Ryu-Ichiro Hata

Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium

A major limitation in developing applications for the use of human embryonic stem cells (HESCs) is our lack of knowledge of their responses to specific cues that control self-renewal, differentiation, and lineage selection. HESCs are most commonly maintained on inactivated mouse embryonic fibroblast feeders in medium supplemented with FCS, or proprietary replacements such as knockout serum-replacement together with FGF-2. These undefined culture conditions hamper analysis of the mechanisms that control HESC behavior. We have now developed a defined serum-free medium, hESF9, for the culture of HESCs on a type I-collagen substrate without feeders. In contrast to other reported media for the culture of HESCs, this medium has a lower osmolarity (292 mosmol/liter), l-ascorbic acid-2-phosphate (0.1 μg/ml), and heparin. Insulin, transferrin, albumin conjugated with oleic acid, and FGF-2 (10 ng/ml) were the only protein components. Further, we found that HESCs would proliferate in the absence of exogenous FGF-2 if heparin was also present. However, their growth was enhanced by the addition of FGF-2 up to 10 ng/ml although higher concentrations were deleterious in the presence of heparin.

Integrins Regulate Mouse Embryonic Stem Cell Self-Renewal

Extracellular matrix (ECM) components regulate stem‐cell behavior, although the exact effects elicited in embryonic stem (ES) cells are poorly understood. We previously developed a simple, defined, serum‐free culture medium that contains leukemia inhibitory factor (LIF) for propagating pluripotent mouse embryonic stem (mES) cells in the absence of feeder cells. In this study, we determined the effects of ECM components as culture substrata on mES cell self‐renewal in this culture medium, comparing conventional culture conditions that contain serum and LIF with gelatin as a culture substratum. mES cells remained undifferentiated when cultured on type I and type IV collagen or poly‐d‐lysine. However, they differentiated when cultured on laminin or fibronectin as indicated by altered morphologies, the activity of alkaline phosphatase decreased, Fgf5 expression increased, and Nanog and stage‐specific embryonic antigen 1 expression decreased. Under these conditions, the activity of signal transducer and activator of transcription (STAT)3 and Akt/protein kinase B (PKB), which maintain cell self‐renewal, decreased. In contrast, the extracellular signal‐regulated kinase (ERK)1/2 activity, which negatively controls cell self‐renewal, increased. In the defined conditions, mES cells did not express collagen‐binding integrin subunits, but they expressed laminin‐ and fibronectin‐binding integrin subunits. The expression of some collagen‐binding integrin subunits was downregulated in an LIF concentration‐dependent manner. Blocking the interactions between ECM and integrins inhibited this differentiation. Conversely, the stimulation of ECM‐integrin interactions by overexpressing collagen‐binding integrin subunits induced differentiation of mES cells cultured on type I collagen. The results of the study indicated that inactivation of the integrin signaling is crucial in promoting mouse embryonic stem cell self‐renewal.

Acidic extracellular pH increases calcium influx‐triggered phospholipase D activity along with acidic sphingomyelinase activation to induce matrix metalloproteinase‐9 expression in mouse metastatic melanoma

Acidic extracellular pH is a common feature of tumor tissues. We have reported that culturing cells at acidic pH (5.4–6.5) induced matrix metalloproteinase‐9 expression through phospholipaseu2003D, extracellular signal regulated kinaseu20031/2 and p38 mitogen‐activated protein kinases and nuclear factor‐κB. Here, we show that acidic extracellular pH signaling involves both pathways of phospholipaseu2003D triggered by Ca2+ influx and acidic sphingomyelinase in mouse B16 melanoma cells. We found that BAPTA‐AM [1,2‐bis(2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid tetrakis (acetoxymethyl) ester], a chelator of intracellular free calcium, and the voltage dependent Ca2+ channel blockers, mibefradil (for T‐type) and nimodipine (for L‐type), dose‐dependently inhibited acidic extracellular pH‐induced matrix metalloproteinase‐9 expression. Intracellular free calcium concentration ([Ca2+]i) was transiently elevated by acidic extracellular pH, and this [Ca2+]i elevation was repressed by EGTA and the voltage dependent Ca2+ channel blockers but not by phospholipaseu2003C inhibitor, suggesting that acidic extracellular pH increased [Ca2+]i through voltage dependent Ca2+ channel. In contrast, SR33557, an L‐type voltage dependent Ca2+ channel blocker and acidic sphingomyelinase inhibitor, attenuated matrix metalloproteinase‐9 induction but did not affect calcium influx. We found that acidic sphingomyelinase activity was induced by acidic extracellular pH and that the specific acidic sphingomyelinase inhibitors (perhexiline and desipramine) and siRNA targeting aSMase/smpd1 could inhibit acidic extracellular pH‐induced matrix metalloproteinase‐9 expression. BAPTA‐AM reduced acidic extracellular pH‐induced phospholipaseu2003D but not acidic sphingomyelinase acitivity. The acidic sphingomyelinase inhibitors did not affect the phosphorylation of extracellular signal regulated kinase 1/2 and p38, but they suppressed nuclear factor‐κB activity. These data suggest that the calcium influx‐triggered phospholipaseu2003D and acidic sphingomyelinase pathways of acidic extracellular pH induced matrix metalloproteinase‐9 expression, at least in part, through nuclear factor‐κB activation.

Roles of Collagen Molecules in Growth and Differentiation of Human Osteoblasts

Collagen is the most abundant organic component of bone. It constitutes a superfamily of extra- cellular matrix proteins whose primary function is structural. The major collagenous component of bone is typeⅠ collagen, but typesⅢ, Ⅴ, and Ⅹ collagens are also found in bone tissue. Recently it was reported that collagen is essential for the growth and differentiation of various kinds of cell including osteoblasts. In order to investigate the function of the respective collagen components in the growth and differentiation of osteoblasts, we employed human osteoblast-like MG-63 cells and normal human osteoblasts, as well as human bone marrow stem cells;cultured them in the presence or absence of ascorbic acid 2-phosphate (Asc 2-P)and/or vitamin D3 (VD3);and examined the relationship between specific collagen types and the growth and differentiation of the cells. The stimulation of cell proliferation by the addition of Asc 2-P or epi- dermal growth factor is closely associated with an increase in the synthesis of typeⅢ collagen. The stimula- tion of osteoblast differentiation by the addition of VD3 resulted in growth retardation of the cells. The inhi- bition of typeⅠ or Ⅲ collagen synthesis by adding the respective siRNAs attenuated the effects of these agents. Employing typeⅠ or Ⅲ collagen as a substratum also regulated the growth and/or differentiation of human osteoblasts. This review reveals that typeⅠ and Ⅲ collagens function as regulatory molecules for the differentiation and proliferation of human osteoblastic cells, and discusses the possibility that collagen metabolism is a pos- sible new target for the treatment of osteoporosis.