No abstract

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 selfrenewal 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.

In this small preliminary study, we found that donor age is the most important factor in predicting G-CSF-induced PBSC yields. Old age and low platelet counts before mobilization might be useful indicators for identifying poor mobilizers. Further validation of these findings in a larger number of donors are needed to establish whether these findings apply to other populations.

We have developed a serum-free medium, designated ESF7, in which leukemia inhibitory factor (LIF) clearly stimulated murine embryonic stem (ES) cell proliferation accompanied by increased expression of nanog and Rex-1 and decreased FGF-5 expression. These effects were dependent on the concentration of LIF. The ES cells maintained in ESF7 medium for more than 2 yr retained an undifferentiated phenotype, as manifested by the expression of the transcription factor Oct-3/4, the stem cell marker SSEA-1, and alkaline phosphatase. Withdrawal of LIF from ESF7 medium resulted in ES cell apoptosis. Addition of serum to ESF7 medium promoted ES cell differentiation. Addition of BMP4 promoted ES cell differentiation into simple epithelial-like cells. In contrast, FGF-2 promoted ES cell differentiation into neuronal and glial-like cells. Under serum-free culture conditions, LIF was sufficient to stimulate cell proliferation, it inhibited cell differentiation, and it maintained self-renewal of ES cells. Because this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent ES cells in vitro, it will allow the elucidation of ES cell responses to growth factors under defined conditions.

The safety and efficacy of harvesting peripheral blood hematopoietic stem cells (PBSC) were evaluated in 38 children weighing 20 kg or less, with the smallest patient weighing 7 kg. The patients had a median age of 42 months and included 26 children with acute leukemias or lymphoma and 12 with various solid tumors. A total of 81 aphereses were performed, mostly in the recovery phase of chemotherapy, with or without granulocyte colony-stimulating factor, using a CS-3000 cell separator and regular procedure no. 3. Blood was withdrawn at a mean rate of 30 mL/min (range, 17 to 46 mL/min) through a temporary radial arterial catheter (20 to 24 guage) and returned through a larger catheter in a peripheral vein. Morbidity related to PBSC harvest was low and all aphereses were completed within 3 hours. The volume of blood per kilogram processed for each apheresis ranged from 85 to 615 mL (median, 270 mL). The median number of colony-forming units-- granulocyte-macrophage (CFU-GM) and CD34+ cells collected were, respectively, 34 x 10(4)/kg and 15 x 10(6)/kg per apheresis and 126 x 10(4)/kg and 31 x 10(6)/kg per patient. Thirty-three patients (87%) required only a single apheresis to collect the minimum requirement of 10 x 10(4) CFU-GM/kg, including 28 patients (74%) from whom 30 x 10(4) CFU-GM/kg was obtained in a single apheresis. Twenty-three of the patients subsequently underwent autografts with PBSC. The median number of days required to achieve an absolute granulocyte count of 0.5 x 10(9)/L and a platelet count of 50 x 10(9)/L were, respectively, 10 (range, 6 to 15) and 14 (range, 9 to 46). The patients remained dependent on platelet transfusion support for a median of 10 days (range, 5 to 35). Thus, harvesting PBSC in very small children with active cancers is effective and safe and does not involve the risk of anesthesia or multiple invasive marrow aspirations.