Progressive Supranuclear palsy (PSP), a rare form of parkinsonism, is a sporadic and progressive neurodegenerative disease linked to the accumulation of Tau protein within neural cells (taupathies). No effective therapy is to date available for this no-option neurological disorder. Bone marrow (BM) mesenchymal stromal cells (MSC) have recently gained attention for therapeutic interventions due to their anti-inflammatory, anti-apoptotic and trophic properties, also for neurological disorders. In this context, our hospital-based GMP-approved facility provides autologous BMMSC manufactured as advanced therapy medicinal products (ATMPs) in a phase I clinical study (NCT01824121) with the aim to evaluate the safety of intra-arterial infusion of autologous BMMSC in patients with PSP. In connection with this project, we set up a parallel in vitro study with the aim to investigate MSC biology, and to disclose if any disease-linked defect occurs also in the non-neuronal mesenchymal compartment.

Idiopathic nephrotic syndrome is generally associated with an abnormal activity of the cellular component of the immune system, requires prolonged medical treatment and, particularly in case of pediatric onset, may seriously affect long-term quality of life. In this context, when immunosuppressive therapies are ineffective, there is an urgent need for innovative approaches, also using advanced therapy medicinal products such as Mesenchymal Stromal Cells (MSCs). In particular, MSC from Cord Blood (CB) are very promising in this regard due to their outmost anti-apoptotic, tissue-regenerating and anti-inflammatory properties. To test the ability of CBMSCs to reduce the damage, they have been used in a standardized in vitro podocytes-endothelial cells co-culture system able to mimic the filtration barrier. In this system, podocytes were damaged with adryamicin to simulate a glomerular damage and the permeability to albumin (BSA) was measured, both in steady-state and after damage. CBMSCs were therefore added to the co-culture system to test their ability to restore the adryamicin induced-alteration of membrane permeability. CBMSCs were able to rescue the normal properties of the filtration barrier in this in vitro system (Figure 1). This preliminary work paves the way for further studies to understand how CBMSC can act in several kidney disease and opens up new paths in the search of the real mechanisms of action of these cells.

Regulatory CD4+CD25+ T lymphocytes (Tregs) can improve the outcome of solid organ transplantation through induction of immunologic tolerance. Due to the low frequency of Tregs in peripheral blood, their enrichment through isolation and ex vivo expansion is required to obtain a suitable quantity of cells for therapeutic use.

Graft-versus-host disease (GvHD) is a severe complication in the setting of allogeneic hematopoietic stem cell transplantation (HSCT). Mesenchymal stromal cells (MSCs) have been extensively used as second-line treatment for acute GvHD. Cord blood (CB) MSCs display low immunogenicity which makes them suitable for an allogeneic use. Herein we reported the infusion of CB-MSCs to treat three patients with severe resistant aGvHD and two patients with overlap syndrome after HSCT.

Bronchopleural fistula (BF) after pneumonectomy is an infrequent but severe condition in which standard reparative approaches are usually frustrating. Under hospital exemption and Ethics Committee approval, our hospital-based GMP-approved facility provided autologous bone marrow (BM) mesenchymal stem cells (MSC) manufactured as advanced therapy medicinal products to treat a BF occurred after extrapleural pneumonectomy in a patient affected by malignant mesothelioma. BM was obtained by puncture of the posterior iliac crest in asepsis and was directly seeded in alphaMEM supplemented with 10% qualified fetal bovine serum in a closed system (Cellstack, Macopharma). At passage 1 cultured cells were harvested, counted and cryopreserved in plastic bags at the target cell dose. The final product was tested for the following quality controls (specifications in brackets): viability with PI- staining (>80%), purity with flow cytometry (CD90+/105+/CD73+/45− > 80%), sterility following Ph. Eu. 2.6.27 (no growth), endotoxins following Ph. Eu. 2.6.14 (<0.25 EU/ml), mycoplasma following Ph. Eu. 2.6.7 (no growth) and kariotyope (46,XY). Before injection, 10x106 BMMSC were thawed at 37°C, washed and resuspended in normal saline solution (5 mL final volume). The final product, in a sterile closed syringe, was sent to the clinical department and injected in the pars membranacea of the right main bronchus stump, as close as possible to the orifice, by an endoscopic needle as already described (Petrella et al, NEJM, 2015). Sixty days later, bronchoscopy disclosed a complete healing of the resection line, with the previous orifice not longer visible. The immunocytochemical stain showed a condition consistent with repair. This application of well-established procedure is an innovative, safe, and non-invasive method for closing small caliber fistula after pneumonectomy, and provides the evidence that BMMSC may be effective in restoring otherwise untreatable airway defects.

In regenerative medicine human cord blood multipotent mesenchymal stromal cells (CBMSC) stand out for their biological peculiarities demonstrated in in vitro and in vivo preclinical studies. During the past 9 years our group extensively investigated and set up protocols for efficient isolation, culture and characterization of CBMSC. To reach this goal a total of 264 CB samples were processed. Characterization for morphological, clonal, differentiation and proliferation properties revealed two divergent CBMSC behaviors. In particular, a cumulative population doublings cut-off (CPD=15) was identified that undoubtedly distinguishes two growth curves. Our findings clearly showed the existence of at least two distinct CBMSC subsets: one mainly short-living and less proliferative (SL-CBMSC), the other long-living, with higher growth rate, and, very importantly, with significantly longer telomere (LL-CBMSC). Based on all the preclinical data obtained by our group in vitro and in animal models of different diseases, from our point of view LL-CBMSC have been considered the best candidate as Good Manufacturing Practice (GMP) advanced therapy medicinal product. Therefore, in our hospital-based GMP facility, authorized by the National Drug Agency (AIFA) in 2007, LL-CBMSC have been expanded and manufactured through a multi-step controlled and well-validated process in GMP conditions. After culture our quality control approach, designed to ensure that controls were implemented and completed satisfactorily during all the manufacturing operations, was applied. Now LL-CBMSC are available as “a drug”, to be distributed for patient doses, much like an off-the-shelf pharmaceutical product. These GMP batches of LL-CBMSC are cryopreserved and available in our biobank in different cell doses to comply any kind of medical needs. Currently several clinical needs can successfully benefit of our GMP LL-CBMSC including renal and lung repair, and GVHD.

Advanced Therapy Medicinal Products should be manufactured in aseptic condition following GMP and their sterility must be evaluated with validated techniques. In case of cryopreserved/thawed cellular products, sterility should be checked on a representative aliquot of thawed product to exclude the occurrence of microbial growth at any phase of the ATMP production, including liquid nitrogen storage. The last European Pharmacopoeia allows the use of rapid and automated colorimetric methods for the detection of microbial contamination in cellular products. Here we describe the validation of a rapid colorimetric method for microbial contamination (BacT/ALERT®, BioMérieux) on fresh and cryopreserved/thawed cord blood mesenchymal stem cells (CBMSC) manufactured in our approved GMP facility (last AIFA authorization 98/2013) following Eu. Ph. 2.6.27. The objectives of this study was to exclude any interference either from any component of the final medicinal products or from the cryopreservation/thawing process on the microbial growth. With this aim, ten microbial strains, as per Eu. Ph., were inoculated into BacT/ALERT® bottles together with the fresh and the cryopreserved/thawed (before and after washing) cellular product in triplicates and the results evaluated after 7 days incubation. Appropriate positive and negative controls were performed. The results demonstrated that the assay was able to detect microbial contamination by all the tested strains with a detectability limit of 10ˆ2 UFC/mL, except for Propionebacterium acnes in wich this limit was 10ˆ3 UFC/mL. This validation study, in compliance with Eu. Ph., demonstrated that there is no interference on the microbial growth revelled by an automated system from any component of either fresh or cryopreserved/thawed/washed cellular products. We recommend to follow appropriate validation studies shaped on the specific manufacturing process before adopting sterility testing for the quality control of ATMPs.

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Mesenchymal stem cells(MSCs)are multipotent cells that can be isolated from many sources, including bone marrow(BM), adipose tissue, umbilical cord blood, Wharton's Jelly and amniotic fluid. The use of these cells in a clinical trial requires that their production complies with Good Manufacturing Practice(GMP).The translation step is even more challenging when MSCs are obtained from an old and or diseased patient such as in the case of autologous approaches for degenerative disorders.Besides the patient/donor–related issues, critical factors in the expansion procedures are starting density, doubling rate, cell confluence, culture duration and use of FBS or other substitutes which could potentially influence the in vitro and in vivo cell properties. With regards to all these parameters, here we describe the GMP procedures and the extensive validation process to obtain BMMSCs suitable for different clinical applications. In particular, two GMP MSC manufacturing processes were validated:the production of BMMSC from healthy donors as off-the-shelf products for allogeneic use and from patients affected by a rare form of parkinsonism (Progressive Supranuclear Palsy) within a specific clinical protocol (NCT01824121).With each donor type, two different culture conditions with either bovine serum or platelet lysate were tested to choice the best culture conditions in consideration of the expected therapeutic effect. With this aim, we established protocols, standard operating procedures, production and quality control processes, we validated specific reagents suitable for clinical applications and clearly defined release specifications and final quality control tests. The evidences from this validation study support the concept that each clinical approach in the context of regenerative medicine needs a keen knowledge of the results that can be obtained by applying different procedures and a critical decision based on the specific clinical needs.

The production of Mesenchymal stem cells (MSC) for cellular therapy should fulfil the requirements of GMP rules. In this contest, the production process and the analytical methods for quality control should be validated to demonstrate standardization and compliance with quality requisites. In our hospital-based GMP facility we produce MSC from different sources and we developed and validated a set of quality control assays for final products: sterility and endotoxins assay (<0,25 EU/mL), absence of mycoplasma and adventitious viruses, cell dose, purity (as percentage of cells CD90+/CD105+/CD45- > 80%) and viability (as percentage of cells PI- > 80%). Sterility was validated following Eu. Ph. 2.6.27 with both the standard microbial strains and those isolated during environmental monitoring. The endotoxin method (Eu. Ph. 2.6.14) was validated at different dilutions of the final product to exclude inhibition. The method to detect mycoplasma was compliant to the Pharmacopoeia requirements (Ph 2.6.7). We also performed a viral validation to exclude the accidentally introduction of respiratory viruses, Cytomegalovirus and Epstein-Barr Virus during production. For cell counting, an automated method (NucleoCounter, Chemometec) was found to be more accurate (R2 > 0.9) and precise (CV < 10%) than a standard manual method (Burker chamber). Moreover, we assessed the repeatability and reproducibility (CV < 5%) of the immunophenotyping analysis of MSC by flow cytometry to determine the purity of our products as the percentage of cells that co-express CD90 and CD105 and is negative to CD45. Finally, cell viability by Annexin-PI analysis was evaluated on MSC after expansion and cryopreservation. All these methods have been included in the Investigational Medicinal Product Dossier of all the MSC-based products currently provided by our Institution in several phase I/II clinical trials already ongoing.