Patent Application: US-201013266845-A

Abstract:
a method for obtaining human microglial precursor cells , comprising : providing a cell population comprising neural precursor cells , wherein the cell population is obtainable from embryoid bodies differentiated from human pluripotent stem cells ; differentiating the cell population comprising neural precursor cells into microglial precursor cells by culturing in medium comprising a growth factor selected from the group consisting of insulin and insulin - like growth factors ; expanding and enriching microglial precursor cells in medium comprising a growth factor selected from the group consisting of insulin and insulin - like growth factors and 10 to 150 ng / ml gm - csf ; and isolating microglial precursor cells comprising cd45 - positive cells .

Description:
for maintenance and expansion of human ifs , cells were cultured in chemically defined medium in the presence of 25 ng / ml recombinant human fibroblast growth factor - 2 ( rhfgf2 ) on a feeder layer . the absence of rhfgf2 and of self - renewal signals produced by feeder leads to spontaneous differentiation into embryoid bodies ( ebs ). ebs were kept in suspension for 8 days for spontaneous differentiation ( fig1 ). then , they were plated on poly - l - ornithin ( plo ) and fibronectin - coated dishes and neural precursors were selected for 14 days in b27 - medium ( gibco / brl / invitrogen ) supplemented with 20 ng / ml rhfgf2 and 5 μg / ml fibronectin or 10 ng / ml laminin to enhance cell survival ( fig2 ). in the selection stage , cells started to grow out and nestin - positive cells were the major population of developing cells ( fig3 ). during expansion of nestin - positive cells in n2 - medium ( gibco / brl / invitrogen ) supplemented with 20 ng / ml rhfgf2 and 10 ng / ml laminin , the number of cells increased . differentiation was initiated by withdrawal of the growth factors after 10 days of expansion . after 2 weeks , different cell types had developed and were characterized for their cell identity by immunocytochemistry . immunocytochemistry for βiii - tubulin after 14 days of differentiation showed developing clusters of βiii - tubulin - positive cells . after 6 weeks of differentiation , cultures were immunolabeled for βiii - tubulin , glial fibrillary acidic protein ( gfap ), cd45 and cd68 to identify the developing cell types . gfap - positive as well as βiii - tubulin - positive cells were detected within the cultures indicating a differentiation of the neural precursors to astrocytes and neurons , respectively . co - immunolabeling for gfap and βiii - tubulin shows no co - expression with cd45 , indicating that the cd45 - positive cell population is distinct from astrocytes and neurons . to enhance development of microglial precursors cells 100 ng / ml recombinant human granulocytemacrophage colony - stimulating factor ( rhgm - csf ), 10 ng / ml recombinant human interleukin - 3 ( rhil - 3 ) and 10 ng / ml recombinant human macrophage colony - stimulating factor ( rhm - csf ) were added to the media . after 2 days , first microglial precursors appeared in the culture ( fig4 ) identified by immunostaining with antibodies directed against the hematopoietic marker protein cd45 . after several days , approximately 2 - 10 % of cells showed immunoreactivity for cd45 . cell colonies developed and microglial precursor lines proliferated in clusters within the mixed neural cultures . for isolation of microglial precursor cells from the differentiated mixed culture a specific method was applied . microglia - like cells identified by morphology were mechanically isolated by a micropipette and expanded on poly - l - lysine ( pll )- coated ( 5 μg / ml ) dishes with the highest density possible for a monolayer . mechanically isolated microglial precursors were cultured in the presence of 100 ng / ml rhgm - csf , 10 ng / ml rhil - 3 and 10 ng / ml rhm - csf . growth factors are required for survival and proliferation of mechanically isolated microglial precursors . after expansion , cells expressing cd45 were sorted by magnetic activated cell sorting ( macs ) or flow cytometry activated cell sorting ( facs ) with antibodies directed against cd45 . sorted cells were cultured on pll - coated dishes in n2 medium supplemented with 100 ng / ml rhgm - csf , 10 ng / ml rhil - 3 and 10 ng / ml rhmcsf . after several days in culture , microglial precursor cell lines started to proliferate ( fig5 ). the phenotype of the cells differed from ramified over bipolar structured till completely rounded cell morphology . cells were isolated by trypsin or cell scraper and split 1 : 3 till 1 : 5 twice a week . cell identity of microglial precursor lines was verified by immunocytochemistry for cd68 and iba1 ( fig6 and 7 ). to obtain clones , single cells were mechanically isolated and transferred into separate pll - coated ( 5 μg / ml ) culture dishes . the isolated microglial precursor cells were cultured in dmem / f12 - medium ( gibco / brl / invitrogen ) containing 100 ng / ml rhgm - csf , 10 ng / ml rhil - 3 and 10 ng / ml rhm - csf . cells were cultured with high density and split 1 : 2 . after splitting , cells recovered and attached again to the new pll - coated dishes . microglial precursors proliferated without addition of growth factors to medium after some passages and were passaged 1 : 3 till 1 : 5 twice a week . microglial precursor cells were expanded to obtain at least 1 × 10 10 cells . cells were analyzed by flow cytometry for expression of cd45 , cd11b , cd11c , cd14 , cd16 , integrin - alpha4 , integrin - beta1 , cx3cr1 and trem2 . in addition , gene transcripts for tnf - alpha , interleukin - 1 beta , nitric oxide synthase - 2 , cx3cl1 , ccl2 , cxcl9 and cxcl10 can be analyzed in the cells under normal and lps - stimulation by real - time rt - pcr . the cells are confirmed by various test systems to be free of pathogens or contaminants ( e . g . viruses etc .). cells were aliquoted and frozen . in vitro experiments with human induced pluripotent stem cell derived microglia and glioma in vitro experiments were performed to confirm the functional activity of human ips - derived microglia using the example of tumor growth . a co - culture of human gfp - transduced ipsdm and the human glioma cell line u87 was carried out using a ratio of 1 : 1 in co - culture . starting the co - culture at day 0 , every day total cell number of co - culture as well as the cell number of glioma in culture alone was counted under the microscope . furthermore , the percentage of glioma and ipsdm in co - 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