Deciphering molecular heterogeneity and dynamics of neural stem cells in human hippocampal development, aging, and injury

Reviewer #1 (Public Review):

In this manuscript, Yao et al. explored the transcriptomic characteristics of neural stem cells (NSCs) in the human hippocampus and their changes under different conditions using single-nucleus RNA sequencing (snRNA-seq). They generated single-nucleus transcriptomic profiles of human hippocampal cells from neonatal, adult, and aging individuals, as well as from stroke patients. They focused on the cell groups related to neurogenesis, such as neural stem cells and their progeny. They revealed genes enriched in different NSC states and performed trajectory analysis to trace the transitions among NSC states and towards astroglial and neuronal lineages in silico. They also examined how NSCs are affected by aging and injury using their datasets and found differences in NSC numbers and gene expression patterns across age groups and injury conditions. One major issue of the manuscript is questionable cell type identification. For example, in Figure 2C, more than 50% of the cells in the astroglial lineage clusters are NSCs, which is extremely high and inconsistent with classic histology studies.

Reviewer #2 (Public Review):

In this manuscript, Yao et al. present a series of experiments aiming at generating a cellular atlas of the human hippocampus across aging, and how it may be affected by injury, in particular, stroke. Although the aim of the study is interesting and relevant for a larger audience, due to the ongoing controversy around the existence of adult hippocampal neurogenesis in humans, a number or technical weaknesses result in poor support for many of the conclusions made from the results of these experiments.

In particular, a recent meta-analysis of five previous studies applying similar techniques to human samples has identified different aspects of sample size as main determinants of the statistical power needed to make significant conclusions. Some of these aspects are the number of nuclei sequenced and subject stratification. These two aspects are of concern in Yao's study. First, the number of sequenced nuclei is lower than the calculated number of nuclei required for detecting rare cell types. However, Yao et al. report succeeding in detecting rare populations, including several types of neural stem cells in different proliferation states, which have been demonstrated to be extremely scarce by previous studies. It would be very interesting to read how the authors interpret these differences. Secondly, the number of donors included in some of the groups is extremely low (n=1) and the miscellaneous information provided about the donors is practically inexistent. As individual factors such as chronic conditions, medication, lifestyle parameters, etc... are considered determinant for the variability of adult hippocampal neurogenesis levels across individuals, this represents a series limitation of the current study. Overall, several technical weaknesses severely limit the relevance of this study and the ability of the authors to achieve their experimental aims.

Author Response

We are very grateful to the editors and reviewers for their valuable comments of our manuscript. We carefully consider all the comments and will provide a revised manuscript with our point-by-point responses as soon as possible. In the meantime, we will try our best to carry out additional experiments to bolster our conclusions. Here, we would like to respond provisionally to the public reviews.

We appreciate the concerns raised by Reviewer 1 regarding the identification of cell types in our study. Specifically, they noted that the high proportion of NSCs within the astroglial lineage clusters is inconsistent with classic histology studies. We apologize for not clearly specifying in the text and figure legend that the data presented in Figure 2C were obtained from neonatal samples, which may explain the higher presence of NSCs. To rectify this issue, we will revise the text to ensure clarity regarding the age group from which the data in Figure 2C were obtained. Additionally, we commit to providing additional UMAP plots and quantitative analysis separately for different age groups to support our findings. This will allow a more accurate representation of the cell type composition, taking into consideration any potential variations that may occur with age.

We appreciate Reviewer 2's acknowledgment that the finding of our study is interesting and relevant to a broader audience. However, he raised two major concerns that could weaken the conclusions drawn from our study. First, the reviewer noted that the number of sequenced nuclei in our study is lower than the calculated number required for detecting rare cell types. We noticed that according to the computational modeling conducted by Tosoni et al. (Neuron, 2023), at least 21 neuroblast cells (NBs) can be identified out of 30,000 granule cells (GCs) from a total of 180,000 dentate gyrus (DG) cells. In our dataset, we sequenced 24,671 GC nuclei and 92,966 total DG cell nuclei, which also includes neonatal samples. The number of nuclei we sequenced is 4.5 times higher than that of Wang et al. (Cell Research, 2022), who also detected NBs. Therefore, it is reasonable to conclude that we were able to detect NBs. Moreover, the presence of these rare cell types has been demonstrated in our study through immunostaining techniques, which provides further evidence. Secondly, Reviewer 2 raised concerns about the low number of donors included in some of the groups, with only one donor (n=1) being represented in certain cases. We acknowledge these limitations and understand that the inclusion of a larger number of donors would strengthen the statistical power and generalizability of our findings. However, due to the scarcity of stroke or neonatal human samples, it is not feasible to collect a larger sample size within the expected timeframe. Although one sample is not enough to show the precise changes in cells and molecular mechanisms caused by stroke, it can provide a typical example to demonstrate our hypothesis that neural stem cells could be activated under conditions of injury. The latter is what we really want to address in the manuscript. Regrading to the donor’s information, we will provide more details about the donors, including any clinical characteristics available, to enhance the transparency of our study. Importantly, we have implemented strict quality control measures to support the reliability of our sequencing data. These measures include: 1) Immediate collection of tissue samples after postmortem (3-4 hrs) to ensure the quality of isolated nuclei. 2) Only nuclei expressing more than 200 genes but fewer than 5000-8600 genes (depending on the peak of enrichment genes) were considered. On average, each cell detected around 3000 genes. 3) The average proportion of mitochondrial genes in each sample was approximately 1.8%, with no sample exceeding 5%.