Referee 1's review:

The authors have isolated a homogenous cell-line of multipotent hNSC from hESC that are expandable and have no chromosomal abnormalities – which they have named SD56. These cells integrate into stroke injured CNS parenchyma and improve function.

SD56 was reported to have the following characteristics:
1) Expandable and uniformly homogenous.
2) Differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro and in vivo.
3) Exhibited no chromosomal abnormalities and did not form tumors after implantation.
4) When transplanted, cells migrated toward ischemia-injured brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation as assayed using the cylinder test.

Major Comments:

p.25
The statistical analysis appears to be incorrect.
The authors compared means using one-way ANOVA; however, they did not list the post-hoc test chosen or even if one was used. I am unsure how, or when, the student’s t-test was used. If by; “inter-group differences was determined by applying Student’s t-test”, the authors mean that multiple t-tests were performed, then the type I error (false positive) is additive for each comparison made. When multiple t-tests are performed there is a lack of independence hence the use of the appropriate post-hoc test. With statistical values close to the alpha error (an example was given of P = 0.052) the null hypothesis may have been rejected when it is actually true.

I think that the authors need to reanalyze the data using the appropriate post-hoc test.

p.5
“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.”
I am not sure what the authors mean by this statement? The study that was referenced states explicably: “Surprisingly, this line could readily differentiate into noncerebellar neurons, astrocytes, and oligodendrocytes both in vitro and in vivo” [J Neurosci Res. 2007 Nov 1]. How are the authors suggesting somatic stem cells are “limited” in this respect?

p.7
“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 2A-D).”

Figure: ..”morphology of NSC derived progeny differentiated into GFAP+ astrocytes”

Before the authors can conclude that the GFAP expressing cells are “astrocytes” they will need to double label with at least nestin and demonstrate that the differentiated GFAP expressing progeny are not a type of GFAP expressing neural progenitor, given that GFAP is expressed by cells other than astrocytes. Especially given that ~37% of the progeny continued to express nestin.

p.8
“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 rarely did only one daughter cell inherits the BrdU and had thus undergone asymmetric segregation of the chromatids (Figure S2).
Figure S2. Asymmetric segregation of BrdU and symmetric expression of Nestin.
The abstract states:
The SD56 hNSCs grew as an adherent monolayer culture, uniformly expressed molecular features of hNSCs including nestin,…”

1) While Figure S2 was given as an example of a rare asymmetric segregation of the chromatids it also shows a nestin negative cell - center-middle. Were all of the cells symmetric in their expression of nestin or where some of the cells nestin negative? Fig2S seems to suggest that there were enough cells that did not express nestin that one showed up in a figure meant to demonstrate asymmetry.
2) Figure 1K has a DAPI counter stain however Figure 1L and M appear to be missing the DAPI counter stain.

p.9
Figure 2A is unclearly written. The authors state that hNSCs were characterized by the expression of NSC specific transcripts:
“RT-PCR analysis confirmed that the transcripts for the neural-specific genes nestin, Notch1 and neural cell adhesion molecule (N-CAM) were expressed by the hNSCs (Figure 2A).”
However, Figure legend 2 states that panel A does not refer to hNSC but to differentiated NSCs, which is to say, their progeny:
“Differentiated hNSCs expressed the neural-specific transcripts nestin and Notch1”

It needs to be made clearer exactly which cells were assayed.

Minor comments:

Grammar:

p8 - inherits should read “inherit”
p9 - (Figure 2A-D) should read B-D
SupplimentS3 “merge” does not have the label “DAPI”
R&D Sytems should read R&D Systems
Novartis Pharmaceeuticals should read Novartis Pharmaceuticals
Figure2 the acronym CNS is not needed.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.