Patent Application: US-9501905-A

Abstract:
the present invention provides a mechanism for obtaining an almost unlimited source of stem cells . a method for the expansion of a population of stem cells that retain their developmental capacity comprises co - culture of a stem cell population with a trophic support , for example , endothelial cells or conditioned medium derived from endothelial cell culture . the method also provides a method for enhancing neurogenesis when neural stem cells are co - cultured with the endothelial cell - derived trophic support to retain their neurogenic potential .

Description:
all patents , applications , publications and other references listed herein are hereby incorporated by reference in their entirety . in the description that follows , certain conventions will be followed as regards the usage of terminology . the term “ stem cell ” as it is known and used in the art refers to an undifferentiated cell having the ability to proliferate and self - renew and ultimately being able to differentiate into a specific cell type , for example , a neuron . the term “ neural stem cell ” refers to an undifferentiated cell derived from the nervous system having the ability to proliferate and self - renew and ultimately being able to differentiate into a one of the cells of the nervous system , for example , a neuron . the term “ trophic support ” refers to a source of exogenous trophic factors and includes without limitation , a cell co - culture which secretes trophic factors , a conditioned medium obtained from a cell culture that secretes trophic factors or a cocktail of purified trophic factors . neural stem cells can be used for transplantation into a heterologous , autologous or xenogeneic host . neural stem cells are isolated from nervous system tissue in accordance with methods known to those of skill in the art and may be obtained from embryonic , post - natal , juvenile or adult neural tissue from human or non - human mammals . the desired resulting cell type , for example , neurons , will determine the developmental stage of the tissue from which the stem cells will be isolated . so for example , to enhance the number of neurons obtained from an expanded stem cell population , the stem cells to be expanded will be obtained from tissue that is at the optimal developmental stage for neuronal differentiation ( for example , e10 - 11 in the mouse .) the stem cells are then co - cultured with endothelial cells or are cultured in a conditioned medium obtained from an endothelial cell culture . cells may be passaged indefinitely or stored frozen in accordance with methods known in the art . to examine a possible functional interaction between neural stem cells and their vascular environment , we co - cultured neural and vascular cells ( fig1 a ). neural stem cells from e10 - 11 mouse cerebral cortex were plated at clonal density on the base of culture wells , while the upper transwell compartment was seeded with purified vascular - associated or other feeder cells : primary bovine pulmonary artery endothelial cells ( bpae ), a mouse brain endothelial cell line ( mbend ), vascular smooth muscle ( vsm ) cells , nih3t3 fibroblasts , or as a control , high density , age - matched cortical cells ( ctx ). fig1 b shows that cd31 stains endothelial cells in the transwells ( upper panel ), but no cd31 + cells are detected below in the cortical cell compartment ( lower panel ). thus , cd31 + ( pecam ) endothelial cells were never found in the lower compartment when bpae or mbend cells were plated in the transwell upper compartment , confirming that the feeder cells cannot migrate through the 0 . 4 82 m diameter membrane pores . as expected ( 6 ), embryonic stem cell clones co - cultured with ctx began producing neurons within a day . most neuron production was over by 7 days , and growth after this time was largely in glial lineages . clones co - cultured with bpae or mbend cells behaved differently ( fig1 d , fig . s 2 ), growing into sheets of largely flattened progeny that maintained tight cell - cell contact , illustrated by strong junctional β - catenin staining ( fig1 c ), with only a few immature , neuron - like cells appearing on top of the sheets . neural stem cell clones grown with endothelial cells were larger , with more primitive progeny ( expressing the progenitor markers nestin and lex ( 20 ), and fewer neurons ( expressing β - tubulin - iii ) than were clones grown with ctx ( fig1 d - g ). hence , endothelial factors facilitate expansion of cortical stem cell clones and inhibit their differentiation . vsm and nih3t3 cells also promoted neural stem cell proliferation ( fig1 f ), but clones were less cohesive and included more glial - like progeny than those in endothelial co - culture . when the transwells were removed , endothelial - expanded stem cell clones continued to proliferate but also began to differentiate , and within 4 days produced βtubulin - iii + neurons ( fig2 b ), which were almost all map - 2 + . approximately 30 % of the neurons had acquired the later neuronal marker neun ( data not shown ). the clones contained up to approximately 10 , 000 progeny , and on average 31 % were neurons . in contrast , in control ctx co - cultures 4 days after transwell removal , stem cell clones ranged up to 4350 cells , and on average only 9 % were neurons ; similarly , e11 cortical cells cultured as neurospheres for 7 days then differentiated in adherent culture for 4 days produced only 7 % neurons . many more stem cell clones growing in bpae co - cultures contained a higher percentage of neurons , up to 64 %, compared to ctx co - culture ( fig2 d , e ), and neuron production was prolonged ( supporting text , fig . s 3 ). increased neurogenesis from endothelial co - cultured neural stem cells does not occur at the expense of gliogenesis : the percentage of gfap + astrocytes generated was similar , and although oligodendrocyte differentiation ( indicated by o4 staining ) was reduced in bpae co - cultures compared to ctx , the difference could not account for the enhancement of neuron generation ( fig2 c , e ). nih3t3 cells enhanced oligodendrocyte generation . co - culture with vsm or nih3t3 cells reduced neurogenesis compared to ctx ( fig2 e ), showing that the endothelial effect is cell - type specific . endothelial cells stimulate proliferation and neurogenesis of neural stem cells from a variety of embryonic cns regions ( not shown ) and from different stages . e15 . 5 cortical and adult svz stem cells grown in endothelial co - culture generated sheets of lex + , nestin + cells . after differentiation , e15 . 5 endothelial - expanded cortical cells and adult svz cells produce more neurons compared to control ( fig2 f , 2h ). neurosphere - expanded stem cells respond to endothelial factors . e15 . 5 cortical cells grown as neurospheres in fgf2 for 7 days were plated in adherent conditions and co - cultured for 3 days with endothelial cells or with age - matched cortical cells , then differentiated by withdrawing feeder cells for 4 days . stem cells exposed to endothelial factors produced 22 % neurons , compared to 2 % neurons in control ctx co - cultures ( fig2 g ). in vivo , most projection neurons are born in the early embryonic period , while glia and interneurons arise later ; adult stem cells are primed to generate interneurons ( 24 , 25 ). to examine the neuron sub - types generated from e10 - 11 cortical stem cells expanded in endothelial co - culture , differentiated clones were stained for gad67 , a gabaergic marker typically expressed in interneurons , or tbr1 , an early pyramidal neuron marker that preferentially labels projection neurons ( 26 ) ( fig3 a ). more stem cell clones growing in bpae co - culture made tbr1 + projection neurons , compared to ctx co - culture ( fig3 b ) or to neurosphere - expanded e10 cells that were subsequently differentiated in adherent culture ( 9 . 95 % versus 2 . 41 %). thus , endothelial cell co - culture supports development of both projection neurons and interneurons . that projection neurons typical of the early embryo arise in e10 - 11 co - cultures after many cell divisions suggests that endothelial factors promote stem cell self - renewal , and inhibit the normal progression in which older stem cells preferentially produce glia or interneurons . we found few tbr1 + neurons produced from e15 . 5 stem cells and none from adult svz cells , indicating that endothelial factors are permissive , not instructive , for this fate : they cannot reverse the restriction . supporting the hypothesis that endothelial factors promote stem cell self - renewal , time - lapse video recording of dividing clones reveals that stem cells grown with endothelial cells undergo symmetric , proliferative divisions generating nestin + progeny , in contrast to the asymmetric division patterns seen in control conditions ( 23 , 27 ) ( fig4 a ). cortical stem cells co - cultured with endothelial cells for 4 days generated more secondary stem cell clones , neurospheres and neuron - generating progenitor cells than did those co - cultured with ctx cells ( fig . s 4 ). the most obvious effect of endothelial factors is that they promote neural stem cell growth as epithelial sheets with extensive junctional contacts ( fig1 c ), which could promote self - renewal by influencing β - catenin signaling pathways ( 28 , 29 ), mode of cell division ( 30 ), and maintaining notch activation ( 31 ). indeed , stem cells co - cultured with endothelial cells and then exposed to γ - secretase inhibitor ii , which inhibits notch1 activation ( 32 ), showed similar extent of cell - cell contact , division and differentiation to those in ctx co - cultures ( fig4 b , fig . s 5 ). in neural stem cells cultured with endothelial factors the notch effector hes1 was up - regulated but hes5 was not ( fig4 c ), consistent with involvement of hes1 in neural stem cell self - renewal ( 33 , 34 ). our results identify endothelial cells as critical components of the neural stem cell niche , as they secrete soluble factors that maintain cns stem cell self - renewal and neurogenic potential . thus , while fgf2 promotes neural stem cell proliferation , it cannot alone maintain their self - renewal ; endothelial factors acting with fgf2 accomplish this . in the presence of endothelial cells , a neural stem cell undergoes symmetric , proliferative divisions to produce undifferentiated stem cell sheets that maintain their multipotency and upon endothelial cell removal generate neurons as well as astrocytes and oligodendrocytes . no cd31 + cells were detected in clones , showing that , at least under these circumstances , neural stem cells do not generate endothelial progeny . growth with endothelial cell - derived factors may be an important tool for promoting neural stem cell self - renewal and neurogenesis , allowing efficient production of neural stem cells and a variety of cns neurons for use in replacement therapies . embryonic cerebral cortices and adult svz were dissected and dissociated as described ( 18 - 20 ). single cells were plated into poly - l - lysine - coated 6 - well plates ( costar ) at clonal density ( 2000 - 4000 cells / well ) and cultured in basal serum - free medium consisting of dmem , b27 , n2 , nac and 10 ng / ml fgf2 ( 19 , 20 ). to test the effects of soluble growth factors ( pdgf , cntf , tgfβ , igf1 , il1 , il6 , g - csf , m - csf , gm - csf , lif , bdnf , steel factor , vegf or egf ), each factor was added to serum - free medium with fgf2 at a range of 10 - 50 ng / ml , and clonal growth ( cell number and generation of neurons and glia ) was assessed ( without co - cultured feeder cells ). lif and vegf were also tested in combination , each added at 10 ng / ml into serum - free medium , with and without fgf2 . for co - culture experiments , bovine pulmonary artery endothelial ( bpae ) cells ( vec technologies inc ., atcc , # ccl - 209 ) or mouse brain endothelial cells ( atcc , # crl - 2299 ) ( 4 ) were used at passage 14 - 16 . three days before co - culturing with cortical cells , endothelial cells were plated into 24 mm transwell membrane inserts ( costar ) at 2000 cells / transwell , in dmem with 10 % fbs . four hours before use , the transwells were well rinsed and transferred to serum - free medium containing 10 ng / ml fgf2 . for control conditions , nih3t3 fibroblasts , vascular smooth muscle ( vsm ) cells or cortical cells were plated in the transwells at the same density and grown in the same manner as endothelial cells . the transwells were placed above freshly plated neural stem cells , and the cultures fed every two days with serum free medium . to differentiate the clones , the transwells were removed and the cells cultured for 4 or 7 days , in some experiments with removal of fgf2 and / or addition of 1 % fbs , 5 μm forskolin , or 1 μm retinoic acid . adult forebrain svz stem cells were grown in adherent culture with endothelial co - culture for 7 days , and for a further 7 days after removal of the transwells . cortical cells were plated at a density of 2000 cells / well into non - coated 6 - well plates in dmem , b27 , n2 , nac and 20 ng / ml fgf for 7 days to generate neurospheres . e10 - e11 neurospheres were transferred to pll - coated plates in medium without mitogen and cultured for 7 days before fixation and staining for β - tubulin - iii and tbr1 . the percentages of β - tubulin - iii + neurons per neurosphere and of tbr1 + neurons were calculated for comparison with endothelial co - cultured adherent e10 - e11 cortical stem cell clones . e15 . 5 neurospheres were transferred to pll - coated 6 - well plates and co - cultured with bpae , ctx or nih3t3 feeder cells in transwells for 3 days , or without feeder cells but with added 1 % fbs , or 5 μm forskolin . differentiation of feeder co - cultures was stimulated by removal of transwells and growth factors for a further 4 days . cultures were then fixed and stained for β - tubulin - iii . sub - cloning to analyse self - renewal and neurogenic potential of stem cell clones e10 - 11 cortical stem cells were cultured for 4 days in either endothelial or ctx co - culture then removed from the wells using trypsin . a sample of each single cell suspension was plated into non - adherent conditions at 1000 cells / well in 6 - well plates and allowed to grow into neurospheres that were counted after 7 days in vitro . the remaining single cells were replated at clonal density in pll - coated 6 - well plates in serum - free medium with 10 ng / ml fgf2 and allowed to grow for 5 days . the clones were then fixed and stained , and total progeny and number of β - tubulin - iii + neuronal progeny were calculated . time - lapse video images ( 18 ) of e10 cortical clones were recorded for 3 - 4 days , then the clones were fixed and stained for nestin and β - tubulin - iii . lineage trees were reconstructed from the recorded cell divisions . the appearance of identified individual progeny was mapped within the lineage trees . this showed the birth of neurons and progenitor cells during clonal development . cells were fixed in 4 % paraformaldehyde in 0 . 1m pbs . primary antibodies were used at the following concentrations : nestin , 1 : 4 ( development studies hybridoma bank ); lex , 1 : 20 ( becton dickinson , cd15 ); β - catenin , 1 : 200 ( becton dickinson ); β - tubulin - iii , 1 : 500 ( sigma ); o4 , 1 : 2 ( development studies hybridoma bank ), or 1 : 100 ( chemicon ); gfap , 1 : 400 ( dako ); gad67 , 1 : 2000 ( chemicon ); tbr1 , 1 : 100 ( a gift from dr . yi - ping hseuh ). secondary antibodies ( alexa fluoro - conjugated , molecular probes ) were used at 1 : 250 , and incubated for 45 minutes at room temperature . cortical stem cells were co - cultured with endothelial cells or cortical cells for 9 days . 10 μg / ml brdu was added to the wells for 24 hours and then washed out . the transwells were removed and the underlying cortical cells were cultured for 2 more days , then fixed . cells were stained for β - tubulin - iii and brdu ( 1 : 10 , becton - dickinson ). γ - secretase inhibitor ii ( calbiochem , 50 μm ) in dmso was added to co - cultured cells after 4 days for 6 hours . the same amount of dmso was used as vehicle control . the cells were then fixed and stained for β - tubulin iii and double - labelled for nestin or β - catenin , and the stained cells were quantified . total rna was isolated from e10 . 5 cortical cells co - cultured with bpae cells or cortical cells for 4 days using rneasy mini kit ( qiagen ) according to manufacturer &# 39 ; s instructions . analysis of gene expression was done using semi - quantitative rt - pcr using superscript one - step rt - pcr with platinum taq ( invitrogen ). the following primers were used : hes1 — tcaacacgacaccggacaaacc ( seq id no . : 1 ) and ggtacttccccaacacgctcgc ( seq id no . : 2 ); hes5 — aagtaccgtggcggtggagat ( seq id no . : 3 ) and gagtaaccctcgctgtagtcc ( seq id no . : 4 ) ( 5 ); gapdh — atgtttgtgatgggtgtgaa ( seq id no . : 5 ) and tgggagttgctgttgaagtc ( seq id no . : 6 ). pcr conditions were 94 ° c . 15 seconds , 57 ° c . 30 seconds , 72 ° c . 30 seconds ; 24 cycles for hes1 and hes5 , 18 cycles for gapdh . rt - pcr gel band densities were calculated using imagequant software . brdu uptake reveals prolonged neurogenesis from neural stem cells in endothelial versus control co - cultures . mouse cortical neurogenesis lasts about 7 days ( from e11 - e18 ) and is followed by gliogenesis ( 23 ). e10 cortical stem cells grown in fgf2 in vitro behave similarly ( 19 ). in contrast , neurogenesis is prolonged in endothelial versus ctx co - cultures , as shown by addition of brdu at 9 days in vitro ( fig7 a ): neurons continue to be generated from dividing progenitors many days after tissue isolation ( rather than simply differentiating from progenitors that had divided earlier ). endothelial factors significantly enhanced self - renewal and neurogenesis compared to other added soluble factors . while retinoic acid , fbs and forskolin can stimulate neuron production from some cell types ( 24 - 27 ), they actually depressed neurogenesis from e10 - e11 cortical stem cells expanded in the presence of endothelial cells ( fig . s 6 a ). similarly , eb15 . 5 neurosphere cells plated in adherent condition produced significantly more neurons ( 22 %) when exposed to endothelial cell factors compared to serum or forskolin ( fig2 g ; fig . s 6 b ). growth on fibronectin had little effect on neurogenesis ( not shown ), and the cortical progenitor cells did not grow well on matrigel ( not shown ), underscoring the significant effect of endothelial - derived factors . the fact that no contact is required between cortical cells and endothelial cells shows that soluble factors are responsible , and similar results were observed using endothelial cell - conditioned medium . fig9 are phase contrast photomicrographs showing stem cells grown in control culture medium ( a ) in contrast to those grown in endothelial cell - conditioned medium ( b ). fig9 c shows cells that have been grown in conditioned medium and have been stained for the progenitor marker , lex . this shows that conditioned medium from endothelial cells supports growth of flattened sheets of mouse neural stem cells that retain expression of lex . endothelial cells secrete numerous bioactive substances . a compilation ( 28 ) includes 26 factors that are known to affect other cell types . of these , we have tested pdgf , cntf , fgf2 , tgfβ , igf1 , il1 , il6 , g - csf , m - csf , gm - csf , lif , bdnf and steel factor , and in addition egf ; none duplicated the result we see from endothelial co - 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