Expansion and angiogenic potential of mesenchymal stem cells from patients with critical limb ischemia.

BACKGROUND: Critical limb ischemia (CLI) is a life- and limb-threatening condition affecting 1% to 10% of the population with peripheral arterial disease. Traditional revascularization options are not possible for up to 50% of CLI patients, in which case, the use of cellular therapies, such as bone marrow-derived mesenchymal stem cells (MSCs), hold great promise as an alternative revascularization therapy. However, no randomized, controlled phase 3 trials to date have demonstrated an improvement in limb salvage with cellular therapies. This may be due to poor cell quality (ie, inability to generate a sufficient number of angiogenic MSCs) or to the inadequate retention and viability of MSCs after delivery, or both. Because concerns remain about the expansion and angiogenic potential of autologous MSCs in the CLI population, the objective of this study was to examine the effect of our novel culture media supplement, pooled human platelet lysate (PL), in lieu of the standard fetal bovine serum (FBS), to improve the expansion potential of MSCs from CLI patients. We also characterized the in vitro angiogenic activity of MSCs from the tibia of amputated CLI limbs compared with MSCs from healthy donors. METHODS: MSCs were obtained from the tibia of four CLI patients (ISC) and four ISC patients with diabetes mellitus (ISC+DM) undergoing major amputation. Healthy MSCs were aspirated from the iliac crest of four young and healthy donors. MSCs were isolated and expanded in culture with PL or FBS. MSCs from passage 3 to 6 were used for phenotypic marker expression and for adipogenic and osteogenic differentiation and were tested for their in vitro angiogenic activity on human microdermal endothelial cells. In parallel MSCs were cultured to passage 11 for population-doubling calculations. RESULTS: MSCs from ISC and ISC+DM patients and from healthy patients exhibited appropriate expression of cell surface markers and differentiation capacity. Population doublings were significantly greater for PL-stimulated compared with FBS-stimulated MSCs in all groups. Biologically active amounts of angiogens were identified in the secretome of all MSCs without consistent trends among groups. PL expansion did not adversely affect the angiogenic activity of MSCs compared with FBS. The ISC and ISC+DM MSCs demonstrated angiogenic effects on endothelial cells similar to those of healthy and ISC MSCs. CONCLUSIONS: PL promotes the rapid expansion of MSCs from CLI and healthy persons. Importantly, MSCs expanded from CLI patients demonstrate the desired angiogenic activity compared with their healthy counterparts. We conclude that autologous MSCs from CLI patients can be sufficiently expanded with PL and be expected to deliver requisite angiogenic effects in vivo. We expect the improved expansion of ISC and ISC+DM with PL to be helpful in improving the successful delivery of autologous MSCs to patients with CLI.

Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing.

We have previously demonstrated that cryopreservation and thawing lead to altered Mesenchymal stromal cells (MSC) functionalities. Here, we further analyzed MSC's fitness post freeze-thaw. We have observed that thawed MSC can suppress T-cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T-cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous Peripheral Blood Mononuclear Cells (PBMCs) and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO-free cryoprotectants or substitution of Human Serum Albumin (HSA) with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ prelicensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1-methyl Tryptophan, an Indoleamine 2,3-dioxygenase (IDO) inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw-induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T-cell cytolysis and affect MSC's cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use. Stem Cells 2016;34:2429-2442.

Platelet lysate obtained via plateletpheresis performed in standing and awake equine donors.

BACKGROUND: Platelet preparations containing growth factors, attachment factors, and enzymes are appealing to enhance healing of injured tissues and as an alternative to xenogenic serum in cell culture media. Plateletpheresis is commonly used to collect platelets in human medicine but has not been validated in horses. STUDY DESIGN AND METHODS: Plateletpheresis to collect platelet concentrate was performed on six female, mixed breed, chemically restrained horses using commercially available apheresis equipment. Before and immediately after plateletpheresis, we performed physical examinations and collected blood for chemistry and coagulation panels and then again at 8, 16, 24, and 48 hours after the procedure. To produce platelet lysate, the platelet concentrate underwent two freeze-thaw cycles followed by centrifugation and filtration processing. The platelet lysate was then analyzed for cellular debris, fibrinogen, and growth factors. RESULTS: The collected platelet concentration contained a mean platelet yield of 390 × 103 /µL. Donor platelet count decreased from a mean of 193 × 103 /µL to 138 × 103 /µL after plateletpheresis, but no individual was at risk for hemorrhage. Pooled platelet lysate had minimal cellular residue and contained growth factor concentrations at 6.1 ng/mL for transforming growth factor-ß1, at 3.5 ng/mL for platelet-derived growth factor-BB, and at 13.8 ng/mL for vascular endothelial growth factor-A. CONCLUSION: Plateletpheresis using commercially available apheresis equipment is a feasible option for collecting platelet concentrate from equine donors. The lysate generated from the apheresis product contains growth factors and has potential to be used as a fetal bovine serum substitute for cell culture.

IDO-independent suppression of T cell effector function by IFN-γ-licensed human mesenchymal stromal cells.

Human bone marrow-derived mesenchymal stromal cells (MSCs) inhibit proliferation of activated T cells, and IFN-γ plays an important role in this process. This IFN-γ-licensed veto property is IDO-dependent. To further decipher the mechanistic underpinnings of MSC veto function on T cells, we investigated the effect of MSCs and IFN-γ-licensed MSCs on T cell effector function as assayed by cytokine secretion of T cells. Although MSCs and IFN-γ-licensed MSCs inhibit T cell proliferation, only IFN-γ-licensed MSCs significantly inhibit Th1 cytokine (IFN-γ, TNF-α, and IL-2) production by T cells. Additionally, IFN-γ-licensed MSCs inhibit T cell degranulation as well as single, double, and triple cytokine-producing T cells. Although IFN-γ-licensed MSCs upregulate their IDO activity, we found that MSC IDO catalytic function is dispensable with regard to MSC-driven inhibition of T cell effector function. Novel flow cytometry based functional screening of MSC-expressed, IFN-γ-licensed inhibitory molecules identified B7H1 and B7DC/PD1 pathways as essential effectors in blocking T cell function. Small interfering RNA-mediated blocking of B7H1 and B7DC reverses the inhibitory potential of IFN-γ-licensed MSCs on T cell effector function. Mechanistic analysis revealed that clustering of MHC and coinhibitory molecules are indispensable for the inhibitory effect of IFN-γ MSCs. Although exogenous IL-2 reverses B7H1-Ig-mediated inhibition of T cell proliferation, it does not affect the veto function of IFN-γ MSCs on both T cell proliferation and effector function. Our results reveal a new immunosuppressive property of IFN-γ-licensed MSCs that inhibits T cell effector function independent of IDO but through the ligands for PD1.

Mesenchymal Stromal Cells Derived From Crohn's Patients Deploy Indoleamine 2,3-dioxygenase-mediated Immune Suppression, Independent of Autophagy.

Autologous bone marrow-derived mesenchymal stromal cells (MSCs) for adoptive cell therapy of luminal Crohn's disease (CD) are being tested in clinical trials. However, CD is associated with dysregulation of autophagy and its effect on MSC's immunobiology is unknown. Here, we demonstrate no quantitative difference in phenotype, in vitro growth kinetics and molecular signatures to IFNγ between MSCs derived from CD and healthy individuals. CD MSCs were indistinguishable from those derived from healthy controls at inhibiting T-cell proliferation through an indoleamine 2,3-dioxygenase (IDO)-dependent mechanism. Upon IFNγ prelicensing, both MSC populations inhibit T-cell effector functions. Neither a single-nucleotide polymorphism (SNP) rs7820268 in the IDO gene, nor a widely reported CD predisposing SNP ATG16L1rs2241880 modulated the suppressive function of MSCs carrying these haplotypes. IFNγ stimulation or coculture with activated T cells upregulated the expression of autophagy genes and/or vacuoles on MSCs. Pharmacological blockade of autophagy pathway did not reverse the immunosuppressive properties and IFNγ responsiveness of MSCs confirming the absence of a functional link between these two cell biochemical properties. We conclude that autophagy, but not IDO and IFNγ responsiveness, is dispensable for MSC's immunosuppressive properties. MSCs from CD subjects are functionally analogous to those of healthy individuals.

Bone Marrow Mesenchymal Stromal Cells from Patients with Acute and Chronic Graft-versus-Host Disease Deploy Normal Phenotype, Differentiation Plasticity, and Immune-Suppressive Activity.

The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is often limited by the development of acute and/or chronic graft-versus-host disease (GVHD). The lack of effective therapies to treat steroid-refractory GVHD patients has bolstered clinical evaluation of mesenchymal stromal cell (MSC) therapy for GVHD. Currently, testing of MSCs for the treatment of GVHD has exclusively used allogeneic MSCs despite emerging evidence that MSCs lose their immunoprivileged status in vivo. We hypothesized that autologous MSCs could be a viable alternative MSC source for treating active GVHD. MSCs were isolated and successfully expanded from the bone marrow of 12 volunteers (ages 2 to 55 years) who had allo-HSCT transplants and subsequently developed GVHD. MSCs from subjects with GVHD demonstrated an initial lag in growth compared with healthy control subjects; however, this lag disappeared with continued ex vivo expansion. Immunophenotype and mesodermal differentiation capacity of MSCs from GVHD patients were indistinguishable from that of healthy control MSCs. In vitro immunomodulatory functional analyses also demonstrated that GVHD MSCs were equivalent to healthy control MSCs with regards to dose dependently suppressing T cell proliferation and up-regulating indoleamine 2,3-dioxygenase expression when primed with IFN-γ. Single tandem repeat chimerism analyses further demonstrated that MSCs expanded from GVHD patients were exclusively recipient derived. Based on these data, we conclude that recipient-derived MSCs from patients with GVHD are analogous to MSCs from healthy volunteers and represent a viable option for clinical testing as an immunomodulatory option for symptomatic GVHD.

Human mesenchymal stromal cells suppress T-cell proliferation independent of heme oxygenase-1.

Mesenchymal stromal cells deploy immune suppressive properties amenable for use as cell therapy for inflammatory disorders. It is now recognized that mesenchymal stromal cells necessitate priming with an inflammatory milieu, in particular interferon-γ, to exert augmented immunosuppressive effects. It has been recently suggested that the heme-catabolizing enzyme heme oxygenase-1 is an essential component of the mesenchymal stromal cell-driven immune suppressive response. Because mesenchymal stromal cells upregulate indoleamine 2,3-dioxygenase expression on interferon-γ priming and indoleamine 2,3-dioxygenase requires heme as a cofactor for optimal catabolic function, we investigated the potential antagonism of heme oxygenase-1 activity on indoleamine 2, 3-dioxygenase and the impact on mesenchymal stromal cell immune plasticity. We herein sought to evaluate the molecular genetic effect of cytokine priming on human mesenchymal stromal cell heme oxygenase-1 expression and its functional role in differentially primed mesenchymal stromal cells. Contrary to previous reports, messenger RNA and protein analyses demonstrated that mesenchymal stromal cells derived from normal subjects (n = 6) do not express heme oxygenase-1 at steady state or after interferon-γ, tumor necrosis factor-α, and/or transforming growth factor-ß priming. Pharmacological inhibition of heme oxygenase-1 with the use of tin protoporphyrin did not significantly abrogate the ability of mesenchymal stromal cells to suppress T-cell proliferation in vitro. Overall, these results unequivocally demonstrate that under steady state and after cytokine priming, human mesenchymal stromal cells immunoregulate T-cell proliferation independent of heme oxygenase-1.

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro.

BACKGROUND AIMS: Multipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown. METHODS: MSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous ß-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to "rescue" the proliferative capacity of MSCs. RESULTS: hPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less ß-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS. CONCLUSIONS: hPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity.

Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing.

Human mesenchymal stromal cells (MSC) can suppress T-cell activation in vitro in an indoleamine 2,3-dioxygenase (IDO)-dependent manner. However, their clinical effects on immune ailments have been inconsistent, with a recent phase III study showing no benefit in acute graft-versus-host disease (GvHD). We here tested the hypothesis that the banked, cryopreserved MSC often used in clinical trials display biologic properties distinct from that of MSC in the log phase of growth typically examined in pre-clinical studies. In freshly thawed cryopreserved MSC derived from normal human volunteers, we observed that MSC up-regulate heat-shock proteins, are refractory to interferon (IFN)-γ-induced up-regulation of IDO, and are compromised in suppressing CD3/CD28-driven T cell proliferation. Immune suppressor activity, IFN-γ responsiveness and induction of IDO were fully restored following 24 h of MSC tissue culture post-thaw. These results highlight a possible cause for the inefficacy of MSC-based immunotherapy reported in clinical trials using cryopreserved MSC thawed immediately prior to infusion.

Cyclooxygenase inhibition in ventilator-induced lung injury.

BACKGROUND: We tested the hypothesis that inhibition of cyclooxygenase (COX) attenuates in vivo ventilator-induced lung injury (VILI) in a prospective, randomized laboratory investigation in a university-affiliated laboratory. Adult male rats were anesthetized and randomized with or without nonselective COX inhibition (ibuprofen) and were subjected to injurious mechanical ventilation (positive end-expiratory pressure = 0; peak inspiratory pressure = 21 mm Hg). METHODS: We investigated the profile of VILI (respiratory mechanics, cytokines, eicosanoids), expression of COX enzymes, and activation of nuclear factor (NF)-κB in ibuprofen- versus vehicle-treated animals. Injurious ventilation caused lung injury (i.e., decrement in compliance, tissue edema, and elevated inflammatory cytokines, eicosanoids, and COX-2). RESULTS: Pretreatment with ibuprofen that effectively inhibited eicosanoid synthesis and COX-2 activity increased survival and attenuated lung edema and decrement in respiratory mechanics. Ibuprofen had no modulatory effect on ventilator-induced activation of NF-κB or inflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1ß, IL-6, GRO/KC [growth-related oncogene/keratinocyte chemoattractant]). COX activity seems important in the pathogenesis of VILI in the in vivo rat. Inhibition of COX provides significant protection (i.e., survival, pulmonary function) in VILI, but without affecting levels of important mediators (tumor necrosis factor-α, IL-1ß, IL-6, GRO/KC) or activation of NF-κB. CONCLUSIONS: These data confirm that nonselective COX inhibition provides partial protection against VILI and that the NF-κB signaling pathway is not exclusively eicosanoid dependent. Studies of COX inhibition in ventilator-associated lung injury might benefit from multimodal targeting that includes a comprehensive focus on inflammatory cytokines and NF-κB.

Evidence for transcriptional regulation of the glucose-6-phosphate transporter by HIF-1alpha: Targeting G6PT with mumbaistatin analogs in hypoxic mesenchymal stromal cells.

Mesenchymal stromal cell (MSC) markers are expressed on brain tumor-initiating cells involved in the development of hypoxic glioblastoma. Given that MSCs can survive hypoxia and that the glucose-6-phosphate transporter (G6PT) provides metabolic control that contributes to MSC mobilization and survival, we investigated the effects of low oxygen (1.2% O(2)) exposure on G6PT gene expression. We found that MSCs significantly expressed G6PT and the glucose-6-phosphatase catalytic subunit beta, whereas expression of the glucose-6-phosphatase catalytic subunit alpha and the islet-specific glucose-6-phosphatase catalytic subunit-related protein was low to undetectable. Analysis of the G6PT promoter sequence revealed potential binding sites for hypoxia inducible factor (HIF)-1alpha and for the aryl hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator (ARNT), AhR:ARNT. In agreement with this, hypoxia and the hypoxia mimetic cobalt chloride induced the expression of G6PT, vascular endothelial growth factor (VEGF), and HIF-1alpha. Gene silencing of HIF-1alpha prevented G6PT and VEGF induction in hypoxic MSCs whereas generation of cells stably expressing HIF-1alpha resulted in increased endogenous G6PT gene expression. A semisynthetic analog of the polyketide mumbaistatin, a potent G6PT inhibitor, specifically reduced MSC-HIF-1alpha cell survival. Collectively, our data suggest that G6PT may account for the metabolic flexibility that enables MSCs to survive under conditions characterized by hypoxia and could be specifically targeted within developing tumors.

Improved autograft survival of mesenchymal stromal cells by plasminogen activator inhibitor 1 inhibition.

Mesenchymal stromal cells (MSCs) display robust reparative properties through their ability to limit apoptosis, enhance angiogenesis, and direct positive tissue remodeling. However, low in vivo survival of transplanted cells limits their overall effectiveness and significantly affects their clinical usage. Consequently, identifying strategies to improve cell survival in vivo are a priority. One explanation for their low survival is that MSCs are often transplanted into ischemic tissue, such as infarcted myocardium, where there is poor blood supply and low oxygen tension. Therefore, we examined how MSCs respond to a hypoxic, nutrient-poor stress environment to identify trophic factors that could be manipulated in advance of MSC transplantation. Combining microarray and proteomic screens we identified plasminogen activator inhibitor 1 (PAI-1) as one factor consistently upregulated in our in vitro ischemia-mimicking conditions. Subsequent genetic and chemical manipulation studies define PAI-1 as a negative regulator of MSC survival in vivo. Mechanistically, MSC-derived PAI-1 does not alter MSC survival through a plasmin-dependent mechanism but rather directly impacts on the adhesiveness of MSCs to their surrounding matrices. Thus we can conclude that post-transplantation, PAI-1 negatively impacts MSC survival by promoting anoikis via matrix detachment.

CD34 expression on murine marrow-derived mesenchymal stromal cells: impact on neovascularization.

OBJECTIVE: CD34 is a sialomucin often expressed by cells with hemangiopoietic potential and widely serves as a surrogate marker of stem cell potential. Mesenchymal stromal cells (MSCs) also express CD34, although the functional significance of its expression remains undefined. In this study, we determined whether CD34(pos) MSCs are functionally distinct from CD34(null) MSCs. MATERIALS AND METHODS: MSCs derived from C57Bl/6 mice were transduced to express the green fluorescent protein (GFP) from which pure CD34(pos) MSC and CD34(null) MSC clones were selected. In vitro, clones were examined by microarray analysis, while in vivo subcutaneous implantation of matrix-embedded MSCs was used to assess cell survival, differentiation, and neovascularization. RESULTS: The flow cytometric phenotype of CD34(pos) and CD34(null) MSCs were similar, as was gene expression of vascular endothelial growth factors (VEGFs) A and B. However, CD34(pos) MSCs upregulated a number of supplementary angiogenesis-associated genes and showed a greater expression of gene associated with vascular differentiation. At 15 days postimplantation, cell survival between CD34(pos) and CD34(null) MSCs was similar, however, CD34(pos) MSCs evoked a significantly greater host-derived response (4.2 +/- 0.7 vs 1.9 +/- 0.5 x 10(6) cells; p < 0.05). GFP-expressing CD34(pos) MSC implants acquired significantly more CD31 expression compared to CD34(null) MSC cells (10.7% +/- 8.4% vs 3.1% +/- 0.6%; p < 0.05), as well as a significantly greater host-derived endothelial cell influx (CD31(+)/CD45(-)). CONCLUSION: CD34 expression by MSCs correlates with enhanced vasculogenic and angiogenic potential in vivo.

Coupling erythropoietin secretion to mesenchymal stromal cells enhances their regenerative properties.

AIMS: Mesenchymal stromal cells (MSCs) possess intrinsic features that identify them as useful for treating ischaemic syndromes. Poor in vivo survival/engraftment of MSCs, however, limits their overall effectiveness. In this work, we tested whether genetically engineering MSCs to secrete erythropoietin (Epo) could represent a better therapeutic platform than MSCs in their native form. METHODS AND RESULTS: MSCs from C57Bl/6 mice were retrovirally transduced with either an empty vector or one that causes the production of Epo and were then analysed for the alterations in angiogenic and survival potential. Using a mouse model of myocardial infarction (MI), the regenerative potential of null MSCs and Epo-overexpressing MSCs (Epo+MSCs) was assessed using serial echocardiogram and invasive haemodynamic measurements. Infarct size, capillary density and neutrophil influx were assessed using histologic techniques. Using in vitro assays coupled with an in vivo Matrigel plug assay, we demonstrate that engineering MSCs to express Epo does not alter their immunophenotype or plasticity. However, relative to mock-modified MSCs [wild-type (WT)-MSCs], Epo+MSCs are more resilient to apoptotic stimuli and initiate a more robust host-derived angiogenic response. We also identify and characterize the autocrine loop established on MSCs by having them secrete Epo. Furthermore, in a murine model of MI, animals receiving intracardiac injections of Epo+MSCs exhibited significantly enhanced cardiac function compared with WT-MSCs and saline-injected control animals post-MI, owing to the increased myocardial capillary density and the reduced neutrophilia. CONCLUSION: Epo overexpression enhances the cellular regenerative properties of MSCs by both autocrine and paracrine pathways.

Fibrinogen-Depleted Equine Platelet Lysate Affects the Characteristics and Functionality of Mesenchymal Stem Cells.

Fetal bovine serum (FBS) is widely used to culture mesenchymal stem cells (MSCs) in the laboratory; however, FBS has been linked to adverse immune-mediated reactions prompting the search for alternative cell culture medium. Platelet lysate (PL) as an FBS substitute has been shown to promote MSCs growth without compromising their functionality. Fibrinogen contained in PL has been shown to negatively impact the immune modulating properties of MSCs; therefore, we sought to deplete fibrinogen from PL and compare proliferation, viability, and immunomodulatory capacities of MSCs in FBS or PL without fibrinogen. We depleted fibrinogen from equine platelet lysate (ePL) and measured platelet-derived growth factor-beta (PDGF-ß), transforming growth factor-beta (TGF-ß) and tumor necrosis factor-alpha (TNF-α) through ELISA. First, we determined the ability of 10% ePL or fibrinogen-depleted lysate (fdePL) compared with 10% FBS to suppress monocyte activation by measuring TNF-α from culture supernatants. We then evaluated proliferation, viability, and immunomodulatory characteristics of bone marrow-derived MSCs (BM-MSCs) cultured in FBS or ePL with or without fibrinogen. Growth factor concentrations decreased in ePL after fibrinogen depletion. Lipopolysaccharide (LPS)-stimulated monocytes exposed to ePL and fdePL produced less TNF-α than LPS-stimulated monocytes in 10% FBS. BM-MSCs cultured in fdePL exhibited lower proliferation rates, but similar viability compared with BM-MSCs in ePL. BM-MSCs in fdePL did not effectively suppress TNF-α expression from LPS-stimulated monocytes compared with BM-MSCs in FBS. Depleting fibrinogen results in a lysate that suppresses TNF-α expression from LPS-stimulated monocytes, but that does not support proliferation and immune-modulatory capacity of BM-MSCs as effectively as nondepleted lysate.

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. METHODS: Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. RESULTS: Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed the release of TNF-α when exposed to LPS-stimulated monocytes similar to those cultured in FBS. CONCLUSION: ePL has the potential to be used for the expansion of MSCs before clinical application, avoiding the concerns associated with the use of FBS.

Innate immune responses of equine monocytes cultured in equine platelet lysate.

Platelet lysate (PL) has been extensively used for the laboratory expansion of human mesenchymal stem cells (MSC) in order to avoid fetal bovine serum (FBS) which has been associated with immune-mediated host reactions and transmission of bovine-origin microbial contaminants. Before suggesting the routine use of PL for MSC culture, we wanted to further investigate whether PL alone might trigger inflammatory responses when exposed to reactive white blood cells such as monocytes. Our objectives were to evaluate the inflammatory profile of equine monocytes cultured with equine PL (ePL) and to determine if ePL can modulate the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated monocytes. In a first experiment, equine monocytes were isolated and incubated with donor horse serum (DHS), FBS, six individual donors ePL or pooled ePL from all horses. In a second experiment, monocytes were stimulated with E. coli LPS in the presence of 1, 5 or 10% DHS and/or pooled ePL. After 6h of incubation, cell culture supernatants were assayed via ELISA for production of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and Interleukin 1ß (IL-1ß) as well as for the anti-inflammatory Interleukin 10 (IL-10). Equine monocytes incubated with pooled ePL produced significantly less TNF-α and significantly more IL-10 than monocytes incubated in FBS. A statistically significant difference was not identified for the production of IL-1ß. The second experiment showed that pooled ePL added to LPS-stimulated equine monocytes resulted in a significant reduction in TNF-α and IL-1ß production. IL-10 production was not significantly upregulated by the addition of ePL to LPS-stimulated monocytes. Finally, the addition of ePL to LPS-stimulated monocytes in the presence of various concentrations of DHS resulted to statistically significant decrease of TNF-α and IL-1ß compared to the control groups. This is the first study to demonstrate that ePL suppresses the release of pro-inflammatory cytokines from stimulated equine monocytes. These results encourage further exploration of PL as a homologous media substitute for FBS but also opens the possibility of investigating its use as means to suppress cell-mediated inflammation.

Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability.

Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC-seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell-seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL-supplemented media resulted in rMSC death, by a process attributable to heat-labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd.

Silencing of the human microsomal glucose-6-phosphate translocase induces glioma cell death: potential new anticancer target for curcumin.

G6P translocase (G6PT) is thought to play a crucial role in transducing intracellular signaling events in brain tumor-derived cancer cells. In this report, we investigated the contribution of G6PT to the control of U-87 brain tumor-derived glioma cell survival using small interfering RNA (siRNA)-mediated suppression of G6PT. Three siRNA constructs were generated and found to suppress up to 91% G6PT gene expression. Flow cytometry analysis of propidium iodide/annexin-V-stained cells indicated that silencing the G6PT gene induced necrosis and late apoptosis. The anticancer agent curcumin, also inhibited G6PT gene expression by more than 90% and triggered U-87 glioma cells death. Overexpression of recombinant G6PT rescued the cells from curcumin-induced cell death. Targeting G6PT expression may provide a new mechanistic rationale for the action of chemopreventive drugs and lead to the development of new anti-cancer strategies.

A novel platelet lysate hydrogel for endothelial cell and mesenchymal stem cell-directed neovascularization.

UNLABELLED: Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration. STATEMENT OF SIGNIFICANCE: Innovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL hydrogel is ideally suited to serve as a cell delivery vehicle for MSCs injected into ischemic tissues to promote vascular regeneration, as demonstrated here in a murine model of hindlimb ischemia.