Comparison of Mesenchymal Stromal Cells From Different Origins for the Treatment of Graft-vs.-Host-Disease in a Humanized Mouse Model.

Mesenchymal stromal cells (MSCs) have potent immunomodulatory properties that make them an attractive tool against graft- vs.-host disease (GVHD). However, despite promising results in phase I/II studies, bone marrow (BM-) derived MSCs failed to demonstrate their superiority over placebo in the sole phase III trial reported thus far. MSCs from different tissue origins display different characteristics, but their therapeutic benefits have never been directly compared in GVHD. Here, we compared the impact of BM-, umbilical cord (UC-), and adipose-tissue (AT-) derived MSCs on T-cell function in vitro and assessed their efficacy for the treatment of GVHD induced by injection of human peripheral blood mononuclear cells in NOD-scid IL-2Rγnull HLA-A2/HHD mice. In vitro, resting BM- and AT-MSCs were more potent than UC-MSCs to inhibit lymphocyte proliferation, whereas UC- and AT-MSCs induced a higher regulatory T-cell (CD4+CD25+FoxP3+)/T helper 17 ratio. Interestingly, AT-MSCs and UC-MSCs activated the coagulation pathway at a higher level than BM-MSCs. In vivo, AT-MSC infusions were complicated by sudden death in 4 of 16 animals, precluding an analysis of their efficacy. Intravenous MSC infusions (UC- or BM- combined) failed to significantly increase overall survival (OS) in an analysis combining data from 80 mice (hazard ratio [HR] = 0.59, 95% confidence interval [CI] 0.32-1.08, P = 0.087). In a sensitivity analysis we also compared OS in control vs. each MSC group separately. The results for the BM-MSC vs. control comparison was HR = 0.63 (95% CI 0.30-1.34, P = 0.24) while the figures for the UC-MSC vs. control comparison was HR = 0.56 (95% CI 0.28-1.10, P = 0.09). Altogether, these results suggest that MSCs from various origins have different effects on immune cells in vitro and in vivo. However, none significantly prevented death from GVHD. Finally, our data suggest that the safety profile of AT-MSC and UC-MSC need to be closely monitored given their pro-coagulant activities in vitro.

Application of Electron Paramagnetic Resonance (EPR) Oximetry to Monitor Oxygen in Wounds in Diabetic Models.

A lack of oxygen is classically described as a major cause of impaired wound healing in diabetic patients. Even if the role of oxygen in the wound healing process is well recognized, measurement of oxygen levels in a wound remains challenging. The purpose of the present study was to assess the value of electron paramagnetic resonance (EPR) oximetry to monitor pO2 in wounds during the healing process in diabetic mouse models. Kinetics of wound closure were carried out in streptozotocin (STZ)-treated and db/db mice. The pO2 was followed repeatedly during the healing process by 1 GHz EPR spectroscopy with lithium phthalocyanine (LiPc) crystals used as oxygen sensor in two different wound models: a full-thickness excisional skin wound and a pedicled skin flap. Wound closure kinetics were dramatically slower in 12-week-old db/db compared to control (db/+) mice, whereas kinetics were not statistically different in STZ-treated compared to control mice. At the center of excisional wounds, measurements were highly influenced by atmospheric oxygen early in the healing process. In pedicled flaps, hypoxia was observed early after wounding. While reoxygenation occurred over time in db/+ mice, hypoxia was prolonged in the diabetic db/db model. This observation was consistent with impaired healing and microangiopathies observed using intravital microscopy. In conclusion, EPR oximetry using LiPc crystals as the oxygen sensor is an appropriate technique to follow wound oxygenation in acute and chronic wounds, in normal and diabetic animals. Nevertheless, the technique is limited for measurements in pedicled skin flaps and cannot be applied to excisional wounds in which diffusion of atmospheric oxygen significantly affects the measurements.

Human Adipose-Derived Mesenchymal Stem Cells in Cell Therapy: Safety and Feasibility in Different "Hospital Exemption" Clinical Applications.

Based on immunomodulatory, osteogenic, and pro-angiogenic properties of adipose-derived stem cells (ASCs), this study aims to assess the safety and efficacy of ASC-derived cell therapies for clinical indications. Two autologous ASC-derived products were proposed to 17 patients who had not experienced any success with conventional therapies: (1) a scaffold-free osteogenic three-dimensional graft for the treatment of bone non-union and (2) a biological dressing for dermal reconstruction of non-healing chronic wounds. Safety was studied using the quality control of the final product (genetic stability, microbiological/mycoplasma/endotoxin contamination) and the in vivo evaluation of adverse events after transplantation. Feasibility was assessed by the ability to reproducibly obtain the final ASC-based product with specific characteristics, the time necessary for graft manufacturing, the capacity to produce enough material to treat the lesion, the surgical handling of the graft, and the ability to manufacture the graft in line with hospital exemption regulations. For 16 patients (one patient did not undergo grafting because of spontaneous bone healing), in-process controls found no microbiological/mycoplasma/endotoxin contamination, no obvious deleterious genomic anomalies, and optimal ASC purity. Each type of graft was reproducibly obtained without significant delay for implantation and surgical handling was always according to the surgical procedure and the implantation site. No serious adverse events were noted for up to 54 months. We demonstrated that autologous ASC transplantation can be considered a safe and feasible therapy tool for extreme clinical indications of ASC properties and physiopathology of disease.

Improvement of subcutaneous bioartificial pancreas vascularization and function by coencapsulation of pig islets and mesenchymal stem cells in primates.

Insufficient oxygenation can limit the long-term survival of encapsulated islets in subcutaneous tissue. Transplantation of coencapsulated pig islets with adipose or bone marrow mesenchymal stem cells (AMSCs or BM-MSCs, respectively) was investigated with regard to implant vascularization, oxygenation, and diabetes correction in primates. The in vivo impact of MSCs on graft oxygenation and neovascularization was assessed in rats with streptozotocin (STZ)-induced diabetes that were subcutaneously transplanted with islets coencapsulated with AMSCs (n = 8) or BM-MSCs (n = 6). Results were compared to islets encapsulated alone (n = 8). STZ diabetic primates were subcutaneously transplanted with islets coencapsulated with BM-MSCs (n = 4) or AMSCs (n = 6). Recipients were monitored metabolically and immunologically, and neoangiogenesis was assessed on explanted grafts. Results were compared with primates transplanted with islets encapsulated alone (n = 5). The cotransplantation of islets with BM-MSCs or AMSCs in diabetic rats showed significantly higher graft oxygenation than islets alone (3% and 3.6% O2 for islets + BM-MSCs or AMSCs, respectively, vs. 2.2% for islets alone). A significantly better glycated hemoglobin correction (28 weeks posttransplantation) was found for primates transplanted with islets and MSCs (7.4% and 8.1%, respectively) in comparison to islets encapsulated alone (10.9%). Greater neoangiogenesis was found in the periphery of coencapsulated islets and AMSCs in comparison to islets alone (p < 0.01). In conclusion, the coencapsulation of pig islets with MSCs can improve significantly the islets' survival/function in vitro. The coencapsulation of islets with MSCs improves implant oxygenation and neoangiogenesis. However, the cotransplantation of islets with MSCs improves only slightly the long-term function of a subcutaneous bioartificial pancreas in a primate preclinical model.

Bioengineered sites for islet cell transplantation.

Although islet transplantation has demonstrated its potential use in treating type 1 diabetes, this remains limited by the need for daily immunosuppression. Islet encapsulation was then proposed with a view to avoiding any immunosuppressive regimen and related side effects. In order to obtain a standard clinical procedure in terms of safety and reproducibility, two important factors have to be taken into account: the encapsulation design (which determines the graft volume) and the implantation site. Indeed, the implantation site should meet certain requirements: (1) its space must be large enough for the volume of transplanted tissues; (2) there must be proximity to abundant vascularization with a good oxygen supply; (3) there must be real-time access to physiologically representative blood glucose levels; (4) there must be easy access for implantation and the reversibility of the procedure (for safety); and finally, (5) the site should have minimal early inflammatory reaction and promote long-term survival. The aim of this article is to review possible preclinical/clinical implantation sites (in comparison with free islets) for encapsulated islet transplantation as a function of the encapsulation design: macro/microcapsules and conformal coating.

Improvement of pig islet function by in vivo pancreatic tissue remodeling: a "human-like" pig islet structure with streptozotocin treatment.

Pig islets demonstrate significantly lower insulin secretion after glucose stimulation than human islets (stimulation index of ∼12 vs. 2 for glucose 1 and 15 mM, respectively) due to a major difference in ß- and α-cell composition in islets (60% and 25% in humans and 90% and 8% in pigs, respectively). This leads to a lower rise in 3',5'-cyclic adenosine monophosphate (cAMP) in pig ß-cells. Since glucagon is the major hormonal effector of cAMP in ß-cells, we modified pig islet structure in vivo to increase the proportion of α-cells per islet and to improve insulin secretion. Selected doses (0, 30, 50, 75, and 100 mg/kg) of streptozotocin (STZ) were intravenously injected in 32 young pigs to assess pancreatic (insulin and glucagon) hormone levels, islet remodeling (histomorphometry for α- and ß-cell proportions), and insulin and glucagon secretion in isolated islets. Endocrine structure and hormonal content of pig islets were compared with those of human islets. The dose of STZ was significantly correlated with reductions in pancreatic insulin content (p< 0.05, r(2) = 0.77) and the proportion of ß-cells (p < 0.05, r(2) = 0.88). A maximum of 50 mg/kg STZ was required for optimal structure remodeling, with an increased proportion of α-cells per islet (26% vs. 48% α-cells per islet for STZ <50 mg/kg vs. >75 mg/kg; p < 0.05) without ß-cell dysfunction. Three months after STZ treatment (30/50 mg/kg STZ), pig islets were isolated and compared with isolated control islets (0 mg/kg STZ). Isolated islets from STZ-treated (30/50 mg/kg) pigs had a higher proportion of α-cells than those from control animals (32.0% vs. 9.6%, respectively, p < 0.05). After in vitro stimulation, isolated islets from STZ-treated pigs demonstrated significantly higher glucagon content (65.4 vs. 21.0 ng/ml, p < 0.05) and insulin release (144 µU/ml) than nontreated islets (59 µU/ml, p < 0.05), respectively. Low-dose STZ (<50 mg/kg) can modify the structure of pig islets in vivo and improve insulin secretion after isolation.

The enhanced performance of bone allografts using osteogenic-differentiated adipose-derived mesenchymal stem cells.

Adipose tissue was only recently considered as a potential source of mesenchymal stem cells (MSCs) for bone tissue engineering. To improve the osteogenicity of acellular bone allografts, adipose MSCs (AMSCs) and bone marrow MSCs (BM-MSCs) at nondifferentiated and osteogenic-differentiated stages were investigated in vitro and in vivo. In vitro experiments demonstrated a superiority of AMSCs for proliferation (6.1±2.3 days vs. 9.0±1.9 days between each passage for BM-MSCs, respectively, P<0.001). A significantly higher T-cell depletion (revealed by mixed lymphocyte reaction, [MLR]) was found for AMSCs (vs. BM-MSCs) at both non- and differentiated stages. Although nondifferentiated AMSCs secreted a higher amount of vascular endothelial growth factor [VEGF] in vitro (between 24 and 72 h of incubation at 0.1-21% O(2)) than BM-MSCs (P<0.001), the osteogenic differentiation induced a significantly higher VEGF release by BM-MSCs at each condition (P<0.001). After implantation in the paraspinal muscles of nude rats, a significantly higher angiogenesis (histomorphometry for vessel development (P<0.005) and VEGF expression (P<0.001)) and osteogenesis (as revealed by osteocalcin expression (P<0.001) and micro-CT imagery for newly formed bone tissue (P<0.05)) were found for osteogenic-differentiated AMSCs in comparison to BM-MSCs after 30 days of implantation. Osteogenic-differentiated AMSCs are the best candidate to improve the angio-/osteogenicity of decellularized bone allografts.

The impact of hyperglycemia and the presence of encapsulated islets on oxygenation within a bioartificial pancreas in the presence of mesenchymal stem cells in a diabetic Wistar rat model.

This study investigates the potential of bone marrow (BM-MSCs) versus adipose mesenchymal stem cells (AMSCs) to potentiate the oxygenation of encapsulated islets in a subcutaneous bioartificial pancreas. Oxygen pressures (inside subcutaneous implants) were followed in vivo (by electronic paramagnetic resonance) in non-diabetic/diabetic rats transplanted with encapsulated porcine islets or empty implants up to 4 weeks post-transplantation. After graft explantation, neoangiogenesis surrounding the implants was assessed by histomorphometry. Angiogenic properties of BM-MSCs and AMSCs were first assessed in vitro by incubation of the cells in hypoxia chambers, under normoxic/hypoxic and hypo-/hyperglycemic conditions, followed by quantification of vascular endothelial growth factor (VEGF) release. Second, the in vivo aspect was studied by subcutaneous transplantation of encapsulated BM-MSCs and AMSCs in diabetic rats and assessment of the cells' angiogenic properties as described above. Diabetic state and islet encapsulation induced a significant decrease of oxygenation of the subcutaneous implant and an increased number of cells expressing VEGF. AMSCs demonstrated a significantly higher VEGF secretion than BM-MSCs in vitro. In vivo, AMSCs improved the implant's oxygenation and vascularization. Diabetes and islet encapsulation significantly reduced the oxygenation of a subcutaneous bioartificial pancreas. AMSCs can improve oxygenation by VEGF release in hypoxia and hyperglycemia states.

In vivo selection of biocompatible alginates for islet encapsulation and subcutaneous transplantation.

Islet encapsulation requires several properties including (1) biocompatibility, (2) immunoprotection, and (3) oxygen diffusion for islet survival and diabetes correction. New chemical alginates were tested in vivo and compared with traditional high-mannuronate and -guluronate alginates. New alginates with coupled peptide sequence (sterile lyophilized high mannuronate [SLM]-RGD3% and sterile lyophilized high guluronate [SLG]-RGD3%), to improve encapsulated cell adherence in the matrix, and alginates with a very low viscosity (VLDM7% and VLDG7%), to reduce implant size by loading a higher number of islets per volume of polymer, were implanted subcutaneously in 70 Wistar rats for comparison with alginates of high viscosity and high content of mannuronic (SLM3%) or guluronic acids (SLG3%). Permeability of alginates to 36-, 75-, and 150-kDa lectins coupled to fluorescein isothiocynate was quantified before implantation and at 2, 4, and 12 weeks after implantation. Biocompatibility (fibrosis, graft stability, immunologic infiltration by CD3/CD68 cells, and neovascularization) was assessed at each explantation time. Permeability to small molecules was found for all alginates. Impermeability to 150-kDa molecules, such as IgG, was observed only for SLM3% before implantation and was maintained up to 12 weeks after implantation. SLM3% and SLG3% demonstrated better graft stability with lower CD3/CD68 recruitment and fibrosis than the other alginates. SLM3% induced a significantly higher angiogenesis and maintained oxygen pressure at approximately 40 mm Hg for up to 4 weeks after implantation as measured by in vivo electronic paramagnetic resonance oximetry. SLM-encapsulated pig islets implanted subcutaneously in rats demonstrated no inflammatory/immunologic reactions and islets functioned for up to 60 days without immunosuppression. A traditional alginate made of high mannuronic content (SLM3%) is an adapted material to immunoprotect islets in subcutaneous tissue. No improvement was found with lower viscosity and use of GRGDSP-peptide sequence.

Quantification of 8 HIV-protease inhibitors and 2 nonnucleoside reverse transcriptase inhibitors by ultra-performance liquid chromatography with diode array detection.

BACKGROUND: Most HPLC-UV methods for therapeutic drug monitoring of anti-HIV drugs have long run times, which reduce their applicability for high-throughput analysis. We developed an ultra-performance liquid chromatography (UPLC)-diode array detection method for the simultaneous quantification of the HIV-protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir (TPV), and the nonnucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. METHODS: Solid-phase extraction of 1 mL plasma was performed with Waters HLB cartridges. After 3 wash steps, we eluted the drugs with methanol, evaporated the alcohol, and reconstituted the residue with 50 microL methanol. We injected a 4-microL volume into the UPLC system (Waters ACQUITY UPLC BEH C8 column maintained at 60 degrees C) and used a linear gradient of 50 mmol/L ammonium acetate and 50 mmol/L formic acid in water versus acetonitrile to achieve chromatographic separation of the drugs and internal standard (A-86093). Three wavelengths (215, 240, and 260 nm) were monitored. RESULTS: All drugs were eluted within 15 min. Calibration curves with concentrations of 0.025-10 mg/L (1.875-75 mg/L for TPV) showed coefficients of determination (r(2)) between 0.993 and 0.999. The lower limits of quantification were well below the trough concentrations reported in the literature. Inter- and intraassay CVs and the deviations between the nominal and measured concentrations were <15%. The method was validated by successful participation in an international interlaboratory QC program. CONCLUSIONS: This method allows fast and simultaneous quantification of all commercially available PIs and NNRTIs for therapeutic drug monitoring.