Placental circulating T cells: a novel, allogeneic CAR-T cell platform with preserved T-cell stemness, more favorable cytokine profile, and durable efficacy compared to adult PBMC-derived CAR-T.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell quality and stemness are associated with responsiveness, durability, and memory formation, which benefit clinical responses. Autologous T cell starting material across patients with cancer is variable and CAR-T expansion or potency can fail during manufacture. Thus, strategies to develop allogeneic CAR-T platforms including the identification and expansion of T cell subpopulations that correspond with CAR-T potency are an active area of investigation. Here, we compared CAR-T cells generated from healthy adult peripheral blood T cells versus placental circulating T (P-T) cells. METHODS: CAR-T cells from healthy adult peripheral blood mononuclear cells (PBMCs) and P-T cells were generated using the same protocol. CAR-T cells were characterized in detail by a combination of multiparameter flow cytometry, functional assays, and RNA sequencing. In vivo antitumor efficacy and persistence of CAR-T cells were evaluated in a Daudi lymphoma xenograft model. RESULTS: P-T cells possess stemness advantages compared with T cells from adult PBMCs. P-T cells are uniformly naïve prior to culture initiation, maintain longer telomeres, resist immune checkpoint upregulation, and resist further differentiation compared with PBMC T cells during CD19 CAR-T manufacture. P-T CD19 CAR-T cells are equally cytotoxic as PBMC-CD19 CAR-T cells but produce less interferon gamma in response to lymphoma. Transcriptome analysis shows P-T CD19 CAR-T cells retain a stem-like gene signature, strongly associate with naïve T cells, an early memory phenotype, and a unique CD4 T cell signature compared with PBMC-CD19 CAR-T cells, which enrich for exhaustion and stimulated memory T cell signatures. Consistent with functional data, P-T CD19 CAR-T cells exhibit attenuated inflammatory cytokine and chemokine gene signatures. In a murine in vivo model, P-T CD19 CAR-T cells eliminate lymphoma beyond 90 days. PBMC-CD19 CAR-T cells provide a non-durable benefit, which only delays disease onset. CONCLUSION: We identified characteristics of T cell stemness enriched in P-T CD19 CAR-T which are deficient in PBMC-derived products and translate into response durability in vivo. Our findings demonstrate that placental circulating T cells are a valuable cell source for allogeneic CAR-T products. Stemness advantages inherent to P-T cells translate to in vivo persistence advantages and long-term durable activity.

A novel immunogenic cell death-related classification indicates the immune landscape and predicts clinical outcome and treatment response in acute myeloid leukemia.

BACKGROUND: Immunogenic cell death (ICD) is closely related to anti-tumor therapy and regulates the tumor microenvironment (TME). This study aims to explore the molecular characteristics of ICD in acute myeloid leukemia (AML) and to analyze the value of ICD-related biomarkers in TME indication, prognosis prediction, and treatment response evaluation in AML. METHODS: Single-sample gene set enrichment analysis was used to calculate the ICD score. LASSO regression was used to construct a prognostic risk score model. We also analyzed differences in clinical characteristics, immune landscape, immunotherapy response, and chemotherapy sensitivity between high-risk and low-risk patients. RESULTS: This study identified two ICD-related subtypes and found significant heterogeneity in clinical prognosis, TME, and immune landscape between different ICD subtypes. Subsequently, a novel ICD-related prognostic risk score model was developed, which accurately predicted the prognosis of AML patients and was validated in nine AML cohorts. Moreover, there were significant correlations between risk scores and clinicopathological factors, somatic mutations, TME characteristics, immune cell infiltration, immunotherapy response, and chemosensitivity. We further validated the model gene expression in a clinically real-world cohort. CONCLUSIONS: The novel ICD-related signatures identified and validated by us can serve as promising biomarkers for predicting clinical outcomes, chemotherapy sensitivity, and immunotherapy response in AML patients, guiding the establishment of personalized and accurate treatment strategies for AML.

Selenium nanoparticles in aquaculture: Unique advantages in the production of Se-enriched grass carp (Ctenopharyngodon idella).

The production of selenium-enriched fish can contribute to alleviating selenium deficiency in human diets. However, it is still unclear which selenium source, as an additive, can efficiently and cost-effectively produce high-quality selenium-enriched fish. This study evaluated the effects of selenium nanoparticles (SeNP), selenite, and selenomethionine (SeMet) on the growth, antioxidant capacity, selenium content, selenium speciation, and meat quality of grass carp. Ten diets were prepared, including a basal diet (BD) and three concentrations (0.1, 0.3, and 0.9 mg/kg) of SeNP, selenite, and SeMet. A total of 600 fish (250.79 ± 1.57 g) were randomly assigned to 30 tanks (3 tanks/group). Fish were fed the experimental diet three times daily for 60 d. In this study, SeNP most significantly promoted the growth and antioxidant capacity of grass carp, with 0.3 mg/kg SeNP identified as the optimal additive concentration. Additionally, SeNP demonstrated equally excellent bioavailability as SeMet and significantly increased the content of SeMet in grass carp (Ctenopharyngodon idella) muscle. Furthermore, compared to SeMet and selenite, dietary SeNP could more significantly enhance the content of selenocysteine (SeCys2) and methylselenocysteine (MeSeCys) in grass carp muscle tissue. In addition, we have demonstrated that SeCys2 and MeSeCys promote apoptosis of cancer cells (HeLa) through the mitochondrial apoptotic pathway (involving Bax and Bcl-2). Furthermore, as an additive, 0.3 mg/kg SeNP significantly improved the flesh quality of grass carp by reducing crude fat and heavy metal content, as well as increasing the levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the ratio of n-3/n-6 polyunsaturated fatty acid (PUFA). In summary, SeNP is the most suitable additive for producing selenium-enriched fish.

Metformin improves fish sperm quality by regulating glucose uptake capacity during in vitro storage.

A suitable additive for fish sperm storage in vitro is necessary for artificial reproduction. In this study, we evaluated the effects of different concentrations (100, 200, 400, and 800 µmol/L) of metformin (Met) on Schizothorax prenanti and Onychostoma macrolepis sperm under storage in vitro for 72 h. Compared with the control group, 400 µmol/L Met was more effective at improving the quality and fertilization capacity of S. prenanti sperm by increasing the adenosine triphosphate (ATP) content within the sperm. Further study found that Met stabilized the ATP level by enhancing the glucose uptake in S. prenanti sperm, and this effect might be associated with the activation of AMP-activated protein kinase (AMPK) in sperm. In this study, we also found that glucose could be absorbed by the sperm of S. prenanti, which was mainly accumulated in the midpiece of S. prenanti sperm, where mitochondria were located. In addition, Compound C significantly inhibited the beneficial effects of Met on the quality and glucose uptake capacity of S. prenanti sperm by inhibiting AMPK phosphorylation. These results revealed that AMPK played an important role in vitro sperm storage, and Met maintained ATP content and increased the storage time of S. prenanti sperm in vitro for 72 h, possibly due to Met enhanced glucose uptake capacity of sperm by activating AMPK. Similarly, the beneficial effects of Met on S. prenanti sperm were also found in O. macrolepis sperm, suggesting that Met may hold great promise for the practice of storing fish in vitro.

Generally, what matters most to fish sperm is a good storage condition in vitro that can both ensure its high motility after fresh water activation and avoid the damage of the nonactivated state. An appropriate additive can provide suitable storage conditions for sperm. Therefore, a suitable additive of fish sperm is necessary for fish to promote effective artificial reproduction. In this study, we evaluated the effects of different concentrations of metformin (Met) on fish sperm under storage in vitro. Compared with the control group, Met was more effective at improving the quality and fertilization capacity of fish by increasing the adenosine triphosphate (ATP) content within the sperm. Further study found that Met stabilized the ATP level by enhancing the glucose uptake in fish sperm, and this effect might be associated with the activation of AMP-activated protein kinase (AMPK) in sperm. The beneficial effects of Met on fish sperm suggest that Met may hold great promise for the practice of storing fish in future vitro storage.

miR-626 Inhibition Enhanced the Radiosensitivity to Oral Squamous Cell Carcinoma via the Downregulation of Nuclear Factor Kappa-B Signaling.

Objective: The effect of miR-626 on the radiosensitivity to oral squamous cell carcinoma (OSCC) was evaluated in this study. Materials and Methods: The level of miR-626 in OSCC patients was determined by analyzing the data of miRNA microarray GSE113956. miR-626 was overexpressed by miR-626 mimics and knockdown were performed by miR-626 inhibitor. The level of miR-626 was detected by quantitative real-time polymerase chain reaction. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays were used to detect the effect of miR-626 on the growth of OSCC cells. Flow cytometry was used to detect the apoptosis of OSCC cells. Western blot and dual luciferase reporter assays were used to explore the underlying mechanism of miR-626 regulating the radiosensitivity to OSCC. The effect of miR-626 on the radiosensitivity to OSCC were examined in an in vivo xenograft model. Results: The serum miR-626 level of OSCC patients was significantly higher than that of healthy controls. miR-626 mimics significantly promoted the OSCC cell growth, but the miR-626 inhibitor significantly suppressed the OSCC cell growth. Radiation combined with the miR-626 inhibitor significantly suppressed the cell proliferation and promoted the apoptosis of SCC-4 and HSC4 cells. Moreover, miR-626 regulates the nuclear factor kappa-B (NF-κB) signaling mediated by TRAF-interacting protein with forkhead-associated domain B. Furthermore, inhibition of miR-626 enhances the radiosensitivity to OSCC in nude mice. Conclusions: miR-626 inhibition enhanced the radiosensitivity to OSCC through the downregulation of NF-κB signaling.

Human placental hematopoietic stem cell derived natural killer cells (CYNK-001) mediate protection against influenza a viral infection.

Influenza A virus (IAV) infections are associated with a high healthcare burden around the world and there is an urgent need to develop more effective therapies. Natural killer (NK) cells have been shown to play a pivotal role in reducing IAV-induced pulmonary infections in preclinical models; however, little is known about the therapeutic potential of adoptively transferred NK cells for IAV infections. Here, we investigated the effects of CYNK-001, human placental hematopoietic stem cell derived NK cells that exhibited strong cytolytic activity against a range of malignant cells and expressed high levels of activating receptors, against IAV infections. In a severe IAV-induced acute lung injury model, mice treated with CYNK-001 showed a milder body weight loss and clinical symptoms, which led to a delayed onset of mortality, thus demonstrating their antiviral protection in vivo. Analysis of bronchoalveolar lavage fluid (BALF) revealed that CYNK-001 reduced proinflammatory cytokines and chemokines highlighting CYNK-001's anti-inflammatory actions in viral induced-lung injury. Furthermore, CYNK-001-treated mice had altered immune responses to IAV with reduced number of neutrophils in BALF yet increased number of CD8+ T cells in the BALF and lung compared to vehicle-treated mice. Our results demonstrate that CYNK-001 displays protective functions against IAV via its anti-inflammatory and immunomodulating activities, which leads to alleviation of disease burden and progression in a severe IAV-infected mice model. The potential of adoptive NK therapy for IAV infections warrants clinical investigation.

CBLB ablation with CRISPR/Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy.

BACKGROUND: Tumors often develop resistance to surveillance by endogenous immune cells, which include natural killer (NK) cells. Ex vivo activated and/or expanded NK cells demonstrate cytotoxicity against various tumor cells and are promising therapeutics for adoptive cancer immunotherapy. Genetic modification can further enhance NK effector cell activity or activation sensitization. Here, we evaluated the effect of the genetic deletion of ubiquitin ligase Casitas B-lineage lymphoma pro-oncogene-b (CBLB), a negative regulator of lymphocyte activity, on placental CD34+ cell-derived NK (PNK) cell cytotoxicity against tumor cells. METHODS: Using CRISPR/Cas9 technology, CBLB was knocked out in placenta-derived CD34+ hematopoietic stem cells, followed by differentiation into PNK cells. Cell expansion, phenotype and cytotoxicity against tumor cells were characterized in vitro. The antitumor efficacy of CBLB knockout (KO) PNK cells was tested in an acute myeloid leukemia (HL-60) tumor model in NOD-scid IL2R gammanull (NSG) mice. PNK cell persistence, biodistribution, proliferation, phenotype and antitumor activity were evaluated. RESULTS: 94% of CBLB KO efficacy was achieved using CRISPR/Cas9 gene editing technology. CBLB KO placental CD34+ cells differentiated into PNK cells with high cell yield and >90% purity determined by CD56+ CD3- cell identity. Ablation of CBLB did not impact cell proliferation, NK cell differentiation or phenotypical characteristics of PNK cells. When compared with the unmodified PNK control, CBLB KO PNK cells exhibited higher cytotoxicity against a range of liquid and solid tumor cell lines in vitro. On infusion into busulfan-conditioned NSG mice, CBLB KO PNK cells showed in vivo proliferation and maturation as evidenced by increased expression of CD16, killer Ig-like receptors and NKG2A over 3 weeks. Additionally, CBLB KO PNK cells showed greater antitumor activity in a disseminated HL60-luciferase mouse model compared with unmodified PNK cells. CONCLUSION: CBLB ablation increased PNK cell effector function and proliferative capacity compared with non-modified PNK cells. These data suggest that targeting CBLB may offer therapeutic advantages via enhancing antitumor activities of NK cell therapies.

Human Placenta-Derived Mesenchymal Stromal-Like Cells Enhance Angiogenesis via T Cell-Dependent Reprogramming of Macrophage Differentiation.

Peripheral arterial disease (PAD) is a leading cause of limb loss and mortality worldwide with limited treatment options. Mesenchymal stromal cell (MSC) therapy has demonstrated positive effects on angiogenesis in preclinical models and promising therapeutic efficacy signals in early stage clinical studies; however, the mechanisms underlying MSC-mediated angiogenesis remain largely undefined. Here, we investigated the mechanism of action of human placenta-derived MSC-like cells (PDA-002) in inducing angiogenesis using mice hind limb ischemia model. We showed that PDA-002 improved blood flow and promoted collateral vessel formation in the injured limb. Histological analysis demonstrated that PDA-002 increased M2-like macrophages in ischemic tissue. Analysis of the changes in functional T cell phenotype in the draining lymph nodes revealed that PDA-002 treatment was associated with the induction of cytokine and gene expression signatures of Th2 response. Angiogenic effect of PDA-002 was markedly reduced in Balb/c nude mice compared with wild type. This reduction in efficacy was reversed by T cell reconstitution, suggesting T cells are essential for PDA-002-mediated angiogenesis. Furthermore, effect of PDA-002 on macrophage differentiation was also T cell-dependent as a PDA-002-mediated M2-like macrophage skewing was only observed in wild type and T cell reconstituted nude mice, but not in nude mice. Finally, we showed that PDA-002-treated animals had enhanced angiogenic recovery in response to the second injury when PDA-002 no longer persisted in vivo. These results suggest that PDA-002 enhances angiogenesis through an immunomodulatory mechanism involving T cell-dependent reprogramming of macrophage differentiation toward M2-like phenotype. Stem Cells 2017;35:1603-1613.

Angiogenic properties of human placenta-derived adherent cells and efficacy in hindlimb ischemia.

OBJECTIVE: Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population from full-term placental tissue and were previously shown to possess anti-inflammatory and immunomodulatory properties. PDACs (formulated as PDA-002) are in clinical trials for peripheral arterial disease with diabetic foot ulcer. In the current study, we examined their angiogenic and tissue reparative properties. METHODS: The effects of PDACs on survival and tube formation of human umbilical vein endothelial cells (HUVECs) were tested using conditioned media and noncontact coculture. Angiogenic effects were assessed in the chick chorioallantoic membrane assay. Hindlimb ischemia (HLI) was induced in mice and rats by femoral artery transection, and blood flow and blood vessel density were monitored in vivo by laser Doppler and angiography in the ischemic and control limbs. Tissue damage and regeneration in HLI were examined in histologic sections of quadriceps muscle stained with hematoxylin and eosin, and newly synthesized blood vessels were detected by indoxyl-tetrazolium staining for alkaline phosphatase. RESULTS: PDACs enhanced the survival of serum-starved HUVECs and stimulated HUVEC tube formation, and in the chick chorioallantoic membrane assay, PDACs stimulated blood vessel formation. In HLI, intramuscular administration of PDACs resulted in improved blood flow and vascular density, and in quadriceps muscle, tissue regeneration and increased numbers of blood vessels were observed. CONCLUSIONS: PDACs exhibited various activities consistent with angiogenesis and tissue repair, supporting the continued investigation of this cell therapy as treatment for vascular disease-related indications.

Human placenta-derived adherent cells prevent bone loss, stimulate bone formation, and suppress growth of multiple myeloma in bone.

Human placenta has emerged as a valuable source of transplantable cells of mesenchymal and hematopoietic origin for multiple cytotherapeutic purposes, including enhanced engraftment of hematopoietic stem cells, modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDACs) are mesenchymal-like stem cells isolated from postpartum human placenta. Multiple myeloma is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. We evaluated the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDACs in the severe combined immunodeficiency (SCID)-rab model of medullary myeloma-associated bone loss. Intrabone injection of PDACs into nonmyelomatous and myelomatous implanted bone in SCID-rab mice promoted bone formation by stimulating endogenous osteoblastogenesis, and most PDACs disappeared from bone within 4 weeks. PDACs inhibitory effects on myeloma bone disease and tumor growth were dose-dependent and comparable with those of fetal human mesenchymal stem cells (MSCs). Intrabone, but not subcutaneous, engraftment of PDACs inhibited bone disease and tumor growth in SCID-rab mice. Intratumor injection of PDACs had no effect on subcutaneous growth of myeloma cells. A small number of intravenously injected PDACs trafficked into myelomatous bone. Myeloma cell growth rate in vitro was lower in coculture with PDACs than with MSCs from human fetal bone or myeloma patients. PDACs also promoted apoptosis in osteoclast precursors and inhibited their differentiation. This study suggests that altering the bone marrow microenvironment with PDAC cytotherapy attenuates growth of myeloma and that PDAC cytotherapy is a promising therapeutic approach for myeloma osteolysis.

Lymphoid enhancer factor 1-mediated Wnt signaling promotes the initiation of trophoblast lineage differentiation in mouse embryonic stem cells.

Embryonic stem (ES) cells can differentiate into all three embryonic germ layers but rarely into trophectoderm (TE) lineages that contribute to the placenta, although TE differentiation can be initiated by genetic manipulation of key genes involved in TE development. We demonstrate that Wnt signaling can initiate TE lineage differentiation by triggering an appropriate cue, caudal-related homeobox 2 (Cdx2). Overexpression and RNA interference knockdown studies indicate that Cdx2 induction in response to Wnt3a is mediated by lymphoid enhancer factor 1, whose expression is regulated by leukemia inhibitory factor (LIF) and bone morphogenetic protein. Removal of LIF, along with addition of Wnt3a, stimulated Cdx2 expression and induced formation of trophoblast stem (TS) cells. These TS cells were able to differentiate into cells with characteristics of spongiotrophoblast and trophoblast giant cells. This is, to our knowledge, the first evidence that TE lineage differentiation can be induced by Wnt signaling in mouse ES cells.

Effects of dimethyl sulfoxide and glycine on cryopreservation induced damage of plasma membranes and mitochondria to striped bass (Morone saxatilis) sperm.

Intact plasma membrane and functional mitochondria are important attributes for the fertilization capacity of fish sperm. In the present study, dimethyl sulfoxide (Me(2)SO) and glycine were investigated in an effort to improve plasma membrane integrity and mitochondrial function in cryopreserved striped bass (Morone saxatilis) sperm. Prior to freezing, no concentration of Me(2)SO (2.5, 5 or 10%) was found to affect (P>0.05) the integrity of plasma membranes after sperm were exposed for 10 min. However, mitochondrial function decreased (P>0.05) with increasing Me(2)SO concentration. Both fluorescent staining and microscopic examination of the ultrastructure of post-thaw plasma membranes indicated that with increasing Me(2)SO concentration, plasma membranes were better protected, and 10% Me(2)SO had the highest percentage of sperm with plasma membranes intact. However, sperm mitochondrial function decreased (P>0.05) with increasing Me(2)SO concentration. The inverse relationship between plasma membrane integrity and mitochondrial function, given the Me(2)SO concentration, suggests that care must be taken to select Me(2)SO concentration that will maximize the protection of both plasma membranes and mitochondrial function. The addition of glycine to the cryomedia increased (P<0.05) the percentage of sperm with post-thaw functional mitochondria and ATP content. However glycine did not provide (P<0.05) protection to post-thaw plasma membrane integrity. The highest percentage of sperm with both intact plasma membranes and functional mitochondria was obtained with 7.5% Me(2)SO and 75 mM glycine.

Activation of sperm motility in striped bass via a cAMP-independent pathway.

The objective of the present study was to identify the effect of osmolality, ions (K+, H+, Ca2+, Mg2+) and cAMP on the initiation of sperm motility in striped bass (Morone saxatilis). Striped bass spermatozoa remained motile in solutions isotonic to seminal plasma (350 mOsm/kg) until osmolality reached 600 mOsm/kg. K+ (0-100 mM) had no effect ( p>0.05 ) on sperm motility, and sperm displayed a high percentage of motility over a wide range of pH (6.0-8.5). Sperm motility could be initiated in Ca2+-free solutions. In contrast, sperm motility was inhibited (P<0.01) by solutions containing > or =10 mM Ca2+, and sperm could not be reactivated by a Ca2+-free solution. This Ca2+ inhibition was not affected by verapamil, a Ca2+ channel blocker. However, if sperm motility was first initiated in a Ca2+-free solution, the addition of Ca2+ solutions, up to 80 mM, failed to inhibit sperm motility, suggesting that Ca2+ inhibited the initiation of motility, but had no control of motile spermatozoa. Mg2+ solutions had similar inhibitory effects on sperm motility as Ca2+ solutions. Therefore, initiation of motility in striped bass sperm may be related to voltage-gated channels across the cell's plasma membrane. Membrane permeable cAMP did not initiate motility of quiescent, intact striped bass spermatozoa, and motility of demembranated sperm could be activated in the absence of cAMP.