Single-Cell Analysis of Regional Differences in Adult V-SVZ Neural Stem Cell Lineages

May combine two claims.

The V-SVZ contained predominantly unramified microglia, although ramified microglia were also present.

“Moreover, the V-SVZ contained predominantly unramified microglia (Figures S1F and S1H), as previously reported (Ribeiro Xavier et al., 2015), although ramified microglia were also present (Figures S1G and S1H).”

• cites Ribeiro Xavier et al., 2015 sourceNot checked yet

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Both Micro.A and Micro.B expressed microglia markers Tmem119, Aif1, and Cx3cr1, whereas Micro.B was also enriched in immediate early transcription factors as well as pro-inflammatory chemokines (e.g., Ccl3 and Ccl4).

“Both Micro.A and Micro.B expressed microglia markers Tmem119, Aif1, and Cx3cr1, whereas Micro.B was also enriched in immediate early transcription factors as well as pro-inflammatory chemokines (e.g., Ccl3 and Ccl4; Friedman et al., 2018; Matcovitch-Natan et al., 2016; Mathys et al., 2017; Figure 1E; Table S1).”

Probably not a substantial claim: may not pass S3 (load-bearing).

A small cluster of cells was detected that are either circulating or resident T lymphocytes.

“We also detected a small cluster of cells, which are either circulating or resident T lymphocytes.”

May combine two claims.

Three major neuronal subclusters were detected in the datasets (3,202 cells), including one enriched in Chat (Neuron 3).

“We detected three major neuronal subclusters in our datasets (3,202 cells; Figure 1E; Table S1), including one enriched in Chat (Neuron 3), which may include ChAT+ local neurons that regulate V-SVZ cells (Paez-Gonzalez et al., 2014).”

May combine two claims.

Astro.2 was depleted for astrocyte gene sets and enriched for "tanycyte 2" markers.

“In contrast, Astro.2 was depleted for astrocyte gene sets and enriched for “tanycyte 2” markers (Campbell et al., 2017; Figure S2A).”

• cites Campbell et al., 2017 sourceNot checked yet

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The distribution of astrocyte subclusters showed clear regional patterns, but not major compositional sex differences.

“The distribution of astrocyte subclusters showed clear regional patterns, but not major compositional sex differences.”

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V-SVZ astrocyte subclusters were differentially distributed in the lateral and septal preparations: Astro.1 was more abundant in lateral wall, Astro.3 and Astro.5 were more abundant in septal wall, and Astro.2 and Astro.4 were more evenly distributed in both walls.

“Interestingly, V-SVZ astrocyte subclusters were differentially distributed in the lateral and septal preparations (Figures 2A and 2B). Astro.1 was more abundant in samples of lateral wall, whereas Astro.3 and Astro.5 were more abundant in those from the septal wall. In contrast, Astro.2 and Astro.4 were more evenly distributed in both lateral and septal walls.”

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Gsx2, Pax6, and Lhx2 were more uniformly expressed across multiple astrocyte subclusters but were depleted in Astro.5.

“Although Gsx2, Pax6, and Lhx2 were more uniformly expressed across multiple astrocyte subclusters, they were depleted in Astro.5 (Figures 2B and S3A; Table S2).”

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Five major subclusters of astrocytes were identified, which were clearly distinct from ependymal cells.

“We identified five major subclusters of astrocytes, which were clearly distinct from ependymal cells (Figure 2A; Table S2).”

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Astrocyte clusters exhibit different connectivity within the trajectory: Astro.2 is connected to both OPCs and neuronal progenitors, whereas Astro.3, which is enriched in the septal wall, is connected only with OPCs.

“Importantly, astrocyte clusters exhibit different connectivity within the trajectory. For example, Astro.2 is connected to both OPCs and neuronal progenitors, whereas Astro.3, which is enriched in the septal wall, is connected only with OPCs (Video S1).”

Classified as probably not a substantial claim. May combine two claims.

Co-expression probability ratios for each transcription factor expressed in the neuronal lineage in individual cells were calculated, and TFs forming significant clusters were isolated to identify TF co-expression modules.

“To investigate the transcriptional regulatory steps defining neuronal lineage differentiation, we calculated the co-expression probability ratios for each transcription factor (TF) expressed in the neuronal lineage in individual cells and isolated TFs forming significant clusters (Figure S5A; Table S4) to identify TF co-expression modules and TFs with mutually exclusive expression profiles.”

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Immunostaining for NG2 confirmed that OPCs were enriched in the septal V-SVZ and were more abundant in the septal V-SVZ in males than in females.

“This confirmed that OPCs were enriched in the septal V-SVZ and, furthermore, that they were more abundant in the septal V-SVZ in males than in females (Figures 4C and 4D).”

May combine two claims.

These transitional cells clustered with Astro.2 and were in a continuous trajectory that progressed via two sequential stages of aNSCs (aNSC1 and aNSC2/TAC) to neuroblasts.

“These transitional cells clustered with Astro.2 and were in a continuous trajectory that progressed via two sequential stages of aNSCs (aNSC1 and aNSC2/TAC) to neuroblasts, suggesting a key role for Astro.2 in NSC activation (Figure 3A).”

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OPCs and COPs were significantly enriched in the septal wall over the lateral wall in both sexes, with OPCs more enriched in males as compared to females.

“OPCs and COPs were significantly enriched in the septal wall over the lateral wall in both sexes, with OPCs more enriched in males as compared to females (Figures 4A and 4B).”

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Several genes with high correlation variability that depend on sex in male versus female comparisons were detected, such as Igfbp2, Slco1c1, and Mfap2, further supporting male and female V-SVZ astrocyte functional gene expression level differences.

“Similarly, we detected several genes with high correlation variability that depend on sex in male versus female comparisons, such as Igfbp2, Slco1c1, and Mfap2, further supporting male and female V-SVZ astrocyte functional gene expression level differences (Figure 5D; Table S5).”

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When lineage potencies of non-dividing astrocytes were compared separately by wall and by sex, NPC-associated genes were enriched in lateral wall astrocytes and OPC-associated genes in septal wall astrocytes.

“Finally, when lineage potencies of non-dividing astrocytes were compared separately by wall and by sex, NPC- associated genes were enriched in lateral wall astrocytes and OPC-associated genes in septal wall astrocytes (Figure 5D; Table S5), consistent with the quantitative differences in NPCs or OPCs in these regions.”

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Several NPC and/or OPC-correlated genes also showed clear regional bias, such as Mt1 or Mt3, which were strong markers of lineage potency in septal astrocytes but did not correlate with either lineage in lateral astrocytes.

“Interestingly, several NPC and/or OPC-correlated genes also showed clear regional bias, such as Mt1 or Mt3, which were strong markers of lineage potency in septal astrocytes but did not correlate with either lineage in lateral astrocytes (Figure 5D).”

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Highly NPC and OPC anti-correlated astrocyte markers, such as Gja1, showed high expression in both the V-SVZ and adjacent parenchymal cells, and Itih3 was more enriched in non-SVZ cells.

“In contrast, highly NPC and OPC anti-correlated astrocyte markers, such as Gja1, showed high expression in both the V-SVZ and adjacent parenchymal cells and Itih3 was more enriched in non-SVZ cells (Figure 5C).”

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Some markers were correlated only with NPCs (e.g., Stmn1) or OPCs (e.g., Cspg5 and Dkk3); anti-correlated markers included classical astrocyte markers such as Gja1, Slc1a3, and Sparcl1.

“Some markers were correlated only with NPCs (e.g., Stmn1) or OPCs (e.g., Cspg5 and Dkk3; Table S5); moreover, our analysis also revealed anti-correlated markers, including classical astrocyte markers, such as Gja1, Slc1a3, and Sparcl1.”

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Top markers that correlated with both NPCs and OPCs included S100a6, Notum, Dbi, and Sfrp1, as well as known V-SVZ qNSC activation and injury-response markers, such as Ascl1, Vim, Thbs4, and Rpl32.

“Top markers that correlated with both NPCs and OPCs included S100a6, Notum, Dbi, and Sfrp1, as well as known V-SVZ qNSC activation and injury-response markers, such as Ascl1, Vim, Thbs4, and Rpl32 (Benner et al., 2013; Dulken et al., 2017; Llorens-Bobadilla et al., 2015; Figure 5B).”

• cites Benner et al., 2013 sourceNot checked yet

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• cites Dulken et al., 2017 sourceNot checked yet

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• cites Llorens-Bobadilla et al., 2015 sourceNot checked yet

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The Astro.2 population showed highest correlation with both NPCs and OPCs, although the septal V-SVZ-enriched Astro.3 was correlated with OPCs only.

“Consistent with the putative lineage order in the force-directed graphs (Figures 3A and 4E), the Astro.2 population showed highest correlation with both NPCs and OPCs, although the septal V-SVZ-enriched Astro.3 was correlated with OPCs only (Figure 5A), in line with the higher proportion of OPCs in the septal wall (Figures 4B and 4C).”

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Dbi, Cpe, Igfbp5, Vim, Marcksl1, Sfrp1, Anxa2, and Dkk3 transcripts showed high enrichment in V-SVZ, including various regional distributions along the dorsal-ventral and rostro-caudal axis.

“Consistent with their high ranking in the lineage potency analysis, Dbi, Cpe, Igfbp5, Vim, Marcksl1, Sfrp1, Anxa2, and Dkk3 transcripts showed high enrichment in V-SVZ, including various regional distributions along the dorsal-ventral and rostro-caudal axis (Figure 5C).”

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Among known markers expressed by adult V-SVZ NSCs, aNSC markers were correlated with NPCs and OPCs (Ascl1, Nes, and Gsx2), whereas qNSC-enriched genes (Vcam1, Cdh2, Nr2e1, S1pr1, and Hes5) were not.

“Finally, among known markers expressed by adult V-SVZ NSCs, aNSC markers were correlated with NPCs and OPCs (Ascl1, Nes, and Gsx2), whereas qNSC-enriched genes (Vcam1, Cdh2, Nr2e1, S1pr1, and Hes5) were not (Figure S5C).”

May combine two claims.

These markers exhibited different regional expression patterns within the V-SVZ walls, highlighting additional V-SVZ astrocyte heterogeneity still to be resolved.

“These markers exhibited different regional expression patterns within the V-SVZ walls, highlighting additional V-SVZ astrocyte heterogeneity still to be resolved.”

May combine two claims.

Several key molecular pathways crucial for NSC maintenance, self-renewal, and proliferation were differentially expressed in male versus female astrocytes, indicating differential effects of these cues and signaling pathways on male and female NSCs.

“In addition to significant regional differences, we also found that several key molecular pathways crucial for NSC maintenance, self-renewal, and proliferation were differentially expressed in male versus female astrocytes, indicating differential effects of these cues and signaling pathways on male and female NSCs.”

May combine two claims.

The most striking sex difference was in OPCs, which were enriched in the septal wall and especially in males.

“The most striking sex difference was in OPCs, which were enriched in the septal wall and especially in males.”

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The study separately profiled cells from the entire lateral and septal wall of male and female adult mice without FACS purification, thereby capturing a broader repertoire of V-SVZ cell types, including cells at multiple stages of differentiation along both the neuronal and oligodendrocyte lineages as well as niche cells.

“Here, we separately profiled cells from the entire lateral and septal wall of male and female adult mice without FACS purification, thereby capturing a broader repertoire of V-SVZ cell types, including cells at multiple stages of differentiation along both the neuronal and oligodendrocyte lineages as well as niche cells.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Adult V-SVZ NSCs are radial cells with many hallmarks of astrocytes.

“Adult V-SVZ NSCs are radial cells with many hallmarks of astrocytes (Doetsch et al., 1999).”

• cites Doetsch et al., 1999 sourceNot checked yet

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Classified as probably not a substantial claim.

Individual cells were randomly sampled without cell sorting to maximally preserve in vivo composition.

“Importantly, individual cells were randomly sampled without cell sorting to maximally preserve in vivo composition.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Adult V-SVZ NSCs co-exist in quiescent (qNSC) and actively dividing (aNSC) states.

“Adult V-SVZ NSCs co-exist in quiescent (qNSC) and actively dividing (aNSC) states (Chaker et al., 2016).”

• cites Chaker et al., 2016 sourceNot checked yet

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Adult V-SVZ NSCs contact the cerebrospinal fluid at the center of pinwheels formed by ependymal cells and extend a long process to the vasculature.

“They contact the cerebrospinal fluid in the ventricles at the center of pinwheels formed by ependymal cells and extend a long process to the vasculature (Mirzadeh et al., 2008; Shen et al., 2008; Tavazoie et al., 2008).”

• cites Mirzadeh et al., 2008 sourceNot checked yet

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• cites Shen et al., 2008 sourceNot checked yet

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• cites Tavazoie et al., 2008 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Upon activation, V-SVZ NSCs give rise to transit amplifying cells (TACs), which generate neuroblasts (NBs) that migrate to the olfactory bulb.

“Upon activation, they give rise to transit amplifying cells (TACs), which in turn generate neuroblasts (NBs) that migrate to the olfactory bulb (Doetsch et al., 1999).”

• cites Doetsch et al., 1999 sourceNot checked yet

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Classified as probably not a substantial claim.

Previous low-throughput single-cell studies have begun to illuminate the stages of qNSC activation in the adult V-SVZ, focusing on NSCs purified by FACS from the lateral wall and the neuronal lineage.

“Previous single-cell studies using low-throughput approaches have begun to illuminate the stages of qNSC activation in the adult V-SVZ (Basak et al., 2018; Dulken et al., 2017; Llorens-Bobadilla et al., 2015; Luo et al., 2015), focusing on NSCs purified by fluorescence-activated cell sorting (FACS) from the lateral wall and the neuronal lineage.”

• cites Basak et al., 2018 sourceNot checked yet

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• cites Dulken et al., 2017 sourceNot checked yet

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• cites Llorens-Bobadilla et al., 2015 sourceNot checked yet

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Classified as probably not a substantial claim.

Oligodendrocytes and astrocytes are also generated in the adult V-SVZ.

“Although oligodendrocytes and astrocytes are also generated in the adult V-SVZ (Menn et al., 2006; Nait-Oumesmar et al., 1999; Ortega et al., 2013; Sohn et al., 2015; Xing et al., 2014), their lineage trajectories are less clear.”

• cites Menn et al., 2006 sourceNot checked yet

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• cites Nait-Oumesmar et al., 1999 sourceNot checked yet

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• cites Ortega et al., 2013 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Adult V-SVZ NSCs are set aside during embryogenesis and remain largely quiescent until adulthood.

“Adult V-SVZ NSCs are set aside during embryogenesis, remaining largely quiescent until adulthood (Fuentealba et al., 2015; Furutachi et al., 2015), and reside in a complex microenvironment comprising diverse cell types and local and long-range signals (Silva-Vargas et al., 2013).”

• cites Fuentealba et al., 2015 sourceNot checked yet

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• cites Furutachi et al., 2015 sourceNot checked yet

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Adult V-SVZ NSCs reside in a complex microenvironment comprising diverse cell types and local and long-range signals.

“Adult V-SVZ NSCs are set aside during embryogenesis, remaining largely quiescent until adulthood (Fuentealba et al., 2015; Furutachi et al., 2015), and reside in a complex microenvironment comprising diverse cell types and local and long-range signals (Silva-Vargas et al., 2013).”

• cites Silva-Vargas et al., 2013 sourceNot checked yet

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Classified as probably not a substantial claim.

To avoid over-interpretation of small compositional differences potentially due to differential sampling, the study focused on compositional differences in cell types with more than 800 cells in follow-up orthogonal validation experiments.

“To avoid over-interpretation of small compositional differences potentially due to differential sampling, we focused on compositional differences in cell types with more than 800 cells in the follow-up orthogonal validation experiments.”

May combine two claims.

Endo.1 may correspond to endothelial capillary cells, Endo.2 to venous endothelial cells, and Endo.3 to arterial endothelial cells.

“Based on marker gene enrichment (Table S1), Endo.1 may correspond to endothelial capillary cells, Endo.2 to venous endothelial cells, and Endo.3 to arterial endothelial cells (Vanlandewijck et al., 2018).”

• cites Vanlandewijck et al., 2018 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Neuron 3 may include ChAT+ local neurons that regulate V-SVZ cells.

“which may include ChAT+ local neurons that regulate V-SVZ cells (Paez-Gonzalez et al., 2014).”

• cites Paez-Gonzalez et al., 2014 sourceNot checked yet

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The lateral and septal walls have different V-SVZ astrocyte compositions, which exhibit significant regional and sex differences at the gene expression level.

“In sum, the lateral and septal walls have different V-SVZ astrocyte compositions, which exhibit significant regional and sex differences at the gene expression level.”

May combine two claims.

This multi-step progression reveals an unappreciated intricacy in neuronal lineage differentiation downstream of qNSC to aNSC transition and TF co-expression profiles that target transcriptionally discrete stages of NSC differentiation.

“This multi-step progression reveals an unappreciated intricacy in neuronal lineage differentiation downstream of qNSC to aNSC transition and TF co-expression profiles that target transcriptionally discrete stages of NSC differentiation.”

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Oligodendrocyte progenitors were also connected to aNSCs in the neuronal trajectory, supporting a model for two lineages of oligodendrocytes.

“Interestingly, oligodendrocyte progenitors were also connected to aNSCs in the neuronal trajectory, supporting a model for two lineages of oligodendrocytes.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Sox2 and Sox21 interaction are regulators of pluripotency and human glioma progression.

“Among these, Sox2 and Sox21 interaction are regulators of pluripotency and human glioma progression (Caglayan et al., 2013; Kuzmichev et al., 2012).”

• cites Caglayan et al., 2013 sourceNot checked yet

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• cites Kuzmichev et al., 2012 sourceNot checked yet

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The higher number of neuronal lineage progenitors in the lateral wall, in contrast to the enrichment of oligodendrocyte progenitors in the septal wall, points to important regional differences in the V-SVZ regarding neurogenesis and gliogenesis.

“In sum, the higher number of neuronal lineage progenitors in the lateral wall, in contrast to the enrichment of oligodendrocyte progenitors in the septal wall, point to important regional differences in the V-SVZ regarding neurogenesis and gliogenesis (Figure S4E).”

May combine two claims.

The analysis revealed that lateral and septal walls have different lineage biases toward neuronal or glial fate at the single-cell level and exhibit some sex differences.

“Our analysis revealed that lateral and septal walls have different lineage biases toward neuronal or glial fate at the single-cell level and exhibit some sex differences.”

Classified as probably not a substantial claim. May combine two claims.

As almost all V-SVZ astrocytes are Slc1a3+, the level of expression of pan-astrocyte genes at the single-cell level may be important to assess the lineage potency of an individual astrocyte.

“As almost all V-SVZ astrocytes are Slc1a3+, the level of expression of these pan-astrocyte genes (e.g., Gja1 and Slc1a3) at the single-cell level may be important to assess the lineage potency of an individual astrocyte (Figure 5B).”

May combine two claims.

The analysis identifies markers correlated with NPC and/or OPC potency and allows the ranking of previously proposed markers in terms of their lineage potency.

“Together, our analysis identifies markers correlated with NPC and/or OPC potency and allows the ranking of previously proposed markers in terms of their lineage potency.”

May combine two claims.

The study provides a platform to identify molecularly distinct pools of stem cells under homeostasis, in response to different physiological states, and that are recruited by injury in each sex.

“Importantly, our study provides a platform to identify molecularly distinct pools of stem cells under homeostasis, in response to different physiological states, and that are recruited by injury in each sex.”

Probably not a substantial claim: may not pass S2 (checkable).

The large-scale census of regionally dissected adult V-SVZ cells will provide a valuable resource for future functional studies, including identification and regulation of regionally distinct adult V-SVZ neural stem cells, their lineages, and interactions with different niche components.

“Finally, our large-scale census of regionally dissected adult V-SVZ cells will provide a valuable resource for future functional studies, including identification and regulation of regionally distinct adult V-SVZ neural stem cells, their lineages, and interactions with different niche components.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In total, putative doublets constituted approximately 7% of cell barcodes, in line with 4% doublets predicted by Poisson statistics at approximately 70% bead loading efficiency.

“In total, the putative doublets constituted cell barcodes ~7% of cell barcodes, in line with 4% doublets predicted by Poisson statistics (~70% bead loading efficiency).”

May combine two claims.

This provides unique insight into regional differences in the lateral and septal walls, including the less-explored oligodendrocyte lineage in the adult V-SVZ, and has allowed identification of transcription factor co-expression modules at the single-cell level.

“Importantly, this provides unique insight into regional differences in the lateral and septal walls, including the less-explored oligodendrocyte lineage in the adult V-SVZ, and has allowed us to identify transcription factor co-expression modules at the single-cell level, which likely delineate transcriptional regulatory changes occurring during neuronal and oligodendrocyte differentiation.”

May combine two claims.

The study detected regional differences in the composition of neuronal and oligodendrocyte progenitors, suggesting that, although both walls can give rise to neurons and glia, the lateral wall is more enriched in neurogenesis and the septal wall in gliogenesis.

“We detected regional differences in the composition of neuronal and oligodendrocyte progenitors, suggesting that, although both walls can give rise to neurons and glia, the lateral wall is more enriched in neurogenesis and the septal wall in gliogenesis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

A similar smoothing method to the neighborhood expression smoothing used here was proposed in a recent study.

“A similar smoothing method was proposed in a recent study (Wagner et al., 2017).”

• cites Wagner et al., 2017Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Phenograph is a modularity-based clustering algorithm that produces robust clusterings on scRNA-seq data.

“Clustering was performed on the resulting k-nearest neighbors graph using Phenograph (Levine et al., 2015), a modularity-based clustering algorithm that produces robust clusterings on scRNA-seq data (Shekhar et al., 2016).”

• cites Levine et al., 2015 sourceNot checked yet

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• cites Shekhar et al., 2016 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

This sex difference may be due to differences in the number and types of stem cells and progenitors in males and females as well as to differences in niche signals.

“This sex difference may be due to differences in the number and types of stem cells and progenitors in males and females as well as to differences in niche signals.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Force-directed graph-based methods have previously been used to examine lineage relationships in single cell data.

“Force-directed graph-based methods have previously been used to examine lineage relationships in single cell data (Weinreb et al., 2018).”

• cites Weinreb et al., 2018 sourceNot checked yet

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Female and male oligodendrocyte levels and myelination are differentially sensitive to steroids, such as progesterone and dihydrotestosterone.

“Female and male oligodendrocyte levels and myelination are differentially sensitive to steroids, such as progesterone and dihydrotestosterone (Swamydas et al., 2009).”

• cites Swamydas et al., 2009 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The second and/or third round of clustering revealed additional putative doublets expressing mature oligodendrocyte and aNSC-NB markers, and astrocyte and endothelial cell markers.

“The second and/or third round of clustering revealed additional putative doublets expressing mature oligodendrocyte and aNSC-NB markers, and astrocyte and endothelial cell markers.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All major cell clusters as well as subclusters were identified in the second replicates except Astro.4.

“All major cell clusters as well as subclusters were identified in the second replicates except Astro.4.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

A small number of choroid plexus cells (4.5%) were detected in one of the four second replicate samples, and formed a separate cluster marked by high enrichment of Sostdc1, Kl, Wfdc2 and Ttr.

“A small number of choroid plexus cells (4.5%) were detected in one of the four second replicate samples, and formed a separate cluster marked by high enrichment of Sostdc1, Kl, Wfdc2 and Ttr.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The authors were not able to validate the additional putative doublet cells in the V-SVZ, so they were excluded from subsequent analysis.

“As we were not able to validate such cells in the V-SVZ, they were also excluded from subsequent analysis (data not shown).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Normalizing each cell's expression prior to calculating neighborhood expression or weighting the neighborhood sum so that the central cell made the greatest contribution yielded similar results.

“Normalizing each cell’s expression prior to calculating neighborhood expression or weighting the neighborhood sum so that the central cell made the greatest contribution yielded similar results.”