epidermal-growth-factor and ascorbate-2-phosphate

We have devised a medium which supports the continuous growth of hepatocytes without losing their replicative potential and differentiation capacity for a longer period. The medium HCGM, contains four key substances in addition to foetal bovine serum. They are epidermal growth factor, nicotinamide, ascorbic acid 2-phosphate and dimethylsulphoxide. When a non-parenchymal cell fraction containing small hepatocytes and non-parenchymal cells was cultured in HCGM, small hepatocytes grew clonally and differentiated into cells expressing either mature hepatocyte marker proteins or biliary cell marker proteins. Thus, for the first time, we showed the presence of a small compartment of bipotent and highly replicative clonogenic hepatocytes in the rat adult liver. HCGM also supported the growth of stellate cells (Ito cells) which were in the original preparation, suggesting the important role of stellate cells for the successful cultivation of hepatocytes. Together, these results suggest that a microenvironment is produced as a result of cooperative interactions between hepatocytes and stellate cells: one which stimulates the growth and differentiation of clonogenic hepatocytes.

Topics: Animals; Ascorbic Acid; Cell Differentiation; Cell Division; Clone Cells; Culture Media; Dimethyl Sulfoxide; Epidermal Growth Factor; Liver; Niacinamide; Rats

This study was designed to investigate the potential of a wound dressing composed of hyaluronic acid (HA) and collagen (Col) spongy sheet containing epidermal growth factor (EGF) and vitamin C derivative (VC). High-molecular-weight HA aqueous solution, hydrolyzed low-molecular-weight HA aqueous solution and heat-denatured Col aqueous solution were mixed, followed by freeze-drying to obtain a spongy sheet. Cross-linkage between Col molecules was induced by UV irradiation of the spongy sheet (C-wound dressing). In a similar manner, three types of spongy sheet containing EGF (EGF-wound dressing), containing VC (VC-wound dressing) or containing EGF and VC (EGF·VC-wound dressing) were prepared by freeze-drying the mixed solution containing the specified components. Cytokine production by fibroblasts was assessed in a wound surface model using a fibroblast-incorporating Col gel sheet (cultured dermal substitute; CDS). CDS was elevated to the air-medium interface, onto which each wound dressing was placed and cultured for 7 days. Fibroblasts in CDS covered with EGF-wound dressing released 3.6 times more VEGF and 3.0 times more HGF, as compared with the C-wound dressing. Fibroblasts in CDS covered with EGF·VC-wound dressing released 4.2 times more VEGF and 6.0 times more HGF, as compared with the C-wound dressing. The efficacy of these wound dressings was evaluated in animal tests using diabetic mice. Each wound dressing was applied to a full-thickness skin defect on the dorsal area measuring 1.5 × 2.0 cm. After 1 week of application, wound conditions were evaluated histologically. The EGF·VC-wound dressing more effectively promoted granulation tissue formation associated with angiogenesis, as compared with other wound dressings.

Topics: Animals; Ascorbic Acid; Bandages; Cell Line; Collagen; Drug Evaluation, Preclinical; Drug Synergism; Epidermal Growth Factor; Fibroblasts; Hepatocyte Growth Factor; Humans; Hyaluronic Acid; Male; Mice; Vascular Endothelial Growth Factor A; Wound Healing

Mesenchymal stem cells (MSCs) have been shown to improve the outcome of acute renal injury models; but whether MSCs can delay renal failure in chronic kidney disease (CKD) remains unclear. In the present study, the were cultured in media containing various concentrations of basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2-phosphate to investigate whether hepatocyte growth factor (HGF) secretion could be increased by the stimulation of these growth factors. Then, TGF-β1-treated renal interstitial fibroblast (NRK-49F), renal proximal tubular cells (NRK-52E) and podocytes were co-cultured with conditioned MSCs in the absence or presence of ascorbic acid 2-phosphate to quantify the protective effects of conditioned MSCs on renal cells. Moreover, male Sprague-Dawley rats were treated with 1 × 10(6) conditioned MSCs immediately after 5/6 nephrectomy and every other week through the tail vein for 14 weeks. It was found that basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2-phosphate promoted HGF secretion in MSCs. Besides, conditioned MSCs were found to be protective against TGF-β1 induced epithelial-to-mesenchymal transition of NRK-52E and activation of NRK-49F cells. Furthermore, conditioned MSCs protected podocytes from TGF-β1-induced loss of synaptopodin, fibronectin induction, cell death and apoptosis. Rats transplanted with conditioned human MSCs had a significantly increase in creatinine clearance rate, decrease in glomerulosclerosis, interstitial fibrosis and increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells counts in splenocytes. Together, our studies indicated that conditioned MSCs preserve renal function by their anti-fibrotic and anti-inflammatory effects. Transplantation of conditioned MSCs may be useful in treating CKD.

Topics: Animals; Apoptosis; Ascorbic Acid; CD4-Positive T-Lymphocytes; Cells, Cultured; Coculture Techniques; Creatinine; Disease Progression; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Female; Fibroblast Growth Factor 2; Fibronectins; Fibrosis; Glomerulosclerosis, Focal Segmental; Hepatocyte Growth Factor; Humans; Kidney; Kidney Tubules, Proximal; Lymphocyte Count; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Microfilament Proteins; Middle Aged; Nephrectomy; Podocytes; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; Transforming Growth Factor beta1; Young Adult

Adipose tissue serves as a source of adipokines and cytokines with both local and systemic actions in health and disease. In this study, we examine the hypothesis that multipotent human adipose-derived stem cells (ASCs), capable of differentiating along the adipocyte, chondrocyte, and osteoblast pathways, contribute to adipose tissue-derived cytokine secretion. Following exposure to basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF), the ASCs significantly increase their secretion of hepatocyte growth factor (HGF), a cytokine implicated in hematopoiesis, vasculogenesis, and mammary epithelial duct formation. Ascorbic acid synergizes with these inductive factors, further increasing HGF levels. Following exposure to lipopolysaccharide, ASCs increase their secretion of both hematopoietic (granulocyte/monocyte, granulocyte, and macrophage colony stimulating factors, interleukin 7) and proinflammatory (interleukins 6, 8, and 11, tumor necrosis factor alpha) cytokines based on ELISA and RT-PCR. In co-cultures established with umbilical cord blood-derived CD34(+) cells, the ASCs support long-term hematopoiesis in vitro. Furthermore, in short-term 12-day co-cultures, the ASC maintain and expand the numbers of both myeloid and lymphoid progenitors. These observations are consistent with the functionality of the secreted cytokines and confirm recent reports by other laboratories concerning the hematopoietic supportive capability of ASCs. We conclude that the ASCs display cytokine secretory properties similar to those reported for bone marrow-derived mesenchymal stem cells (MSCs).

Topics: Adipocytes; Adipose Tissue; Adult; Adult Stem Cells; Angiogenic Proteins; Ascorbic Acid; Cell Differentiation; Cell Proliferation; Cells, Cultured; Coculture Techniques; Cytokines; Endothelial Cells; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Stem Cells; Hepatocyte Growth Factor; Humans; Inflammation Mediators; Interleukin-11; Interleukin-6; Interleukin-7; Interleukin-8; Lipopolysaccharides; Middle Aged; Multipotent Stem Cells; Paracrine Communication; RNA, Messenger; Time Factors; Tumor Necrosis Factor-alpha

The present study succeeded for the first time in cultivating for more than 2 months human normal hepatocytes which showed a high growth potential and expressed their differentiated phenotypes. Constituents of culture medium were critical for this culture, and the medium optimized for their growth contained fresh human serum, fetal bovine serum, Swiss 3T3-cell conditioned medium, L-ascorbic acid 2-phosphate, epidermal growth factor, nicotinamide, and dimethyl sulfoxide. Hepatocytes steadily replicated and formed colonies which continued to increase in size up to around 35 days. The number of hepatocytes in the most replicative colonies increased 17-fold during 31 days. Cells in colonies expressed normal differentiated hepatocytic phenotypes for as long as 35 days. These hepatocytes retained normal liver functions at least for 70 days such as to secrete albumin, and to metabolize lidocaine and D-galactose.

Topics: 3T3 Cells; Adult; Aged; Albumins; Animals; Ascorbic Acid; Biomarkers; Blood Proteins; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Size; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Dimethyl Sulfoxide; DNA; Epidermal Growth Factor; Female; Galactose; Humans; Lidocaine; Liver; Male; Mice; Middle Aged; Niacinamide; Time Factors

The present study succeeded in cultivating normal adult rat hepatocytes for at least 85 days without losing their replicative potential and differentiation capacity. Small pieces of hepatocyte aggregates (clusters) were prepared from the primary culture of hepatocytes and used as starting material for the growth experiment. Some of the hepatocytes started to proliferate at 3 days when the clusters were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 10 mmol/L nicotinamide, 0.2 mmol/L L-ascorbic acid 2-phosphate, and 1% dimethylsulfoxide. Clusters continued to grow and formed colonies. All the cells covering colonies expressed normal hepatocyte-specific proteins. The number of albumin-expressing cells in the most replicative colonies increased sixfold during 32 days. Most of the cells were mononucleate and small in size and some of them expressed immature hepatocyte markers such as alpha-fetoprotein. Electron microscopy of cells in colonies revealed the presence of peroxisomes in the cytoplasm and desmosomes, tight junctions, and bile canaliculus-like structures between the cells. Depletion of one of the additives inhibited the growth of hepatocytes. The culture medium used also supported the growth of stellate cells (Ito cells) that had contaminated the original preparation in small numbers and seems to cooperatively stimulate a proliferative population of hepatocytes.

Topics: Albumins; alpha-Fetoproteins; Animals; Ascorbic Acid; Cell Aggregation; Cell Culture Techniques; Cell Division; Culture Media; Dimethyl Sulfoxide; Epidermal Growth Factor; Immunohistochemistry; Liver; Male; Microscopy, Electron; Niacinamide; Phenotype; Rats; Rats, Inbred F344; Time Factors

Small hepatocytes existed in the supernatant following low-speed centrifugation of the cell suspension after collagenase liver perfusion. The cells proliferated for more than 2 months and formed colonies in the Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide, 10% fetal bovine serum, 1 mM ascorbic 2-phosphate, and 10 ng/ml epidermal growth factor. One small cell finally proliferated to several hundred cells. In addition, some cells in the colonies were shown to differentiate into mature hepatocytes that had a large cytoplasm and sometimes two nuclei. The secretion of albumin in the medium by the hepatocytes increased with time in culture, and the cells possessed connexin 32 in their cell membrane and many peroxisomes. Thus, the small hepatocytes may be "committed progenitor cells" which can further differentiate into mature hepatocytes.

Topics: Animals; Ascorbic Acid; Cell Differentiation; Cell Division; Cell Separation; Cells, Cultured; Collagenases; Epidermal Growth Factor; Liver; Male; Niacinamide; Rats; Rats, Sprague-Dawley; Serum Albumin

The effect of ascorbate and L-ascorbic acid 2-phosphate (P-Asc), a stable derivative of ascorbate, on the growth of cultured rabbit keratocytes was examined. In a 12-day culture, P-Asc (0.05-0.1 mM) and ascorbate (0.05 mM) enhanced the growth of the cells, whereas 0.1-1.0 mM ascorbate was cytotoxic to the cells. An increase in the concentration of P-Asc up to 1.0 mM resulted in no further change in the cell growth. The effect of 0.1 mM P-Asc was observed after the confluency had been achieved in the culture. The lower toxicity of P-Asc may be due to its stability. The presence of both 0.1 mM P-Asc and mouse epidermal growth factor in the medium stimulated cell growth to a higher degree than each factor alone. The results suggested that these two factors enhance the growth of keratocytes in a different manner.

Topics: Animals; Ascorbic Acid; Cell Count; Cell Division; Cell Survival; Cells, Cultured; Connective Tissue; Cornea; DNA; DNA Replication; Epidermal Growth Factor; Male; Rabbits

Recombinant human epidermal growth factor (EGF, 2-10 ng/ml) stimulated growth and production of non-collagenous proteins, but inhibited production of collagen by 60% in cultured human skin fibroblasts. Type analysis of the collagen produced indicated that inhibition of the collagen production observed was mainly a reflection of a reduction in type I collagen. The accumulation of pro alpha 1(I) and pro alpha 2(I) mRNAs and the transcriptional activity of these genes were determined in human skin fibroblasts in order to investigate site(s) of regulation of type I collagen production by human EGF in the absence and presence of L-ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative. Human EGF (10 ng/ml) used alone reduced the steady state levels of mRNAs for pro alpha 1(I) and pro alpha 2(I) chains and transcriptional activity of these genes in vitro by 45%. Asc 2-P (0.2 mM) alone, on the other hand, raised production of type I collagen and the steady state levels of mRNAs for pro alpha 1(I) and pro alpha 2(I) collagen chains as well as stimulated transcriptional activity of these genes. Human EGF attenuated these stimulative effects of Asc 2-P. These results indicate that human EGF regulates type I collagen synthesis at the transcriptional level in cultured fibroblasts in the presence and absence of Asc 2-P. The possibility that human EGF plays a role as a regulator of type I collagen genes in vivo was discussed.

Topics: Ascorbic Acid; Cells, Cultured; Collagen; DNA; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Fibroblasts; Humans; Nucleic Acid Hybridization; RNA, Messenger; Skin; Transcription, Genetic