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Fracture remains one of the most common traumatic conditions in orthopedic surgery. The use of mesenchymal stem cells (MSCs) to augment fracture repair is promising. Leucine-rich repeat-containing GPCR 5 (Lgr5), a transmembrane protein, has been identified as a novel adult stem cell marker in various organs and tissues. However, the roles of Lgr5 in MSCs are not fully understood. In this study, we investigated cellular functions of Lgr5 in MSCs and its potential implications in treating fracture. Lgr5-overexpressing MSCs (MSC Lgr5 ) were established in murine SV40 promoter-driven luciferase reporter MSC line through virus transfection. Results of real-time quantitative PCR and Western blot analysis confirmed the increased expression of Lgr5 in MSC Lgr5 . MSC Lgr5 exhibited increased osteogenic capacity, which may result from elevated expression of β-catenin and phosphorylated ERK1/2 within the nuclear region of cells. In contrast, inhibition of Lgr5 expression decreased the osteogenic differentiation ability of MSCs, accompanied with increased mitochondrial fragmentation and reduced expression of β-catenin. Local transplantation of MSC Lgr5 at fracture sites accelerated fracture healing via enhanced osteogenesis and angiogenesis. MSC Lgr5 stimulated the tube formation capacity of HUVECs in a Matrigel coculture system in vitro significantly. Taken together, results suggest that Lgr5 is implicated in the cellular processes of osteogenic differentiation of MSCs through regulation of Wnt and ERK signaling pathways and mitochondrial dynamics in fusion and fission. Inhibition of Lgr5 expression induced increased mitochondrial fragmentation and suppression of osteogenesis. MSC Lgr5 exhibited enhanced therapeutic efficacy for fracture healing, which may serve as a superior cell source for bone tissue repair.-Lin, W., Xu, L., Pan, Q., Lin, S., Feng, L., Wang, B., Chen, S., Li, Y., Wang, H., Li, Y., Wang, Y., Lee, W. Y. W., Sun, D., Li, G. Lgr5-overexpressing mesenchymal stem cells augment fracture healing through regulation of Wnt/ERK signaling pathways and mitochondrial dynamics.

Stem cell-based therapies exhibit profound therapeutic potential for treating various human diseases, including cancer. Among the cell types that can be used for this purpose, mesenchymal stem cells (MSCs) are considered as promising source of stem cells in personalized cell-based therapies. The inherent tumor-tropic property of MSCs can be used to target cancer cells. Although the impacts of MSCs on tumor progression remain elusive, they have been genetically modified or engineered as targeted anticancer agents which could inhibit tumor growth by blocking different processes of tumor. In addition, there are close interactions between MSCs and cancer stem cells (CSCs). MSCs can regulate the growth of CSCs through paracrine mechanisms. This review aims to focus on the current knowledge about MSCs-based tumor therapies, the opportunities and challenges, as well as the prospective of its further clinical implications.

Cell therapy holds promise for treating a variety of diseases. Seeking available source of adult stem cells remains a great challenge in cell therapy. Urine is considered as an ideal source of adult stem cells which can be easily acquired by noninvasive methods. However, specific cell types in urine have not been well documented. Here, the aim of our study is to identify cell types in urine, and isolate and expand progenitor/stem cells from human urine and further evaluate their multipotency. Urine samples were collected from healthy donors. The cell suspension was seeded and selected because of plastic adherence. Colonies with two different morphologies appeared 7 days later. One type of colony was spindle-shaped and fibroblast-like; the other cell type displayed rounder shape. Cells that displayed fibroblast-like shape were selectively enriched using a cloning cylinder. Then multidifferentiation induction assays and immunophenotyping assays were applied. Characterization assays indicated that adherent cells possessed potent trilineage differentiation capacity and expressed CXCR4 and Nanog, as well as some mesenchymal stem cell surface antigens (including CD90 and CD44). Taken together, at least two cell populations exist in human urine. A stem cell subpopulation with trilineage differentiation capacity from human urine can be selectively enriched using the cloning cylinder method. Urine may become an ideal source of adult stem cells for cell therapy and further clinical implications., (© 2019 The Authors.)

Background: A promising approach of bone repair is to use stem cells, such as mesenchymal stem cells (MSCs). Seeking available source of MSCs still remains a great challenge in tissue engineering and cell therapy. Peripheral blood (PB) emerges as an alternative source of MSCs which can be easily acquired with minimal invasiveness. This study was undertaken to evaluate the multipotency of PB-MSCs and effects of human PB-MSCs transplantation on ectopic bone regeneration in nude mice., Methods: Human venous blood collected was mixed with heparin and then red blood cells were removed using red blood cell lysis buffer. Cell suspension was cultured in normoxia-culture and hypoxia-culture conditions, respectively. The non-adherent cells were removed by half changing culture media every three days. Cells were selected due to plastic adherence. The adherent cells were then passaged and subjected to multi-differentiation induction assays in vitro and in vivo ectopic bone formation assay., Results: Characterization assays indicated that cells cultured under hypoxia possessed potent multi-lineage differentiation capacity and expressed Nanog and Lgr5, as well as a series of MSC surface antigens (including CD29, CD90, CD105, and CD73). Additionally, regenerated bone tissues by transplantation of human PB-MSCs in vivo were confirmed by histological examinations of ectopic osteogenesis assay. A purified population of MSCs can be obtained within a short period of time using this protocol with a successful rate of 60%., Conclusion: We reported an effective and reliable method to harvest highly purified MSCs with potent multi-differentiation potential from human peripheral blood. Lgr5 may be a potential biomarker for identification of a subpopulation of PB-MSCs., The Translational Potential of This Article: PB-MSCs is an alternative cell source for cell therapy, which may be harvested, culture expanded and PB-MSCs loaded with β-tricalcium phosphate (β-TCP) may be used to promote bone repair., (© 2019 Chinese University of Hong Kong.)

Objective: To observe and compare the clinical efficacy of 1-year trial fitting and software fitting orthokeratology lenses., Methods: One hundred myopes who received vision correction with the use of orthokeratology lenses form July 2016 to September 2017 were included in this study. Subjects were assigned randomly into the two groups: the trial fitting group (group A) and the software fitting group (group B). For the right eye of each subject, measurements, such as uncorrected visual acuity (UCVA, logarithm of minimal angle of resolution), refractive error, corneal topography, ocular health status, and the fitting situation, were obtained at baseline, 1 week, 1 month, 3 months, 6 months, and 12 months after lens wear. Axial length and corneal endothelium cells (CECs) were also measured at baseline and 12 months after wearing the lens., Results: Compared with the baseline, the spherical equivalent refraction, UCVA, and central corneal curvature changed significantly after orthokeratology (OK) lens wear (all P<0.05). Between groups A and B, the parameters aforementioned were insignificant at each time point (all P>0.05). Axial length and CECs showed no significant changes during the first year of OK treatment (all P>0.05). Rate of corneal staining between two groups revealed no difference during 1-year visit (P<0.05)., Conclusion: Both the trial lens fitting and software fitting approaches were effective in temporarily reducing myopia, providing good UCVA and delaying the elongation of axial length for moderate and high myopic adolescents. Both the two approaches can be combined in OK lens fitting.

Phenylalanine ammonia-lyase (PAL) gene plays an important role in the synthesis of flavones, lignin, and other bioactive compounds in living organisms. Inonotus baumii, the only known flavone-producing filamentous fungus, is of great importance in the investigation of flavone metabolic pathways. To study the function of PAL enzyme in I. baumii flavone synthesis, a full-length cDNA of pal gene was cloned from I. baumii using DOP-PCR and RACE-PCR. The 2502-bp PAL coding region encodes an 833 amino acid protein with an approximate MW of 88.2kDa. Three introns and four exons are present in the DNA sequence of IbPAL. Amino acid sequence alignment showed that IbPAL shares 76% similarity with PALs of Inonotus fungi. The three-dimensional structure of IbPAL showed that it is composed of an MIO domain, a core domain and an inserted shielding domain. On this basis, the IbPAL was expressed and purified using the prokaryotic expression vector pSMART-V with a 6xHis-tag in Escherichia coli, and its enzymatic activity was subsequently detected. Our results will aid in understanding the enzymatic properties of PAL and further confirm the mechanism of flavone synthesis in I. baumii., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cell therapy continues to attract growing interest as a promising approach to treat a variety of diseases. Mesenchymal stem cells (MSCs) have been one of the most intensely studied candidates for cell therapy. Since the homing capacity of MSCs is an important determinant of effective MSC-based therapy, the enhancement of homing efficiency is essential for optimizing the therapeutic outcome. Furthermore, trafficking of endogenous MSCs to damaged tissues, also referred to as endogenic stem cell homing, and the subsequent participation of MSCs in tissue regeneration are considered to be a natural self-healing response. Therefore, strategies to stimulate and reinforce the mobilisation and homing of MSCs have become a key point in regenerative medicine. The current review focuses on advances in the mechanisms and factors governing trafficking of MSCs, and the relationship between MSC mobilisation and skeletal diseases, providing insights into strategies for their potential translational implications.

Developing a recombinant vector for noninvasively delivering biological macromolecules into the brain is important. This study constructed and purified a protein complex based on the cholera toxin (CT) molecular structure. Enhanced green fluorescent protein (EGFP)-modified A2 subunits of CT (CTA2) were used as tracer molecules for introduction of transactivator of transcription (TAT) through the A subunit into cells. The protein complex EGFP-CTA2-TAT/(CTB)5 (CTB: B subunit of CT) was obtained using an in vitro recombination method and verified by monosialoganglioside-enzyme-linked immunosorbent assay and high performance liquid chromatography assay. The protein complexes bound more strongly to monosialoganglioside (GM1) than (CTB)5 at low concentrations (0.625-1.25 μg/mL). In vitro assays revealed that the transmembrane function of TAT was also maintained. The GM1-binding activity and cell membrane-penetrating ability suggested that a CT structure-based protein complexes could be used to design a delivery carrier for intranasal administration through GM1 binding. The expression vector introduced in this study provides a feasible expression frame for constructing several new macromolecular protein drugs for effective cell penetration., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Green tea polyphenol (GTP) is one of the most promising chemopreventive agent for cancer; it can inhibit cancer cell proliferation and induce apoptosis through p53-dependent cell signaling pathways. Unfortunately, many tumor cells lack the functional p53, and little is known about the effect of GTP on the p53-deficient/mutant cancer cells. To understand the p53-independent mechanisms in GTP-treated p53-dificient/mutant cancer cells, we have now examined GTP-induced cytotoxicity in human hepatoma Hep3B cells (p53-deficient). The results showed that GTP could induce Bax and Bak activation, cytochrome c release, caspase activation, and necroptosis of Hep3B cells. Bax and Bak, two key molecules of mitochondrial permeability transition pore (MPTP), were interdependently activated by GTP, with translocation and homo-oligomerization on the mitochondria. Bax and Bak induce cytochrome c release. Importantly, cytochrome c release and necroptosis were diminished in Hep3B cells (Bax(-/-)) and Hep3B cells (Bak(-/-)). Furthermore, overexpression of Bcl-2 could ameliorate GTP-induced cytochrome c release and necroptosis. Together, the findings suggested that GTP-induced necroptosis was modulated by the p53-independent pathway, which was related to the translocation of Bax and Bak to mitochondria, release of cytochrome c, and activation of caspases.

According to the characteristics of OCT images for macula edema, we studied a method for segmentation of the macula edema. Based on the Chan-Vese model, we proposed an improved level-set algorithm. With defining the integer-valued signed function directly, the curve could evolute outward or inward by changing the inside neighboring rid points and outside neighboring grid points into each other. We realized image segmentation which is much faster than the method of Chan-Vese model and smoothness regularization. We segmented 45 images and extracted the macula edema of each image. After achieving good segmentation results, we estimated the volume of the macular edema. The method provides quantitative analytic tools for clinical diagnosis and therapy.