivity to BBB permeability following TBI in comparison to Evans Blue. Significant differences in sample size were not detected between Evans Blue and Alexa Fluor 680; there were significant differences found throughout year to year analysis at the lower range of thresholds. SUMMARY STATEMENT: This work provides a comparative analysis of BBB permeability assay techniques after CCI model of injury in rats., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Systemic administration of marrow stromal cells (MSCs) leads to the release of a broad range of factors mediating recovery in rodent stroke models. The release of these factors could depend on the various cell types within the peripheral blood as they contact systemically administered MSCs. In this study, we assessed the immunomodulatory interactions of MSCs with peripheral blood derived monocytes (Mϕ) collected from acute stroke patients., Methods: Peripheral blood from stroke patients was collected at 5-7 days ( N = 5) after symptom onset and from age-matched healthy controls ( N = 5) using mononuclear cell preparation (CPT) tubes. After processing, plasma and other cellular fractions were removed, and Mϕ were isolated from the mononuclear fraction using CD14 microbeads. Mϕ were then either cultured alone or co-cultured with MSCs in a trans-well cell-culture system. Secretomes were analyzed after 24 h of co-cultures using a MAGPIX reader., Results: Our results show that there is a higher release of IFN-γ and IL-10 from monocytes isolated from peripheral blood at day 5-7 after stroke compared with monocytes from healthy controls. In trans-well co-cultures of MSCs and monocytes isolated from stroke patients, we found statistically significant increased levels of IL-4 and MCP-1, and decreased levels of IL-6, IL-1β, and TNF-α. Addition of MSCs to monocytes increased the secretions of Fractalkine, IL-6, and MCP-1, while the secretions of TNF-α decreased, as compared to the secretions from monocytes alone. When MSCs were added to monocytes from stroke patients, they decreased the levels of IL-1β, and increased the levels of IL-10 significantly more as compared to when they were added to monocytes from control patients., Conclusion: The systemic circulation of stroke patients may differentially interact with MSCs to release soluble factors integral to their paracrine mechanisms of benefit. Our study finds that the effect of MSCs on Mϕ is different on those derived from stroke patients blood as compared to healthy controls. These findings suggest immunomodulation of peripheral immune cells as a therapeutic target for MSCs in patients with acute stroke., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Satani, Parsha, Davis, Gee, Olson, Aronowski and Savitz.)

Valine-citrulline is a protease-cleavable linker commonly used in many drug delivery systems, including antibody-drug conjugates (ADC) for cancer therapy. However, its suboptimal in vivo stability can cause various adverse effects such as neutropenia and hepatotoxicity, leading to dose delays or treatment discontinuation. Here, we report that glutamic acid-glycine-citrulline (EGCit) linkers have the potential to solve this clinical issue without compromising the ability of traceless drug release and ADC therapeutic efficacy. We demonstrate that our EGCit ADC resists neutrophil protease-mediated degradation and spares differentiating human neutrophils. Notably, our anti-HER2 ADC shows almost no sign of blood and liver toxicity in healthy mice at 80 mg kg-1. In contrast, at the same dose level, the FDA-approved anti-HER2 ADCs Kadcyla and Enhertu show increased levels of serum alanine aminotransferase and aspartate aminotransferase and morphologic changes in liver tissues. Our EGCit conjugates also exert greater antitumor efficacy in multiple xenograft tumor models compared with Kadcyla and Enhertu. This linker technology could substantially broaden the therapeutic windows of ADCs and other drug delivery agents, providing clinical options with improved efficacy and safety., (©2022 American Association for Cancer Research.)

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed. Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

Mesenchymal stromal cells (MSC) undergo functional maturation upon their migration from bone marrow and introduction to a site of injury. This inflammatory licensing leads to heightened immune regulation via cell-to-cell interaction and the secretion of immunomodulatory molecules, such as anti-inflammatory mediators and antioxidants. Pro-inflammatory cytokines are a recognized catalyst of inflammatory licensing; however, biomechanical forces, such as fluid shear stress, are a second, distinct class of stimuli that incite functional maturation. Here we show mechanotransduction, achieved by exposing MSC to various grades of wall shear stress (WSS) within a scalable conditioning platform, enhances the immunomodulatory potential of MSC independent of classical pro-inflammatory cytokines. A dose-dependent effect of WSS on potency is evidenced by production of prostaglandin E2 (PGE 2 ) and indoleamine 2,3 dioxygenase 1 (IDO1), as well as suppression of tumor necrosis factor-α (TNF- α) and interferon-γ (IFN-γ) production by activated immune cells. Consistent, reproducible licensing is demonstrated in adipose tissue and bone marrow human derived MSC without significant impact on cell viability, cellular yield, or identity. Transcriptome analysis of WSS-conditioned BM-MSC elucidates the broader phenotypic implications on the differential expression of immunomodulatory factors. These results suggest mechanotransduction as a viable, scalable pre-conditioning alternative to pro-inflammatory cytokines. Enhancing the immunomodulatory capacity of MSC via biomechanical conditioning represents a novel cell therapy manufacturing approach., Competing Interests: SO has received research support from Athersys, CBR Systems, Hope Bio, Generate Life Sciences, Cellvation, and Biostage. CC has received research support from Athersys, CBR Systems, Hope Bio, Biostage, Generate Life Sciences, and Cellvation, and is on the Scientific Advisory Board of Cellvation and Generate Life Sciences. BG is on the Scientific Advisory Board of Cellvation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Skibber, Olson, Prabhakara, Gill and Cox.)

Abstract: Platelets are subcellular anucleate components of blood primarily responsible for initiating and maintaining hemostasis. After injury to a blood vessel, platelets can be activated via several pathways, resulting in changed shape, adherence to the injury site, aggregation to form a plug, degranulation to initiate activation in other nearby platelets, and acceleration of thrombin formation to convert fibrinogen to fibrin before contracting to strengthen the clot. Platelet function assays use agonists to induce and measure one or more of these processes to identify alterations in platelet function that increase the likelihood of bleeding or thrombotic events. In severe trauma, these assays have revealed that platelet dysfunction is strongly associated with poor clinical outcomes. However, to date, the mechanism(s) causing clinically significant platelet dysfunction remain poorly understood. We review the pros, cons, and evidence for use of many of the popular assays in trauma, discuss limitations of their use in this patient population, and present approaches that can be taken to develop improved functional assays capable of elucidating mechanisms of trauma-induced platelet dysfunction. Platelet dysfunction in trauma has been associated with need for transfusions and mortality; however, most of the current platelet function assays were not designed for evaluating trauma patients, and there are limited data regarding their use in this population. New or improved functional assays will help define the mechanisms by which platelet dysfunction occurs, as well as help optimize future treatment., Competing Interests: C.S.C. has the following relationships to disclose: Coagulex, Inc. sponsored research funding and equity/royalty (interest via The University of Texas Health Science Center at Houston [UTHealth]). B.S.G. has the following relationships to disclose: Coagulex, Inc. sponsored research funding and equity/royalty (interest via UTHealth). UTHealth has an institutional conflict of interest for holding equity in Coagulex. C.E.W. receives funding from the William Stamps Farish Fund, the Howell Family Foundation, and the James H. “Red” Duke Professorship. Unrelated to the present effort, C.E.W. holds a financial interest in Dicisio Health LLC and receives grants from Grifols and Athersys. J.C.C. has funding from the Department of Defense, Aniara, and has received speaker honoraria and research funding from Grifols. S.D.O. is supported by National Institute of Neurological Diseases and Stroke of the National Institutes of Health award number R21NS116302. J.B.S. is supported by a T32 fellowship from the National Institute of General Medical Sciences of the National Institutes of Health under award number T32GM008792., (Copyright © 2022 by the Shock Society.)

Traumatic brain injury (TBI) results in a cascade of cellular responses, which produce neuroinflammation, partly due to the activation of microglia. Accurate identification of microglial populations is key to understanding therapeutic approaches that modify microglial responses to TBI and improve long-term outcome measures. Notably, previous studies often utilized an outdated convention to describe microglial phenotypes. We conducted a temporal analysis of the response to controlled cortical impact (CCI) in rat microglia between ipsilateral and contralateral hemispheres across seven time points, identified microglia through expression of activation markers including CD45, CD11b/c, and p2y12 receptor and evaluated their activation state using additional markers of CD32, CD86, RT1B, CD200R, and CD163. We identified unique sub-populations of microglial cells that express individual or combination of activation markers across time points. We further portrayed how the size of these sub-populations changes through time, corresponding to stages in TBI response. We described longitudinal changes in microglial population after CCI in two different locations using activation markers, showing clear separation into cellular sub-populations that feature different temporal patterns of markers after injury. These changes may aid in understanding the symptomatic progression following TBI and help define microglial subpopulations beyond the outdated M1/M2 paradigm., (© 2022. The Author(s).)

The number of mesenchymal stromal/stem cell (MSC) therapeutics and types of clinical applications have greatly diversified during the past decade, including rapid growth of poorly regulated "Stem Cell Clinics" offering diverse "Unproven Stem Cell Interventions." This product diversification necessitates a critical evaluation of the reliance on the 2006 MSC minimal criteria to not only define MSC identity but characterize MSC suitability for intravascular administration. While high-quality MSC therapeutics have been safely administered intravascularly in well-controlled clinical trials, repeated case reports of mild-to-more-severe adverse events have been reported. These are most commonly related to thromboembolic complications upon infusion of highly procoagulant tissue factor (TF/CD142)-expressing MSC products. As TF/CD142 expression varies widely depending on the source and manufacturing process of the MSC product, additional clinical cell product characterization and guidelines are needed to ensure the safe use of MSC products. To minimize risk to patients receiving MSC therapy, we here propose to supplement the minimal criteria used for characterization of MSCs, to include criteria that assess the suitability of MSC products for intravascular use. If cell products are intended for intravascular delivery, which is true for half of all clinical applications involving MSCs, the effects of MSC on coagulation and hemocompatibility should be assessed and expression of TF/CD142 should be included as a phenotypic safety marker. This adjunct criterion will ensure both the identity of the MSCs as well as the safety of the MSCs has been vetted prior to intravascular delivery of MSC products., (© The Author(s) 2022. Published by Oxford University Press.)

Introduction: Traumatic brain injury is a leading cause of injury-related death and morbidity. Multiple clinical and pre-clinical studies have reported various results regarding sex-based differences in TBI. Our accepted rodent model of traumatic brain injury was used to identify sex-based differences in the pathological features of TBI., Methods: Male and female Sprague-Dawley rats were subjected to either controlled-cortical impact (CCI) or sham injury; brain tissue was harvested at different time intervals depending on the specific study. Blood-brain barrier (BBB) analysis was performed using infrared imaging to measure fluorescence dye extravasation. Microglia and splenocytes were characterized with traditional flow cytometry; microglia markers such as CD45, P2Y12, CD32, and CD163 were analyzed with t-distributed stochastic neighbor embedding (t-SNE). Flow cytometry was used to study tissue cytokine levels, and supplemented with ELISAs of TNF-⍺, IL-17, and IL-1β of the ipsilateral hemisphere tissue., Results: CCI groups of both sexes recorded a higher BBB permeability at 72 hours post-injury than their respective sham groups. There was significant difference in the integrated density value of BBB permeability between the male CCI group and the female CCI group (female CCI mean = 3.08 x 108 ± 2.83 x 107, male CCI mean = 2.20 x 108 ± 4.05 x 106, p = 0.0210), but otherwise no differences were observed. Traditional flow cytometry did not distinguish any sex-based difference in regards to splenocyte cell population after CCI. t-SNE did not reveal any significant difference between the male and female injury groups in the activation of microglia. Cytokine analysis after injury by flow cytometry and ELISA was limited in differences at the time point of 6 hours post-injury., Conclusion: In our rodent model of traumatic brain injury, sex-based differences in pathology and neuroinflammation at specified time points are limited, and only noted in one specific analysis of BBB permeability., Competing Interests: CC is on the Scientific Advisory Board of Cellvation and CBR. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Scott, Prabhakara, Walters, Olson and Cox.)

Introduction: Resection and reconstruction of the esophagus remains fraught with morbidity and mortality. Recently, data from a porcine reconstruction model revealed that segmental esophageal reconstruction using an autologous mesenchymal stromal cell-seeded polyurethane graft (Cellspan esophageal implant [CEI]) can facilitate esophageal regrowth and regeneration. To this end, a patient requiring a full circumferential esophageal segmental reconstruction after a complex multiorgan tumor resection was approved for an investigational treatment under the Food and Drug Administration Expanded Access Use (Investigational New Drug 17402)., Methods: Autologous adipose-derived mesenchymal stromal cells (Ad-MSCs) were isolated from the Emergency Investigational New Drug patient approximately 4 weeks before surgery from an adipose tissue biopsy specimen. The Ad-MSCs were grown and expanded under current Good Manufacturing Practice manufacturing conditions. The cells were then seeded onto a polyurethane fiber mesh scaffold (Cellspan scaffold) and cultured in a custom bioreactor to manufacture the final CEI graft. The cell-seeded scaffold was then shipped to the surgical site for surgical implantation. After removal of a tumor mass and a full circumferential 4 cm segment of the esophagus that was invaded by the tumor, the CEI was implanted by suturing the tubular CEI graft to both ends of the remaining native esophagus using end-to-end anastomosis., Results: In this case report, we found that a clinical-grade, tissue-engineered esophageal graft can be used for segmental esophageal reconstruction in a human patient. This report reveals that the graft supports regeneration of the esophageal conduit. Histologic analysis of the tissue postmortem, 7.5 months after the implantation procedure, revealed complete luminal epithelialization and partial esophageal tissue regeneration., Conclusions: Autologous Ad-MSC seeded onto a tubular CEI tissue-engineered graft stimulates tissue regeneration following implantation after a full circumferential esophageal resection., (© 2021 THE AUTHORS.)

Proper formation and maintenance of the mitotic spindle is required for faithful cell division. Although much work has been done to understand the roles of the key molecular components of the mitotic spindle, identifying the consequences of force perturbations in the spindle remains a challenge. We develop a computational framework accounting for the minimal force requirements of mitotic progression. To reflect early spindle formation, we model microtubule dynamics and interactions with major force-generating motors, excluding chromosome interactions that dominate later in mitosis. We directly integrate our experimental data to define and validate the model. We then use simulations to analyze individual force components over time and their relationship to spindle dynamics, making it distinct from previously published models. We show through both model predictions and biological manipulation that rather than achieving and maintaining a constant bipolar spindle length, fluctuations in pole-to-pole distance occur that coincide with microtubule binding and force generation by cortical dynein. Our model further predicts that high dynein activity is required for spindle bipolarity when kinesin-14 (HSET) activity is also high. To the best of our knowledge, our results provide novel insight into the role of cortical dynein in the regulation of spindle bipolarity., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Traumatic brain injury (TBI) causes both physical disruption of the blood brain barrier (BBB) and altered immune responses that can lead to significant secondary brain injury and chronic inflammation within the central nervous system (CNS). Cell therapies, including mesenchymal stromal cells (MSC), have been shown to restore BBB integrity and augment endogenous splenic regulatory T cells (Treg), a subset of CD4+ T cells that function to regulate immune responses and prevent autoimmunity. We have recently shown that infusion of human cord blood-derived Treg decreased neuroinflammation after TBI in vivo and in vitro. However, while both cells have demonstrated anti-inflammatory and regenerative potential, they likely utilize differing, although potentially overlapping, mechanisms. Furthermore, studies investigating these two cell types together, as a combination therapy, are lacking. In this study, we compared the ability of Treg+MSC combination therapy, as well as MSC and Treg monotherapies, to improve BBB permeability in vivo and suppress inflammation in vitro. While Treg+MSC combination did not significantly augment potency in vivo, our in vitro data demonstrates that combination therapy may augment therapeutic potency and immunosuppressive potential compared to Treg or MSC monotherapy., Competing Interests: The authors have read the journal’s policy and have the following competing interests: SDO was a Guest Editor on the “Stem Cell Plasticity in Tissue Repair and Regeneration” Call for Papers for PLOS ONE. CSC has received research support from Athersys, CBR Systems, Hope Bio, Biostage, and is on the Scientific Advisory Board of Cellvation, Biostage, and CBR. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Introduction: Microglia is the resident tissue macrophages of the central nervous system. Prolonged microglial activation often occurs after traumatic brain injury and is associated with deteriorating neurocognitive outcomes. Resolution of microglial activation is associated with limited tissue loss and improved neurocognitive outcomes. Limiting the prolonged pro-inflammatory response and the associated secondary tissue injury provides the rationale and scientific premise for considering microglia as a therapeutic target., Areas Covered: In this review, we discuss markers of microglial activation, such as immunophenotype and microglial response to injury, including cytokine/chemokine release, free radical formation, morphology, phagocytosis, and metabolic shifts. We compare the origin and role in neuroinflammation of microglia and monocytes/macrophages. We review potential therapeutic targets to shift microglial polarization. Finally, we review the effect of cell therapy on microglia., Expert Opinion: Dysregulated microglial activation after neurologic injury, such as traumatic brain injury, can worsen tissue damage and functional outcomes. There are potential targets in microglia to attenuate this activation, such as proteins and molecules that regulate microglia polarization. Cellular therapeutics that limit, but do not eliminate, the inflammatory response have improved outcomes in animal models by reducing pro-inflammatory microglial activation via secondary signaling. These findings have been replicated in early phase clinical trials.

The inflammatory response after traumatic brain injury (TBI) can lead to significant secondary brain injury and chronic inflammation within the central nervous system. Cell therapies, including mesenchymal stromal cells (MSC), have led to improvements in animal models of TBI and are under investigation in human trials. One potential mechanism for the therapeutic potential of MSC is their ability to augment the endogenous response of immune suppressive regulatory T cells (Treg). We have recently shown that infusion of human cord blood Treg decreased chronic microgliosis after TBI and altered the systemic immune response in a rodent model. These cells likely use both overlapping and distinct mechanisms to modulate the immune system; therefore, combining Treg and MSC as a combination therapy may confer therapeutic benefit over either monotherapy. However, investigation of Treg + MSC combination therapy in TBI is lacking. In this study, we compared the ability MSC + Treg combination therapy, as well as MSC and Treg monotherapies, to inhibit the neuroinflammatory response to TBI in vivo and in vitro. Treg + MSC combination therapy demonstrated increased potency to reduce the neuro- and peripheral inflammatory response compared to monotherapy; furthermore, the timing of infusion proved to be a significant variable in the efficacy of both MSC monotherapy and Treg + MSC combination therapy in vivo and in vitro., (©AlphaMed Press 2020.)

To advance our understanding of the movement of elastic microstructures in a viscous fluid, techniques that utilize available data to estimate model parameters are necessary. Here, we describe a Bayesian uncertainty quantification framework that is highly parallelizable, making parameter estimation tractable for complex fluid-structure interaction models. Using noisy in silico data for swimmers, we demonstrate the methodology's robustness in estimating fluid and elastic swimmer parameters, along with their uncertainties. We identify correlations between model parameters and gain insight into emergent swimming trajectories of a single swimmer or a pair of swimmers. Our proposed framework can handle data with a spatiotemporal resolution representative of experiments, showing that this framework can be used to aid in the development of artificial micro-swimmers for biomedical applications, as well as gain a fundamental understanding of the range of parameters that allow for certain motility patterns.

Objective: Abnormal pulmonary vasculature directly affects the development and progression of congenital diaphragmatic hernia (CDH)-associated pulmonary hypertension (PH). Though overarching structural and cellular changes in CDH-affected pulmonary arteries have been documented, the precise role of the extracellular matrix (ECM) in the pulmonary artery (PA) pathophysiology remains undefined. Here, we quantify the structural, compositional, and mechanical CDH-induced changes in the main and distal PA ECM and investigate the efficacy of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) as a therapy to ameliorate pathological vascular ECM changes., Methods: Pregnant Sprague-Dawley rodents were administered nitrofen to induce CDH-affected pulmonary vasculature in the offspring. A portion of CDH-affected pups was treated with intravenous infusion of MSC-EVs (1 × 10 10 /mL) upon birth. A suite of histological, mechanical, and transmission electron microscopic analyses were utilized to characterize the PA ECM., Results: The CDH model main PA presented significantly altered characteristics-including greater vessel thickness, greater lysyl oxidase (LOX) expression, and a relatively lower ultimate tensile strength of 13.6 MPa compared to control tissue (25.1 MPa), suggesting that CDH incurs ECM structural disorganization. MSC-EV treatment demonstrated the potential to reverse CDH-related changes, particularly through rapid inhibition of ECM remodeling enzymes (LOX and MMP-9). Additionally, MSC-EV treatment bolstered structural aspects of the PA ECM and mitigated pathological disorganization as exhibited by increased medial wall thickness and stiffness that, while not significantly altered, trends away from CDH-affected tissue., Conclusions: These data demonstrate notable ECM remodeling in the CDH pulmonary vasculature, along with the capacity of MSC-EVs to attenuate pathological ECM remodeling, identifying MSC-EVs as a potentially efficacious therapeutic for CDH-associated pulmonary hypertension., (© 2020 Wiley Periodicals LLC.)

Congenital diaphragmatic hernia (CDH) leads to pathophysiologic pulmonary vasoreactivity. Previous studies show that mesenchymal stromal cell-derived extracellular vesicles (MSCEv) inhibit lung inflammation and vascular remodeling. We characterize MSCEv and human pulmonary artery endothelial cell (HPAEC) interaction, as well as the pulmonary artery (PA) response to MSCEv treatment. HPAECs were cultured with and without exposure to nitrofen (2,4-dichloro-phenyl-p-nitrophenylether) and treated with MSCEv. HPAEC viability, architecture, production of reactive oxygen species (ROS), endothelial dysfunction-associated protein levels (PPARγ, LOX-1, LOX-2, nuclear factor-κB [NF-κB], endothelial NO synthase [eNOS], ET-1 [endothelin 1]), and the nature of MSCEv-cellular interaction were assessed. Newborn rodents with and without CDH (nitrofen model and Sprague-Dawley) were treated with intravascular MSCEv or vehicle control, and their PAs were isolated. Contractility was assessed by wire myography. The contractile (KCL and ET-1) and relaxation (fasudil) responses were evaluated. HPAEC viability correlated inversely with nitrofen dose, while architectural compromise was directly proportional. There was a 2.1 × increase in ROS levels in nitrofen HPAECs ( P  < 0.001), and MSCEv treatment attenuated ROS levels by 1.5 × versus nitrofen HPAECs ( P  < 0.01). Nitrofen-induced alterations in endothelial dysfunction-associated proteins are shown, and exposure to MSCEv restored more physiologic expression. Nitrofen HPAEC displayed greater MSCEv uptake (80% increase, P  < 0.05). Adenosine, a clathrin-mediated endocytosis inhibitor, decreased uptake by 46% ( P  < 0.05). CDH PA contraction was impaired with KCL (108.6% ± 1.4% vs. 112.0% ± 1.4%, P  = 0.092) and ET-1 (121.7% ± 3.0% vs. 131.2% ± 1.8%, P  < 0.01). CDH PA relaxation was impaired with fasudil (32.2% ± 1.9% vs. 42.1% ± 2.2%, P  < 0.001). After MSCEv treatment, CDH PA contraction improved (125.9% ± 3.4% vs. 116.4 ± 3.5, P  = 0.06), and relaxation was unchanged (32.5% ± 3.2% vs. 29.4% ± 3.1%, P  = 0.496). HPAEC exposure to nitrofen led to changes consistent with vasculopathy in CDH, and MSCEv treatment led to a more physiologic cellular response. MSCEv were preferentially taken up by nitrofen-treated cells by clathrin-dependent endocytosis. In vivo, MSCEv exposure improved PA contractile response. These data reveal mechanisms of cellular and signaling alterations that characterize MSCEv-mediated attenuation of pulmonary vascular dysfunction in CDH-associated pulmonary hypertension.

Traumatic brain injury (TBI) causes a profound inflammatory response within the central nervous system and peripheral immune system, which contributes to secondary brain injury and further morbidity and mortality. Preclinical investigations have demonstrated that treatments that downregulate microglia activation and polarize them toward a reparative/anti-inflammatory phenotype have improved outcomes in preclinical models. However, no therapy to date has translated into proven benefits in human patients. Regulatory T cells (Treg) have been shown to downregulate pathologic immune responses of the innate and adaptive immune system across a variety of pathologies. Furthermore, cellular therapy has been shown to augment host Treg responses in preclinical models; yet, studies investigating the use of Treg as a therapeutic for TBI are lacking. In a rodent TBI model, we demonstrate that human umbilical cord blood Treg modulate the central and peripheral immune response after injury in vitro and in vivo., (© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Traumatic brain injury (TBI) results in a cascade of cellular responses, which produce neuroinflammation, partly due to microglial activation. Transforming from surveying to primed phenotypes, microglia undergo considerable molecular changes. However, specific microglial profiles in rat remain elusive due to tedious methodology and limited availability of reagents. Here, we present a flow cytometry-based analysis of rat microglia 24 h after TBI using the controlled cortical impact model, validated with a bioinformatics approach. Isolated microglia are analyzed for morphological changes and their expression of activation markers using flow cytometry, traditional gating-based analysis methods and support the data by employing bioinformatics statistical tools. We use CD45, CD11b/c, and p2y12 receptor to identify microglia and evaluate their activation state using CD32, CD86, RT1B, CD200R, and CD163. The results from logic-gated flow cytometry analysis was validated with bioinformatics-based analysis and machine learning algorithms to detect quantitative changes in morphology and marker expression in microglia due to activation following TBI.

Secondary alveolar bone grafts (ABGs) are the standard treatment for the alveolar defect in patients with cleft lip and palate (CLP), but remain invasive and have several disadvantages such as delayed timing of alveolar repair, donor-site complications, graft resorption, and need for multiple surgeries. Earlier management of the alveolar defect (primary ABG) would be ideal, but is limited by the minimal bony donor sites available in the infant. In this study we used a critical-size alveolar bone defect model in the rat to investigate the use of Wharton's Jelly (WJ), the stem cell-rich connective tissue matrix of the umbilical cord, to generate bone within the alveolar cleft. Human WJ was isolated and implanted into a critical-size alveolar bone defect model representative of secondary cleft ABG surgery in 10-11-week-old male Sprague-Dawley rats. The defects were monitored with CT imaging of living animals to evaluate bone regrowth and healing over 24 weeks, followed by histomorphometric evaluation at 24 weeks, after the last CT scan. CT data confirmed that the defect size was critical and did not lead to the union of the bones in the control animals ( n  = 12) for the entire duration of the study. New bone growth was stimulated leading to partial-to-full closure of the defect in the animals treated with WJ ( n  = 12). Twenty four weeks postoperatively, the percent increase in new bone formation in the WJ-treated group (156.58% ± 20.67%) was markedly higher than that in the control group (50.36% ± 21.07%) ( p  < 0.05). Histomorphometric data also revealed significantly greater new bone formation in WJ-treated versus control animals, confirming CT findings. qPCR analysis of human Alu elements was unable to detect any appreciable long-term persistence of human cells in the new bone, indicating that WJ may enhance bone growth by mediating osteoinduction in the host tissue, rather than through osteogenic differentiation of WJ-embedded cells. Impact statement In this study, Wharton's Jelly enhanced bone growth in a preclinical alveolar defect model, indicating its potential use as a natural adjunct in the repair of the alveolar cleft defect in patients with cleft lip and palate (CLP). The clinical success of this approach would represent a paradigm shift in the treatment of patients with CLP by reducing or eliminating the need for subsequent secondary alveolar bone graft and reducing their number of lifetime surgeries.

In the U.S., approximately 1.7 million people suffer traumatic brain injury each year, with many enduring long-term consequences and significant medical and rehabilitation costs. The primary injury causes physical damage to neurons, glia, fiber tracts and microvasculature, which is then followed by secondary injury, consisting of pathophysiological mechanisms including an immune response, inflammation, edema, excitotoxicity, oxidative damage, and cell death. Most attempts at intervention focus on protection, repair or regeneration, with regenerative medicine becoming an intensively studied area over the past decade. The use of stem cells has been studied in many disease and injury models, using stem cells from a variety of sources and applications. In this study, human adipose-derived mesenchymal stromal cells (MSCs) were administered at early (3 days) and delayed (14 days) time points after controlled cortical impact (CCI) injury in rats. Animals were routinely assessed for neurological and vestibulomotor deficits, and at 32 days post-injury, brain tissue was processed by flow cytometry and immunohistochemistry to analyze neuroinflammation. Treatment with HB-adMSC at either 3d or 14d after injury resulted in significant improvements in neurocognitive outcome and a change in neuroinflammation one month after injury., Competing Interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests: KAR, HP, and AD are paid employees of Hope Bio and have received salary support for their role in this study. SDO and CSC have both received research support from sponsored research agreements between the University of Texas Health Science Center at Houston and Hope Bio. SDO is a Guest Editor on the “Stem Cell Plasticity in Tissue Repair and Regeneration” Call for Papers for PLoS ONE. Hope Bio produces and markets HB-AdMSCs and HB-AdMSC-related products. There are no other patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Clinical trials in trauma populations are exploring the use of clinical cellular therapeutics (CCTs) like human mesenchymal stromal cells (MSC) and mononuclear cells (MNC). Recent studies demonstrate a procoagulant effect of these CCTs related to their expression of tissue factor (TF). We sought to examine this relationship in blood from severely injured trauma patients and identify methods to reverse this procoagulant effect. Human MSCs from bone marrow, adipose, and amniotic tissues and freshly isolated bone marrow MNC samples were tested. TF expression and phenotype were quantified using flow cytometry. CCTs were mixed individually with trauma patients' whole blood, assayed with thromboelastography (TEG), and compared with healthy subjects mixed with the same cell sources. Heparin was added to samples at increasing concentrations until TEG parameters normalized. Clotting time or R time in TEG decreased relative to the TF expression of the CCT treatment in a logarithmic fashion for trauma patients and healthy subjects. Nonlinear regression curves were significantly different with healthy subjects demonstrating greater relative decreases in TEG clotting time. In vitro coadministration of heparin normalized the procoagulant effect and required dose escalation based on TF expression. TF expression in human MSC and MNC has a procoagulant effect in blood from trauma patients and healthy subjects. The procoagulant effect is lower in trauma patients possibly because their clotting time is already accelerated. The procoagulant effect due to MSC/MNC TF expression could be useful in the bleeding trauma patient; however, it may emerge as a safety release criterion due to thrombotic risk. The TF procoagulant effect is reversible with heparin., (© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

To accelerate the development of strategies for cartilage tissue engineering, models are necessary to investigate the interactions between cellular dynamics and the local microenvironment. We use a discrete framework to capture the individual behavior of cells, modeling experiments where cells are seeded in a porous scaffold or hydrogel and over the time course of a month, the scaffold slowly degrades while cells divide and synthesize extracellular matrix constituents. The movement of cells and the ability to proliferate is a function of the local porosity, defined as the volume fraction of fluid in the surrounding region. A phenomenological approach is used to capture a continuous profile for the degrading scaffold and accumulating matrix, which will then change the local porosity throughout the construct. We parameterize the model by first matching total cell counts in the construct to chondrocytes seeded in a polyglycolic acid scaffold (Freed et al. in Biotechnol Bioeng 43:597-604, 1994). We investigate the influence of initial scaffold porosity on the total cell count and spatial profiles of cell and ECM in the construct. Cell counts were higher at day 30 in scaffolds of lower initial porosity, and similar cell counts were obtained using different models of scaffold degradation and matrix accumulation (either uniform or cell-specific). Using this modeling framework, we study the interplay between a phenomenological representation of scaffold architecture and porosity as well as the potential continuous application of growth factors. We determine parameter regimes where large cellular aggregates occur, which can hinder matrix accumulation and cellular proliferation. The developed modeling framework can easily be extended and can be used to identify optimal scaffolds and culture conditions that lead to a desired distribution of extracellular matrix and cell counts throughout the construct.

Changes in calcium concentration along the sperm flagellum regulate sperm motility and hyperactivation, characterized by an increased flagellar bend amplitude and beat asymmetry, enabling the sperm to reach and penetrate the ovum (egg). The signalling pathways by which calcium increases within the flagellum are well established. However, the exact mechanisms of how calcium regulates flagellar bending are still under investigation. We extend our previous model of planar flagellar bending by developing a fluid-structure interaction model that couples the 3D motion of the flagellum in a viscous Newtonian fluid with the evolving calcium concentration. The flagellum is modelled as a Kirchhoff rod: an elastic rod with preferred curvature and twist. The calcium dynamics are represented as a 1D reaction-diffusion model on a moving domain, the flagellum. The two models are coupled assuming that the preferred curvature and twist of the sperm flagellum depend on the local calcium concentration. To investigate the effect of calcium on sperm motility, we compare model results of flagellar bend amplitude and swimming speed for three cases: planar, helical (spiral with equal amplitude in both directions), and quasi-planar (spiral with small amplitude in one direction). We observe that for the same parameters, the planar swimmer is faster and a turning motion is more clearly observed when calcium coupling is accounted for in the model. In the case of flagellar bending coupled to the calcium concentration, we observe emergent trajectories that can be characterized as a hypotrochoid for both quasi-planar and helical bending., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Institute of Mathematics and its Applications. All rights reserved.)

Background: Human multipotent adult progenitor cells (MAPC®) are an emerging therapy for traumatic brain injury (TBI); however, clinically translating a therapy involves overcoming many factors in vivo which are not present in pre-clinical testing. In this study we examined clinical parameters in vitro that may impact cell therapy efficacy., Methods: MAPC were infused through varying gauged needles and catheters with and without chlorhexidine, and their viability tested with trypan blue exclusion. MAPC were co-cultured with phenytoin and celecoxib at relevant clinical concentrations for 1 h and 24 h. Anti-inflammatory potency was tested using a stimulated rat splenocyte co-culture and ELISA for TNF-α production. MAPC were cultured under different osmolar concentrations and stained with propidium iodide for viability. Anti-inflammatory potency was tested by co-culture of MAPC with naïve lymphocytes activated by CD3/CD28 beads, and Click-iT® Plus EdU was used to quantify proliferation by flow cytometry., Results: The mean viability of the MAPC infused via needles was 95 ± 1%; no difference was seen with varying flow rate, but viability was notably reduced by chlorhexidine. MAPC function was not impaired by co-culture with phenytoin, celecoxib, or combination with both. Co-culture with phenytoin showed a decrease in TNF-α production as compared to the MAPC control. MAPC cultured at varying osmolar concentrations all had viabilities greater than 90% with no statistical difference between them. Co-culture of MAPC with CD3/CD28 activated PBMCs showed a significant reduction in proliferation as measured by EdU uptake., Discussion: Needle diameter, phenytoin, celecoxib, and a relevant range of osmolarities do not impair MAPC viability or anti-inflammatory potency in vitro ., (© 2019 The Author(s).)

Background and Objective: Most stroke patients are prescribed aspirin (ASA) to adjust blood coagulability. Marrow stromal cells (MSCs) are being tested in clinical trials for stroke patients who likely are prescribed aspirin. One of the principal mechanisms of action of MSCs and ASA is modulation of the inflammatory response, including those mediated by monocytes (Mo). Thus, here we tested if aspirin can modify anti-inflammatory properties of MSCs or Mo alone, and in combination., Methods: Mo were isolated at 24 h of stroke onset from ischemic stroke patients with NIHSS ranging from 11 to 20 or from healthy controls. Human bone marrow-derived MSCs from healthy subjects were used at passage 3. Mo, MSCs, and MSCs-Mo co-cultures were exposed to ASA at clinically relevant doses. The secretome profile of inflammatory mediators was measured using Magpix multiplex cytokine array. Viability was measured using MTT assay. Linear mixed effect model was used for statistical analysis., Results: Overall Mo from control subjects exposed to ASA showed increased secretion of IL-1RA, IL-8, MCP-1, and TNF-α and Mo from stroke patients showed greater release of IL-1RA and MCP-1. In MSCs-Mo co-cultures, ASA added to co-cultures of control Mo reduced fractalkine secretion while it increased the fractalkine secretion when added to Mo from stroke patients. In addition, in co-cultures independent of Mo origin, ASA reduced IL-6, IL-8, MCP-1, and TNF-α., Conclusions: Aspirin in acute stroke patients may modulate the secretome profile of Mo and MSCs, thus potentially modulating immune and inflammatory responses associated with stroke. Our results suggest that stroke trials involving the use of intravenous MSCs should consider the effect of aspirin as a confounding factor., (Copyright © 2019 Elsevier B.V. All rights reserved.)

For several decades, multipotent mesenchymal stromal cells (MSCs) have been extensively studied for their therapeutic potential across a wide range of diseases. In the preclinical setting, MSCs demonstrate consistent ability to promote tissue healing, down-regulate excessive inflammation and improve outcomes in animal models. Several proposed mechanisms of action have been posited and demonstrated across an array of in vitro models. However, translation into clinical practice has proven considerably more difficult. A number of prominent well-funded late-phase clinical trials have failed, thus calling out for new efforts to optimize product delivery in the clinical setting. In this review, we discuss novel topics critical to the successful translation of MSCs from pre-clinical to clinical applications. In particular, we focus on the major routes of cell delivery, aspects related to hemocompatibility, and potential safety concerns associated with MSC therapy in the different settings.

Cartilage tissue engineering is commonly initiated by seeding cells in porous materials such as hydrogels or scaffolds. Under optimal conditions, the resulting engineered construct has the potential to fill regions where native cartilage has degraded or eroded. Within a cell-seeded scaffold supplied by nutrients and growth factors, extracellular matrix accumulation should occur concurrently with scaffold degradation. At present, the interplay between cell-mediated synthesis and linking of matrix constituents and the evolving scaffold properties is not well understood. We develop a computational model of extracellular matrix accumulation in a cell-seeded scaffold based on a continuum reaction-diffusion system with inhomogeneous inclusions representing individual cells. The effects of porosity on engineered tissue outcomes is accounted for via the use of mixture variables capturing the spatiotemporal dynamics of both bound and unbound system constituents. The unbound constituents are the nutrients and unlinked extracellular matrix, while the bound constituents are the scaffold and the linked extracellular matrix. The linking model delineates binding of matrix constituents to either existing bound extracellular matrix or to scaffold. Results on a representative domain exhibit bound matrix trapping (vs spreading) around cells in scaffolds with lower (vs higher) initial porosity, similar to experimental results obtained by Erickson et al. (Osteoarthr Cartil 17:1639-1648, 2009). Significant alterations in the spatiotemporal accumulation of bound matrix are observed when, among the set of all model parameters, only the initial scaffold porosity is varied. The model presented herein proposes a methodology to investigate coupling between cell-mediated biosynthesis and linking of extracellular matrix in porous, cell-seeded scaffolds that has the potential to aid in the design of optimal tissue-engineered cartilage constructs.

Aim: Traumatic brain injury is a complex condition consisting of a mechanical injury with neurovascular disruption and inflammation with limited clinical interventions available. A growing number of studies report systemic delivery of human umbilical cord blood (HUCB) as a therapy for neural injuries. Materials & methods: HUCB cells from five donors were tested to improve blood-brain barrier integrity in a traumatic brain injury rat model at a dose of 2.5 × 10 7 cells/kg at 24 or 72 h postinjury and for immunomodulatory activity in vitro . Results & Conclusion: We observed that cells delivered 72 h postinjury significantly restored blood-brain barrier integrity. HUCB cells reduced the amount of TNF-α and IFN-γ released by activated primary rat splenocytes, which correlated with the expression of COX2 and IDO1.

Clinical cellular therapeutics (CCTs) have shown preliminary efficacy in reducing inflammation after trauma, preserving cardiac function after myocardial infarction, and improving functional recovery after stroke. However, most clinically available cell lines express tissue factor (TF) which stimulates coagulation. We sought to define the degree of procoagulant activity of CCTs as related to TF expression. CCT samples from bone marrow, adipose, amniotic fluid, umbilical cord, multi-potent adult progenitor cell donors, and bone marrow mononuclear cells were tested. TF expression and phenotype were quantified using flow cytometry. Procoagulant activity of the CCTs was measured in vitro with thromboelastography and calibrated thrombogram. Fluorescence-activated cell sorting (FACS) separated samples into high- and low-TF expressing populations to isolate the contribution of TF to coagulation. A TF neutralizing antibody was incubated with samples to demonstrate loss of procoagulant function. All CCTs tested expressed procoagulant activity that correlated with expression of tissue factor. Time to clot and thrombin formation decreased with increasing TF expression. High-TF expressing cells decreased clotting time more than low-TF expressing cells when isolated from a single donor using FACS. A TF neutralizing antibody restored clotting time to control values in some, but not all, CCT samples. CCTs demonstrate wide variability in procoagulant activity related to TF expression. Time to clot and thrombin formation decreases as TF load increases and this procoagulant effect is neutralized by a TF blocking antibody. Clinical trials using CCTs are in progress and TF expression may emerge as a safety release criterion. Stem Cells Translational Medicine 2018;7:731-739., (© 2018 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Background: Understanding the embryonic stem cell (ESC) fate decision between self-renewal and proper differentiation is important for developmental biology and regenerative medicine. Attention has focused on mechanisms involving histone modifications, alternative pre-messenger RNA splicing, and cell-cycle progression. However, their intricate interrelations and joint contributions to ESC fate decision remain unclear., Results: We analyze the transcriptomes and epigenomes of human ESC and five types of differentiated cells. We identify thousands of alternatively spliced exons and reveal their development and lineage-dependent characterizations. Several histone modifications show dynamic changes in alternatively spliced exons and three are strongly associated with 52.8% of alternative splicing events upon hESC differentiation. The histone modification-associated alternatively spliced genes predominantly function in G2/M phases and ATM/ATR-mediated DNA damage response pathway for cell differentiation, whereas other alternatively spliced genes are enriched in the G1 phase and pathways for self-renewal. These results imply a potential epigenetic mechanism by which some histone modifications contribute to ESC fate decision through the regulation of alternative splicing in specific pathways and cell-cycle genes. Supported by experimental validations and extended datasets from Roadmap/ENCODE projects, we exemplify this mechanism by a cell-cycle-related transcription factor, PBX1, which regulates the pluripotency regulatory network by binding to NANOG. We suggest that the isoform switch from PBX1a to PBX1b links H3K36me3 to hESC fate determination through the PSIP1/SRSF1 adaptor, which results in the exon skipping of PBX1., Conclusion: We reveal the mechanism by which alternative splicing links histone modifications to stem cell fate decision.

Despite intensive research and clinical trials with numerous therapeutic treatments, traumatic brain injury (TBI) is a serious public health problem in the United States. There is no effective FDA-approved treatment to reduce morbidity and mortality associated with TBI. Inflammation plays a pivotal role in the pathogenesis of TBI. We looked to re-purpose existing drugs that reduce immune activation without broad immunosuppression. Teriflunomide, an FDA-approved drug, has been shown to modulate immunological responses outside of its ability to inhibit pyrimidine synthesis in rapidly proliferating cells. In this study, we tested the efficacy of teriflunomide to treat two different injury intensities in rat models of TBI. Our results show that teriflunomide restores blood-brain barrier integrity, decreases inflammation, and increases neurogenesis in the subgranular zone of the hippocampus. While we were unable to detect neurocognitive effects of treatment on memory and special learning abilities after treatment, a 2-week treatment following injury was sufficient to reduce neuroinflammation up to 120 days later., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Traumatic brain injury (TBI) is a major cause of death and disability. TBI results in a prolonged secondary central neuro-inflammatory response. Previously, we have demonstrated that multiple doses (2 and 24 h after TBI) of multipotent adult progenitor cells (MAPC) delivered intravenously preserve the blood-brain barrier (BBB), improve spatial learning, and decrease activated microglia/macrophages in the dentate gyrus of the hippocampus. In order to determine if there is an optimum treatment window to preserve the BBB, improve cognitive behavior, and attenuate the activated microglia/macrophages, we administered MAPC at various clinically relevant intervals., Methods: We administered two injections intravenously of MAPC treatment at hours 2 and 24 (2/24), 6 and 24 (6/24), 12 and 36 (12/36), or 36 and 72 (36/72) post cortical contusion injury (CCI) at a concentration of 10 million/kg. For BBB experiments, animals that received MAPC at 2/24, 6/24, and 12/36 were euthanized 72 h post injury. The 36/72 treated group was harvested at 96 h post injury., Results: Administration of MAPC resulted in a significant decrease in BBB permeability when administered at 2/24 h after TBI only. For behavior experiments, animals were harvested post behavior paradigm. There was a significant improvement in spatial learning (120 days post injury) when compared to cortical contusion injury (CCI) in groups when MAPC was administered at or before 24 h. In addition, there was a significant decrease in activated microglia/macrophages in the dentate gyrus of hippocampus of the treated group (2/24) only when compared to CCI., Conclusions: Intravenous injections of MAPC at or before 24 h after CCI resulted in improvement of the BBB, improved cognitive behavior, and attenuated activated microglia/macrophages in the dentate gyrus.

No current clinical intervention can alter the course of acute spinal cord injury (SCI), or appreciably improve neurological outcome. Mesenchymal stromal cells (MSCs) have been shown to modulate the injury sequelae of SCI largely via paracrine effects, although the mechanisms remain incompletely understood. One potential modality is through secretion of extracellular vesicles (EVs). In this study, we investigate whether systemic administration of EVs isolated from human MSCs (MSCEv) has the potential to be efficacious as an alternative to cell-based therapy for SCI. Additionally, we investigate whether EVs isolated from human MSCs stimulated with pro-inflammatory cytokines have enhanced anti-inflammatory effects when administered after SCI. Immunohistochemistry supported the quantitative analysis, demonstrating a diminished inflammatory response with apparent astrocyte and microglia disorganization in cord tissue up to 10 mm caudal to the injury site. Locomotor recovery scores showed significant improvement among animals treated with MSCEv. Significant increases in mechanical sensitivity threshold were observed in animals treated with EVs from either naïve MSC (MSCEv wt ) or stimulated MSC (MSCEv + ), with a statistically significant increase in threshold for MSCEv + -treated animals when compared to those that received MSCEv wt . In conclusion, these data show that treatment of acute SCI with extracellular vesicles derived from human MSCs attenuates neuroinflammation and improves functional recovery.

Although it is well known that nitrofen induces congenital diaphragmatic hernia (CDH), including CDH-associated lung hypoplasia and pulmonary hypertension (PH) in rodents, the mechanism of pathogenesis remains largely unclear. It has been reported that pulmonary artery (PA) endothelial cell (EC) dysfunction contributes to the development of PH in CDH. Thus, we hypothesized that there is significant alteration of endothelial dysfunction-associated proteins in nitrofen-induced CDH PAs. Pregnant SD rats received either nitrofen or olive oil on gestational day 9.5. The newborn rats were sacrificed and divided into a CDH (n = 81) and a control (n = 23) group. After PA isolation, the expression of PA endothelial dysfunction-associated proteins was assessed on Western blot and immunostaining. We demonstrate that the expression of C-reactive protein and endothelin-1 and its receptors, ETA and ETB, were significantly increased in the CDH PAs. Levels of phosphorylated myosin light chain were significantly elevated, but those of phosphorylated endothelial nitric oxide synthase, caveolin-1, and mechanistic target of rapamycin were significantly decreased in the CDH PAs. In this work, we elucidate alterations in the expression of endothelial dysfunction-associated proteins specific to nitrofen-induced CDH rodent PAs, thereby advancing our understanding of the critical role of endothelial dysfunction-associated pathways in the pathogenesis of nitrofen-induced CDH., (© 2017 S. Karger AG, Basel.)