Simulated microgravity impairs human NK cell cytotoxic activity against space radiation-relevant leukemic cells.

Natural killer (NK) cells are an important first-line of defense against malignant cells. Because of the potential for increased cancer risk from astronaut exposure to space radiation, we determined whether microgravity present during spaceflight affects the body's defenses against leukemogenesis. Human NK cells were cultured for 48 h under normal gravity and simulated microgravity (sµG), and cytotoxicity against K-562 (CML) and MOLT-4 (T-ALL) cells was measured using standard methodology or under continuous sµG. This brief exposure to sµG markedly reduced NK cytotoxicity against both leukemias, and these deleterious effects were more pronounced in continuous sµG. RNA-seq performed on NK cells from two additional healthy donors provided insight into the mechanism(s) by which sµG reduced cytotoxicity. Given our prior report of space radiation-induced human T-ALL in vivo, the reduced cytotoxicity against MOLT-4 is striking and raises the possibility that µG may increase astronaut risk of leukemogenesis during prolonged missions beyond LEO.

Simulated Microgravity Impairs Human NK Cell Cytotoxic Activity Against Space Radiation-Relevant Leukemic Cells.

Natural killer (NK) cells are important effectors of the innate immune system. Unlike T cells, NK cells do not require antigen-priming, making them an important first-line of defense against malignant cells. Because of the potential for increased cancer risk as a result of astronaut exposure to space radiation, we performed studies to determine whether conditions of microgravity present during spaceflight affects the body's natural defenses against leukemogenesis. Human NK cells were cultured for 48 hours under normal gravity and simulated microgravity (sµG), and cytotoxicity against K-562 (CML) and MOLT-4 (T-ALL) cell lines was measured using standard methodology or under continuous conditions of sµG. Even this brief exposure to sµG markedly reduced NK cytotoxicity against both leukemic cells using standard assay procedures, and these deleterious effects were even more pronounced in continuous sµG. RNA-seq performed on NK cells from two healthy donors provided insight into the mechanism(s) by which sµG reduced cytotoxicity. Given our prior report that human HSC exposed to simulated space radiation gave rise to T-ALL in vivo , the reduced cytotoxicity against MOLT-4 is striking and raises the possibility that µG may add to astronaut risk of leukemogenesis during prolonged missions beyond LEO.

Immediate effects of acute Mars mission equivalent doses of SEP and GCR radiation on the murine gastrointestinal system-protective effects of curcumin-loaded nanolipoprotein particles (cNLPs).

Introduction: Missions beyond low Earth orbit (LEO) will expose astronauts to ionizing radiation (IR) in the form of solar energetic particles (SEP) and galactic cosmic rays (GCR) including high atomic number and energy (HZE) nuclei. The gastrointestinal (GI) system is documented to be highly radiosensitive with even relatively low dose IR exposures capable of inducing mucosal lesions and disrupting epithelial barrier function. IR is also an established risk factor for colorectal cancer (CRC) with several studies examining long-term GI effects of SEP/GCR exposure using tumor-prone APC mouse models. Studies of acute short-term effects of modeled space radiation exposures in wildtype mouse models are more limited and necessary to better define charged particle-induced GI pathologies and test novel medical countermeasures (MCMs) to promote astronaut safety. Methods: In this study, we performed ground-based studies where male and female C57BL/6J mice were exposed to γ-rays, 50 MeV protons, or 1 GeV/n Fe-56 ions at the NASA Space Radiation Laboratory (NSRL) with histology and immunohistochemistry endpoints measured in the first 24 h post-irradiation to define immediate SEP/GCR-induced GI alterations. Results: Our data show that unlike matched γ-ray controls, acute exposures to protons and iron ions disrupts intestinal function and induces mucosal lesions, vascular congestion, epithelial barrier breakdown, and marked enlargement of mucosa-associated lymphoid tissue. We also measured kinetics of DNA double-strand break (DSB) repair using gamma-H2AX- specific antibodies and apoptosis via TUNEL labeling, noting the induction and disappearance of extranuclear cytoplasmic DNA marked by gamma-H2AX only in the charged particle-irradiated samples. We show that 18 h pre-treatment with curcumin-loaded nanolipoprotein particles (cNLPs) delivered via IV injection reduces DSB-associated foci levels and apoptosis and restore crypt villi lengths. Discussion: These data improve our understanding of physiological alterations in the GI tract immediately following exposures to modeled space radiations and demonstrates effectiveness of a promising space radiation MCM.

Autologous bone marrow-derived MSCs engineered to express oFVIII-FLAG engraft in adult sheep and produce an effective increase in plasma FVIII levels.

Introduction: Hemophilia A (HA) is the most common X-linked bleeding disorder, occurring in 1 in 5,000 live male births and affecting >1 million individuals worldwide. Although advances in protein-based HA therapeutics have improved health outcomes, current standard-of-care requires infusion 2-3 times per week for life, and 30% of patients develop inhibitors, significantly increasing morbidity and mortality. There are thus unmet medical needs requiring novel approaches to treat HA. Methods: We tested, in a highly translational large animal (sheep) model, whether the unique immunological and biological properties of autologous bone marrow (BM)-derived mesenchymal stromal cells (MSCs) could enable them to serve as cellular delivery vehicles to provide long-term expression of FVIII, avoiding the need for frequent infusions. Results: We show that autologous BM-MSCs can be isolated, transduced with a lentivector to produce high levels of ovine (o)FVIII, extensively expanded, and transplanted into adult animals safely. The transplanted cells engraft in multiple organs, and they stably produce and secrete sufficient quantities of FVIII to yield elevated plasma FVIII levels for at least 15 weeks. Discussion: These studies thus highlight the promise of cellular-based gene delivery approaches for treating HA.

Curcumin Nanodiscs Improve Solubility and Serve as Radiological Protectants against Ionizing Radiation Exposures in a Cell-Cycle Dependent Manner.

Curcumin, a natural polyphenol derived from the spice turmeric (Curcuma longa), contains antioxidant, anti-inflammatory, and anti-cancer properties. However, curcumin bioavailability is inherently low due to poor water solubility and rapid metabolism. Here, we further refined for use curcumin incorporated into "biomimetic" nanolipoprotein particles (cNLPs) consisting of a phospholipid bilayer surrounded by apolipoprotein A1 and amphipathic polymer scaffolding moieties. Our cNLP formulation improves the water solubility of curcumin over 30-fold and produces nanoparticles with ~350 µg/mL total loading capacity for downstream in vitro and in vivo applications. We found that cNLPs were well tolerated in AG05965/MRC-5 human primary lung fibroblasts compared to cultures treated with curcumin solubilized in DMSO (curDMSO). Pre-treatment with cNLPs of quiescent G0/G1-phase MRC-5 cultures improved cell survival following 137Cs gamma ray irradiations, although this finding was reversed in asynchronously cycling log-phase cell cultures. These findings may be useful for establishing cNLPs as a method to improve curcumin bioavailability for administration as a radioprotective and/or radiomitigative agent against ionizing radiation (IR) exposures in non-cycling cells or as a radiosensitizing agent for actively dividing cell populations, such as tumors.

Regenerative medicine: prenatal approaches.

This two-paper Series focuses on recent advances and applications of regenerative medicine that could benefit paediatric patients. Innovations in genomic, stem-cell, and tissue-based technologies have created progress in disease modelling and new therapies for congenital and incurable paediatric diseases. Prenatal approaches present unique opportunities associated with substantial biotechnical, medical, and ethical obstacles. Maternal plasma fetal DNA analysis is increasingly adopted as a noninvasive prenatal screening or diagnostic test for chromosomal and monogenic disorders. The molecular basis for cell-free DNA detection stimulated the development of circulating tumour DNA testing for adult cancers. In-utero stem-cell, gene, gene-modified cell (and to a lesser extent, tissue-based) therapies have shown early clinical promise in a wide range of paediatric disorders. Fetal cells for postnatal treatment and artificial placenta for ex-utero fetal therapies are new frontiers in this exciting field.

Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII.

Introduction: Placenta-derived mesenchymal cells (PLCs) endogenously produce FVIII, which makes them ideally suited for cell-based fVIII gene delivery. We have previously reported that human PLCs can be efficiently modified with a lentiviral vector encoding a bioengineered, expression/secretion-optimized fVIII transgene (ET3) and durably produce clinically relevant levels of functionally active FVIII. The objective of the present study was to investigate whether CRISPR/Cas9 can be used to achieve location-specific insertion of a fVIII transgene into a genomic safe harbor, thereby eliminating the potential risks arising from the semi-random genomic integration inherent to lentiviral vectors. We hypothesized this approach would improve the safety of the PLC-based gene delivery platform and might also enhance the therapeutic effect by eliminating chromatin-related transgene silencing. Methods: We used CRISPR/Cas9 to attempt to insert the bioengineered fVIII transgene "lcoET3" into the AAVS1 site of PLCs (CRISPR-lcoET3) and determined their subsequent levels of FVIII production, comparing results with this approach to those achieved using lentivector transduction (LV-lcoET3) and plasmid transfection (Plasmid-lcoET3). In addition, since liver-derived sinusoidal endothelial cells (LSECs) are the native site of FVIII production in the body, we also performed parallel studies in human (h)LSECs). Results: PLCs and hLSECs can both be transduced (LV-lcoET3) with very high efficiency and produce high levels of biologically active FVIII. Surprisingly, both cell types were largely refractory to CRISPR/Cas9-mediated knockin of the lcoET3 fVIII transgene in the AAVS1 genome locus. However, successful insertion of an RFP reporter into this locus using an identical procedure suggests the failure to achieve knockin of the lcoET3 expression cassette at this site is likely a function of its large size. Importantly, using plasmids, alone or to introduce the CRISPR/Cas9 "machinery", resulted in dramatic upregulation of TLR 3, TLR 7, and BiP in PLCs, compromising their unique immune-inertness. Discussion: Although we did not achieve our primary objective, our results validate the utility of both PLCs and hLSECs as cell-based delivery vehicles for a fVIII transgene, and they highlight the hurdles that remain to be overcome before primary human cells can be gene-edited with sufficient efficiency for use in cell-based gene therapy to treat HA.

Functional characterization of the immunomodulatory properties of human urine-derived stem cells.

BACKGROUND: Urine-derived stem cells (USCs) have been widely researched as a novel cell source for stem cell therapy, but their immunomodulatory characteristics remain to be investigated. This study aimed to characterize the immunomodulatory properties of human USCs. METHODS: Human USCs were isolated from fresh voiding urine samples from healthy male donors and expanded. Their cell surface markers were characterized by flow cytometry analysis and the telomerase activities for several USCs clones were determined. The immunosuppressive potential of USCs was evaluated by the performing the mixed lymphocyte reaction (MLR) [co-culture with peripheral blood mononuclear cells (PBMNCs)] and natural killer cells (NK) cytotoxicity assay. USCs cytokines release profile was determined by using human cytokine proteome array. RESULTS: USCs exhibited high cell surface expression of embryonic/mesenchymal stem cells (MSCs) markers CD29, CD44, CD54, CD73, CD90, CD146, and CD166, while lacked expression of hematopoietic stem cell markers CD11, CD14, CD19, CD31, CD34, CD45, B cell marker CD79, and co-stimulatory factors CD80 and CD86, thus, exhibiting the phenotype of MSCs. MLR indicated that USCs significantly inhibited the proliferation of PBMNCs, as compared to that of the human smooth muscle cells (SMCs). In cell cytotoxicity assays, NK cells displayed less cytotoxicity against USCs than against bone marrow mesenchymal stem cells (BMSCs) and SMCs. Furthermore, upon PBMNCs stimulation, USCs secreted higher levels of immunomodulatory cytokines, including IL-6, IL-8, MCP-1, RANTES, GROα, and GM-CSF, compared to those of BMSCs, especially when directly contact mix-culture with PBMNCs. CONCLUSIONS: USCs secreted immunoregulatory cytokines and possessed immunomodulatory properties, comparable to those of BMSCs.

Gene and Stem Cell Therapies for Fetal Care: A Review.

Importance: Gene and stem cell therapies have become viable therapeutic options for many postnatal disorders. For select conditions, prenatal application would provide improved outcomes. The fetal state allows for several theoretical advantages over postnatal therapy, including immune immaturity and cellular niche accessibility. Observations: Advances in prenatal diagnostic accuracy and surgical precision, as well as improvements in stem cell and gene therapy methods, have made prenatal gene and stem cell therapy realistic. Studies in mouse models and early human trials demonstrate the feasibility of these approaches. Additional efforts are under way to streamline fetal applications of stem cell and gene therapy while carefully considering best ethical practice and following established regulatory pathways. Conclusions and Relevance: Fetal stem cell and gene therapy bring important therapeutic opportunities for select disorders that present in the fetal and neonatal periods. While this field is in its infancy, these therapies are starting to be available clinically, and clinicians should be aware of their benefits and challenges.

Therapeutic Mesenchymal Stromal Cells for Immunotherapy and for Gene and Drug Delivery.

Mesenchymal stromal cells (MSCs) possess several fairly unique properties that, when combined, make them ideally suited for cellular-based immunotherapy and as vehicles for gene and drug delivery for a wide range of diseases and disorders. Key among these are: (1) their relative ease of isolation from a variety of tissues; (2) the ability to be expanded in culture without a loss of functionality, a property that varies to some degree with tissue source; (3) they are relatively immune-inert, perhaps obviating the need for precise donor/recipient matching; (4) they possess potent immunomodulatory functions that can be tailored by so-called licensing in vitro and in vivo; (5) the efficiency with which they can be modified with viral-based vectors; and (6) their almost uncanny ability to selectively home to damaged tissues, tumors, and metastases following systemic administration. In this review, we summarize the latest research in the immunological properties of MSCs, their use as immunomodulatory/anti-inflammatory agents, methods for licensing MSCs to customize their immunological profile, and their use as vehicles for transferring both therapeutic genes in genetic disease and drugs and genes designed to destroy tumor cells.

Soluble Fas affects erythropoiesis in vitro and acts as a potential predictor of erythropoiesis-stimulating agent therapy in patients with chronic kidney disease.

Serum soluble Fas (sFas) levels are associated with erythropoietin (Epo) hyporesponsiveness in patients with chronic kidney disease (CKD). Whether sFas could predict the need for erythropoiesis-stimulating agent (ESA) usage and its influence in erythropoiesis remain unclear. We evaluated the relation between sFas and ESA therapy in patients with CKD with anemia and its effect on erythropoiesis in vitro. First, we performed a retrospective cohort study with 77 anemic patients with nondialysis CKD. We performed in vitro experiments to investigate whether sFas could interfere with the behavior of hematopoietic stem cells (HSCs). HSCs were isolated from umbilical cord blood and incubated with recombinant sFas protein in a dose-dependent manner. Serum sFas positively correlated with Epo levels (r = 0.30, P = 0.001) but negatively with hemoglobin (r = -0.55, P < 0.001) and glomerular filtration rate (r = -0.58, P < 0.001) in patients with CKD at baseline. Elevated sFas serum levels (4,316 ± 897 vs. 2,776 ± 749, P < 0.001) with lower estimated glomerular filtration rate (26.2 ± 10.1 vs. 33.5 ± 14.3, P = 0.01) and reduced hemoglobin concentration (11.1 ± 0.9 vs. 12.5 ± 1.2, P < 0.001) were identified in patients who required ESA therapy compared with patients with non-ESA. Afterward, we detected that the sFas level was slight correlated with a necessity of ESA therapy in patients with nondialysis CKD and anemia. In vitro assays demonstrated that the erythroid progenitor cell frequency negatively correlated with sFas concentration (r = -0.72, P < 0.001). There was decreased erythroid colony formation in vitro when CD34+ HSCs were incubated with a higher concentration of sFas protein (1.56 ± 0.29, 4.33 ± 0.53, P < 0.001). Our findings suggest that sFas is a potential predictor for ESA therapy in patients with nondialysis CKD and that elevated sFas could affect erythropoiesis in vitro.

Deconstructed Microfluidic Bone Marrow On-A-Chip to Study Normal and Malignant Hemopoietic Cell-Niche Interactions.

Human hematopoietic niches are complex specialized microenvironments that maintain and regulate hematopoietic stem and progenitor cells (HSPC). Thus far, most of the studies performed investigating alterations of HSPC-niche dynamic interactions are conducted in animal models. Herein, organ microengineering with microfluidics is combined to develop a human bone marrow (BM)-on-a-chip with an integrated recirculating perfusion system that consolidates a variety of important parameters such as 3D architecture, cell-cell/cell-matrix interactions, and circulation, allowing a better mimicry of in vivo conditions. The complex BM environment is deconvoluted to 4 major distinct, but integrated, tissue-engineered 3D niche constructs housed within a single, closed, recirculating microfluidic device system, and equipped with cell tracking technology. It is shown that this technology successfully enables the identification and quantification of preferential interactions-homing and retention-of circulating normal and malignant HSPC with distinct niches.

Bone Marrow Endothelial Cells Influence Function and Phenotype of Hematopoietic Stem and Progenitor Cells after Mixed Neutron/Gamma Radiation.

The bone marrow (BM) microenvironment plays a crucial role in the maintenance and regeneration of hematopoietic stem (HSC) and progenitor cells (HSPC). In particular, the vascular niche is responsible for regulating HSC maintenance, differentiation, and migration of cells in and out of the BM. Damage to this niche upon exposure to ionizing radiation, whether accidental or as a result of therapy, can contribute to delays in HSC recovery and/or function. The ability of BM derived-endothelial cells (BMEC) to alter and/or protect HSPC after exposure to ionizing radiation was investigated. Our data show that exposure of BMEC to ionizing radiation resulted in alterations in Akt signaling, increased expression of PARP-1, IL6, and MCP-1, and decreased expression of MMP1 and MMP9. In addition, global analysis of gene expression of HSC and BMEC in response to mixed neutron/gamma field (MF) radiation identified 60 genes whose expression was altered after radiation in both cell types, suggesting that a subset of genes is commonly affected by this type of radiation. Focused gene analysis by RT-PCR revealed two categories of BMEC alterations: (a) a subset of genes whose expression was altered in response to radiation, with no additional effect observed during coculture with HSPC, and (b) a subset of genes upregulated in response to radiation, and altered when cocultured with HSPC. Coculture of BMEC with CD34+ HSPC induced HSPC proliferation, and improved BM function after MF radiation. Nonirradiated HSPC exhibited reduced CD34 expression over time, but when irradiated, they maintained higher CD34 expression. Nonirradiated HSPC cocultured with nonirradiated BMEC expressed lower levels of CD34 expression compared to nonirradiated alone. These data characterize the role of each cell type in response to MF radiation and demonstrate the interdependence of each cell's response to ionizing radiation. The identified genes modulated by radiation and coculture provide guidance for future experiments to test hypotheses concerning specific factors mediating the beneficial effects of BMEC on HSPC. This information will prove useful in the search for medical countermeasures to radiation-induced hematopoietic injury.

Targeting Mesenchymal Stromal Cells/Pericytes (MSCs) With Pulsed Electromagnetic Field (PEMF) Has the Potential to Treat Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of synovium (synovitis), with inflammatory/immune cells and resident fibroblast-like synoviocytes (FLS) acting as major players in the pathogenesis of this disease. The resulting inflammatory response poses considerable risks as loss of bone and cartilage progresses, destroying the joint surface, causing joint damage, joint failure, articular dysfunction, and pre-mature death if left untreated. At the cellular level, early changes in RA synovium include inflammatory cell infiltration, synovial hyperplasia, and stimulation of angiogenesis to the site of injury. Different angiogenic factors promote this disease, making the role of anti-angiogenic therapy a focus of RA treatment. To control angiogenesis, mesenchymal stromal cells/pericytes (MSCs) in synovial tissue play a vital role in tissue repair. While recent evidence reports that MSCs found in joint tissues can differentiate to repair damaged tissue, this repair function can be repressed by the inflammatory milieu. Extremely-low frequency pulsed electromagnetic field (PEMF), a biophysical form of stimulation, has an anti-inflammatory effect by causing differentiation of MSCs. PEMF has also been reported to increase the functional activity of MSCs to improve differentiation to chondrocytes and osteocytes. Moreover, PEMF has been demonstrated to accelerate cell differentiation, increase deposition of collagen, and potentially return vascular dysfunction back to homeostasis. The aim of this report is to review the effects of PEMF on MSC modulation of cytokines, growth factors, and angiogenesis, and describe its effect on MSC regeneration of synovial tissue to further understand its potential role in the treatment of RA.

Mesenchymal stem cells in myeloproliferative disorders - focus on primary myelofibrosis.

Primary myelofibrosis (PMF) is the most aggressive Philadelphia-negative (Ph-) myeloproliferative neoplasm (MPN), characterized by bone marrow (BM) insufficiency, myelofibrosis (MF), osteosclerosis, neoangiogenesis, and extramedullary hematopoiesis (EMH) in spleen and liver. Presently, there is no curative treatment for this disease and therapy consists primarily of symptom relief and, in selected cases, allogeneic hematopoietic stem cell transplant (alloHSCT). PMF's major defining characteristics, as well as several recently described aspects of its cellular and molecular pathophysiology all support a critical role for dysregulated cell-cell/cell-extracellular matrix interactions and cytokine/chemokine signaling within the BM niche in the natural history of this disease. This review will highlight current data concerning the involvement of the BM niche, particularly of mesenchymal stem cells (MSC), in PMF, and will then discuss the rationale for a stroma-directed treatment, and the advantages such an approach would offer over the current treatments focused on targeting the malignant clone.

Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Selectively recruiting bone marrow (BM)-derived stem and progenitor cells to injury sites is a promising therapeutic approach. The coordinated action of soluble factors is thought to trigger the mobilization of stem cells from the BM and recruit them to lesions to contribute to tissue regeneration. Nevertheless, the temporal response profile of the major cellular players and soluble factors involved in priming the BM and recruiting BM-derived cells to promote regeneration is unknown. We show that injury alters the BM cellular composition, introducing population-specific fluctuations during tissue regeneration. We demonstrate that injury causes an immediate, transient response of mesenchymal stromal cells and endothelial cells followed by a nonoverlapping increase in hematopoietic stem and progenitor cells. Moreover, BM reaction is identical whether the injury is inflicted on skin and muscle or also involves a bone defect, but these 2 injury paradigms trigger distinct systemic cytokine responses. Together, our results indicate that the BM response to injury in the early stages of regeneration is independent of the tissue-of-injury based on the 2 models used, but the injured tissue dictates the systemic cytokine response.-Leitão, L., Alves, C. J., Alencastre, I. S., Sousa, D. M., Neto, E., Conceição, F., Leitão, C., Aguiar, P., Almeida-Porada, G., Lamghari, M. Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Mesenchymal Stromal Cell Secretome: Influencing Therapeutic Potential by Cellular Pre-conditioning.

Mesenchymal stromal cells (MSCs) are self-renewing, culture-expandable adult stem cells that have been isolated from a variety of tissues, and possess multipotent differentiation capacity, immunomodulatory properties, and are relatively non-immunogenic. Due to this unique set of characteristics, these cells have attracted great interest in the field of regenerative medicine and have been shown to possess pronounced therapeutic potential in many different pathologies. MSCs' mode of action involves a strong paracrine component resulting from the high levels of bioactive molecules they secrete in response to the local microenvironment. For this reason, MSCs' secretome is currently being explored in several clinical contexts, either using MSC-conditioned media (CM) or purified MSC-derived extracellular vesicles (EVs) to modulate tissue response to a wide array of injuries. Rather than being a constant mixture of molecular factors, MSCs' secretome is known to be dependent on the diverse stimuli present in the microenvironment that MSCs encounter. As such, the composition of the MSCs' secretome can be modulated by preconditioning the MSCs during in vitro culture. This manuscript reviews the existent literature on how preconditioning of MSCs affects the therapeutic potential of their secretome, focusing on MSCs' immunomodulatory and regenerative features, thereby providing new insights for the therapeutic use of MSCs' secretome.

Correction: A human bone marrow mesodermal-derived cell population with hemogenic potential.

At the time of publication the funding information was omitted from the article - this has now been corrected in both the HTML and the PDF.