In vitro transdifferentiation of corneal epithelial-like cells from human skin-derived precursor cells.

The damage of human corneal cells encounter with the problem of availability of corneal cells for replacement. Limitation of the source of corneal cells has been realized. An attempt of development of corneal epithelial-like cells from the human skin-derived precursor (hSKPs) has been made in this study. Combination of three essential growth factors: epidermal growth factor (EGF), keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) could demonstrate successfully induction of hSKPs to differentiation into corneal cells.The induced cells expressed the appearance of markers of corneal epithelial cells as shown by the presence of keratin 3 (K3) by antibody label and Western blot assay. The K3 gene expression of induced hSKPs cells as shown by reverse transcription-polymerase chain reaction (RT-PCR) technology was also demonstrated. The presence of these markers at both gene and protein levels could lead to our conclusion that the directional transdifferentiation of hSKPs cells into corneal epithelial cells was successfully done under this cell induction protocol. The finding shows a newly available stem cell source can be obtained from easily available skin. Cells from autologous human skin might be used for corneal disorder treatment in future clinical application.

Exploring stemness gene expression and vasculogenic mimicry capacity in well- and poorly-differentiated hepatocellular carcinoma cell lines.

Vasculogenic mimicry (VM) is the phenomenon where cancer cells mimic endothelial cells by forming blood vessels. A stem cell-like phenotype has been proposed to be involved in this tumor plasticity. VM seems to correlate with metastasis rate, but there have been no reports on the effects of pro-metastatic and pro-angiogenic factors or hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) on VM formation of hepatocellular carcinoma (HCC) cells. Here, we determine VM capacity and expression of stemness genes (Oct4, Sox2, Nanog and CD133) in well- and poorly-differentiated HCC cell lines. The poorly-differentiated cell line SK-Hep-1 with mesenchymal features (high invasiveness and expressing Vimentin, with no E-cadherin) could form VM in vitro, while the well-differentiated cell line HepG2 did not form VM. There was no correlation between expression of stemness genes and intrinsic VM capacity. However, HGF but not VEGF, could induce VM formation in HepG2, concomitant with epithelial-mesenchymal transition (EMT), de-differentiation and increased expression of stemness genes. Our results show that the role of stemness genes in VM capacity of HCC cells is likely to depend on differentiation status.

Cadmium bioavailability from vegetable and animal-based foods assessed with in vitro digestion/caco-2 cell model.

BACKGROUND: Chronic dietary cadmium (Cd) exposure results in kidney dysfunction and decrease in bone mineral density. OBJECTIVE: To determine and compare the bioavailability of Cd from vegetable and animal-based foods. MATERIAL AND METHOD: Caco-2 cells were exposed to Cd in boiled pig kidney, ark shell, kale, raw kale, mixed boiled pig kidney with raw kale and CdCl2 after in vitro digestion. Then cellular Cd uptake from the digests and reference CdCl2 solution was measured by atomic absorption spectrometry. RESULTS: Cd bioavailability from animal-based foods was higher than that from vegetable-based foods. In addition, raw kale exhibited an inhibitory effect on Cd bioavailability when mixed with boiled pig kidney. However Cd in kale was increasingly absorbed after boiling. CONCLUSION: Cd binding to different molecular species, other food components in vegetable and animal-based foods, food combination, as well as cooking processes influenced the uptake of dietary Cd. A relative bioavailability factor accounted for the food matrix might be necessary for exposure assessment and consequently for estimation and prevention of the risk of dietary Cd.

Parathyroid hormone enhances osteoblast differentiation from human skin derived precursor cells in vitro.

Parathyroid hormone (PTH), a new effective treatment for osteoporosis patients which promotes the anabolic effect in vivo, can enhance the differentiation of osteoblasts derived from the human skin-derived precursor cells (hSKPs) in vitro culture. This research investigated the effects of PTH by studying the gene expressions and other markers of osteoblast differentiation along with the induction of hSKPs to osteoblast in two experiment groups, i.e. the osteogenic induction medium (OM) only and the OM plus PTH (OM + PTH). The results of each type were compared between these two groups. Both groups expressed the Cbfa1 gene, a regulator of osteoblasts and also one of the most osteoblast specific genes. The findings were that the OM + PTH group showed more intense alkaline phosphatase staining than the other. The gene expressions of protein showing the mature osteoblasts like osteocalcin (OCN) and bone sialoprotein (BSP) in the OM + PTH group expressed higher and faster (Day 14) than the OM group. Moreover, the gene expression of osteoprotegerin (OPG) possessing the protein produced by the mature osteoblasts showed a higher level in the OM + PTH group on the same day as OCN and BSP occurred. This protein performs a function in inhibiting osteoclast maturation. The present study found that PTH enhanced the differentiation of osteoblasts derived from hSKPs by promoting the maturation of osteoblasts in vitro. It possibly concerns with the anabolic effect of PTH in a treatment for osteoporosis patients. Additionally, hSKPs are the interesting sources for osteoporosis treatments when combining with PTH.

Juice of eclipta prostrata inhibits cell migration in vitro and exhibits anti-angiogenic activity in vivo.

BACKGROUND: The invasion of cancer cells is critical for metastasis. The effects of Eclipta prostrata, a Thai medicinal plant, on invasion, migration and adhesion of cancer cells were investigated and the anti-angiogenic activity in vivo was evaluated. MATERIALS AND METHODS: In vitro invasion and migration assays were performed in modified Boyden chambers. In vivo anti-angiogenic activity was determined using the chick chorioallantoic membrane (CAM) assay. RESULTS: E. prostrata juice inhibited cancer invasion and migration, without affecting cell adhesion. Cell migration was inhibited in a variety of cancer cell types and in endothelial cells, with IC50 values of 31-70 microg/ml, much lower than the IC50 values for cytotoxicity of 203-1,217 microg/ml for cancer cells and >4,000 microg/ml for endothelial cells. Fifty percent inhibition of angiogenesis by E. prostrata juice was observed at 200 microg/egg. CONCLUSION: E. prostrata juice inhibited cancer and endothelial cell migration in vitro and also showed in vivo anti-angiogenic activity.

Osteoblast differentiation and bone formation gene expression in strontium-inducing bone marrow mesenchymal stem cell.

Osteoblastic differentiation from human mesenchymal stem cell (hMSCs) is an important step of bone formation. We studied the in vitro induction of hMSCs by using strontium ranelate, a natural trace amount in water, food and human skeleton. The mRNA synthesis of various osteoblast specific genes was assessed by means of reverse transcription polymerase chain reaction (RT-PCR). In the hMSCs culture, strontium ranelate could enhance the induction of hMSCs to differentiate into osteoblasts. Cbfa1 gene was earlier expressed on day 4 of cell culture (the control group, on day 14) and osteonectin on day 11 (control, on day 21). The early Cbfa1 expression indicates that strontium could enhance osteoblastic differentiation. The detection of osteonectin using strontium induction indicates the role of strontium in enhancing bone remodeling, bone structure stabilization of hydroxyapatite molecule and collagen fibril organization. The cultured hMSCs in the presence of strontium expressed genes of bone extracellular matrix: collagen type I, bone sialoprotein and osteocalcin on the same days as control (same medium with no strontium). Concentration of strontium ranelate has been recommended to be optimized in between 0.2107 - 21.07 microg/ml whereas the high concentration up to 210.7 microg/ml have delayed effect on osteoblastic differentiation with delayed expression on Cbfa1 and osteonectin, and inhibitory effect on bone sialoprotein expression. In addition, strontium could help cell expansion by maintaining cell proliferation rate of hMSCs and osteoblast lineage. We recommend that the strontium is an important factor for inducing mesenchymal stem cells to differentiate into osteoblasts with further enhancement on bone formation. This model might provide a useful cell source for tissue engineering and bone repair.

In vitro osteogenesis from human skin-derived precursor cells.

Embryonic tissue and organ development are initiated from three embryonic germ layers: ectoderm (skin and neuron), mesoderm (blood, bone, muscle, cartilage and fat) and endoderm (respiratory and digestive tract). In former times, it was believed that cell types in each germ layer are specific and do not cross from one to another throughout life. A new finding is that one tissue lineage can differentiate across to another tissue lineage, and this is termed transdifferentiation. We were interested in studying the transdifferentiation of skin-derived precursor cells (ectoderm layer) to osteoblastic cells (mesoderm layer). Human skin-derived precursor cells (hSKP) were isolated and induced into an osteoblastic lineage using osteogenic induction medium (alpha-MEM plus 10% fetal bovine serum supplemented with ascorbic acid, beta-glycerophosphate and dexamethasone). The specific characteristics of osteoblastic cells, including the expression of enzyme alkaline phosphatase, the deposition of mineral and the expression of osterix, bone sialoprotein and osteocalcin, were detected only from the inductive group. The results in our study show that SKP from human skin are a practically available source for osteogenesis. The samples are easily obtainable for autologous use with a high expansion capacity.

Increased smooth muscle actin expression from bone marrow stromal cells under retinoic acid treatment: an attempt for autologous blood vessel tissue engineering.

Vascular replacement in vital organs is sometimes necessary for human life for example because of atherosclerosis. Blood vessel tissue engineering is applied for autologous transplantations to avoid graft rejections. Stem cells are used for blood vessel tissue engineering because they are the origin of smooth muscle cells, endothelial cells and fibroblasts. This paper shows that bone marrow stromal cells (BMSCs) can be induced to differentiate into the early stage of smooth muscle cells by using 0.01 microM retinoic acid. The differentiation of BMSCs to smooth muscle cells was detected by the expression of smooth muscle alpha actin (SM alpha-actin), the earliest smooth muscle cell marker. The SM alpha-actin marker expression was demonstrated using indirect immunofluorescence technique and Western blot analysis. The induction of BMSC to form early stages of smooth muscle cells in this study is appropriate for blood vessel tissue engineering because the early stage smooth muscle cells may be stimulated to develop vascular walls with endothelial cells using a co-culture system.

Enhanced maturation and proliferation of beta-thalassemia/Hb E erythroid precursor cells in culture.

Upon erythroid cell maturation in vivo, beta-thalassemic erythroid cells accumulate unmatched unstable alpha-globin chains that are believed to be a causal factor in such cell destruction. This study showed that beta-thalassemia/Hb E erythroid precursor cells from peripheral blood had accelerated maturation, and could mature to the terminal erythroid stage. During the early period of cell culture, erythroid precursor cells derived from subjects with the more severe form of beta-thalassemia/Hb E had higher rate of erythroid maturation. In addition, peripheral blood mononuclear cells from beta-thalassemia/Hb E subjects had higher erythroid proliferative potential than cells derived from normal controls. Erythroid proportion in the more severe beta-thalassemia/Hb E cases was less than that of the milder cases. Premature apoptosis was not observed during the 15 days of erythroid cell culture from both beta-thalassemia/Hb E and normal subjects.

Growth factor combination for chondrogenic induction from human mesenchymal stem cell.

During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-beta3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-beta3 and BMP-6 or TGF-beta3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction.

Ex vivo cytotoxicity of the Bacillus thuringiensis Cry4B delta-endotoxin to isolated midguts of Aedes aegypti larvae.

The pathological effect of the Bacillus thuringiensis Cry delta- endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.

Increased circulating activated endothelial cells, vascular endothelial growth factor, and tumor necrosis factor in thalassemia.

An increased number of circulating endothelial cells (CECs) was demonstrated in alpha- and beta-thalassemic patients, beta-thalassemia/hemoglobin E (BE), both splenectomized (BE[S]) and non-splenectomized (BE[NS]), had higher numbers of CECs than alpha-thalassemia, both HbH (alpha-thal l/alpha-thal 2; H) and HbH with hemoglobin Constant Spring (alpha-thal 1/CS; H/CS). CECs were also increased in heterozygous HbE (EA) and homozygous HbE (EE). The highest level of tumor necrosis factor-alpha (TNF-alpha) was found in HbH/CS patients, whereas the highest levels of vascular endothelial growth factor (VEGF) was observed in BE[S] patients. Significant decreases, in protein C and protein S levels were found in both alpha- and beta-thalassemia compared with normal. Good correlations between the numbers of CEC and TNF-alpha, VEGF, protein C, and protein S levels were demonstrated in this study. In addition, markers for endothelial cell activation and injury (intercellular adhesion molecule-1, ICAM-1/CD54; vascular cell adhesion molecule-1, VCAM-1/CD106; and E-selectin, ELAM-1/CD62E) were detected on the surface of isolated CECs using immunofluorescence technique. Appearance of CECs with markers for endothelial cell activation, together with increased levels of TNF-alpha and VEGF and decreased levels of protein C and protein S in the circulation, may account for the propensity of vascular perturbation in thalassemic subjects.