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Neuroinflammation, a key contributor to neurodegenerative diseases, results from excessive microglial activation. Microglia that respond to pathogenic molecules switch to the M1 type and secrete various immune cytokines, which can cause neuronal damage. Therefore, our study focused on molecules that can enhance the neuroprotective role of microglia and reduce neuronal damage. The adipocyte enhancer-binding protein 1 (AEBP1) gene is known for its role in regulating immune responses in macrophages. However, its role in neuroinflammation has not been fully explored. Therefore, we investigated the role of AEBP1 in microglial cells activated by lipopolysaccharide (LPS). First, we confirmed that AEBP1 is expressed in LPS-activated microglia and demonstrated that downregulation of AEBP1 using shRNA in activated microglia reduced the immune response via the nuclear factor-kappa-B (NFκB) pathway. These results promote a shift toward neuroprotective M2 microglia, thereby reducing neuronal damage. Next, we confirmed that the expression of AEBP1 was elevated in the brains of Alzheimer’s disease (AD) mice. Additionally, animal experiments to assess the therapeutic effects of AEBP1 showed that microglia gathered around amyloid beta (Aβ) and reduced its size. Taken together, our results provide the first evidence that AEBP1 can reduce inflammatory activity in microglia, suggesting its potential as a target molecule for immunotherapy.

Mesenchymal stem cells (MSCs) promote therapeutic angiogenesis to cure serious vascular disorders. However, their survival period and cytokine-secretory capacity are limited. Although hepatocyte growth factor (HGF) can accelerate the rate of angiogenesis, recombinant HGF is limited because of its very short half-life (<3–5 minutes). Thus, continuous treatment with HGF is required to obtain an effective therapeutic response. To overcome these limitations, we produced genome-edited MSCs that secreted HGF upon drug-specific induction. The inducible HGF expression cassette was integrated into a safe harbor site in an MSC chromosome using the TALEN system, resulting in the production of TetOn-HGF/human umbilical cord blood-derived (hUCB)-MSCs. Functional assessment of the TetOn-HGF/hUCB-MSCs showed that they had enhanced mobility upon the induction of HGF expression. Moreover, long-term exposure by doxycycline (Dox)-treated TetOn-HGF/hUCB-MSCs enhanced the anti-apoptotic responses of genome-edited MSCs subjected to oxidative stress and improved the tube-formation ability. Furthermore, TetOn-HGF/hUCB-MSCs encapsulated by arginine-glycine-aspartic acid (RGD)-alginate microgel induced to express HGF improved in vivo angiogenesis in a mouse hindlimb ischemia model. This study showed that the inducible HGF-expressing hUCB-MSCs are competent to continuously express and secrete HGF in a controlled manner. Thus, the MSCs that express HGF in an inducible manner are a useful therapeutic modality for the treatment of vascular diseases requiring angiogenesis.

Human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSCs) exhibit potency for the regeneration of infarcted hearts. Vascular endothelial growth factor (VEGF) is capable of inducing angiogenesis and can boost stem cell‐based therapeutic effects. However, high levels of VEGF can cause abnormal blood vessel growth and hemangiomas. Thus, a controllable system to induce therapeutic levels of VEGF is required for cell therapy. We generated an inducible VEGF‐secreting stem cell (VEGF/hUCB‐MSC) that controls the expression of VEGF and tested the therapeutic efficacy in rat myocardial infarction (MI) model to apply functional stem cells to MI. To introduce the inducible VEGF gene cassette into a safe harbor site of the hUCB‐MSC chromosome, the transcription activator‐like effector nucleases system was used. After confirming the integration of the cassette into the locus, VEGF secretion in physiological concentration from VEGF/hUCB‐MSCs after doxycycline (Dox) induction was proved in conditioned media. VEGF secretion was detected in mice implanted with VEGF/hUCB‐MSCs grown via a cell sheet system. Vessel formation was induced in mice transplanted with Matrigel containing VEGF/hUCB‐MSCs treated with Dox. Moreover, seeding of the VEGF/hUCB‐MSCs onto the cardiac patch significantly improved the left ventricle ejection fraction and fractional shortening in a rat MI model upon VEGF induction. Induced VEGF/hUCB‐MSC patches significantly decreased the MI size and fibrosis and increased muscle thickness, suggesting improved survival of cardiomyocytes and protection from MI damage. These results suggest that our inducible VEGF‐secreting stem cell system is an effective therapeutic approach for the treatment of MI. Stem Cells Translational Medicine 2017;6:1040–1051

Neuron damage by microglia, which act as macrophage cells in the brain, can result in various brain diseases. However, the function of pro-inflammatory or anti-inflammatory microglia in the neurons remains controversial. Guanylate-binding protein-2 (GBP2) is expressed and activated in the microglia in the early phase of the inflammatory response and plays an important role in controlling immune responses. In this study, we evaluated whether GBP2 initially reduces the immune response induced by microglia, and whether microglia induce pro-inflammatory functions in neurons via GBP2 expression. In lipopolysaccharide (LPS)-stimulated microglia, we assessed the expression of GBP2 and how it affects neurons via activated microglia. The biological functions of microglia due to the downregulation of the GBP2 gene were examined using short hairpin RNA (shRNA)-RNA-GBP2. Downregulated GBP2 affected the function of mitochondria in the microglia and showed reduced neuronal damage when compared to the control group in the co-culture system. Furthermore, this protein was observed to be highly expressed in the brains of dementia mice. Our results are the first to report that the downregulation of GBP2 in activated microglia has an anti-inflammatory function. This study suggests that the GBP2 gene can be used as a therapeutic target biomarker for inflammation-related neurodegenerative diseases.

Atherosclerotic plaques within the vasculature may eventually lead to heart failure. Currently, cardiac stenting is the most effective and least invasive approach to treat this disease. However, in-stent restenosis is a complex chronic side effect of stenting treatment. This study used coronary stents coated with stem cells secreting angiogenic growth factors via an inducible genome-editing system to reduce stent restenosis and induce re-endothelialization within the artery. The characteristics of the cells and their adhesion properties on the stents were confirmed, and the stents were transplanted into a swine model to evaluate restenosis and the potential therapeutic use of stents with stem cells. Restenosis was evaluated using optical coherence tomography (OCT), microcomputed tomography (mCT) and angiography, and re-endothelialization was evaluated by immunostaining after cardiac stent treatment. Compared to a bare metal stent (BMS) or a parental umbilical cord blood-derived mesenchymal stem cell (UCB-MSC)-coated stent, the stents with stem cells capable of the controlled release of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) successfully reduced restenosis within the stent and induced natural re-endothelialization. Furthermore, UCB-MSCs exhibited the ability to differentiate into endothelial cells in Matrigel, and HGF and VEGF improved this differentiation. Our study indicates that stents coated with UCB-MSCs secreting VEGF/HGF reduce the restenosis side effects of cardiac stenting with improved re-endothelialization. Heart disease: Coated stents reduce repeated blocking of arteries Coating stents with modified stem cells may reduce the risk of repeated artery blocking in the treatment of heart disease. Currently, tubular stents are used to re-open arteries narrowed or blocked by the build-up of fatty deposits. However, if the artery is damaged by the stent or tries to reject it, the resulting inflammatory immune response can result in restenosis, or re-blocking. Je-Yoel Cho at Seoul National University, Republic of Korea, and co-workers trialed stents coated with modified stem cells in swine models. The stem cells are designed to secrete controlled levels of two growth factors, enabling them to differentiate into blood vessel lining cells and build a protective layer around the stent. In comparison with standard stents and stents coated with unmodified stem cells, the modified stents improved blood flow and reduced restenosis.

Late endothelial progenitor cells (LEPCs) are derived from mononuclear cells (MNCs) and are thought to directly incorporate into blood vessels and differentiate into mature endothelial cells (ECs). Using transcriptome and proteome analysis, we identified distinctive LEPC profiles and found that Hedgehog-interacting protein (HIP) is strongly expressed in LEPCs. Inhibition of HIP by lentiviral knockdown activated canonical hedgehog signaling in LEPCs, while it activated non-canonical hedgehog signaling in ECs. In LEPCs, HIP knockdown induced much enhanced tube formation and resistance to apoptosis under oxidative stress conditions via canonical hedgehog signaling. Although HIP is strongly expressed in proliferating LEPCs, HIP expression is down-regulated during angiogenesis and under oxidative stress condition. Moreover, when LEPCs are treated with angiogenic triggers such as VEGF and FGF2, HIP expression is reduced. Our findings suggest that HIP blocks LEPC angiogenesis and regulate survival when there is no angiogenic stimulation. HIP inhibition in LEPCs enhanced tube formation and reduced apoptosis, resulting in improved angiogenesis.

The integration of innovative medical technologies and interdisciplinary collaboration could improve the treatment of cancer, a globally prevalent and often deadly disease. Despite recent advancements, current cancer therapies fail to specifically address recurrence and target cancer stem cells (CSCs), which contribute to relapse. In this study, we utilized three types of cancer cells, from which three types of CSCs were further derived, to conduct a proteomic analysis. Additionally, shared cell surface biomarkers were identified as potential targets for a comprehensive treatment strategy. The selected biomarkers were evaluated through short hairpin RNA treatment, which revealed contrasting functions in cancer cells and CSCs. Knockdown of the identified proteins revealed that they regulate the epithelial–mesenchymal transition (EMT) and stemness via the ERK signaling pathway. Resistance to anticancer agents was consequently reduced, ultimately enhancing the overall anticancer effects of the treatment. Additionally, the significance of these biomarkers in clinical patient outcomes was confirmed using bioinformatics. Our study suggests a novel cancer treatment strategy that addresses the limitations of current anticancer therapies.

Breast cancer in women is one of the most common life-threatening malignancies. Despite of the development for the improved treatment, there are still many limitations to overcome. Among them, cancer stem cells (CSCs) are well known for tumor formation, development, cellular heterogeneity, and cancer recurrence. Therefore, to completely cure breast cancer, treatment of both cancer and CSC is required. To selectively target CSCs, we generated a liposome-based smart nano complex using CEACAM 6 (CD66c) antibody (Ab), a novel cell-surface biomarker of breast-derived CSCs (BCSCs) discovered in our previous research. Selective and increased cellular uptake was observed in BCSCs treated with CD66c Ab-conjugated rhodamine-labeled liposomes (CDRHOL) depending on the expression level of CD66c. CD66c Ab-conjugated doxorubicin (DOX)-loaded liposomes (CDDOXL) selectively showed increased cell killing effects in BCSCs with high CD66c expression levels. In an in vivo animal study, CDRHOL showed enhanced accumulation in xenografted BCSC tumors with low delivery into non-target organs. Moreover, mice treated with CDDOXL have assessed the decreased induction ability of immune response by low expression levels of pro-inflammatory cytokines and reduced liver toxicity by histopathological analysis. Finally, the improved antitumor effect of CDDOXL was evaluated in a metastatic BCSC mouse model via systemic administration. Collectively, our study is the first to demonstrate that a multi-functional nano complex using a novel surface biomarker of BCSC may be a more effective therapeutic agent for the treatment of cancer and CSCs.

Polycystic Kidney Disease (PKD) is a disorder that affects the kidneys and other organs, and its major forms are encoded by polycystin-1 (PC1) and polycystin-2 (PC2), as PKD1 and PKD2. It is located sandwiched inside and outside cell membranes and interacts with other cells. This protein is most active in kidney cells before birth, and PC1 and PC2 work together to help regulate cell proliferation, cell migration, and interactions with other cells. The molecular relationship and the function between PKD1 and cancer is well known, such as increased or decreased cell proliferation and promoting or suppressing cell migration depending on the cancer cell type specifically. However, its function in stem cells has not been revealed. Therefore, in this study, we investigated the biological function of PC1 and umbilical cord blood-derived mesenchymal stem cell (UCB-MSC). Furthermore, we assessed how it affects cell migration, proliferation, and the viability of cells when expressed in the PKD1 gene. In addition, we confirmed in an ex vivo artificial tooth model generated by the three-dimension printing technique that the ability to differentiate into osteocytes improved according to the expression level of the stemness markers when PKD1 was expressed. This study is the first report to examine the biological function of PKD1 in UCB-MSC. This gene may be capable of enhancing differentiation ability and maintaining long-term stemness for the therapeutic use of stem cells.

The combination of therapeutic nucleic acids and chemotherapeutic drugs has shown great promise for cancer therapy. In this study, asialoglycoprotein receptors (ASGPR) targeting-ligand-based liposomes were tested to determine whether they can co-deliver vimentin siRNA and doxorubicin to hepatocellular carcinoma (HCC) selectively. To achieve this goal, we developed an ASGPR receptor targeted co-delivery system called gal-doxorubicin/vimentin siRNA liposome (Gal-DOX/siRNA-L). The Gal-DOX/siRNA-L was created via electrostatic interaction of galactose linked-cationic liposomal doxorubicin (Gal-DOX-L) on vimentin siRNA. Previous studies have shown that Gal-DOX/siRNA-L inhibited tumor growth by combined effect of DOX and vimentin siRNA than single delivery of either DOX or vimentin siRNA. These Gal-DOX/siRNA-Ls showed stronger affinity to human hepatocellular carcinoma cells (Huh7) than other cells (lung epithelial carcinoma, A549). These liposomes also have demonstrated that novel hepatic drug/gene delivery systems composed of cationic lipid (DMKE: O,O’-dimyristyl-N-lysyl glutamate), cholesterol, galactosylated ceramide, POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), and PEG2000-DSPE (distearoyl phosphatidyl ethanolamine) at 2:1:1:1:0.2 (moral ratios) can be used as an effective drug/gene carrier specifically targeting the liver in vivo. These results suggest that Gal-DOX-siRNA-L could effectively target tumor cells, enhance transfection efficacy and subsequently achieve the co-delivery of DOX and siRNA, demonstrating great potential for synergistic anti-tumor therapy.