No abstract

Small extracellular vesicles (sEVs) from mesenchymal stromal/stem cells (MSCs) are transiting rapidly towards clinical applications. However, discrepancies and controversies about the biology, functions, and potency of MSC-sEVs have arisen due to several factors: the diversity of MSCs and their preparation; various methods of sEV production and separation; a lack of standardized quality assurance assays; and limited reproducibility of in vitro and in vivo functional assays. To address these issues, members of four societies (SOCRATES, ISEV, ISCT and ISBT) propose specific harmonization criteria for MSC-sEVs to facilitate data sharing and comparison, which should help to advance the field towards clinical applications. Specifically, MSC-sEVs should be defined by quantifiable metrics to identify the cellular origin of the sEVs in a preparation, presence of lipid-membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. For practical purposes, new MSC-sEV preparations might also be measured against a well-characterized MSC-sEV biological reference. The ultimate goal of developing these metrics is to map aspects of MSC-sEV biology and therapeutic potency onto quantifiable features of each preparation.

Mesenchymal stromal cells (MSCs) as a pharmaceutical for ailments characterized by pathogenic autoimmune, alloimmune and inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic sponsored studies. There is a broad consensus that despite different tissue sourcing and varied culture expansion protocols, human MSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repair functionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methods of measurements of such would benefit the field. This would satisfy both mechanistic research as well as development of release potency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual registration. In response to this unmet need, the International Society for Cellular Therapy (ISCT) addressed the issue at an international workshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused on discussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune disorders. We here provide consensus perspective arising from this forum. We propose that focused analysis of selected MSC markers robustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Regulatory Authorities. Workshop participants identified three preferred analytic methods that could inform a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome. We also advocate that potency assays acceptable to the Regulatory Authorities be rendered publicly accessible in an “open-access” manner, such as through publication or database collection.

The immune suppression inherent in allogeneic stem cell transplantation (SCT) offers a favorable environment for infection by opportunistic agents, such as human cytomegalovirus (CMV). Despite the application of potent antiviral prophylaxis, patients remain at risk for CMV infection until adequate immunity is restored. CMV-specific CD8(+) T cell counts were monitored, using HLA-A2 tetrameric complexes, to establish the level of immune response to the viral phosphoprotein UL83 in patients after allogeneic SCT. Correlating this with viral replication and clinical status shows that the level of tetramer-positive T cells provides an assessment of CMV immune reconstitution after stem cell transplantation. Most patients with seropositive donors did reconstitute long-term CMV immunity, unless prolonged immunosuppression to control graft-versus-host disease was induced. Together with polymerase chain reaction testing, this technique provides measurable parameters that can be a guide to therapeutic decision making and can form the basis of CMV immunotherapy.

Adult ECMO patients with lower Hb(nadir) require more daily red blood cell and FFP. Hypertension increases daily FFP requirements. Recent antiplatelet agents, larger Hb decline and longer ECMO duration increase daily platelet requirements. Patients with sepsis or on ECMO for medical reasons have longer ECMO duration, which is associated with total transfusion requirements. Some of these factors may be identified early to optimize blood product support.

Robust and rapid induction of interferon-␤ (IFN-␤) in monocytes after pathogenic stimulation is a hallmark of innate immune responses. Here, we reveal the molecular mechanism underlying this key property that is exclusive to human blood monocytes. We found that IFN-␤ was produced rapidly in primary human monocytes as a result of cooperation between the myeloid-specific transcription factor IRF8 and the ubiquitous transcription factor IRF3. Knockdown of IRF8 in monocytes abrogated IFN-␤ transcription, whereas reintroduction of IRF8 into the IRF8 ؊/؊ 32Dcl3 murine myeloid cell line reinstated IFN-␤ transcription. Moreover, we provide evidence that IRF8 constitutively binds to the ETS/IRF composite element of the IFN-␤ promoter region together with PU.1 in vivo. Furthermore we uncovered a requirement for IRF3, a master regulator of IFN-␤ production, as a previously un-indentified interaction partner of IRF8. We mapped the protein- IntroductionProduction of interferon-␤ (IFN-␤) by the host is an essential first line of defense against both viral and nonviral pathogens. [1][2][3] As well as its involvement in innate immunity, IFN-␤ is an important initiator and modulator of the adaptive immune response. [4][5][6] For example, IFN-␤ influences the differentiation, maturation, and migration of dendritic cells. 7 In addition, it regulates antibody production and antigen-mediated apoptosis of B cells, 8 alongside promoting the generation of CD4 ϩ Th1 responses, 9 and the expansion of antigen-specific CD8 ϩ T cells. 10 Interestingly, Stockinger et al have recently demonstrated that IFN-␤ production contributes to Listeria monocytogenes-induced lethality in mice. 11 As such, IFN-␤ seems able to induce immunopathology as well as influence the resolution of infections, thus making it important to understand the mechanisms linking pathogen stimulation of immune cells with the production of IFN-␤.IFN-␤ transcription is initiated after cellular recognition of pathogen-associated molecular patterns by pattern recognition receptors. 12 Many cell types express IFN-␤ after microbial exposure, [13][14][15] although the underlying mechanisms vary. In murine embryonic fibroblasts, IFN-␤ induction is mediated by the activation of both IRF3 and IRF7. 16,17 In contrast, induction of IFN-␤ in murine plasmacytoid dendritic cells occurs after CpG recognition by Toll-like receptor 9 and is entirely dependent on IRF7, 18 whereas in murine macrophages transcription of IFN-␤ is dependent on IRF3 alone. 19 IRF3 is a ubiquitous cytosolic transcription factor that translocates to the nucleus on activation, 20 where it is able to induce IFN-␤ expression. However, this process takes more than 6 hours to produce detectable levels of IFN-␤ mRNA in HeLa cells, with levels peaking even later at 9 to 19 hours. 21 Monocytes are myeloid cells of the immune system that play a key role in the rapid innate response to pathogens, including the production of IFN-␤. 11,22 However, the molecular mechanisms underlying the rapid induction of IFN-␤ in monocytes hav...

A state‐of‐the‐art workshop focused on the use of human platelet lysate (HPL) for cell therapy. The meeting established that HPL is used mainly as an adjunct material for ex vivo expansion of mesenchymal stem/progenitor cells (MSCs), where it is successfully used as a substitute for fetal bovine serum. HPL manufacturing as a cell expansion supplement is currently not yet uniformly standardized with regard to platelet source and production methodology. There are very few reports of HPL preparations manufactured specifically for direct clinical use. There exists an urgent need for controlled clinical studies for HPL and for standardization of product definition. Workshop participants also stated a need for consensus minimum release criteria to allow for better product definition and to limit variability in performance. The increasing use of cell‐based therapies including MSCs has led to an increasing demand for HPL, either produced in blood establishments or large‐scale manufacture by biopharmaceutical companies. The use of pooled donor platelets for HPL production may require the implementation of pathogen inactivation procedures and/or removal steps to improve the safety of advanced cell therapy products. There should also be a requirement for thorough risk assessments and risk mitigation steps, including the qualification of suppliers and identification of ingredients as well as meticulous monitoring of product quality and safety profiles. State‐of‐the‐art regulatory approaches for HPL used for human cell propagation and PRP in direct clinical applications were reviewed.

Cell and gene therapies (CGTs) are progressively entering into clinical practice in different parts of the world. The International Society for Cell & Gene Therapy (ISCT), a global scientific society, has been committed since 1992 to supporting and developing knowledge on clinical applications of CGTs. Considering the number of products that have been progressively approved and, in some cases, withdrawn in recent years, the ISCT would like to present a brief annual report on CGTs with marketing authorization (MA) in different regions. This article reflects the dynamic momentum around authorized CGTs coinciding with the parallel increase of unproven approaches where cells are delivered without appropriate and rigorous scientific and regulatory assessment and authorization. This is intended to be a living document with a yearly update linked to a dedicated section of the ISCT website for faster adjustments. The aim is to ultimately inform, by periodic snapshots, the scientific community, healthcare stakeholders and patient associations on authorized CGT products as a way to increase communication around the approved therapeutic approaches charged with heightened expectations.