Objective-Mesenchymal stem cells (MSCs) are capable of modulating the immune system through interaction with a wide range of immune cells. This study investigates the hypothesis that interaction of MSCs with macrophages could play a significant role in their anti-inflammatory/ immune modulatory effects.Materials and Methods-MSCs were derived from bone marrow and monocytes were isolated from peripheral blood of healthy donors. We cultured human monocytes for seven days without any added cytokines to generate macrophages, and then co-cultured them for three more days with culture-expanded MSCs. We used cell surface antigen expression and intracellular cytokine expression patterns to study the immunophenotype of macrophages at the end of this co-culture period, and phagocytic assays to investigate their functional activity in vitro.Results-Macrophages co-cultured with MSCs consistently showed high level expression of CD206, a marker of alternatively activated macrophages. Furthermore, these macrophages expressed high levels of IL-10 and low levels of IL-12, as determined by intracellular staining, typical of alternatively activated macrophages. However, macrophages co-cultured with MSCs also expressed high levels of IL-6 and low levels of TNF-α, compared to controls. Functionally, macrophages cocultured with MSCs showed a higher level of phagocytic activity.Conclusions-We describe a novel type of human macrophage generated in vitro after co-culture with MSCs that assume an immunophenotype defined as IL-10 high, IL-12 low, IL-6 high and TNF-α low secreting cells. These MSC-educated macrophages may be a unique and novel type of alternatively activated macrophages with potentially significant role in tissue repair. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Keywords Conflict of Interest Disclosure:No financial interest/relationships with financial interest relating to the topic of this article have been declared. NIH Public Access Materials and methods Cell cultureWe used human blood and bone marrow to derive monocytes and MSCs, respectively. All protocols were approved by the Health Sciences Institutional Review Board of University of Wisconsin-Madison School of Medicine and Public Health. Monocytes were isolated from human peripheral blood by using magnetic bead separation methods according to manufacturers' protocol. Briefly, peripheral blood mononuclear cells were collected from blood of healthy donors by density gradient separation using Percoll (GE Healthcare Biosciences, Piscataway, NJ, USA). Red blood cells were lysed by incubating cells in ACK lysis buffe...

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.

Tisagenlecleucel is a CD19 chimeric antigen receptor (CAR) T-cell therapy approved for treatment of pediatric and young adult patients with relapsed/refractory acute lymphoblastic leukemia (ALL) and adults with non-Hodgkin lymphoma (NHL). The initial experience with tisagenlecleucel in a real-world setting from a cellular therapy registry is presented here. As of January 2020, 511 patients were enrolled from 73 centers, and 410 patients had follow-up data reported (ALL, n = 255; NHL, n = 155), with a median follow-up of 13.4 and 11.9 months for ALL and NHL, respectively. Among patients with ALL, the initial complete remission (CR) rate was 85.5%. Twelve-month duration of response (DOR), event-free survival, and overall survival (OS) rates were 60.9%, 52.4%, and 77.2%, respectively. Among adults with NHL, the best overall response rate was 61.8%, including an initial CR rate of 39.5%. Six-month DOR, progression-free survival, and OS rates were 55.3%, 38.7%, and 70.7%, respectively. Grade ≥3 cytokine release syndrome and neurotoxicity were reported in 11.6% and 7.5% of all patients, respectively. Similar outcomes were observed in patients with in-specification and out-of-specification products as a result of viability <80% (range, 61% to 79%). This first report of tisagenlecleucel in the real-world setting demonstrates outcomes with similar efficacy and improved safety compared with those seen in the pivotal trials.

Mesenchymal stromal/stem cells (MSCs) are multipotent stem cells present in most fetal and adult tissues. Ex vivo culture-expanded MSCs are being investigated for tissue repair and immune modulation, but their full clinical potential is far from realization. Here we review the role of oxidative stress in MSC biology, as their longevity and functions are affected by oxidative stress. In general, increased reactive oxygen species (ROS) inhibit MSC proliferation, increase senescence, enhance adipogenic but reduce osteogenic differentiation, and inhibit MSC immunomodulation. Furthermore, aging, senescence, and oxidative stress reduce their ex vivo expansion, which is critical for their clinical applications. Modulation of sirtuin expression and activity may represent a method to reduce oxidative stress in MSCs. These findings have important implications in the clinical utility of MSCs for degenerative and immunological based conditions. Further study of oxidative stress in MSCs is imperative in order to enhance MSC ex vivo expansion and in vivo engraftment, function, and longevity.

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34+ cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.

Although transplant practices have changed over the last decades there is no information on trends in incidence and outcome of cGVHD over time. This study utilized the central database of the Center for International Blood and Marrow Transplant Research (CIBMTR) to describe the time trends for cGVHD incidence, non-relapse mortality, and the risk factors for cGVHD. The 12-year period was divided into three intervals: 1995-1999, 2000-2003, 2004-2007, and included 26,563 patients with acute leukemia, chronic myeloid leukemia and myelodysplastic syndrome. In the multivariate analysis, the incidence of cGVHD was shown to be increased in more recent years (odds ratio= 1.19, p<0.0001) and this trend was still seen when adjusting for donor type, graft type, or conditioning intensity. In patients with cGVHD, non-relapse mortality has decreased over time, but at 5-years there were no significant differences among different time periods. Risk factors for cGVHD were in line with previous studies. This is the first comprehensive characterization of the trends in cGVHD incidence and underscores the mounting need for addressing this major late complication of transplantation in future research.

Objective-We have previously shown the simultaneous generation of CD73+ mesenchymal stromal cells (MSCs) along with CD34+ hematopoietic cells from human embryonic stem cells (ESCs) when they are co-cultured with OP9 murine stromal cells. We investigated whether MSCs can be derived from human ESCs without co-culturing with OP9 cells, and if such cells exhibit immunological properties similar to MSCs derived from adult human bone marrow (BM).Materials and Methods-Our starting populations were undifferentiated ESCs cultured on matrigel-coated plates without feeder cells. The differentiated fibroblast-looking cells were tested for expression of MSC markers and their potential for multi-lineage differentiation. We investigated surface expression of HLA molecules on these MSCs before and after treatment with interferon gamma (IFN-γ). We also tested the proliferative response of T-lymphocytes towards MSCs and the effects of MSCs in mixed lymphocyte reaction (MLR) assays. Conclusions-MSCs can be derived from human ESCs without feeder cells. These human ESCderived MSCs have cell surface markers, differentiation potentials, and immunological properties in vitro that are similar to adult BM-derived MSCs. Results-We

IntroductionThe ability to transfer genes into repopulating hematopoietic stem cells ex vivo and to achieve regulated expression in specific lineages following hematopoietic reconstitution would create many therapeutic opportunities. 1 Although the initial use of murine oncoretroviral vectors to transfer genes into primitive murine hematopoietic cells was reported 20 years ago, 2 translation of this approach to clinical application has been slow and has required considerable effort. Despite progress being achieved in the murine system with correction of single gene defects in murine models of human immunodeficiencies [3][4][5][6] and chronic granulomatous disease, 7,8 the much lower efficiency of gene transfer into human stem cells 1 has hampered success. The necessity for high-level oncoretroviral vector gene transfer to achieve therapeutic benefit, however, was circumvented in 2 recent clinical trials designed to cure severe combined immunodeficiency due to a deficiency of the common ␥-chain of the lymphoid cytokine receptor 9 or adenosine deaminase. 10 In these trials, a potent selective repopulating advantage of the gene-corrected lymphoid cells resulted in therapeutically relevant numbers of functional lymphocytes.Despite this success, 2 barriers appear to limit the ability of murine oncoretroviral vectors to achieve adequate transduction of primitive hematopoietic stem cells for treatment of other disorders in which the gene-corrected cells do not have a selective advantage. Because the human homolog of the receptor for murine ecotropic vector particles does not interact with the ecotropic envelope protein, amphotropic particles have been used in both human studies and in large animal models. The amphotropic receptor, however, is expressed at low levels on human stem cells. 11 Alternative envelopes have been tested, such as those derived from the gibbon ape leukemia virus, 12 feline endogenous virus (RD114), 13 or feline leukemia virus type C, 14 the receptors for which are expressed at higher levels on primitive hematopoietic cell populations. However, large animal studies have failed to clearly identify a superior pseudotype that consistently yields high-level stem cell gene transfer. These data suggest that a second barrier-namely, the requirement for mitosis to allow integration of the oncoretroviral vector genome 15 -along with the relative instability of the preintegration complex 16 may be the main limitations of oncoretroviral-mediated stem cell gene transfer. In an effort to induce stem cell cycling, cytokines such as stem cell factor (SCF), Flt-3 ligand (Flt3-L), and megakaryocyte growth and development factor (MGDF) 17,18 are added to culture medium and a fragment of fibronectin is used to colocalize vector particles and target cells, 19 leading to improved stem cell transduction efficiency in large animal models. 12,20,21 Nonetheless, there remains significant variability among animals, with many animals having low marking and only occasional animals having proportions of genetically modifi...