Persistence of Metarhizium brunneum (Ascomycota: Hypocreales) in the Soil Is Affected by Formulation Type as Shown by Strain-Specific DNA Markers.

The genus Metarhizium has an increasingly important role in the development of Integrated Pest Control against Tephritid fruit flies in aerial sprays targeting adults and soil treatments targeting preimaginals. Indeed, the soil is considered the main habitat and reservoir of Metarhizium spp., which may be a plant-beneficial microorganism due to its lifestyle as an endophyte and/or rhizosphere-competent fungus. This key role of Metarhizium spp. for eco-sustainable agriculture highlights the priority of developing proper monitoring tools not only to follow the presence of the fungus in the soil and to correlate it with its performance against Tephritid preimaginals but also for risk assessment studies for patenting and registering biocontrol strains. The present study aimed at understanding the population dynamics of M. brunneum strain EAMb 09/01-Su, which is a candidate strain for olive fruit fly Bactrocera oleae (Rossi, 1790) preimaginal control in the soil, when applied to the soil at the field using different formulations and propagules. For this, strain-specific DNA markers were developed and used to track the levels of EAMb 09/01-Su in the soil of 4 field trials. The fungus persists over 250 days in the soil, and the levels of the fungus remained higher when applied as an oil-dispersion formulation than when applied as a wettable powder or encapsulated microsclerotia. Peak concentrations of EAMb 09/01-Su depend on the exogenous input and weakly on environmental conditions. These results will help us to optimize the application patterns and perform accurate risk assessments during further development of this and other entomopathogenic fungus-based bioinsecticides.

Derivation of Multipotent Mesenchymal Stromal Cells from Ovine Bone Marrow.

In the field of orthopedics, translational research of novel therapeutic approaches involves the use of large animal models (such as sheep, goat, pig, dog, and horse) due to the similarities with humans in weight, size, joint structure, and bone/cartilage healing mechanisms. Particularly in the development of cell-based therapies, the lack of manageable immunocompromised preclinical large animal models prevents the use of human cells, which makes it necessary to produce equivalent homologous cell types for the study of their pharmacodynamics, pharmacokinetics, and toxicology. The methods described herein allow for the isolation, expansion, manipulation, and characterization of fibroblastic-like ovine bone marrow-derived multipotent mesenchymal stromal cells (BM-MSC) that, similar to human BM-MSC, adhere to standard plastic surfaces; express specific surface markers such as CD44, CD90, CD140a, CD105, and CD166; and display trilineage differentiation potential in vitro. Homogeneous cell cultures result from a 3-week bioprocess yielding cell densities in the range of 2-4 × 104 MSC/cm2 at passage 2, which corresponds to ∼8 cumulative population doublings. Large quantities of BM-MSC resulting from following this methodology can be readily used in proof of efficacy and safety studies in the preclinical development stage. © 2018 by John Wiley & Sons, Inc.

Optimisation of a potency assay for the assessment of immunomodulative potential of clinical grade multipotent mesenchymal stromal cells.

Clinical use of multipotent Mesenchymal Stromal Cell (MSC)-based medicinal products requires their production in compliance with Good Manufacturing Practices, thus ensuring that the final drug product meets specifications consistently from batch to batch in terms of cell viability, identity, purity and potency. Potency relates to the efficacy of the medicine in its target clinical indication, so adequate release tests need to be defined and validated as quality controls. Herein we report the design and optimisation of parameters affecting the performance of an in vitro cell-based assay for assessing immunomodulatory potential of clinical grade MSC for human use, based on their capacity to inhibit proliferation of T lymphocytes under strong polyclonal stimuli. The resulting method was demonstrated to be reproducible and relatively simple to execute. Two case studies using clinical grade MSC are presented as examples to illustrate the applicability of the methodology described in this work.

Clinical translation of a mesenchymal stromal cell-based therapy developed in a large animal model and two case studies of the treatment of atrophic pseudoarthrosis.

Pseudoarthrosis is a relatively frequent complication of fractures, in which the lack of mechanical stability and biological stimuli results in the failure of bone union, most frequently in humerus and tibia. Treatment of recalcitrant pseudoarthrosis relies on the achievement of satisfactory mechanical stability combined with adequate local biology. Herein we present two cases of atrophic pseudoarthrosis that received a tissue-engineering product (TEP) composed of autologous bone marrow-derived mesenchymal stromal cells (BM-MSC) combined with deantigenized trabecular bone particles from a tissue bank. The feasibility of the treatment and osteogenic potential of the cell-based medicine was first demonstrated in an ovine model of critical size segmental tibial defect. Clinical-grade autologous BM-MSC were produced following a good manufacturing practice-compliant bioprocess. Results were successful in one case, with pseudoarthrosis resolution, and inconclusive in the other one. The first patient presented atrophic pseudoarthrosis of the humeral diaphysis and was treated with osteosynthesis and TEP resulting in satisfactory consolidation at month 6. The second case presented a recalcitrant pseudoarthrosis of the proximal tibia and the Masquelet technique was followed before filling the defect with the TEP. This patient presented a neuropathic pain syndrome unrelated to the treatment that forced the amputation of the extremity 3 months later. In this case, the histological analysis of the tissue formed at the defect site provided evidence of neovascularization but no overt bone remodelling activity. It is concluded that the use of expanded autologous BM-MSC to treat pseudoarthrosis was demonstrated to be feasible and safe, provided that no clinical complications were reported, and early signs of effectiveness were observed. Copyright © 2016 John Wiley & Sons, Ltd.

Assessment of biodistribution using mesenchymal stromal cells: Algorithm for study design and challenges in detection methodologies.

BACKGROUND AIMS: Biodistribution of candidate cell-based therapeutics is a critical safety concern that must be addressed in the preclinical development program. We aimed to design a decision tree based on a series of studies included in actual dossiers approved by competent regulatory authorities, noting that the design, execution and interpretation of pharmacokinetics studies using this type of therapy is not straightforward and presents a challenge for both developers and regulators. METHODS: Eight studies were evaluated for the definition of a decision tree, in which mesenchymal stromal cells (MSCs) were administered to mouse, rat and sheep models using diverse routes (local or systemic), cell labeling (chemical or genetic) and detection methodologies (polymerase chain reaction [PCR], immunohistochemistry [IHC], fluorescence bioimaging, and magnetic resonance imaging [MRI]). Moreover, labeling and detection methodologies were compared in terms of cost, throughput, speed, sensitivity and specificity. RESULTS: A decision tree was defined based on the model chosen: (i) small immunodeficient animals receiving heterologous MSC products for assessing biodistribution and other safety aspects and (ii) large animals receiving homologous labeled products; this contributed to gathering data not only on biodistribution but also on pharmacodynamics. PCR emerged as the most convenient technique despite the loss of spatial information on cell distribution that can be further assessed by IHC. DISCUSSION: This work contributes to the standardization in the design of biodistribution studies by improving methods for accurate assessment of safety. The evaluation of different animal models and screening of target organs through a combination of techniques is a cost-effective and timely strategy.

A reproducible method for the isolation and expansion of ovine mesenchymal stromal cells from bone marrow for use in regenerative medicine preclinical studies.

The use of multipotent mesenchymal stromal cells (MSCs) as candidate medicines for treating a variety of pathologies is based on their qualities as either progenitors for the regeneration of damaged tissue or producers of a number of molecules with pharmacological properties. Preclinical product development programmes include the use of well characterized cell populations for proof of efficacy and safety studies before testing in humans. In the field of orthopaedics, an increasing number of translational studies use sheep as an in vivo test system because of the similarities with humans in size and musculoskeletal architecture. However, robust and reproducible methods for the isolation, expansion, manipulation and characterization of ovine MSCs have not yet been standardised. The present study describes a method for isolation and expansion of fibroblastic-like, adherent ovine MSCs that express CD44, CD90, CD140a, CD105 and CD166, and display trilineage differentiation potential. The 3-week bioprocess proposed here typically yielded cell densities of 1.4 × 104 MSCs/cm2 at passage 2, with an expansion factor of 37.8 and approximately eight cumulative population doublings. The osteogenic potential of MSCs derived following this methodology was further evaluated in vivo in a translational model of osteonecrosis of the femoral head, in which the persistence of grafted cells in the host tissue and their lineage commitment into osteoblasts and osteocytes was demonstrated by tracking enhanced green fluorescent protein-labelled cells. Copyright © 2016 John Wiley & Sons, Ltd.

Clinical-scale expansion of CD34+ cord blood cells amplifies committed progenitors and rapid scid repopulation cells.

Umbilical cord blood (UCB) transplantation is associated with long periods of aplastic anaemia. This undesirable situation is due to the low cell dose available per unit of UCB and the immaturity of its progenitors. To overcome this, we present a cell culture strategy aimed at the expansion of the CD34+ population and the generation of granulocyte lineage-committed progenitors. Two culture products were produced after either 6 or 14days of in vitro expansion, and their characteristics compared to non-expanded UCB CD34+ controls in terms of phenotype, colony-forming activity and multilineage repopulation potential in NOD-scid IL2Rγnull mice. Both expanded cell products maintained rapid SCID repopulation activity similar to the non-expanded control, but 14-day cultured cells showed impaired long term SCID repopulation activity. The process was successfully scaled up to clinically relevant doses of 89×106 CD34+ cells committed to the granulocytic lineage and 3.9×109 neutrophil precursors in different maturation stages. Cell yields and biological properties presented by the cell product obtained after 14days in culture were superior and therefore this is proposed as the preferred production setup in a new type of dual transplant strategy to reduce aplastic periods, producing a transient repopulation before the definitive engraftment of the non-cultured UCB unit. Importantly, human telomerase reverse transcriptase activity was undetectable, c-myc expression levels were low and no genetic abnormalities were found, as determined by G-banding karyotype, further confirming the safety of the expanded product.

Evaluation of a cell-banking strategy for the production of clinical grade mesenchymal stromal cells from Wharton's jelly.

BACKGROUND AIMS: Umbilical cord (UC) has been proposed as a source of mesenchymal stromal cells (MSCs) for use in experimental cell-based therapies provided that its collection does not raise any risk to the donor, and, similar to bone marrow and lipoaspirates, UC-MSCs are multipotent cells with immuno-modulative properties. However, some of the challenges that make a broader use of UC-MSCs difficult include the limited availability of fresh starting tissue, time-consuming processing for successful derivation of cell lines, and the lack of information on identity, potency and genetic stability in extensively expanded UC-MSCs, which are necessary for banking relevant cell numbers for preclinical and clinical studies. METHODS: Factors affecting the success of the derivation process (namely, time elapsed from birth to processing and weight of fragments), and methods for establishing a two-tiered system of Master Cell Bank and Working Cell Bank of UC-MSCs were analyzed. RESULTS: Efficient derivation of UC-MSCs was achieved by using UC fragments larger than 7 g that were processed within 80 h from birth. Cells maintained their immunophenotype (being highly positive for CD105, CD90 and CD73 markers), multi-potentiality and immuno-modulative properties beyond 40 cumulative population doublings. No genetic abnormalities were found, as determined by G-banding karyotype, human telomerase reverse transcriptase activity was undetectable and no toxicity was observed in vivo after intravenous administration of UC-MSCs in athymic rats. DISCUSSION: This works demonstrates the feasibility of the derivation and large-scale expansion of UC-MSCs from small and relatively old fragments of UC typically discarded from public cord blood banking programs.

Use of a chronic model of articular cartilage and meniscal injury for the assessment of long-term effects after autologous mesenchymal stromal cell treatment in sheep.

Regenerative therapies using adult stem cells have attracted great interest in the recent years and offer a promising alternative to current surgical practices. In this report, we evaluated the safety and efficacy of an autologous cell-based treatment of osteoarthritis using mesenchymal stromal cells expanded from bone marrow aspirates that were administered intra-articularly. Ten 2-year old ewes were divided in two groups (for analysis at 6 and 12 months, respectively). Full thickness articular cartilage defects of approximately 60mm(2) were created arthroscopically in the medial femorotibial condyles and a meniscal tear in the anterior horn of the medial meniscus in the 20 hind legs. Intra-articular injection of 4 mL of either treatment (a suspension of cells) or control (same as treatment, without cells) were applied one month after generating a chronic condition similar to human pathology. Animals were monitored radiographically, by MRI and ultrasound scanning; and macroscopic and histological analyses were conducted at 6 and 12 months. Furthermore a full necropsy was performed at 12 months post-treatment. The intra-articular injection of autologous MSC was safe, as judged by the lack of local or systemic adverse effects during the clinical follow-up and by a full necropsy performed at 12 months post-treatment. Evidence of regeneration of articular cartilage and meniscus was case-dependent but statistically significant improvement was found in specific macroscopic and histological parameters. Such parameters included colour, rigidity, cell distribution and hyaline quality of the refill tissue as well as the structure of subchondral bone.

Treatment of femoral head osteonecrosis with advanced cell therapy in sheep.

BACKGROUND: The purpose of this study was to evaluate the efficacy of core decompression associated with advanced cell therapy for the treatment of femoral head osteonecrosis in an established sheep model. METHODS: Early stage osteonecrosis of the right hip was induced cryogenically in 15 mature sheep. At 6 weeks, the sheep were divided into three groups, Group A: core decompression only; Group B: core decompression followed by implantation of an acellular bone matrix scaffold; Group C: core decompression followed by implantation of a cultured BMSC loaded bone matrix scaffold. At 12 weeks, MRI hip studies were performed and then the proximal femur was harvested for histological analysis. RESULTS: In the group of advanced cell therapy, Group C, there was a tendency to higher values of the relative surface of newly formed bone with a mean of 20.3 versus 11.27 % in Group A and 13.04 % in Group B but it was not statistically significant. However, the mean relative volume of immature osteoid was 8.6 % in Group A, 14.97 in Group B, and 53.49 % in Group C (p < 0.05), revealing a greater capacity of osteoid production in the sheep treated with BMSCs. MRI findings were not conclusive due to constant bone edema artifact in all cases. CONCLUSIONS: Our findings indicate that a BMCSs loaded bone matrix scaffold is capable of stimulating bone regeneration more effectively than isolated core decompression or in association with an acellular scaffold in a preclinical femoral head osteonecrosis model in sheep.