Xeno-free and feeder-free culture and differentiation of human embryonic stem cells on recombinant vitronectin-grafted hydrogels.

Recombinant vitronectin-grafted hydrogels were developed by adjusting surface charge of the hydrogels with grafting of poly-l-lysine for optimal culture of human embryonic stem cells (hESCs) under xeno- and feeder-free culture conditions, with elasticity regulated by crosslinking time (10-30 kPa), in contrast to conventional recombinant vitronectin coating dishes, which have a fixed stiff surface (3 GPa). hESCs proliferated on the hydrogels for over 10 passages and differentiated into the cells derived from three germ layers indicating the maintenance of pluripotency. hESCs on the hydrogels differentiated into cardiomyocytes under xeno-free culture conditions with much higher efficiency (80% of cTnT+ cells) than those on conventional recombinant vitronectin or Matrigel-coating dishes just only after 12 days of induction. It is important to have an optimal design of cell culture biomaterials where biological cues (recombinant vitronectin) and physical cues (optimal elasticity) are combined for high differentiation of hESCs into specific cell lineages, such as cardiomyocytes, under xeno-free and feeder-free culture conditions.

Rheumatoid arthritis is associated with rs17337023 polymorphism and increased serum level of the EGFR protein.

OBJECTIVE: We have previously described the association of rheumatoid arthritis (RA) prevalence and two epidermal growth factor receptor (EGFR) SNPs (rs17337023 and rs2227983) among the Taiwanese population. This present study aimed to elucidate whether the SNPs can alter the expression of EGFR in the progression of RA. METHODS: The cohort study included 366 Taiwan's Han Chinese RA patients and 326 age and gender matched healthy controls. Blood samples collected from the participants were analyzed to determine their serum EGFR levels and to identify EGFR SNPs from their genomic DNA. Genotyping for EGFR SNPs was performed by restriction fragment length polymorphism (RFLP) assay. The relationship between EGFR SNP and the clinical manifestations of RA was evaluated. RESULTS: Our results showed that a statistically significant difference in genotype frequency distributions at rs17337023 SNP for RA patients and controls (p ˂ 0.05). In addition, compared with the haplotype frequencies between case and control groups, the RA patient with the GT haplotype appeared to be a significant "protective" haplotype compared with other haplotypes (OR: 0.73, 95% CI: 0.59-0.91; p = 0.005). Furthermore, the increased serum level of EGFR was also observed in RA patients (p ˂ 0.001). CONCLUSION: Our study showed that RA is associated with rs17337023 SNP in EGFR gene and increased serum level of the EGFR protein. These findings suggest EGFR is worthy of further investigation as a therapeutic target for RA.

Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods.

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 µm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not.

Data of continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surfaces.

This data article contains two figures and one table supporting the research article entitled: "Continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surface" [1]. The table shows coating conditions of three copolymers, poly(styrene-co-acrylic acid) grafted with oligovitronectin, poly(styrene-co-N-isopropylacrylamide) and poly(styrene-co-polyethylene glycol methacrylate) to prepare thermoresponsive surface. XPS spectra show the nitrogen peak of the polystyrene surface coated with poly(styrene-co-acrylic acid) grafted with oligovitronectin. The surface coating density analyzed from sorption of poly(styrene-co-acrylic acid) grafted with oligovitronectin by UV-vis spectroscopy is also presented.

Pluripotency maintenance of amniotic fluid-derived stem cells cultured on biomaterials.

The stem cell fates of pluripotency and differentiation are regulated by not only soluble biological cues but also insoluble biochemical cues (i.e., extracellular matrix (ECM)) and the physical cues of cell culture biomaterials (i.e., elasticity). We investigated the maintenance of pluripotency and the differentiation lineages of human amniotic fluid-derived stem cells (hAFSCs) cultured on poly(vinyl alcohol-co-itaconic acid) (PVA) hydrogels grafted with several types of ECM and corresponding oligopeptides in expansion medium. hAFSCs cultured on soft PVA hydrogels (12.2 kPa) that were grafted with oligopeptides derived from fibronectin and vitronectin showed high pluripotency, which was evaluated by Oct4, Sox2 and Nanog expression. The hAFSCs grown on soft PVA hydrogels (12.2 kPa) grafted with each oligopeptide showed higher pluripotency, as assessed by Oct4 and Nanog expression, than hAFSCs grown on stiff PVA hydrogels (25.3 kPa) grafted with the same oligopeptides and a much higher pluripotency than those grown on rigid tissue-culture polystyrene dishes. Soft biomaterials appeared to be adequate to maintain the pluripotency of hAFSCs. Surprisingly, hAFSCs that showed higher pluripotency on PVA hydrogels grafted with oligopeptides derived from fibronectin and vitronectin also expressed higher levels of early differentiation markers for three germ layers in expansion medium. This result suggests that hAFSCs are heterogeneous and that this population contains highly pluripotent stem cells and stem cells that can be easily differentiated.

Genetic susceptibility to idiopathic membranous nephropathy in high-prevalence Area, Taiwan.

Idiopathic membranous nephropathy (MN) is one common cause of idiopathic nephrotic syndrome in adults; 25% of MN patients proceed to end-stage renal disease. In adults, membranous nephropathy is a lead cause of nephrotic syndrome, with about 75% of the cases idiopathic. Secondary causes include autoimmune disease, infection, drugs and malignancy. Three hypotheses about pathogenesis have surfaced: preformed immune complex, in situ immune complex formation, and auto-antibody against podocyte membrane antigen. Pathogenesis does involve immune complex formation with later deposition in sub-epithelial sites, but definite mechanism is still unknown. Several genes were recently proven associated with primary membranous nephropathy in Taiwan: IL-6, NPHS1, TLR-4, TLR-9, STAT4, and MYH9 . These may provide a useful tool for diagnosis and prognosis. This article reviews epidemiology and lends new information on KIRREL2 (rs443186 and rs447707) polymorphisms as underlying causes of MN; polymorphisms revealed by this study warrant further investigation.

Purification of human adipose-derived stem cells from fat tissues using PLGA/silk screen hybrid membranes.

The purification of human adipose-derived stem cells (hADSCs) from human adipose tissue cells (stromal vascular fraction) was investigated using membrane filtration through poly(lactide-co-glycolic acid)/silk screen hybrid membranes. Membrane filtration methods are attractive in regenerative medicine because they reduce the time required to purify hADSCs (i.e., less than 30 min) compared with conventional culture methods, which require 5-12 days. hADSCs expressing the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the permeation solution from the hybrid membranes. Expression of the surface markers CD44, CD73, and CD99 on the cells in the permeation solution from the hybrid membranes, which were obtained using 18 mL of feed solution containing 50 × 104 cells, was statistically significantly higher than that of the primary adipose tissue cells, indicating that the hADSCs can be purified in the permeation solution by the membrane filtration method. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the permeation solution, whereas CD34⁺ cells could not be purified by the conventional culture method. The hADSCs in the permeation solution demonstrated a superior capacity for osteogenic differentiation based on their alkali phosphatase activity, their osterix gene expression, and the results of mineralization analysis by Alizarin Red S and von Kossa staining compared with the cells from the suspension of human adipose tissue. These results suggest that the hADSCs capable of osteogenic differentiation preferentially permeate through the hybrid membranes.

Suppression of cancer-initiating cells and selection of adipose-derived stem cells cultured on biomaterials having specific nanosegments.

Cancer-initiating cells [cancer stem cells (CSCs)] in colon cancer cells can be selectively suppressed when they are cultured on Pluronic (nanosegment)-grafted dishes, whereas CSCs are maintained on conventional tissue culture dishes and extracellular matrix-coated dishes. CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumorigenic clones. The purification or depletion (suppression) of CSCs should be useful for analyzing CSC characteristics and for clinical application. CSCs can be selectively suppressed from colon cancer cells containing adipose-derived stem cells (ADSCs) on Pluronic-grafted dishes, while ADSCs remain on the dishes. ADSCs on Pluronic-grafted dishes after the suppression of the CSCs can differentiate into osteoblasts, chondrocytes, adipocytes, cardiomyocytes, and neuronal cells. The CSCs and ADSCs exhibited different characteristics. The selection of ADSCs was possible on Pluronic-grafted dishes that suppressed the CSCs from the fat tissues of cancer patients (i.e., cell-sorting dishes), which was explained by specific biomedical characteristics of Pluronic.

The combined influence of substrate elasticity and surface-grafted molecules on the ex vivo expansion of hematopoietic stem and progenitor cells.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem and progenitor cells (HSPCs) for transplantation. However, the low number of HSPCs from a single UCB donor limits the direct transplantation of UCB to patients. Because little is known about the effects of the physical microenvironment on HSPC expansion, we investigated the ex vivo expansion of HSPCs cultured on biomaterials with different elasticities and grafted with different nanosegments. Polyvinylalcohol-co-itaconic acid (PVA-IA)-coated dishes with different stiffnesses ranging from a 3.7 kPa to 30.4 kPa storage modulus were used. Fibronectin or an oligopeptide (CS1, EILDVPST) was grafted onto the PVA-IA substrates. High ex vivo fold expansion of HSPCs was observed in the PVA-IA dishes grafted with fibronectin or CS1, which displayed an intermediate stiffness ranging from 12.2 kPa to 30.4 kPa. The fold expansion was more than 1.4 times higher than that cultured in tissue culture polystyrene dishes (TCPS, 12 GPa). Furthermore, HSPCs cultured in fibronectin or CS1-grafted PVA-IA-coated dishes with a stiffness of 12.2-30.4 kPa generated more pluripotent colony-forming units (CFU-GM and CFU-GEMM) than those in TCPS dishes. This result indicates that both the physical and biological properties of biomaterials affect the ex vivo expansion of HSPCs.

The isolation and differentiation of human adipose-derived stem cells using membrane filtration.

Human adipose-derived stem cells (hADSCs) were purified from a suspension of human adipose tissue cells (stromal vascular fraction) by the conventional culture method and by membrane filtration through polyurethane (PU) foam membranes. hADSCs can be obtained from a suspension of human adipose tissue cells using the membrane filtration method in less than 30 min, whereas the conventional culture method requires 5-12 days. hADSCs that express the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the recovery solution from the PU membranes; no hADSCs were isolated in the permeate. After filtration, the cells expressing the mesenchymal stem cell markers were 3-4.5 times more concentrated than in the initial suspension of human adipose tissue cells, with the level of concentration depending on the surface modification of the PU membrane. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the recovery solutions, whereas CD34(+) cells could not be purified by the conventional culture method. The hADSCs in the recovery solution demonstrated a superior capacity for osteogenic differentiation than did the cells in the suspension of human adipose tissue cells. These results suggested that the hADSCs with the capability for osteogenic differentiation adhered to the PU membranes.

Effect of the surface density of nanosegments immobilized on culture dishes on ex vivo expansion of hematopoietic stem and progenitor cells from umbilical cord blood.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem and progenitor cells for hematopoietic stem cell (HSC) transplantation. However, the low number of HSCs obtainable from a single donor of UCB limits direct transplantation of UCB to the treatment of pediatric patients. In this study, we investigated the ex vivo expansion of HSCs cultured on biomaterials grafted with several nanosegments, i.e. polyamine, fibronectin, RGDS, and CS1 (EILDVPST), at several surface densities. No direct correlation was found between fold expansion of HSCs and physical parameters of the culture dishes, i.e. surface roughness and water contact angle of the culture dishes. However, a small amount of grafted amino groups, less than 0.8 residual µmol cm(-2), on the dishes was effective for the ex vivo expansion of HSCs. A high amount of grafted amino groups hindered the ex vivo expansion of HSCs on the dishes. HSCs cultured on dishes with a high concentration of CS1 (2.40 residual µmol cm(-2)) showed greater expansion of HSCs and more pluripotent colony-forming units (i.e. colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte (CFU-GEMM)) than those on fibronectin-grafted and polyamine-grafted dishes. These data suggest that the specific interaction between HSCs and CS1 helps to maintain the pluripotency of HSCs during the ex vivo expansion of HSCs.

Serum adiponectin and leptin levels in Taiwanese breast cancer patients.

The role of two adipocytokines, adiponectin and leptin, in Taiwanese breast cancer patients remains to be determined. In this study, we analyzed the correlations between the serum levels of adiponectin and leptin and the various clinicopathological parameters in 100 newly diagnosed, histologically confirmed breast cancer patients and 100 controls. We found serum levels were decreased significantly for adiponectin in the breast cancer patients, in comparison to controls (Student t-test, P=0.003), while serum levels were increased significantly for leptin in the breast cancer patients in comparison to controls (Student t-test, P=0.025). Leptin/adiponectin (L/A ratio) were increased significantly in the breast cancer patients, in comparison to controls (Student t-test, P=0.009). Among the clinicopathological parameters, estrogen receptor, progesterone receptor, HER2/neu, lymph node metastasis, tumor stage, and tumor grade all showed no effect on the serum levels of adiponectin and leptin. BMI was negatively and positively correlated to serum adiponectin and leptin levels, respectively (Spearman's correlation, r=-0.333 and 0.323, respectively; P<0.001 for both). Intriguingly, serum L/A ratio disclosed a positive correlation to tumor size (r=0.21, P=0.036). In summary, our results suggest that low serum adiponectin levels and high serum leptin levels are associated with an increased risk for breast cancer. Also, independent of the effect of BMI, the increased serum ratio of L/A may indicate the presence of aggressive breast cancers.

Decreased expression of phosphorylated JNK in breast infiltrating ductal carcinoma is associated with a better overall survival.

Phosphorylation/activation of c-jun NH2-terminal kinase (JNK) has an ambivalent role, pro-proliferation or antiproliferation, in human cancers, which is determined by different cell types and by its crosstalk with other kinases. So far, the role of phosphorylated JNK (p-JNK) in breast cancer is mostly undefined. In this study, we analyzed the expression of p-JNK, as well as p-ERK1/2 and p-38, in the pair of cancer and noncancer breast tissues, by using immunoblotting techniques. These results were further correlated with the clinicopathological characteristics and overall survival. Decreased p-JNK1/2 expression in cancer tissues was observed in 48.5% of breast infiltrating ductal carcinoma (IDC) cases and was correlated significantly with the increased tumor grade and the decreased age at diagnosis (p = 0.030 and 0.029). Interestingly, the Kaplan-Meier survival curve showed that the decreased p-JNK1/2 expression was associated with a better overall survival of IDC (p = 0.004). The expression of p-JNK1/2 was positively correlated with p-p38 (p = 0.002), but not p-ERK1/2. Furthermore, co-expressed p-JNK1/2 and p-p38 was associated with a poor overall survival of IDC (p = 0.007). In conclusion, our results indicate that the aberrant p-JNK1/2 expression and the co-expressed p-JNK1/2 and p-p38 in breast tissues may play a role in the carcinogenesis of breast IDC.

The correlation between renal function and systolic or diastolic blood pressure in severe preeclamptic women.

OBJECTIVE: To evaluate the correlation between renal function and systolic or diastolic blood pressure in preeclamptic mothers. METHODS: In this prospective study from August 1998 to September 2002, 28 women >or= 28 weeks gestation with severe preeclampsia were selected. Meanwhile, 56 normotensive pregnant women without proteinuria or edema served as the control group. Urine was collected for 24 hours for all subjects. The concentration of uric acid, blood urea nitrogen, creatinine, sodium, calcium, and albumin in the 24-hour urine and blood of both groups were examined. Neonatal outcome also was evaluated. RESULTS: The serum and 24-hour urine concentration of blood urea nitrogen, creatinine, and albumin were significantly higher in severe preeclamptic women. Serum uric acid and urinary albumin/creatinine ratio was significantly higher in severe preeclamptic women compared with that in normotensive mothers and showed positive correlation with systolic or diastolic blood pressure. On the other hand, serum calcium/creatinine ratio was significantly lower in the severe preeclamptic group and negatively correlated to blood pressure. In multiple regressions, systolic or diastolic blood pressure was dependent on serum uric acid, albumin/creatinine, and calcium/creatinine ratios. Fetal birth weight was significantly lower in women with severe preeclampsia and with a lower Apgar score < 7 at 1 minute and 5 minutes and more preterm delivery compared with that in normotensive women. CONCLUSION: Renal function in women with severe preeclampsia was significantly impaired and highly correlated with systolic or diastolic blood pressure.

Urinary cyclic guanosine 3',5'-monophosphate and cyclic adenosine 3',5'-monophosphate changes in spontaneous and induced onset active labor.

BACKGROUND: The aim of this prospective, randomized study was to investigate the changes in urinary cyclic guanosine 3',5'-monophosphate (cGMP) and cyclic adenosine 3',5'-monophosphate (cAMP) between the latent and the active phases of spontaneous and prostaglandin E(1) (PGE(1))-induced labor. METHODS: Seventy singleton pregnant women at 36-41(+) weeks' gestation without signs of fetal distress were enrolled. The first group consisted of 35 pregnant women in whom labor was induced by PGE(1) applied intravaginally. The second group consisted of 35 women who had spontaneous active labor. Clinical data of the two groups were assessed as labor progressed. RESULTS: After the onset of active labor, urinary cGMP/creatinine (U cGMP/Cr) decreased in both groups with the percentage decline of 35.2 and 9.7, respectively, but this difference was only significant in the PGE(1)-induced group (P=0.033). After the onset of active labor, urinary cAMP/creatinine (U cAMP/Cr) decreased in both groups with the percentage decline of 36.5 and 15.6, respectively, but this difference was only significant in the PGE(1)-induced group (P=0.001). The duration of the latent phase was significantly shortened in the PGE(1)-induced group compared with the spontaneous labor group (P<0.05). CONCLUSIONS: Decreased U cGMP/Cr and U cAMP/Cr may be a transition from the latent to the active phase in PGE(1)-induced labor. Our results suggest that U cGMP/Cr and U cAMP/Cr can serve as easily obtained secondary messenger markers of myometrial contractility and cervical ripening at the onset of active labor. The NO-cGMP system and the G-protein alpha-cAMP system in the human uterus may concomitantly contribute to uterine quiescence during pregnancy and show downregulation in U cGMP/Cr and U cAMP/Cr at the initiation of active labor.

Serum vascular endothelial growth factor165 levels and uterine fibroid volume.

BACKGROUND: Vascular endothelial growth factor165 (VEGF165) demonstrates increased expression in uterine fibroids. This study aimed to investigate the relationship between serum VEGF165 levels and uterine fibroid volume. METHODS: On a prospective observational basis, 80 women with symptomatic uterine fibroids underwent hysterectomy. Uterine weight was determined after hysterectomy. Six hours before and 48 h after hysterectomy, serum VEGF165 levels were measured using an enzyme-linked immunosorbent assay. RESULTS: Logistic regression analysis showed no correlation between serum VEGF165 levels and uterine weight (r=0.0037; P>0.05). The mean serum VEGF165 level declined significantly from 716.31+/-457.99 to 581.81+/-403.32 pg/mL after hysterectomy (P<0.0001). Controlling for age, body mass index, uterine weight, proliferative or secretory phase, and parity, only parous patients (n=58) were found to have significantly decreased serum VEGF165 levels after hysterectomy (P=0.000035), in contrast to nulliparous patients (n=22; P=0.15). CONCLUSIONS: Serum VEGF165 levels do not correlate with uterine fibroid volume, but demonstrate a significant decrease after hysterectomy. The decrease in serum VEGF165 levels after hysterectomy was significant in parous but not in nulliparous patients with uterine fibroids. Serum VEGF165 levels may not predict uterine fibroid development.

Prospective randomized comparison of single-dose versus 1-day cefazolin for prophylaxis in gynecologic surgery.

BACKGROUND: The purpose of this prospective, randomized study was to compare the efficacy of single-dose versus 1-day cefazolin prophylaxis for the prevention of postoperative gynecologic infections. METHODS: From June 2001 to January 2003, 548 patients were randomized to receive either single-dose (1 g of cefazolin intravenously before surgery, 273 patients) or 1-day cefazolin (1 g intravenously before surgery and three more doses every 6 hr after surgery, 275 patients) prophylaxis. RESULTS: A total of 531 (267 patients in the single-dose group and 264 in the 1-day group) completed the study. Only one of 267 (0.37%) patients in the single-dose group developed a trocar wound infection and one of 264 (0.37%) patients in the 1-day group developed a vaginal cuff infection. Had a single dose of prophylactic antibiotics been administered to all patients, the antibiotic cost would have been reduced by 75-80%. CONCLUSIONS: The use of single-dose preoperative cefazolin prophylaxis was as effective as four doses of cefazolin for preventing serious infectious morbidity among our patients. Shortening the duration of antibiotics prophylaxis also reduced medical costs and microorganism resistance.