Our results indicate that a self-renewable and homogenous population of hNSCs, SD56, was derived from hESCs using defined media supplemented with a specific combination of growth factors. The SD56 hNSCs grew as an adherent monolayer culture, uniformly expressed molecular features of hNSCs including nestin, vimentin and the radial glial marker 3CB2, and did not express the pluripotency markers Oct4 or Nanog. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. They exhibited no chromosomal abnormalities and demonstrated non-tumorigenic properties after implantation into ischemic brains and into naïve nude rat brains and flanks. Furthermore, the transplanted SD56 hNSCs migrated toward the stroke-damaged adult brain parenchyma, engrafted and improved the independent use of the stroke-impaired forelimb.
Maintenance of stem cells requires symmetrical and asymmetrical cell divisions to both expand and to give rise to specialized progeny of a specific tissue (reviewed in 
). In vivo, a complex cellular micro-environment or niche ensures the self-maintenance property of NSCs 
. In vitro, defined growth factors and extracellular matrices support stem cell self-renewal 
. The embryonic stem cells can propagate in a predominantly proliferative symmetrical mode, leading to homogeneous cell cultures growing relatively quickly with minimal cell differentiation 
. Tissue specific stem cells, however, self-renew in a predominant asymmetric mode to maintain them selves and compensate for the loss of differentiated cells due to disease or injury. Thus, NSCs isolated from developing or adult brain grow as a mixture of undifferentiated and differentiated cells due the predominant asymmetrical mode of cell division 
. A recent study has reported that a murine ESC-derived NSC line (LC1) is propagated as an adherent homogenous culture with a dominant mode of symmetrical self-renewal 
. A combination of EGF and FGF2 was sufficient to propagate these NSCs as an adherent monolayer. However, the SD56 hNSC line described here required the combination of EGF, bFGF and LIF for self-maintenance. Although there are morphological and molecular similarities between our hNSCs and the NSCs previously described 
, the methods of isolation and growth are different. In addition to the different combination of growth factors used, the hNSC line we have isolated did not go through the rosette-structure stage. The in vitro analysis of BrdU incorporation and nestin expression indicated that our hNSCs divide predominantly symmetrically. This type of growth pattern is characteristic of primitive normal stem cells undergoing mostly symmetric cell division to increase the stem cell pool at the early stage of development or during tissue regeneration after injury 
. RT-PCR and immunocytochemistry analysis demonstrated that these undifferentiated SD56 cells did not express any pluripotency, endodermal or mesodermal markers. Furthermore, the SD56 hNSCs described here exhibited the multipotential characteristic to differentiate into neurons, astrocytes and oligodendrocytes both in vitro and upon transplantation. Together these findings suggest that the hNSC line we isolated are appropriately programmed and share similar characteristics with the definitive NSCs of the developing brain.
The SD56 hNSCs demonstrated a remarkable ability to migrate toward the stroke-damaged parenchyma of the adult rat brain. This directed migration by the majority of the grafted cells could be due to their uniform cellular composition, which results in an equal response to the host microenvironment.
In previous studies, subpopulations of transplanted hESCs that were enriched in neural cells migrated in host microenvironments conducive to cell migration, such as the developing brain or in structures such as the rostral migratory stream 
. In the adult lesioned brain, the grafted hESC-derived neural cells proliferated and formed rosettes 
, teratomas 
or a cellular mass that induced a gliotic host response whereby local astrocytes demarcated the grafts 
. Enriched neural cultures derived from mouse 
and monkey ESCs 
have produced behavioral improvements when transplanted into animal models of stroke and brain injury. However, in these cases, the transplanted non-human ESCs also formed a mass with signs of overgrowth in the core, as well as deformations 
. ESCs plated at low density acquire neural identity within few hours after plating 
. Interestingly, nearly all viable cells expressed nestin, the early neural fate marker Sox1, and the pluripotency marker Oct4. Together, these studies are seminal and suggest that complete neuralization may not be achieved through certain enrichment processes, consequently the neural cells could revert to a pluripotent stage 
. The dispersion of the grafted hNSCs within host parenchyma may allow for more graft-host interactions that could stabilize differentiation, inhibit growth and prevent gliotic host response.
In the present study, SD56 hNSC-transplanted animals demonstrated a stable improvement in the sensorimotor function when evaluated for spontaneous exploratory activity in the cylinder test that detects long-lasting deficits in forelimb use in the experimental models of stroke 
. The transplantation of hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 8 weeks post transplantation. This is the first report demonstrating that the transplantation of hNSCs derived from hESCs can improve neurologic behavior after experimental stroke. Together, these findings are encouraging and suggest that these cells are promising for future development. In addition to their therapeutic application, the hNSCs isolated under the reported conditions offer a means to interrogate host environments and unravel mechanistic features of self-renewal, non-tumorigenicity and functional engraftability in animal models of neurological disorders.