HIV-associated thrombotic thrombocytopenic purpura (HIV-TTP): A practical guide and review of the literature.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP), a serious thrombotic microangiopathy (TMA), is prevalent in the South African HIV-infected population. The exact pathogenesis of HIV-associated TTP (HIV-TTP) is however still unclear with diagnostic and therapeutic inconsistancies. METHODS: A systematic review of the published literature regarding HIV-TTP was performed. RESULTS: HIV-TTP is still associated with significant morbidity and mortality in Africa despite the availability of anti-retroviral therpy (ART). Diagnosis of HIV-TTP requires the presence of a micro-angiopathic haemolytic anaemia with significant red blood cell schistocytes and thrombocytopenia in the absence of another TMA but background activation of the coagulation system and inflammation in HIV infected people can result in diagnostic anbiguity. Plasma therapy in the form of infusion or exchange is successful but expensive, associated with side-effects and not widely available. Adjuvant immunosuppression therapy may of benefit in patients with HIV-TTP and ART must always be optimised. Endothelial dysfunction caused by chronic inflammation and complement activation most likely contributes to the development of HIV-TTP. CONCLUSION: The role of adjuvant immunomodulating therpy, the therapeutic targets and pathogenic contribution from endothelial dysfunction in HIV-TTP requires further investigation.

Distinguishing and overlapping laboratory results of thrombotic microangiopathies in HIV infection: Can scoring systems assist?

BACKGROUND: Patients with Human Immunodeficiency Virus (HIV) infection are at risk of thrombotic microangiopathies (TMAs) notably thrombotic thrombocytopenic purpura (TTP) and disseminated intravascular coagulation (DIC). Overlap between laboratory results exists resulting in diagnostic ambiguity. METHODS: Routine laboratory results of 71 patients with HIV-associated TTP (HIV-TTP) and 81 with DIC with concomitant HIV infection (HIV-DIC) admitted between 2015 and 2021 to academic hospitals in Johannesburg, South Africa were retrospectively reviewed. Both the PLASMIC and the International Society of Thrombosis and Haemostasis (ISTH) DIC scores were calculated. RESULTS: Patients with HIV-TTP had significantly (P < .001) increased schistocytes and features of hemolysis including elevated lactate dehydrogenase (LDH)/upper-limit-of-normal ratio (median of 9 (interquartile range [IQR] 5-12) vs 3 (IQR 2-5)) but unexpectedly lower fibrinogen (median 2.8 (IQR 2.2-3.4) vs 4 g/L (IQR 2.5-9.2)) and higher D-dimer (median 4.8 (IQR 2.4-8.1) vs 3.6 g/L (IQR 1.7-6.2)) levels vs the HIV-DIC cohort. Patients with HIV-DIC were more immunocompromised with frequent secondary infections, higher platelet and hemoglobin levels, more deranged coagulation parameters and less hemolysis. Overlap in scoring systems was however observed. CONCLUSION: The laboratory parameter overlap between HIV-DIC and HIV-TTP might reflect a shared pathogenesis including endothelial dysfunction and inflammation and further research is required. Fibrinogen in DIC may be elevated as an acute phase reactant and D-dimers may reflect the extensive hemostatic activation in HIV-TTP. Inclusion of additional parameters in TMA scoring systems such the LDH/upper-limit-of-normal ratio, schistocytes count and wider access to ADAMTS-13 testing may enhance diagnostic accuracy and ensure appropriate utilization of plasma.

Fungi populate deep-sea coral gardens as well as marine sediments in the Irish Atlantic Ocean.

Fungi populate deep Oceans in extreme habitats characterized by high hydrostatic pressure, low temperature and absence of sunlight. Marine fungi are potential major contributors to biogeochemical events, critical for marine communities and food web equilibrium under climate change conditions and a valuable source of novel extremozymes and small molecules. Despite their ecophysiological and biotechnological relevance, fungal deep-sea biodiversity has not yet been thoroughly characterized. In this study, we describe the culturable mycobiota associated with the deepest margin of the European Western Continental Shelf: sediments sampled at the Porcupine Bank and deep-water corals and sponges sampled in the Whittard Canyon. Eighty-seven strains were isolated, belonging to 43 taxa and mainly Ascomycota. Ten species and four genera were detected for the first time in the marine environment and a possible new species of Arachnomyces was isolated from sediments. The genera Cladosporium and Penicillium were the most frequent and detected on both substrates, followed by Candida and Emericellopsis. Our results showed two different fungal communities: sediment-associated taxa which were predominantly saprotrophic and animal-associated taxa which were predominantly symbiotic. This survey supports selective fungal biodiversity in the deep North Atlantic, encouraging further mycological studies on cold water coral gardens, often overexploited marine habitats.

Deep-Sea Coral Garden Invertebrates and Their Associated Fungi Are Genetic Resources for Chronic Disease Drug Discovery.

Chronic diseases characterized by bone and cartilage loss are associated with a reduced ability of progenitor cells to regenerate new tissues in an inflammatory environment. A promising strategy to treat such diseases is based on tissue repair mediated by human mesenchymal stem cells (hMSCs), but therapeutic outcomes are hindered by the absence of small molecules to efficiently modulate cell behaviour. Here, we applied a high-throughput drug screening technology to bioprospect a large library of extracts from Irish deep-sea organisms to induce hMSC differentiation toward musculoskeletal lineages and reduce inflammation of activated macrophages. The library included extracts from deep-sea corals, sponges and filamentous fungi representing a novel source of compounds for the targeted bioactivity. A validated hit rate of 3.4% was recorded from the invertebrate library, with cold water sea pens (octocoral order Pennatulacea), such as Kophobelemnon sp. and Anthoptilum sp., showing the most promising results in influencing stem cell differentiation toward osteogenic and chondrogenic lineages. Extracts obtained from deep-sea fungi showed no effects on stem cell differentiation, but a 6.8% hit rate in reducing the inflammation of activated macrophages. Our results demonstrate the potential of deep-sea organisms to synthetize pro-differentiation and immunomodulatory compounds that may represent potential drug development candidates to treat chronic musculoskeletal diseases.

Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells Are Functionally and Genetically Different From Bone Marrow-Derived Mesenchymal Stromal Cells.

There has been considerable interest in the generation of functional mesenchymal stromal cell (MSC) preparations from induced pluripotent stem cells (iPSCs) and this is now regarded as a potential source of unlimited, standardized, high-quality cells for therapeutic applications in regenerative medicine. Although iMSCs meet minimal criteria for defining MSCs in terms of marker expression, there are substantial differences in terms of trilineage potential, specifically a marked reduction in chondrogenic and adipogenic propensity in iMSCs compared with bone marrow-derived (BM) MSCs. To reveal the cellular basis underlying these differences, we conducted phenotypic, functional, and genetic comparisons between iMSCs and BM-MSCs. We found that iMSCs express very high levels of both KDR and MSX2 compared with BM-MSCs. In addition, BM-MSCs had significantly higher levels of PDGFRα. These distinct gene expression profiles were maintained during culture expansion, suggesting that prepared iMSCs are more closely related to vascular progenitor cells (VPCs). Although VPCs can differentiate along the chondrogenic, osteogenic, and adipogenic pathways, they require different inductive conditions compared with BM-MSCs. These observations suggest to us that iMSCs, based on current widely used preparation protocols, do not represent a true alternative to primary MSCs isolated from BM. Furthermore, this study highlights the fact that high levels of expression of typical MSC markers such as CD73, CD90, and CD105 are insufficient to distinguish MSCs from other mesodermal progenitors in differentiated induced pluripotent stem cell cultures. Stem Cells 2019;37:754-765.

A Novel High-Throughput Screening Platform Identifies Itaconate Derivatives from Marine Penicillium antarcticum as Inhibitors of Mesenchymal Stem Cell Differentiation.

Worldwide diffused diseases such as osteoarthritis, atherosclerosis or chronic kidney disease are associated with a tissue calcification process which may involve unexpected local stem cell differentiation. Current pharmacological treatments for such musculoskeletal conditions are weakly effective, sometimes extremely expensive and often absent. The potential to develop new therapies is represented by the discovery of small molecules modulating resident progenitor cell differentiation to prevent aberrant tissue calcification. The marine environment is a rich reserve of compounds with pharmaceutical potential and many novel molecules are isolated from macro and microorganisms annually. The potential of small molecules synthetized by marine filamentous fungi to influence the osteogenic and chondrogenic differentiation of human mesenchymal stem/stromal cells (hMSCs) was investigated using a novel, high-throughput automated screening platform. Metabolites synthetized by the marine-derived fungus Penicillium antarcticum were evaluated on the platform. Itaconic acid derivatives were identified as inhibitors of calcium elaboration into the matrix of osteogenically differentiated hMSCs and also inhibited hMSC chondrogenic differentiation, highlighting their capacity to impair ectopic calcification. Bioactive small molecule discovery is critical to address ectopic tissue calcification and the use of biologically relevant assays to identify naturally occurring metabolites from marine sources represents a strategy that can contribute to this effort.

Mesenchymal stromal cell therapy: progress in manufacturing and assessments of potency.

Mesenchymal stromal cell (MSC) therapies have been pursued for a broad spectrum of indications but mixed reports on clinical efficacy have given rise to some degree of skepticism regarding the effectiveness of this approach. However, recent reports of successful clinical outcomes and regulatory approvals for graft-versus-host disease, Crohn's disease and critical limb ischemia have prompted a shift in this perspective. With hundreds of clinical trials involving MSCs currently underway and an increasing demand for large-scale manufacturing protocols, there is a critical need to develop standards that can be applied to processing methods and to establish consensus assays for both MSC processing control and MSC product release. Reference materials and validated, uniformly applied tests for quality control of MSC products are needed. Here, we review recent developments in MSC manufacturing technologies, release testing and potency assays. We conclude that, although MSCs hold considerable promise clinically, economies of scale have yet to be achieved although numerous bioreactor technologies for scalable production of MSCs exist. Additionally, rigorous disease-specific product testing and comprehensive understanding of mechanisms of action, which are linked to relevant process and product release potency assays, will be required to ensure that these therapies continue to be successful.

Proceedings of the signature series symposium "cellular therapies for orthopaedics and musculoskeletal disease proven and unproven therapies-promise, facts and fantasy," international society for cellular therapies, montreal, canada, may 2, 2018.

The Signature Series Symposium "Cellular Therapies for Orthopaedics and Musculoskeletal Disease Proven and Unproven Therapies-Promise, Facts and Fantasy" was held as a pre-meeting of the 26th International Society for Cellular Therapy (ISCT) annual congress in Montreal, Canada, May 2, 2018. This was the first ISCT program that was entirely dedicated to the advancement of cell-based therapies for musculoskeletal diseases. Cellular therapies in musculoskeletal medicine are a source of great promise and opportunity. They are also the source of public controversy, confusion and misinformation. Patients, clinicians, scientists, industry and government share a commitment to clear communication and responsible development of the field. Therefore, this symposium convened thought leaders from around the world in a forum designed to catalyze communication and collaboration to bring the greatest possible innovation and value to patients with musculoskeletal conditions.

Syndecan-2-positive, Bone Marrow-derived Human Mesenchymal Stromal Cells Attenuate Bacterial-induced Acute Lung Injury and Enhance Resolution of Ventilator-induced Lung Injury in Rats.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Human mesenchymal stromal cells demonstrate promise for acute respiratory distress syndrome, but current studies use highly heterogenous cell populations. We hypothesized that a syndecan 2 (CD362)-expressing human mesenchymal stromal cell subpopulation would attenuate Escherichia coli-induced lung injury and enhance resolution after ventilator-induced lung injury. METHODS: In vitro studies determined whether CD362 human mesenchymal stromal cells could modulate pulmonary epithelial inflammation, wound healing, and macrophage phagocytosis. Two in vivo rodent studies determined whether CD362 human mesenchymal stromal cells attenuated Escherichia coli-induced lung injury (n = 10/group) and enhanced resolution of ventilation-induced injury (n = 10/group). RESULTS: CD362 human mesenchymal stromal cells attenuated cytokine-induced epithelial nuclear factor kappa B activation, increased epithelial wound closure, and increased macrophage phagocytosis in vitro. CD362 human mesenchymal stromal cells attenuated Escherichia coli-induced injury in rodents, improving arterial oxygenation (mean ± SD, 83 ± 9 vs. 60 ± 8 mmHg, P < 0.05), improving lung compliance (mean ± SD: 0.66 ± 0.08 vs. 0.53 ± 0.09 ml · cm H2O, P < 0.05), reducing bacterial load (median [interquartile range], 1,895 [100-3,300] vs. 8,195 [4,260-8,690] colony-forming units, P < 0.05), and decreasing structural injury compared with vehicle. CD362 human mesenchymal stromal cells were more effective than CD362 human mesenchymal stromal cells and comparable to heterogenous human mesenchymal stromal cells. CD362 human mesenchymal stromal cells enhanced resolution after ventilator-induced lung injury in rodents, restoring arterial oxygenation (mean ± SD: 113 ± 11 vs. 89 ± 11 mmHg, P < 0.05) and lung static compliance (mean ± SD: 0.74 ± 0.07 vs. 0.45 ± 0.07 ml · cm H2O, P < 0.05), resolving lung inflammation, and restoring histologic structure compared with vehicle. CD362 human mesenchymal stromal cells efficacy was at least comparable to heterogenous human mesenchymal stromal cells. CONCLUSIONS: A CD362 human mesenchymal stromal cell population decreased Escherichia coli-induced pneumonia severity and enhanced recovery after ventilator-induced lung injury.

Combination of acquired von Willebrand syndrome (AVWS) and Glanzmann thrombasthenia in monoclonal gammopathy of uncertain significance (MGUS), a case report.

BACKGROUND: Autoimmune paraphenomena, are associated with B-cell lymphoproliferative disorders, including monoclonal gammopathy of uncertain significance. These paraphenomena can rarely include acquired bleeding disorders. CASE PRESENTATION: This case study reports an unusual clinical presentation of 2 acquired bleeding disorders, Acquired von Willebrand syndrome (disease) and Acquired Glanzmann's thrombasthenia, in an elderly patient with monoclonal gammopathy of uncertain significance. CONCLUSIONS: Acquired bleeding disorders are often underdiagnosed and a high degree of clinical suspicion is required. The patient in this study demonstrated platelet aggregometry which was atypical for isolated Glanzmann's thrombosthenia because of the severe concomitant endogenous decrease in von Willebrand factor. There was an absence of platelet aggregation to all tested agonists including ristocetin. Once the diagnosis was made, however, the patient showed a partial response to intravenous immunoglobulin confirming the immunological pathogenesis in this case. This case highlights the need to consider acquired bleeding disorders in patients with a possible predisposing factor.

Differentiation of Vascular Stem Cells Contributes to Ectopic Calcification of Atherosclerotic Plaque.

The cellular and molecular basis of vascular calcification (VC) in atherosclerosis is not fully understood. Here, we investigate role of resident/circulating progenitor cells in VC and contribution of inflammatory plaque environment to this process. Vessel-derived stem/progenitor cells (VSCs) and mesenchymal stem cells (MSCs) isolated from atherosclerotic ApoE(-/-) mice showed significantly more in vitro osteogenesis and chondrogenesis than cells generated from control C57BL/6 mice. To assess their ability to form bone in vivo, cells were primed chondrogenically or cultured in control medium on collagen glycosaminoglycan scaffolds in vitro prior to subcutaneous implantation in ApoE(-/-) and C57BL/6 mice using a crossover study design. Atherosclerotic ApoE(-/-) MSCs and VSCs formed bone when implanted in C57BL/6 mice. In ApoE(-/-) mice, these cells generated more mature bone than C57BL/6 cells. The atherosclerotic in vivo environment alone promoted bone formation by implanted C57BL/6 cells. Un-primed C57BL/6 VSCs were unable to form bone in either mouse strain. Treatment of ApoE(-/-) VSC chondrogenic cultures with interleukin (IL)-6 resulted in significantly increased glycosaminoglycan deposition and expression of characteristic chondrogenic genes at 21 days. In conclusion, resident vascular cells from atherosclerotic environment respond to the inflammatory milieu and undergo calcification. IL-6 may have a role in aberrant differentiation of VSCs contributing to vascular calcification in atherosclerosis.

Bone Marrow-Derived Mesenchymal Stem Cells Have Innate Procoagulant Activity and Cause Microvascular Obstruction Following Intracoronary Delivery: Amelioration by Antithrombin Therapy.

Mesenchymal stem cells (MSCs) are currently under investigation as tools to preserve cardiac structure and function following acute myocardial infarction (AMI). However, concerns have emerged regarding safety of acute intracoronary (IC) MSC delivery. This study aimed to characterize innate prothrombotic activity of MSC and identify means of its mitigation toward safe and efficacious therapeutic IC MSC delivery post-AMI. Expression of the initiator of the coagulation cascade tissue factor (TF) on MSC was detected and quantified by immunofluorescence, FACS, and immunoblotting. MSC-derived TF antigen was catalytically active and capable of supporting thrombin generation in vitro. Addition of MSCs to whole citrated blood enhanced platelet thrombus deposition on collagen at arterial shear, an effect abolished by heparin coadministration. In a porcine AMI model, IC infusion of 25 × 10(6) MSC during reperfusion was associated with a decrease in coronary flow reserve but not when coadministered with an antithrombin agent (heparin). Heparin reduced MSC-associated thrombosis incorporating platelets and VWF within the microvasculature. Heparin-assisted therapeutic MSC delivery also reduced apoptosis in the infarct border zone at 24 hours, significantly improved infarct size, left ventricular (LV) ejection fraction, LV volumes, wall motion, and attenuated histologic evidence of scar formation at 6 weeks post-AMI. Heparin alone or heparin-assisted fibroblast control cell delivery had no such effect. Procoagulant TF activity of therapeutic MSCs is associated with reductions in myocardial perfusion when delivered IC may be successfully managed by heparin coadministration. This study highlights an important mechanistic insight into safety concerns associated with therapeutic IC MSC delivery for AMI.

Human mesenchymal stromal cells decrease the severity of acute lung injury induced by E. coli in the rat.

BACKGROUND: Mesenchymal stromal cells (MSCs) demonstrate considerable promise in preclinical acute respiratory distress syndrome models. We wished to determine the efficacy and mechanisms of action of human MSCs (hMSCs) in the setting of acute lung injury induced by prolonged Escherichia coli pneumonia in the rat. METHODS: Adult male Sprague Dawley rats underwent intratracheal instillation of E. coli bacteria in all experiments. In Series 1, animals were randomised to intravenous administration of: (1) vehicle (phosphate buffered saline (PBS), 300 µL); (2) 1×10(7) fibroblasts/kg; (3) 1×10(7) hMSCs/kg or (4) 2×10(7) hMSCs/kg. Series 2 determined the lowest effective hMSC dose. Series 3 compared the efficacy of intratracheal versus intravenous hMSC administration, while Series 4 examined the efficacy of cryopreserved hMSC. Series 5 examined the efficacy of the hMSC secretome. Parallel in vitro experiments further assessed the potential for hMSCs to secrete LL-37 and modulate macrophage phagocytosis. RESULTS: hMSC therapy reduced the severity of rodent E. coli pneumonia, improving survival, decreasing lung injury, reducing lung bacterial load and suppressing inflammation. Doses as low as 5×10(6) hMSCs/kg were effective. Intratracheal hMSC therapy was as effective as intravenous hMSC. Cryopreserved hMSCs were also effective, while the hMSC secretome was less effective in this model. hMSC therapy enhanced macrophage phagocytic capacity and increased lung and systemic concentrations of the antimicrobial peptide LL37. CONCLUSIONS: hMSC therapy decreased E. coli induced pneumonia injury and reduced lung bacterial burden, potentially via enhanced macrophage phagocytosis and increased alveolar LL-37 concentrations.

Therapeutic efficacy of human mesenchymal stromal cells in the repair of established ventilator-induced lung injury in the rat.

BACKGROUND: Rodent mesenchymal stem/stromal cells (MSCs) enhance repair after ventilator-induced lung injury (VILI). We wished to determine the therapeutic potential of human MSCs (hMSCs) in repairing the rodent lung. METHODS: In series 1, anesthetized rats underwent VILI (series 1A, n = 8 to 9 per group) or protective ventilation (series 1B, n = 4 per group). After VILI, they were randomized to intravenous administration of (1) vehicle (phosphate-buffered saline); (2) fibroblasts (1 × 10 cells/kg); or (3) human MSCs (1 × 10 cells/kg) and the effect on restoration of lung function and structure assessed. In series 2, the efficacy of hMSC doses of 1, 2, 5, and 10 million/kg was examined (n = 8 per group). Series 3 compared the efficacy of both intratracheal and intraperitoneal hMSC administration to intravascular delivery (n = 5-10 per group). Series 4 examined the efficacy of delayed hMSC administration (n = 8 per group). RESULTS: Human MSC's enhanced lung repair, restoring oxygenation (131 ± 19 vs. 103 ± 11 vs. 95 ± 11 mmHg, P = 0.004) compared to vehicle or fibroblast therapy, respectively. hMSCs improved lung compliance, reducing alveolar edema, and restoring lung architecture. hMSCs attenuated lung inflammation, decreasing alveolar cellular infiltration, and decreasing cytokine-induced neutrophil chemoattractant-1 and interleukin-6 while increasing keratinocyte growth factor concentrations. The lowest effective hMSC dose was 2 × 10 hMSC/kg. Intraperitoneal hMSC delivery was less effective than intratracheal or intravenous hMSC. hMSCs enhanced lung repair when administered at later time points after VILI. CONCLUSIONS: hMSC therapy demonstrates therapeutic potential in enhancing recovery after VILI.

Thermoresponsive Substrates Used for the Growth and Controlled Differentiation of Human Mesenchymal Stem Cells.

This communication outlines the advances made in the development of thermoresponsive substrates for human mesenchymal stem cell (hMSC) expansion and subsequent controlled specific and multilineage differentiation from a previous study performed by this group. Previously, the development of an inexpensive and technically accessible method for hMSC expansion and harvesting was reported, using the solvent casting deposition method and thermoresponsive poly(N-isopropylacrylamide). Here, the logical continuation of this work is reported with the multipassage expansion of hMSCs with phenotypic maintenance followed by induced adipogenic, osteogenic, and chondrogenic differentiation. Interestingly, 1 µm thick solvent cast films are not only capable of hosting an expanding population of phenotypically preserved hMSCs similar to tissue culture plastic controls, but also the cells detached via temperature control better maintain their ability to differentiate compared to conventionally trypsinized cells. This approach to hMSC expansion and differentiation can be highly attractive to stem cell researchers where clinical therapies have seen a collective deviation away from the employment of animal derived products such as proteolytic trypsin.

Chondrogenic differentiation increases antidonor immune response to allogeneic mesenchymal stem cell transplantation.

Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3(+) and CD68(+) infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use.

Impact of mesenchymal stem cell secreted PAI-1 on colon cancer cell migration and proliferation.

Mesenchymal Stem Cells are known to engraft and integrate into the architecture of colorectal tumours, with little known regarding their fate following engraftment. This study aimed to investigate mediators of Mesenchymal Stem Cell (MSC) and colon cancer cell (CCC) interactions. Mesenchymal Stem Cells and colon cancer cells (HT29 and HCT-116) were cultured individually or in co-culture on 3-dimensional scaffolds. Conditioned media containing all secreted factors was harvested at day 1, 3 and 7. Chemokine secretion and expression were analyzed by Chemi-array, ELISA (Macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor type 1 (PAI-1)) and RQ-PCR. Colon cancer cell migration and proliferation in response to recombinant PAI-1, MSCs and MSCs+antibody to PAI-1 was analyzed using Transwell inserts and an MTS proliferation assay respectively. Chemi-array revealed secretion of a wide range of factors by each cell population, including PAI-1 and MIF. ELISA analysis revealed Mesenchymal Stem Cells to secrete the highest levels of PAI-1 (MSC mean 10.6 ng/mL, CCC mean 1.01 ng/mL), while colon cancer cells were the principal source of MIF. MSC-secreted PAI-1 stimulated significant migration of both CCC lines, with an antibody to the chemokine shown to block this effect (67-88% blocking,). A cell-line dependant effect on CCC proliferation was shown for Mesenchymal Stem Cell-secreted PAI-1 with HCT-116 cells showing decreased proliferation at all concentrations, and HT29 cells showing increased proliferation in the presence of higher PAI-1 levels. This is the first study to identify PAI-1 as an important mediator of Mesenchymal Stem Cell/colon cancer cell interactions and highlights the significant functional impact of Mesenchymal Stem Cell-secreted PAI-1 on colon cancer cells.

Effects of intratracheal mesenchymal stromal cell therapy during recovery and resolution after ventilator-induced lung injury.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been demonstrated to attenuate acute lung injury when delivered by intravenous or intratracheal routes. The authors aimed to determine the efficacy of and mechanism of action of intratracheal MSC therapy and to compare their efficacy in enhancing lung repair after ventilation-induced lung injury with intravenous MSC therapy. METHODS: : After induction of anesthesia, rats were orotracheally intubated and subjected to ventilation-induced lung injury (respiratory rate 18(-1) min, P insp 35 cm H2O,) to produce severe lung injury. After recovery, animals were randomized to receive: (1) no therapy, n = 4; (2) intratracheal vehicle (phosphate-buffered saline, 300 µl, n = 8); (3) intratracheal fibroblasts (4 × 10 cells, n = 8); (4) intratracheal MSCs (4 × 10(6) cells, n = 8); (5) intratracheal conditioned medium (300 µl, n = 8); or (6) intravenous MSCs (4 × 10(6) cells, n = 4). The extent of recovery after acute lung injury and the inflammatory response was assessed after 48 h. RESULTS: Intratracheal MSC therapy enhanced repair after ventilation-induced lung injury, improving arterial oxygenation (mean ± SD, 146 ± 3.9 vs. 110.8 ± 21.5 mmHg), restoring lung compliance (1.04 ± 0.11 vs. 0.83 ± 0.06 ml · cm H2O(-1)), reducing total lung water, and decreasing lung inflammation and histologic injury compared with control. Intratracheal MSC therapy attenuated alveolar tumor necrosis factor-α (130 ± 43 vs. 488 ± 211 pg · ml(-1)) and interleukin-6 concentrations (138 ± 18 vs. 260 ± 82 pg · ml(-1)). The efficacy of intratracheal MSCs was comparable with intravenous MSC therapy. Intratracheal MSCs seemed to act via a paracine mechanism, with conditioned MSC medium also enhancing lung repair after injury. CONCLUSIONS: Intratracheal MSC therapy enhanced recovery after ventilation-induced lung injury via a paracrine mechanism, and was as effective as intravenous MSC therapy.

Induced pluripotent stem cells in companion animals: how can we move the field forward?

Following a one medicine approach, the development of regenerative therapies for human patients leads to innovative treatments for animals, while pre-clinical studies on animals provide knowledge to advance human medicine. Among many different biological products under investigation, stem cells are among the most prominent. Mesenchymal stromal cells (MSCs) are extensively investigated, but they present challenges such as senescence and limited differentiation ability. Embryonic stem cells (ESCs) are pluripotent cells with a virtually unlimited capacity for self-renewal and differentiation, but the use of embryos carries ethical concerns. Induced pluripotent stem cells (iPSCs) can overcome all of these limitations, as they closely resemble ESCs but are derived from adult cells by reprogramming in the laboratory using pluripotency-associated transcription factors. iPSCs hold great potential for applications in therapy, disease modeling, drug screening, and even species preservation strategies. However, iPSC technology is less developed in veterinary species compared to human. This review attempts to address the specific challenges associated with generating and applying iPSCs from companion animals. Firstly, we discuss strategies for the preparation of iPSCs in veterinary species and secondly, we address the potential for different applications of iPSCs in companion animals. Our aim is to provide an overview on the state of the art of iPSCs in companion animals, focusing on equine, canine, and feline species, as well as to identify which aspects need further optimization and, where possible, to provide guidance on future advancements. Following a "step-by-step" approach, we cover the generation of iPSCs in companion animals from the selection of somatic cells and the reprogramming strategies, to the expansion and characterization of iPSCs. Subsequently, we revise the current applications of iPSCs in companion animals, identify the main hurdles, and propose future paths to move the field forward. Transferring the knowledge gained from human iPSCs can increase our understanding in the biology of pluripotent cells in animals, but it is critical to further investigate the differences among species to develop specific approaches for animal iPSCs. This is key for significantly advancing iPSC application in veterinary medicine, which at the same time will also allow gaining pre-clinical knowledge transferable to human medicine.

Media matters: culture medium-dependent hypervariable phenotype of mesenchymal stromal cells.

BACKGROUND: Despite a long history of investigation and sustained efforts in clinical testing, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration. Several barriers are impeding the clinical progression of MSC therapies, to the forefront of these is a lack of standardised manufacturing protocols which is further compounded by an absence of biologically meaningful characterisation and release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, among other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action. METHODS: This work analysed the influence of five widely used culture media formulations on the colony-forming potential, proliferation kinetics, trilineage differentiation potential and immunomodulatory potential of human bone marrow-derived MSCs (BM-MSCs). The surface marker expression profiles were also characterised using a high-content flow cytometry screening panel of 243 markers. RESULTS: Significant differences in the biological attributes of BM-MSCs including clonogenicity, proliferation, differentiation propensity and immunomodulatory capacity were revealed in response to the composition of the culture medium. Despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs: CD73, CD90 and CD105. Immunophenotypic profiling revealed that the culture medium also had a significant influence on the surface proteome, with one-third of tested markers exhibiting variable expression profiles. Principal component analysis demonstrated that BM-MSCs isolated and expanded in a proprietary xeno- and serum-free medium displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate and foetal bovine serum-containing media. CONCLUSIONS: These data suggest that media composition has a highly significant impact on the biological attributes of MSCs, but standard surface marker tests conceal these differences. The results indicate a need for (1) standardised approaches to manufacturing, with an essential focus on defined media and (2) new biologically relevant tests for MSC characterisation and product release.