Chitosan-assisted differentiation of porcine adipose tissue-derived stem cells into glucose-responsive insulin-secreting clusters

<div><p>The unique advantage of easy access and abundance make the adipose-derived stem cells (ADSCs) a promising system of multipotent cells for transplantation and regenerative medicine. Among the available sources, porcine ADSCs (pADSCs) deserve especial attention due to the close resemblance of human and porcine physiology, as well as for the upcoming availability of humanized porcine models. Here, we report on the isolation and conversion of pADSCs into glucose-responsive insulin-secreting cells. We used the stromal-vascular fraction of the dorsal subcutaneous adipose from 9-day-old male piglets to isolate pADSCs, and subjected the cells to an induction scheme for differentiation on chitosan-coated plates. This one-step procedure promoted differentiation of pADSCs into pancreatic islet-like clusters (PILC) that are characterized by the expression of a repertoire of pancreatic proteins, including pancreatic and duodenal homeobox (Pdx-1), insulin gene enhancer protein (ISL-1) and insulin. Upon glucose challenge, these PILC secreted high amounts of insulin in a dose-dependent manner. Our data also suggest that chitosan plays roles not only to enhance the differentiation potential of pADSCs, but also to increase the glucose responsiveness of PILCs. Our novel approach is, therefore, of great potential for transplantation-based amelioration of type 1 diabetes.</p></div

Isolation of multipotent Adipose-Derived Stem Cells (ADSCs) from pig subcutaneous fat depot.

<p>(A). Flow cytometric analysis of porcine (p) ADSCs. 1 x 10⁶ cells were analyzed for stem cells markers. Numbers indicate the percentage of stained cells in the population (black) compared with the unstained control (gray). The x-axes represent the relative fluorescence intensity. (B). Multipotency of pADSCs. pADSCs were differentiated into adipocytes (left panel), osteocytes (middle panel) and chondrocytes (right panel) and stained as described in the text. Images were taken at 100 x magnification using phase contrast microscopy.</p

Chitosan enhances differentiation of pADSCs into insulin-producing Pancreatic Islet-Like Clusters (PILC).

<p>(A). Representative phase contrast microscopy analysis of the morphology of pADSC-derived PILC. Cells were differentiated for indicated times on regular (left panels) or chitosan-coated (right panels) plates as described in the text. (B). Representative immunofluorescence analysis of β-cell differentiation-associated markers, including Pdx-1, ISL-1 and insulin, in pADSC-derived PILC. PILC grown on regular and chitosan plates on day 12 or 15 were analyzed using antibodies against indicated markers and observed under a confocal microscope. (C). Effects of chitosan on the differential production of insulin by pADSC-derived PILC. The same procedure as in (B) was performed on pADSC differentiation from day 0 to 15. (D). Quantification of the effects of chitosan on the mRNA expression of β-cell differentiation-associated genes in pADSC-derived PILC. Total RNA was extracted from the clusters and analyzed by real-time PCR as described in the text. Values were normalized to β-actin and expressed as mean ± SEM, n = 6. *, <i>P</i> < 0.05. (Abbreviations: Pdx-1: pancreatic and duodenal homeobox-1; ISL-1: insulin gene enhancer protein-1; PAX4: pair box gene 4; GLUT2: glucose transporter 2)</p

The expression of pADSC-derived insulin-secreting cells and clusters after withdrawing the PILC differentiation medium for 9 days.

<p>(A) Quantification of the effects of withdrawing the PILC differentiation medium on the mRNA expression of β-cell differentiation-associated genes in pADSC-derived PILC. The differentiation medium was withdrawn from 0 to 9 days. Total RNA was extracted from the clusters and analyzed by real-time PCR as described in the text. Values were normalized to β-actin and expressed as mean ± SEM, n = 6. *, P < 0.05. (B) Quantification of insulin secretion level from PILCs in response to a glucose challenge on withdrawal day 9. PILCs from day 9 of withdrawal were starved overnight and treated with the indicated concentrations of glucose for one hour. Values were obtained by ELISA analysis as described in the text, normalized against protein concentrations and expressed as mean ± SEM, n = 5. *, P < 0.05.</p

Glucose responsiveness of pADSC-derived insulin-secreting clusters.

<p>Quantification of insulin secretion level from PILCs in response to a glucose challenge. PILC from day 15 of differentiation were starved overnight and treated with the indicated concentrations of glucose for one hour. Insulin values were obtained by ELISA analysis as described in the text, normalized against protein concentrations and expressed as mean ± SEM, n = 6. *, <i>P</i> < 0.05.</p

A Novel Detection Platform for Shrimp White Spot Syndrome Virus Using an ICP11-Dependent Immunomagnetic Reduction (IMR) Assay

<div><p>Shrimp white spot disease (WSD), which is caused by white spot syndrome virus (WSSV), is one of the world’s most serious shrimp diseases. Our objective in this study was to use an immunomagnetic reduction (IMR) assay to develop a highly sensitive, automatic WSSV detection platform targeted against ICP11 (the most highly expressed WSSV protein). After characterizing the magnetic reagents (Fe₃O₄ magnetic nanoparticles coated with anti ICP11), the detection limit for ICP11 protein using IMR was approximately 2 x 10⁻³ ng/ml, and the linear dynamic range of the assay was 0.1~1 x 10⁶ ng/ml. In assays of ICP11 protein in pleopod protein lysates from healthy and WSSV-infected shrimp, IMR signals were successfully detected from shrimp with low WSSV genome copy numbers. We concluded that this IMR assay targeting ICP11 has potential for detecting the WSSV.</p></div

IMR (%)–ϕ_ICP11 (spiked-rICP11-concentration in PBS) curve showing concentration-dependent IMR signals for ICP11 with standard deviations (duplicate measurements).

<p>IMR (%)–ϕ_ICP11 (spiked-rICP11-concentration in PBS) curve showing concentration-dependent IMR signals for ICP11 with standard deviations (duplicate measurements).</p

Linear correlation between actual ICP11 concentration (ϕ_ICP11) and ICP11 concentration (ϕ_ICP11-c) as measured by IMR in Fig 5.

<p>Linear correlation between actual ICP11 concentration (ϕ_ICP11) and ICP11 concentration (ϕ_ICP11-c) as measured by IMR in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138207#pone.0138207.g005" target="_blank">Fig 5</a>.</p

IMR detection of ICP11 protein in shrimp.

<p>(A) Measured ICP11 protein concentration (ϕ_ICP11-IMR) in non-challenged shrimp (normal control) and in shrimp with a light or severe WSSV infection. The cut-off value (the dashed line) was based on ROC curve analysis of the IMR results. (B) Sensitivity and specificity of the IMR assay as determined by ROC curve analysis.</p

Correlation between detected ICP11-IMR concentration and WSSV copy number.

<p>Pleopod samples were collected from shrimp and subjected to IMR assay and real-time PCR. Dots indicate shrimp belonging to the non-challenged control group (0.08 ~ 7 WSSV copies/mg tissue); crosses indicate light infection (10 ~ 3100 WSSV copies/mg tissue); triangles indicate severe infection (1.57 x 10⁴ ~ 2.83 x 10⁵ WSSV copies/mg tissue).</p