A Roadmap for the Production of a GMP-Compatible Cell Bank of Allogeneic Bone Marrow-Derived Clonal Mesenchymal Stromal Cells for Cell Therapy Applications

Background: Allogeneic mesenchymal stromal cells (MSCs) have been used extensively in various clinical trials. Nevertheless, there are concerns about their efficacy, attributed mainly to the heterogeneity of the applied populations. Therefore, producing a consistent population of MSCs is crucial to improve their therapeutic efficacy. This study presents a good manufacturing practice (GMP)-compatible and cost-effective protocol for manufacturing, banking, and lot-release of a homogeneous population of human bone marrow-derived clonal MSCs (cMSCs). Methods: Here, cMSCs were isolated based on the subfractionation culturing method. Afterward, isolated clones that could reproduce up to passage three were stored as the seed stock. To select proliferative clones, we used an innovative, cost-effective screening strategy based on lengthy serial passaging. Finally, the selected clones re-cultured from the seed stock to establish the following four-tired cell banking system: initial, master, working, and end of product cell banks (ICB, MCB, WCB, and EoPCB). Results: Through a rigorous screening strategy, three clones were selected from a total of 21 clones that were stored during the clonal isolation process. The selected clones met the identity, quality, and safety assessments criteria. The validated clones were stored in the four-tiered cell bank system under GMP conditions, and certificates of analysis were provided for the three-individual ready-to-release batches. Finally, a stability study validated the EoPCB, release, and transport process of the frozen final products. Conclusion: Collectively, this study presents a technical and translational overview of a GMP-compatible cMSCs manufacturing technology that could lead to the development of similar products for potential therapeutic applications. Graphical Abstract: [Figure not available: see fulltext.

Two Decades of Global Progress in Authorized Advanced Therapy Medicinal Products: An Emerging Revolution in Therapeutic Strategies

The introduction of advanced therapy medicinal products (ATMPs) to the global pharma market has been revolutionizing the pharmaceutical industry and has opened new routes for treating various types of cancers and incurable diseases. In the past two decades, a noticeable part of clinical practices has been devoting progressively to these products. The first step to develop such an ATMP product is to be familiar with other approved products to obtain a general view about this industry trend. The present paper depicts an overall perspective of approved ATMPs in different countries, while reflecting the degree of their success in a clinical point of view and highlighting their main safety issues and also related market size as a whole. In this regard, published articles regarding safety, efficacy, and market size of approved ATMPs were reviewed using the search engines PubMed, Scopus, and Google Scholar. For some products which the related papers were not available, data on the relevant company website were referenced. In this descriptive study, we have introduced and classified approved cell, gene, and tissue engineering-based products by different regulatory agencies, along with their characteristics, manufacturer, indication, approval date, related regulatory agency, dosage, product description, price and published data about their safety and efficacy. In addition, to gain insights about the commercial situation of each product, we have gathered accessible sale reports and market size information that pertain to some of these products

Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation-mediated instability using small molecules

Regulatory T (T-reg) cell therapy is a promising approach for immune tolerance induction in autoimmunity conditions and cell/organ transplantations. Insufficient isolation yields and impurity during downstream processes and T-reg instability after adoptive transfer in inflammatory conditions are major limitations to T-reg therapy, and indicate the importance of seeking a valid, reliable method for de-novo generation of T-regs. In this research, we evaluated T-reg-like cells obtained from different T-reg differentiation protocols in terms of their yield, purity and activity. Differentiation was performed on naive CD4(+) cells and a naive CD4(+)/T-reg co-culture by using three different protocols - ectopic expression of forkhead box protein P3 (E-FoxP3), soluble transforming growth factor beta (S-TGF) and small molecules N-acetyl puromycin and SR1555 (N-Ac/SR). The results showed that a high yield of a homogeneous population of T-reg-like cells could be achieved by the N-Ac/SR method under a T helper type 17 (Th17)-polarizing condition, particularly interleukin (IL)-6 and TGF-beta, when compared with the E-FoxP3 and S-TGF methods. Surprisingly, SR completely inhibited the differentiation of IL-17-producing cells and facilitated T-reg generation in the inflammatory condition and had highly suppressive activity against T cell proliferation without T-reg-specific demethylase region (TSDR) demethylation. For the first time, to our knowledge, we report the generation of efficient, pure T-reg-like cells by using small molecules during in-vitro inflammatory conditions. Our results suggested that the N-Ac/SR method has several advantages for T-reg generation when compared with the other methods, including a higher purity of T-regs, easier procedure, superior suppressive activity during the inflammatory condition and decreased cost

Inducible VEGF Expression by Human Embryonic Stem Cell-Derived Mesenchymal Stromal Cells Reduces the Minimal Islet Mass Required to Reverse Diabetes

International audienceIslet transplantation has been hampered by loss of function due to poor revascularization. We hypothesize that co-transplantation of islets with human embryonic stem cell-derived mesenchymal stromal cells that conditionally overexpress VEGF (hESC-MSC:VEGF) may augment islet revascularization and reduce the minimal islet mass required to reverse diabetes in mice. HESC-MSCs were transduced by recombinant lentiviruses that allowed conditional (Dox-regulated) overexpression of VEGF. HESC-MSC:VEGF were characterized by tube formation assay. After co-transplantation of hESC-MSC:VEGF with murine islets in collagen-fibrin hydrogel in the omental pouch of diabetic nude mice, we measured blood glucose, body weight, glucose tolerance and serum C-peptide. As control, islets were transplanted alone or with non-transduced hESC-MSCs. Next, we compared functional parameters of 400 islets alone versus 200 islets co-transplanted with hESC-MSC:VEGF. As control, 200 islets were transplanted alone. Metabolic function of islets transplanted with hESC-MSC:VEGF significantly improved, accompanied by superior graft revascularization, compared with control groups. Transplantation of 200 islets with hESC-MSC:VEGF showed superior function over 400 islets alone. We conclude that co-transplantation of islets with VEGF-expressing hESC-MSCs allowed for at least a 50% reduction in minimal islet mass required to reverse diabetes in mice. This approach may contribute to alleviate the need for multiple donor organs per patient

A complex approach to noninvasive estimation of microcirculatory tissue impairments in feet of patients with diabetes mellitus using spectroscopy

The possibility of a complex approach for studying changes in the system of blood microcirculation and metabolic processes in the biotissue of lower extremities using optical noninvasive methods of laser doppler flowmetry (LDF), fluorescence spectroscopy, and diffuse reflectance spectroscopy in combination with different modes of heating tests has been assessed. Seventy-six patients with type 2 diabetes mellitus, with 14 patients having visible trophic foot impairments, and 48 healthy volunteers have been examined. The parameters of LDF signals and spectra of fluorescence intensity and diffuse reflectance for foot skin have been analyzed. Statistically significant differences in the recorded parameters between the groups under study have been found. It has been concluded that combined application of noninvasive methods of spectroscopy could be used for diagnostics of complications both upon the occurrence of preliminary symptoms of diabetes, when pathological changes are still reversible, and in the presence of impairments to prevent aggravation of the disease and select an adequate correction of the treatment