starting from an initial population of 1 million cells, the cumulative cell number was calculated at each passage as the fold of increase×the total cell number and plotted as logarithm with base 2 in function of time ( g ). the cell perpetuation ( g ) and population doubling ( h ) analysis demonstrated the continuous and stable growth of the hnscs. ( i ) rt - pcr analysis showing the down regulation of the pluripotency transcripts oct4 and nanog in secondary neurospheres and in expanded hnscs at passage 8 ( p8 ). ( j ) cytogenetic evaluation of the sd56 hnscs line at passage 12 by standard g - banding was performed. twenty metaphase cells were analyzed and showed a normal female chromosome complement ( 46, xx ). isolated and expanded hnscs expressed the neural precursor cell markers nestin ( k ), vimentin ( l ) and the radial glial cell marker 3cb2 ( m ) in virtually all the progeny. ( n - p ) clonal self - renewal ability of the isolated hnscs through symmetrical cell division. single ( n ), two - cell stage ( o ) and four - cell stage ( p ) of a hnsc proliferating over a 3 - day culture period. note the symmetrical segregation of brdu and nestin in the progeny. bars: ( a, b, c, d ) 200 µm; ( e, f ) 100 µm; ( k – m ) 20 µm; ( n – p ) 10 µm. to ascertain self - renewal ability under clonal conditions, a single cell suspension was plated at clonal density ( 1 – 2 cell / 10 µl ). to determine if the hnscs divide symmetrically, we pulsed cultures with the thymidine analog, bromodeoxyuridine ( brdu ), after plating and looked for the expression of nestin in the progeny. cultures were fixed after 1, 2 or 3 div ( figure 1n – p ). after 2 days, plated single cells first underwent a symmetric cell division and gave rise to daughter cells that were both positive for brdu and nestin. the clone of cells continued to grow over the 3 div and all the progeny expressed nestin. brdu labeling demonstrated that it was rare for only one daughter cell to inherit the brdu and thus had undergone asymmetric segregation of the chromatids ( figure s2 ). g - band karyotyping of these hnscs confirmed the normal, non - transformed nature of cells after 12 passages ( figure 1j ). we named the derived hnscs sd56 ( intermittently referred to as sd56 hnscs or hnscs ). under these defined growth conditions, the hnscs showed stable growth with a 2. 7±0. 2 fold increase every 5 to 7 days ( figure 1g ). the population doubling at each passage averaged at 1. 4±0. 1 ( figure 1h ). the viability of hnscs at each passage was consistent at the approximate value of 98 %. the projected cumulative cell numbers demonstrated that trillions of cells could be generated over a period of 5 months ( figure 1g ). we expanded the isolated hnscs lines for over 20 passages with a stable phenotype. an initial cell bank of 75 vials containing 2 to 5 million cells each was generated and cryopreserved. upon removal of the mitogenic factors and plating on a coverslip pre - coated with poly-l-ornithine ( plo ) substrate, the hnscs spontaneously differentiated into neurons, astrocytes and oligodendrocytes, a property that is consistent with normal multipotent hnscs ( figure 2 ). after 2 div, hnscs expressed transcripts for the neural - specific genes nestin, notch1 and neural cell adhesion molecule ( n - cam ) ( figure 2a ) and for the lineage specific markers β - tubulin class iii, medium - size neurofilament ( nf - m ) and microtubule - associated protein 2 ( map - 2 ) for neurons, gfap for astrocytes and myelin basic protein ( mbp ) for oligodendrocytes ( figure 2a ). transcripts for the gabaergic cell marker glutamic acid decarboxylase ( gad ) were expressed, but transcripts for the tyrosine hydroxylase ( th ), a marker for dopaminergic neurons, were undetectable. immunocytochemical analysis ( figure 2b – f ) of 10 day - old cultures demonstrated that the proportion of nestin + cells was 36. 6±2. 7 %, 62. 5±2. 8 % expressed the neuronal marker tuj1, 1. 9±0. 3 % expressed the astrocytic marker gfap and 7. 1±0. 4 % were oligodendrocytes and expressed galactocerebrocide ( gc ) ( figure 2f ). a subset ( 9. 8±1. 6 % ) of the gfap + astrocytes co - expressed nestin. 10. 1371 / journal. pone. 0001644. g002figure 2hesc - derived hnscs spontaneously differentiated into the 3 principal central nervous system cell types. dissociated hnscs were washed free of growth factors and plated on poly-l-onithine coated glass coverslips. differentiated cultures were either harvested after 2 div for total rna extraction and rt - pcr analysis or fixed after 10 div and processed for indirect immunocytochemistry. ( a ) differentiated hnscs expressed the neural - specific transcripts nestin and notch1 as well as transcripts: for neurons ( β - tubulin class iii, map - 2, ncam and medium - size neurofilament, nf - m ), for astrocytes ( gfap ) and for oligodendrocytes ( mbp ). the hnscs expressed transcripts for gad, but not for th. b, c & d, morphology of nsc - derived progeny differentiated into gfap + astrocytes ( b ), gc - expressing oligodendrocytes ( c ) and tuj1 + neurons ( d ), dapi ( blue ) show life cell nuclei. ( e ) photo showing cultures double-immunostained for tuj1 ( green ) and nestin ( red ) ( dapi, blue ). ( f ) quantitative analysis of immunostained 10 day - old cultures for the 3 neural cell types. of three independent experiments, each performed in duplicate. bars: ( c ) 20 µm; ( d, e ) 10 µm. the isolated hnscs are normal and do not form tumors in normal nude ratsthe self - renewal and pluripotent abilities of the hescs are also associated with tumorigenic properties. therefore, the first critical step toward developing therapeutic hnscs is to verify that they are non-tumorigenic. the monolayer culture of sd56 hnscs clearly demonstrated contact inhibition of growth, a normal karyotype and did not express the pluripotency transcripts oct - 4 and nanog. removal of mitogenic factors and continued culture on plastic resulted in cell senescence that is characteristic of non - transformed cells. to determine whether sd56 hnscs form tumors in vivo, we transplanted them at high density ( see methods ) into the forebrain and subcutaneously into the flank of nude rats. the animals were kept for a two - month post - transplant survival period. to label mitotically active cells in vivo during s-phase, the rats were injected ip with brdu ( 50 mg / kg ) every 8 hours during the last 24 hours before euthanasia. the transit amplifying endogenous precursors located in the subventricular zone ( svz ) were labeled ( figure s3 ); however, we were unable to detect grafted sd56 hnscs co - localizing the human - specific nuclear marker hnuc and brdu ( figure s3 ). no surviving sd56 hnscs were detected in the flank of the transplanted animals suggesting that the grafted cells are not tumorigenic. transplanted cells survived, migrated toward and engrafted into the stroke - damaged host tissueto investigate the survival and functional engraftment in an injury environment, hnscs ( 4×105 ) were transplanted into the ischemic boundary zone in the rat striatum one week after the middle cerebral artery occlusion ( mcao ) was performed. animals were euthanized two months later and the brains processed for histo - pathology and immunocytochemistry. grafted sd56 hnscs, identified with hnuc, demonstrated a 37. 0±15. 8 % survival rate and a remarkable dispersion toward the stroke - damaged tissue with no sign of overgrowth or tumorigenesis. the majority of grafted cells ( 61. 2±4. 7 % ) migrated at least 200 µm away from the injection site and penetrated an average distance of 806. 4±49. 3 µm into the stroke - damaged tissue ( figure 3a – c ). immunostaining with the blood vessel marker, glut1, revealed dilated vessels in the infarcted striatum and a close association between vessels and the grafted hnscs ( figure 3b, 3c ).