Superior protective effects of PGE2 priming mesenchymal stem cells against LPS-induced acute lung injury (ALI) through macrophage immunomodulation.

BACKGROUND: Mesenchymal stem cells (MSCs) have demonstrated remarkable therapeutic promise for acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS). MSC secretomes contain various immunoregulatory mediators that modulate both innate and adaptive immune responses. Priming MSCs has been widely considered to boost their therapeutic efficacy for a variety of diseases. Prostaglandin E2 (PGE2) plays a vital role in physiological processes that mediate the regeneration of injured organs. METHODS: This work utilized PGE2 to prime MSCs and investigated their therapeutic potential in ALI models. MSCs were obtained from human placental tissue. MSCs were transduced with firefly luciferase (Fluc)/eGFP fusion protein for real-time monitoring of MSC migration. Comprehensive genomic analyses explored the therapeutic effects and molecular mechanisms of PGE2-primed MSCs in LPS-induced ALI models. RESULTS: Our results demonstrated that PGE2-MSCs effectively ameliorated lung injury and decreased total cell numbers, neutrophils, macrophages, and protein levels in bronchoalveolar lavage fluid (BALF). Meanwhile, treating ALI mice with PGE2-MSCs dramatically reduced histopathological changes and proinflammatory cytokines while increasing anti-inflammatory cytokines. Furthermore, our findings supported that PGE2 priming improved the therapeutic efficacy of MSCs through M2 macrophage polarization. CONCLUSION: PGE2-MSC therapy significantly reduced the severity of LPS-induced ALI in mice by modulating macrophage polarization and cytokine production. This strategy boosts the therapeutic efficacy of MSCs in cell-based ALI therapy.

Enhancement of hair growth through stimulation of hair follicle stem cells by prostaglandin E2 collagen matrix.

Prostaglandin metabolism is involved in the regulation of the periodic process of hair follicles. Preliminary research data reported that prostaglandin E2 (PGE2) exhibits potential in hair growth. However, the relevant evidence is still insufficient. Herein, we prepared a PGE2 matrix by conjugating PGE2 with collagen via crosslinkers to avoid rapid degradation of PGE2 molecules in vivo. First, we measured the physical properties of the PGE2 matrix. A mouse model of hair loss was established, and PGE2 matrix subcutaneous injection was applied to evaluate hair growth. Under different treatments with the PGE2 matrix, the morphology of hair follicles, the dynamic expression of hair follicle stem cell markers and key regulators in the hair growth cycle were explored. Our data revealed that the PGE2 matrix increased the proportion of developing hair follicles at the early growth stage. Improvements in hair follicle stem cells, such as Sox9+ and Lgr5+ cells, have also been confirmed as therapeutic effects of PGE2 to stimulate hair follicle growth. Our study indicated that PGE2 exhibits effective roles in hair development during anagen. Furthermore, the results also highlight the potential of the PGE2 delivery system as a novel therapeutic strategy for the treatment of hair disorders in the future.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Attenuate Radiation-Induced Lung Injury via miRNA-214-3p.

Aims: Radiotherapy is an effective treatment for thoracic malignancies, but it can cause pulmonary injury and may lead to respiratory failure in a subset of patients. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are now recognized as a new candidate for cell-free treatment of lung diseases. Here, we investigated whether MSC-derived EVs (MSC-EVs) could ameliorate radiation-induced lung injury. Results: We exposed mice to thoracic radiation with a total dose of 15 Gy and assessed the protective effects of MSC-EVs on endothelial cells damage, vascular permeability, inflammation, and fibrosis. We found that MSC-EVs attenuated radiation-induced lung vascular damage, inflammation, and fibrosis. Moreover, MSC-EVs reduced the levels of radiation-induced DNA damage by downregulating ATM/P53/P21 signaling. Our results confirmed that the downregulation of ataxia telangiectasia mutated (ATM) was regulated by miR-214-3p, which was enriched in MSC-EVs. Further analysis demonstrated that MSC-EVs inhibited the senescence-associated secretory phenotype development and attenuated the radiation-induced injury of endothelial cells. Innovation and Conclusion: Our study reveals that MSC-EVs can reduce pulmonary radiation injury through transferring miR-214-3p, providing new avenues to minimize lung injury from radiation therapy. Antioxid. Redox Signal. 35, 849-862.

Extracellular vesicles derived from human placental mesenchymal stem cells alleviate experimental colitis in mice by inhibiting inflammation and oxidative stress.

Mesenchymal stem cells (MSCs) are pluripotent cells that can be applied to the treatment of immune disorders, including inflammatory bowel disease (IBD). The therapeutic effects of MSCs have been mostly attributed to the secretion of soluble factors with paracrine actions, such as extracellular vesicles (EVs), which may play a relevant role in the repair of damaged tissues. In the present study, a mouse model of colitis was induced with the use of trinitrobenzene sulfonic acid (TNBS). EVs derived from human placental mesenchymal stem cells (hP­MSCs) were used for the treatment of colitis by in situ injection. Clinical scores were applied to verify the therapeutic effects of EVs on mice with colitis. Inflammation in the colon was evaluated by measuring the levels of various inflammatory cytokines. The content of reactive oxygen species (ROS) was detected by the use of molecular imaging methods for real­time tracking and the therapeutic effects of EVs on mucosal healing in mice with colitis were evaluated. The results revealed that the injection of EVs regulated the balance of pro­inflammatory and anti­inflammatory cytokines in colon tissue. Treatment with EVs also suppressed oxidative stress by decreasing the activity of myeloperoxidase (MPO) and ROS. Histological analysis further confirmed that the EVs significantly promoted mucosal healing, as reflected by the promotion of the proliferation of colonic epithelial cells and the maintenance of tight junctions. Taken together, the findings of the present study demonstrated that EVs derived from hP­MSCs alleviated TNBS­induced colitis by inhibiting inflammation and oxidative stress. These findings may provide a novel theoretical basis for the EV­based treatment of IBD.

Novel cyclin-dependent kinase 9 (CDK9) inhibitor with suppression of cancer stemness activity against non-small-cell lung cancer.

A series of novel, highly potent, selective CDK9 inhibitors with cancer stem cells (CSCs) inhibition activity were designed and synthesized for non-small-cell lung cancer (NSCLC) therapy. Structure-activity relationship analysis based on enzymatic and cellular activities led to the discovery of a promising inhibitor 21e. 21e potently inhibited CDK9 with IC50 value of 11 nM and suppressed the stemness properties of NSCLC effectively. It could decrease the stemness phenotypes of NSCLC cells, including tumor sphere formation, side-population and stemness markers abundance. 21e displayed good selectivity over the CDK family kinases and kinase profiling assay against 381 kinases. In addition, 21e inhibited cell proliferation, colony-formation, and cell cycle progression and induced apoptosis in NSCLC. In H1299 xenograft mouse model, a once-daily dose of compound 21e at 20 mg/kg significantly suppressed the tumor growth without obvious toxicity. Studies of mechanisms of action indicated that 21e efficiently inhibited CDK9 signaling pathway and stemness both in vitro and in vivo. Collectively, 21e as a novel CDK9 inhibitor with CSCs inhibition properties could be a promising agent for the treatment of NSCLC.

Molecular Imaging of Tumor Angiogenesis and Therapeutic Effects with Dual Bioluminescence.

BACKGROUND: Angiogenesis is critical for the growth of tumor by supplying nutrients and oxygen that exacerbates the metastasis and progression of cancer. Noninvasive imaging of angiogenesis during the tumor therapeutic processes may provide novel opportunities for image-guided tumor management. OBJECTIVE: Here, we want to develop a mouse animal model for assessing cancer progression and angiogenesis in the same individuals by molecular imaging. METHODS: Breast cancer model was developed with mouse breast cancer cell line 4T1 carrying a reporter system encoding a triple fusion (TF) reporter gene consisting of renilla luciferase (Rluc), red fluorescent protein (RFP) and herpes simplex virus truncated thymidine kinase (HSV-ttk) in transgenic mice, which expressed firefly luciferase (Fluc) under the promoter of vascular endothelial growth factor receptor 2 (Vegfr2-luc). The mice were subsequently treated with ganciclovir (GCV) and the tumor angiogenesis was tracked by Fluc imaging and the growth status of tumor was monitored by imaging of Rluc simultaneously. CONCLUSION: Overall, this traceable breast cancer model can simultaneously image the tumor growth and angiogenesis in single individual, which may facilitate a better understanding the mechanisms of angiogenesis in the progression and regression of tumor.

Identification of Stem-Like Cells in Atrial Myxoma by Markers CD44, CD19, and CD45.

Atrial myxoma is the most frequent tumor arising mainly in atrial septum and its origin remains uncertain. It has been reported that a subpopulation of stem-like cells are present in benign tumors and responsible for tumor initiation and maintenance. In this study, we investigated whether stem-like cells could contribute to the atrial cardiac myxoma. Immunohistology data confirmed that a population of cells bearing the surface markers CD19, CD45, and CD44 resided in a mucopolysaccharide-rich matrix of myxoma. Moreover, we isolated myxoma cells with phase-bright culture method and confirmed that myxoma derived cells express robust level of CD19, CD45, and CD44. Furthermore, the pluripotency of this population of cells also was validated by cardiomyocytes and smooth muscle cells differentiation in vitro. Our results indicate that primary cardiac myxoma may arise from mesenchymal stem cells with the ability to generate tumors with multilineage differentiation. In conclusion, this study for the first time verified that stem-like cells are present in atrial myxoma and this population of cells may have the capacity for myxoma initiation and progression.

Human embryonic stem cell-derived endothelial cells as cellular delivery vehicles for treatment of metastatic breast cancer.

Endothelial progenitor cells (EPCs) have shown tropism towards primary tumors or metastases and are thus potential vehicles for targeting tumor therapy. However, the source of adult EPCs is limited, which highlights the need for a consistent and renewable source of endothelial cells for clinical applications. Here, we investigated the potential of human embryonic stem cell-derived endothelial cells (hESC-ECs) as cellular delivery vehicles for therapy of metastatic breast cancer. In order to provide an initial assessment of the therapeutic potency of hESC-ECs, we treated human breast cancer MDA-MB-231 cells with hESC-EC conditioned medium (EC-CM) in vitro. The results showed that hESC-ECs could suppress the Wnt/ß-catenin signaling pathway and thereby inhibit the proliferation and migration of MDA-MB-231 cells. To track and evaluate the possibility of hESC-EC-employed therapy, we employed the bioluminescence imaging (BLI) technology. To study the therapeutic potential of hESC-ECs, we established lung metastasis models by intravenous injection of MDA-MB-231 cells labeled with firefly luciferase (Fluc) and green fluorescent protein (GFP) to NOD/SCID mice. In mice with lung metastases, we injected hESC-ECs armed with herpes simplex virus truncated thymidine kinase (HSV-ttk) intravenously on days 11, 16, 21, and 26 after MDA-MB-231 cell injection. The NOD/SCID mice were subsequently treated with ganciclovir (GCV), and the growth status of tumor was monitored by Fluc imaging. We found that MDA-MB-231 tumors were significantly inhibited by intravenously injected hESC-ECs. The tumor-suppressive effects of the hESC-ECs, by inhibiting Wnt/ß-catenin signaling pathway and inducing tumor cell death through bystander effect in human metastatic breast cancer model, provide previously unexplored therapeutic modalities for cancer treatment.

Assessment of therapeutic efficacy of liposomal nanoparticles mediated gene delivery by molecular imaging for cancer therapy.

The inadequate treatment efficacy, suboptimal cancer detection and disease monitoring in anticancer therapies have led to the quest for clinically relevant, innovative multifaceted solutions such as development of targeted and traceable approaches. Molecular imaging technologies with the versatility of liposomal nanoparticles platform offer tangible options to better guide treatment delivery and monitor outcome. In this study, we introduced noninvasive, quantitative and functional imaging techniques with reporter gene methods to probe breast cancer processes with liposomal nanoparticles by bioluminescence imaging (BLI). A breast cancer model was applied for therapy by injecting 5.0 x 10(5) 4T1 cells carrying a reporter system encoding a double fusion reporter gene consisting of firefly luciferase (Fluc) and green fluorescent protein (GFP) into BALB/c mice. Liposomal nanoparticles loaded with a triple fusion gene containing the herpes simplex virus truncated thymidine kinase (HSV-ttk) and renilla luciferase (Rluc) and red fluorescent protein (RFP) were applied by in situ injection for monitoring and evaluating gene therapy. The BALB/c mice were subsequently treated with ganciclovir (GCV) and the growth status of tumor was monitored by bioluminescence imaging of Fluc and the treatment delivery of liposomal nanoparticle was efficiently tracked by Rluc imaging. In fact, TF plasmids were shown to be useful for monitoring and evaluating targeting efficacy and gene therapy by non-invasive molecular imaging. In conclusion, the combination of noninvasive imaging techniques and liposomal nanoparticle can provide a practical and clinically useful way for gene delivery and monitoring the level of gene expression over time and treatment response in patients undergoing gene therapy.

CD44 antibody-targeted liposomal nanoparticles for molecular imaging and therapy of hepatocellular carcinoma.

Most hepatocellular carcinoma (HCC) therapies fail to target cancer stem cells (CSCs) and monitor cancer progression or regression. The purpose of this study was to evaluate the possibility of cancer imaging and simultaneously monitoring targeted therapy in a single animal by anti-CD44 antibody-mediated liposomal nanoparticle. In this study, an in situ liver tumor model was applied for therapy by injecting 1.0 × 10(6) HepG2 cells carrying a reporter system encoding a double fusion (DF) reporter gene consisting of firefly luciferase (Fluc) and green fluorescent protein (GFP) into the liver of NOD/SCID mice. A strategy was developed which specifically targeted HCC via anti-CD44 antibody-mediated liposomal nanoparticle delivery, loaded of either doxorubicin (Dox) or a triple fusion (TF) gene containing the herpes simplex virus truncated thymidine kinase (HSV-ttk) and renilla luciferase (Rluc) and red fluorescent protein (RFP). The NOD/SCID mice were subsequently treated with ganciclovir (GCV) and the growth status of tumor was monitored by optical bioluminescence imaging (BLI) of Fluc and specific targeting of the liposomal nanoparticle was tracked by Rluc imaging. CD44 antibody-mediated liposomal nanoparticle, loaded of TF plasmids, were shown to be useful for monitoring and evaluating targeting efficacy and gene therapy by non-invasive molecular imaging. Here, we demonstrate the time intensive preclinical steps involved in molecular target identification, validation, and characterization by dual molecular imaging. This targeted and traceable therapeutic strategy has potential advantages to overcome the problems of conventional tumor therapy and may open a new application for the treatment of HCC by targeting CSCs.

[Differentiation of human embryonic stem cells to endothelial cells via improved three-dimension approach].

OBJECTIVE: To establish an improved three-dimension (3D) and serum-free approach to differentiate human embryonic stem cells (hESCs) into endothelial cells, and detect the endothelial functions of the obtained cells. METHODS: We cultured undifferentiated H9 human embryonic stem cell line in low-adhesion dishes to form embryonic bodies (EBs). After 12 days, EBs were harvested, re-suspended into rat tail collagen type I, and put into the incubator (37℃). After 30 minutes, EGM-2 culture medium was added to the solidified collagen, and the EBs were cultured for another 3 days to form embryonic body-sproutings (EB-sproutings). EB-sproutings were digested with 0.25% collagenase I and 0.56 U/ml Liberase Blendzyme for 20 minutes respectively, and the CD31(+) cells were sorted by FACS. The endothelial functions were tested by Dil-ac-LDL uptake assay and tube formation assay. RESULTS: This approach raised the efficiency of endothelial differentiation to 18%, and also avoided the contamination with animal materials. The obtained hESC-derived endothelial cells (hESC-ECs) had the similar pattern of surface biomarkers as human umbilical vein endothelial cells (HUVECs), and their endothelial functions were confirmed by the uptake of Dil-ac-LDL and the tube formation on Matrigel. CONCLUSIONS: The improved 3D approach can enhance the efficiency of differentiation from hESCs into endothelial cells. Furthermore, serum free differentiation system may be applied in future hESC-based therapies for various ischemic diseases.

Bioluminescence reporter gene imaging characterize human embryonic stem cell-derived teratoma formation.

Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However, teratoma formation can be a major obstacle for hES-mediated cell therapy. Therefore, tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study, hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology, we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover, FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA-4, and the subcultured SSEA-4(+) cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA-4(+) cells derived from teratoma exhibited multipotency, retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers.