Isolation and Characterization of Novel Murine Epiphysis Derived Mesenchymal Stem Cells

BACKGROUND: While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments. CONCLUSIONS/SIGNIFICANCES: These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities

Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase

The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a [FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGACThydA) using a log-phase expression promoter PpckA to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum [FeFe]- hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38 �C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40 �C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic [FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight

The clinical impact of supraclavicular lymph node metastasis in patients with locally advanced esophageal squamous cell carcinoma receiving curative concurrent chemoradiotherapy.

BACKGROUND:To evaluate the clinical significance of supraclavicular lymph node (SCLN) in patients with locally advanced esophageal squamous cell carcinoma (ESCC) receiving curative concurrent chemoradiotherapy (CCRT). MATERIALS AND METHODS:We retrospectively analyzed all 369 locally advanced ESCC patients treated with CCRT between 2000 and 2015, including 70 patients with SCLN metastasis and 299 patients without SCLN metastasis. RESULTS:For these locally advanced ESCC patients treated with curative CCRT, N0-2 were significantly associated with superior progression-free survival (PFS) and overall survival (OS) in univariate and multivariable analyses. However, there were no significant differences in PFS and OS between the SCLN metastasis and non-SCLN metastasis groups; a subgroup analysis also revealed there was no significant differences in PFS and OS between patients with and without SCLN metastasis either in the N0-2 or in the N3 subgroup analysis. CONCLUSIONS:Our study suggests that SCLN metastasis is not a prognostic factor in locally advanced ESCC patients receiving curative CCRT, and that SCLNs should be considered to be regional LNs and treated with curative intent

Univariate and multivariate analyses results of PFS and OS in in 369 locally advanced esophageal squamous cell carcinoma patients receiving curative CCRT.

<p>Univariate and multivariate analyses results of PFS and OS in in 369 locally advanced esophageal squamous cell carcinoma patients receiving curative CCRT.</p

The survival curves of 70 esophageal squamous cell carcinoma patients with supraclavicular lymph node (SCLN) metastasis compared to the 299 patients without SCLN metastasis.

<p>(A) Progression-free survival. (B) Overall survival.</p

Comparison of survival curves of esophageal squamous cell carcinoma patients with or without SCLN metastasis in different N statuses.

<p>(A) N0-2 status, progression-free survival (B) N0-2 status, overall survival. (C) N3 status, progression-free survival (D) N3 status, overall survival. SCLN: supraclavicular lymph node.</p

Clinicopathological parameters in 369 locally advanced esophageal squamous cell carcinoma patients with/without supraclavicular lymph node (SCLN) metastasis receiving curative CCRT.

<p>Clinicopathological parameters in 369 locally advanced esophageal squamous cell carcinoma patients with/without supraclavicular lymph node (SCLN) metastasis receiving curative CCRT.</p

Prevention of skin flap necrosis in mice by EMSCs injection.

<p>(A) Ischemic flap showed necrosis in the control mice and near complete healing in the EMSCs (at the fifth passage) injected mice after 6 days of surgery. (B) Percentage of necrosis was quantified by planimetry. Percentage of necrotic area is significantly larger in the control group than in the EMSCs injected group (n = 4). (C) The skin structure was evaluated by H&E staining. Skin thickness is decreased and mucinous layer is preserved in the EMSCs injected group as compared to the control group. (D) In higher magnification, extra vascular erythrocytes and hemorrhage (arrowhead) can be observed in the control group. (E) Immunostaining of von Willebrand factor (vWF) of skin flap, and (F) its quantification showed significantly more vWF expressed cells (arrowhead) in the EMSCs injected group than the control group. **represents <i>P</i><0.01. Scale bars represent 100 µm. Data are presented as mean ± s.d. and analyzed with Student's <i>t</i>-test. PM: platysma muscle; ML: mucinous layer; Control: complete medium injection-group; EMSC: EMSCs-injection group.</p

Repair of bone fracture in mice after transplantation of EMSCs.

<p>(A) Fracture healing was assessed by X-ray after 14 days with or without EMSCs (at the fifth passage) transplantation. Arrowheads indicate the site of fracture. (B) The bone density of fracture site (dotted box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036085#pone-0036085-g008" target="_blank">Figure 8A</a>) was quantified (n = 4). EMSCs transplantation significantly improved osteocalcification as compared to the control group. (C) The morphology of fracture sites were examined by H&E staining. Tissue disorganization was showed in the control group while the EMSCs transplanted group newly formed bone tissue (arrow) and capillaries (arrowhead). * represents <i>P</i><0.05. Scale bars represent 100 µm. Data are presented as mean ± s.d. and analyzed with Student's <i>t</i>-test. Control: SPONGOSTAN carried with complete medium-transplantation group; EMSC: SPONGOSTAN carried with EMSCs-transplantation group.</p

Establishment of EMSCs and BMMSCs.

<p>(A) Schematic protocol for the establishment of cultures of EMSCs and BMMSCs. (B) Phase-contrast micrograph of EMSCs in primary culture after seven days. Arrow heads indicate triangle, spindle-shaped, fibroblast-like EMSCs. Lymphohematopoietic cells are indicated by arrows. (C) Phase-contrast micrograph of BMMSCs in primary culture after seven days. Arrow heads indicate flat, fibroblast-like BMMSCs and arrows indicate lymphohematopoietic cells. (D) Phase contrast micrograph of EMSCs at the fifth passage showing that EMSCs maintained their shape during propagating. (E) Phase contrast micrograph of BMMSCs upon the fifth passage showing that BMMSCs became flatted with increasing passages. All the scale bars represent 100 µm.</p