any methods, additional references, nature portfol io reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10. 1038 / s41588 - 025 - 02182 - 6. backgroundhuman embryonic stem cells ( hescs ) offer a virtually unlimited source of neural cells for structural repair in neurological disor ders, such as stroke. neural cells can be derived from hescs either by direct enrichment, or by isolating specific growth factor - responsive and expandable populations of human neural stem cells ( hnscs ). studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo. methods / principal findingswe isolated an expandable and homogenous population of hnscs ( named sd56 ) from hescs using a defined media supplemented with epidermal growth factor ( egf ), basic fibroblast growth factor ( bfgf ) and leukemia inhibitory growth factor ( lif ). these hnscs grew as an adherent monolayer culture. they were fully neuralized and uniformly expressed molecular features of nscs, including nestin, vimentin and radial glial markers. these hnscs did not express the pluripotency markers oct4 or nanog, nor did they express markers for the mesoderm or endod erm lineages. the self - renewal property of the hnscs was characterized by a predominant symmetrical mode of cell division. the sd56 hnscs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. together, these criteria confirm the definitive nsc identity of the sd56 cell line. importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naive nude rat brains and flanks. furthermore, hnscs isolated under these conditions migrated toward the ischemia - injured adult brain parenchyma and improved the independent use of the stroke - impaired forelimb two months post - transplantation. conclusions / significancethe sd56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. these properties indicate that this hnsc line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research. to date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke. current therapeutic approaches, such as the use of thrombolytics, benefit only 1 to 4 % of patients [ 1 ]. consequently, the majority of stroke patients experience progression of ischemia associated with debilitating neurological deficits. recent evidence has suggested that the transplantation of cells derived from cord blood, bone marrow or brain tissue ( fetal and adult ) enhances sensorimotor function in experimental models of stroke [ 2 ], [ 3 ]. however, the normal human - derived somatic stem cells used in these studies have a limited capacity to differentiate into the diverse neural cell types optimal for structural and physiological tissue repair and are not amenable for large - scale cell production. unlike other sources of stem cells, hesc lines possess a nearly unlimited self - renewal capacity and the developmental potential to differentiate into virtually any cell type of the organism. as such, they constitute an ideal source of cells for regenerative medicine. the successful derivation of hesc lines from the inner cell mass of preimplantation embryos and their long - term maintenance in vitro over multiple passages has been demonstrated [ 4 ] and standar diz ed. differentiation and enrichment processes that direct hescs towards a neural lineage have also been achieved. to promote neuralization, escs were cultured in a defined media supplemented with morphogens or growth factors [ 5 ], [ 6 ], [ 7 ] or cultured under conditions that promote “ rosettes ”, structures morphologically similar to the developing neural tube [ 8 ], [ 9 ]. this neuralization process has proven invaluable in understanding the specification of hesc - derived neural tissue [ 10 ], [ 11 ], [ 12 ]. however, the enriched neural progeny derived displayed overgrowth and limited migration after grafting into normal newborn mice [ 13 ] and lesioned adult rat striatum [ 12 ], [ 14 ], [ 15 ], [ 16 ]. the inner cores of these grafts contained tumorigenic precursor cells ( reviewed in [ 17 ] ). these findings suggest that neural cells generated by acute exposure to growth factors and / or morphogens may still be heterogeneous and potentially tumorigenic. we report an alternative method for the isolation and the perpetuation of a multipotent hnsc line from the hescs with a primitive mode of self - renewal. we also demonstrate their long - term expansion, non-tumorigenic properties and functional engraftability in an experimental model of stroke. in vitro isolation, growth and differentiation of hesc - derived hnscsthe hescs were maintained and expanded on mouse feeder layer in media supplemented with bfgf ( figure 1a ). after cell dissociation, a portion of the hescs was cultured in serum free medium containing egf, bfgf and lif. these factors are known to stimulate the proliferation of human fetal - derived nscs [ 18 ], [ 19 ]. after 3 days in vitro ( div ), there was selective survival and growth of cells that aggregated in clusters or spheres ( figure 1b ). these primary spheres were harvested and replated in fresh media. during the following week, the spheres attached to the flask and a fibroblast - like cell population began to migrate out ( figure 1c ). secondary spheres ( 2° spheres ) were generated from these cultures and lifted off by the end of the week leaving a hollow in the middle of the attached cells ( figure 1d ). the floating 2° spheres were collected and replated in fresh growth medium for 2 weeks. the cultures were then passaged by collagenase cell dissociation every 7 div for an additional 4 passages ( figure s1 ). at the 5th and 6th passages, spheres were dissociated into a single - cell suspension using trypsin - edta. at this stage there was a change in the hnscs ' adherent properties, and the cells began to grow as a monolayer with multiple foci of cells throughout the culture ( figure 1f ). the adherent hnsc culture stained uniformly for nestin ( figure 1k ), vimentin ( figure 1l ) and with the radial glial marker 3cb2 ( figure 1m ) indicating their homogeneity and nsc identity. under these culture conditions, it is noteworthy that we did not observe the formation of rosettes which has been previously reported to occur under certain conditions during neuralization of hescs [ 8 ], [ 20 ], [ 21 ]. rt - pcr analysis confirmed that these hnscs did not express the pluripotency transcripts oct - 4 and nanog ( figure 1i ). furthermore, the hnscs did not express transcripts for brachyury and foxa2, marker genes for mesoderm and endod erm respectively ( negative result, data not shown ). 10. 1371 / journal. pone. 0001644. g001figure 1isolation and purification of hnscs from the hescs. the hescs were grown on a mouse feeder layer ( a ). primary neurospheres ( b ) were isolated and replated to eliminate other non - neural cells. the selectively harvested secondary neurospheres ( arrow in c ), left behind hollow cores in the surface area ( star in d ) where they attached earlier. they were perpetuate d for an additional 5 passages ( e ). these 2° spheres were then passaged using a single cell dissociation protocol ( f ). arrow in f shows an example of a focus of proliferating cells. ( g, h ) the hnscs were passaged every 5 – 7 days, as described in the methods section.