A feeder-free and efficient production of functional neutrophils from human embryonic stem cells.

A novel, feeder-free hematopoietic differentiation protocol was established for highly efficient production of neutrophils from human embryonic stem cells (hESCs). For the induction of differentiation, spheres were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin-coated plates. After approximately 2 weeks, a sac-like structure filled with abundant round cells emerged at the center of flattened spheres. After cutting off this sac-like structure, round cells actively proliferated, either floating in the supernatant or associated weakly with the adherent cells. Almost all of these round cells were CD45-positive hematopoietic cells with myeloid phagocytic markers (CD33 and CD11b), and approximately 30%-50% of the round cells were mature neutrophils, as judged from morphology, cytochemical characteristics (myeloperoxidase and neutrophil alkaline phosphatase), and neutrophil-specific cell surface markers (CD66b, CD16b, and GPI-80). In addition, hESC-derived neutrophils had chemotactic capacity in response to the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalanine and neutrophil-specific chemokine interleukin (IL)-8. Using "semipurified" neutrophils migrated to IL-8, both phagocytic and respiratory burst activities were demonstrated. Finally, it was shown that hESC-derived neutrophils had chemotactic activity in vivo in a murine air-pouch inflammatory model. The present results indicate successful induction of functional mature neutrophils from hESCs via highly efficient feeder-free differentiation culture system of human hematopoietic cells.

A feeder-free hematopoietic differentiation system with generation of functional neutrophils from feeder- and cytokine-free primate embryonic stem cells.

We have established a novel feeder- and recombinant cytokine-free culture system for the maintenance of primate embryonic stem (ES) cells along with a feeder-free hematopoietic differentiation protocol for high efficiency CD45-positive cell production. In our system, cynomolgus monkey ES cells were properly maintained in an undifferentiated state with high immature marker expressions and teratoma-producing activities. Embryoid bodies (EBs) were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin-coated plates. After about 2 weeks, a sac-like structure filled with abundant round cells emerged at the center of flattened EB. Then total cells were collected and transferred onto new gelatin-coated plates, where cells were firmly attached and actively proliferated to confluence. After another few days culture, abundant floating cells were detected in the culture supernatant. These cells expressed high levels of CD45 (>90%), while adherent cells expressed low levels of CD45 (<10%). The former consisted of various differentiated stages of myeloid cells from immature myeloblasts to mature polymorphonuclear neutrophils and macrophages. Although the percentages of neutrophils varied between 10 to 20 depending on experiments, their mature phenotype was reproducibly confirmed by specific staining and functional assays. Our protocol provides the minimum essence for primate ES cell maintenance and hematopoietic differentiation that is beneficial from economical and clinical points of view.

Abnormal telomerase activity and telomere length in T and B cells from patients with systemic lupus erythematosus.

OBJECTIVE: To evaluate the clinical significance of telomerase activity and telomere length in T and B lymphocytes from patients with systemic lupus erythematosus (SLE). METHODS: CD3+ (T cell) and CD19+ (B cell) lymphocytes were isolated from the peripheral blood of SLE patients and healthy controls by means of magnetic bead-coupled antibodies. SLE patients were classified as active or inactive cases according to the SLE Disease Activity Index (SLEDAI). Telomere activity of lymphocytes was measured by telomeric-repeat amplification protocol. Telomere length was measured by flow cytometry-fluorescence in situ hybridization. RESULTS: T cell telomerase activity was significantly higher in patients with both active and inactive SLE than in controls, but was lower than B cell telomerase activity in patients with active SLE, and was not correlated with SLEDAI results. B cell telomerase activity was only significantly higher than in controls in patients with active SLE, and was strongly correlated with SLEDAI. Four laboratory results, anti-dsDNA antibody titer, IgG level, C3 level, and CH50 level, were correlated with B cell telomerase activity. Telomere length in T cells was significantly shorter than in controls. In contrast, the telomere length in B cells did not differ significantly from controls. CONCLUSION: In patients with SLE, many T cells divide continuously. Their telomerase activity was higher than that in control T cells, but not so high as to prevent telomere shortening. In contrast, B cells do not divide abnormally in the inactive phase of SLE, but divide rapidly in the active phase.