A Good Manufacturing Practice-grade standard protocol for exclusively human mesenchymal stromal cell-derived extracellular vesicles.

BACKGROUND AIMS: Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal-derived xenogenic additives and depleted of EVs. METHODS: Depletion of EVs from complete growth medium was achieved by centrifugation at 120 000 g for 3 h, which reduced RNA-containing pHPL EVs to below the detection limit. RESULTS: Bone marrow (BM)-derived MSCs propagated in this medium retained the characteristic surface marker expression, cell morphology, viability and in vitro osteogenic and adipogenic differentiation potential. The proliferation rate was not significantly affected after 48 h but was decreased by 13% after 96 h. EVs collected from BM-MSCs cultured in EV-depleted medium revealed a similar RNA pattern as EVs generated in standard pHPL EV-containing medium but displayed a more clearly defined pattern of proteins characteristic for EVs. Reduction of pHPL content from 10% to 2% or serum-/pHPL-free conditions strongly altered MSC characteristics and RNA content of released EV. CONCLUSIONS: The 10% pHPL-based EV-depleted medium is appropriate for purification of exclusively human MSC-derived EVs. With this Good Manufacturing Practice-grade protocol, characterization and establishment of protein and RNA profiles from MSC-derived EVs can now be achieved to identify active components in therapeutic EVs for future clinical application.

An alternative mini buffy coat preparation method for adult patients with extracorporeal photopheresis contraindications.

BACKGROUND: Extracorporeal photopheresis (ECP) is an important cell-based therapy for various diseases but is limited to patients eligible for apheresis. We developed an alternative mini buffy coat (BC) preparation method using the Spectra Optia® apheresis system and compared its efficacy of white blood cell (WBC) recovery with the standard mini BC preparation method already established for pediatric patients. METHODS: Whole blood (450 ± 45 mL) samples were collected from 30 randomly selected healthy volunteer blood donors and divided into two groups. In the first group, WBCs were separated with a fully automated separator device (Compomat G4® ). In the second group, BCs were separated with the bone marrow processing program of the Spectra Optia apheresis system. RESULTS: There were no significant differences in total leukocyte counts per product between the two groups. In contrast, lymphocyte counts per product were significantly higher (P < 0.001) in BCs separated from apheresis. CONCLUSION: Our novel technique resulted in similar WBC yields but higher lymphocyte yields than the standard mini BC preparation method. This method can serve as an alternative to WBC collection in conventional ECP for adult patients with apheresis contraindications. J. Clin. Apheresis 32:12-15, 2017. © 2016 Wiley Periodicals, Inc.

A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair.

RNA trans-splicing represents an auspicious option for the correction of genetic mutations at RNA level. Mutations within COL7A1 causing strong reduction or absence of type VII collagen are associated with the severe skin blistering disease dystrophic epidermolysis bullosa. The human COL7A1 mRNA constitutes a suitable target for this RNA therapy approach, as only a portion of the almost 9 kb transcript has to be delivered into the target cells. Here, we have proven the feasibility of 5' trans-splicing into the Col7a1 mRNA in vitro and in vivo. We designed a 5' RNA trans-splicing molecule, capable of replacing Col7a1 exons 1-15 and verified it in a fluorescence-based trans-splicing model system. Specific and efficient Col7a1 trans-splicing was confirmed in murine keratinocytes. To analyze trans-splicing in vivo, we used gene gun delivery of a minicircle expressing a FLAG-tagged 5' RNA trans-splicing molecule into the skin of wild-type mice. Histological and immunofluorescence analysis of bombarded skin sections revealed vector delivery and expression within dermis and epidermis. Furthermore, we have detected trans-spliced type VII collagen protein using FLAG-tag antibodies. In conclusion, we describe a novel in vivo nonviral RNA therapy approach to restore type VII collagen expression for causative treatment of dystrophic epidermolysis bullosa.

Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate.

BACKGROUND: Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin. METHODS: We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages. RESULTS: The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure. CONCLUSIONS: Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

Matrix-degrading podosomes in smooth muscle cells.

Activation of protein kinase C by phorbol esters triggers the remodelling of the actin cytoskeleton and the formation of podosomes in smooth muscle cells (SMCs). Regional control of actin dynamics at specialised microdomains results in a local reduction in contractile forces. The molecular basis for this local inhibition of contractility includes the clustering of cortactin during podosome formation (which precedes the rapid, local dispersion of myosin, tropomyosin and h1 calponin), and the specific recruitment of 110-kDa actin filament-associated protein (AFAP-110) and 190-kDa Rho-specific GTPase-activating protein (p190RhoGAP) to the microdomains. Podosome formation also correlates with cell polarisation, the induction of cell motility, and local degradation of the extracellular matrix. These findings may provide explanations for the complex mechanisms underlying SMC invasion in the course of the development of atherosclerotic lesions and restenosis, and support the concept that matrix degradation and the concomitant engagement of the molecular machinery initiating actin-based cell motility drive tissue invasion in smooth muscle.

Expression profiling of aging in the human skin.

During the last years it was shown that the aging process is controlled by specific genes in a large number of organisms (C. elegans, Drosophila, mouse or humans). To investigate genes involved in the natural aging process of the human skin we applied cDNA microarray analysis of naturally aged human foreskin samples. For the array experiments a non-redundant set of 2135 pre-selected EST clones was used. These arrays were used to probe the patterns of gene expression in naturally aged human skin of five young (3-4 years of age) and five old (68-72 years of age) healthy persons. We found that in total 105 genes change their expression over 1.7-fold during the aging process in the human skin. Of these 43 genes were shown to be down-regulated in contrast to 62 up-regulated genes. Expression of regulated genes was confirmed by real-time PCR. These results suggest that the aging process in the human skin is connected with the deregulation of various cellular processes, like cell cycle control, cytoskeletal changes, inflammatory response, signaling and metabolism.

The role of calponin in the gene profile of metastatic cells: inhibition of metastatic cell motility by multiple calponin repeats.

Metastasis of diseased cells is the basic event leading to death in individuals with cancer. Establishment of metastasis requires that tumour cells migrate from the site of the primary tumour into the circulation system, escape from the vasculature and form secondary tumours at novel sites. These processes depend to a large degree on cytoskeletal remodeling. We show here that multiple copies of the short actin-binding module CLIK(23) from human or Caenorhabditis elegans calponin proteins effectively inhibit cell motility on two dimensional matrices and suppress soft agar colony formation of metastatic melanoma and adenocarcinoma cells of murine and human origin. Ectopic expression of CLIK(23) modules for 30 days results in the formation of multinucleated cells. The repeat displays true modular behaviour, resulting in increased cytoskeletal effects in direct correlation with the increase in number of modules. Our results demonstrate that the role of calponin in the signature profile of metastasising cells is that of a mechanical stabiliser of the actin cytoskeleton, which interferes with actin turnover by binding at a unique interface along the actin filament.