They expressed well-described canonical markers of lamina propria fibs.
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They expressed well-described canonical markers of lamina propria fibs.
I think this is correct. The ADAMDEC1hi, CCL8, CCL13, CCL11 profile is like in the Kinchen et al. paper (2018) and a lot of the following milestone papers.
Refer to resolution Leiden Lineage L1. These submucosal fibroblasts could be divided in two clusters: cluster 2 and cluster 8: Cluster 2 has high expression of SFRP2, C3, EFEMP1, CFD, FBLN2, among others. Present in intestinal biopsies. However, cluster 8 has high expression of C7, KCNN3, PDGFRL. Potentially, these could be fibroblasts from deeper layers
These are submucosal PI16+CD34+CD55 fibroblasts, previously also described as "adventitial-like" or "universal" (https://pmc.ncbi.nlm.nih.gov/articles/PMC11196929/; ; https://www.nature.com/articles/s41590-025-02267-8) fibroblasts. Also trophocytes (CD81, RSPO3, GREM1...) are likely in here. I think it is much more likely that the very most of these cells come from the submucosa (or possibly to an extent from under the intestinal crypts (https://www.pnas.org/doi/10.1073/pnas.1620059114), i.e. are not subserosal. Check e.g. submucosal fibroblast profiles here: https://www.sciencedirect.com/science/article/pii/S009286742400254X https://www.biorxiv.org/content/10.1101/2025.07.29.667377v2.full.pdf https://academic.oup.com/jimmunol/article-abstract/201/1/215/7952082?redirectedFrom=fulltext&login=false 10.1084/jem.20250471
This cluster expresses VSTM2A, characterising them as S2a/bottom crypt
Markers of subepithelial F3, SOX6 wnt5b expressed by this cluster.
Although the cluster seems to contain other cell types, the top genes being GJA5 and SEMA3G does indicate majority of cells being arteriole endothelium. Litviňuková M, et al. Cells of the adult human heart. Nature. 2020;588:466–472. doi: 10.1038/s41586-020-2797-4 Phansalkar R, et al. Coronary blood vessels from distinct origins converge to equivalent states during mouse and human development. eLife. 2021 doi: 10.7554/elife.70246.
Some of these are RGS5+ NOTCH3. Are they pericytes? They would correspond to cluster 17 from Leiden_lineage_L1
This cluster contains a mix of cells. One of the markers mentioned in the metadata as specific is PLVAP (not expressed by half of the cells in this cluster). I would suggest selectin leiden L1 cluster 14 (as the arterial endothelium using GJA5 and SEMA3G as markers). Cluster 14, however, lacks PLVAP (highly expressed in clusters 3 and 17) which I would annotate as capilary (based on PLVAP expression).
Based on the literature review, the high expression of ACKR1 and CD36 suggests Venule/capillary phenotype.
This cluster shows expression of adhesion molecules MADCAM, SELEP and SELE normally described in inflammed endothelium but also described in venular endothelium. It also expresses another marker of venular endothelium lrg1.
Split in two clusters, the one belonging to the lamina propria fibroblasts (cluster 6_Leiden lineage L1) and the other belonging to the submucosal (cluster 2_Leiden_lineage_L1)
My guess would be that this is a mixed cluster of submucosal (or here "subserosal") fibroblasts and ADAMDEC1+ interstitial cells, given the presence of key signature markers such as PI16, CD34, SFRP2 on one hand and ADAMDEC1, CCL11, CCL13 etc on the other hand. Is there any location data for these cells that would prove these would sit in the muscular propria?
This cluster expresses NPY and NRG1, markers of S2b/crypt top
this cluster also expressed NRG1, demonstrated to be crypt top (validated in https://doi.org/10.1038/s41467-023-40156-6 by ISH).
Based on Leiden_lineage_L1, cells at the top of this cluster are cluster 21. This cluster expresses WT1, PLIN2 - are they adipocytes? If not clear, merge cluster 21 with cluster 2 (share more marker genes)
These lack classic submucosal and trophocyte markers. CD34, CD55, PI16, CD81, RSPO3, GREM1.... https://academic.oup.com/jimmunol/article-abstract/201/1/215/7952082?redirectedFrom=fulltext&login=false 10.1084/jem.20250471 https://www.sciencedirect.com/science/article/pii/S009286742400254X https://www.biorxiv.org/content/10.1101/2025.07.29.667377v2.full.pdf
Smooth muscle cell signature markers expressed such as ACTA2, ACTG2, TAGLN, MYH11...
This population is expressing CD36 and CA4, a typical set of marker genes involved in capillary endothelium. Litviňuková M, et al. Cells of the adult human heart. Nature. 2020;588:466–472. doi: 10.1038/s41586-020-2797-4. Gillich A, et al. Capillary cell-type specialization in the alveolus. Nature. 2020;586:785–789. doi: 10.1038/s41586-020-2822-7.
Both vascular endothelial cells could be annotated as Lymphatic endothelium (CCL21, LYVE1, MMRN1). However, there is an heterogeneity within this cluster, although I don't think the current separation is correct. Based on Leiden_lineage_L2, cluster 13_3 expresses specifically NTS and ACKR4, is this a subtype of lymphatic endothelial cells?
Agree with the overall annotation, as PROX1, FLT4 as "lymphatic endothelial cells (LEC)". Although the cell number is low there is heterogeneity in some canonical markers that would suggest the presence of LEC subsets within the cluster annotated now as "Vascular Endothelial Lymphatic". For instance, not all express LYVE1 or CCL21. Indeed, cells lacking expression of these two markers express ACKR4 (Leiden lineage L2 cluster 13_3) could be annotated as Ceiling subcapsular sinus LECs (https://doi.org/10.1016/j.immuni.2019.06.027) . Also clusters 13_0 and 13_1 do express LYVE1, CCL21 as well as CLEC4M marker of medullary sinus LECs (https://doi.org/10.1016/j.immuni.2019.06.027).
Clearly LYVE-1+, PROX1+ lymphatic endothelial cells (LEC). https://www.nature.com/articles/nrgastro.2017.79 But please delete "vascular" as this is indicative that is NOT a lymphatic cell, but a blood endothelial cell (BEC).
I think pericytes and SMCs are overclustered. In my opinion, the clusters in "Leiden_Lineage_L1" of these cells make more sense. Based on that resolution, clusters would be annotated as: - Cluster 15: Contractile pericytes (RERGL, KCNA5, RGS5) - Cluster 16: Pericytes (HIGD1B, STEAP4, CD36) - Cluster 18: SMCs GREM2+ (seen in Kong et al, doi: 10.1016/j.immuni.2023.01.002., although for them they are myofibroblasts) - Cluster 11: SMCs as they are now (SOSTDC1, NPNT, HHIP, MYOCD) Maybe there could be more granularity in the pericytes, it could be helpful if someone knows more marker genes. If not, I would just have two clusters of pericytes
Uncertain on this population, albeit showing both venule and capillary marker genes, there is a mixture of arteriole markers. Trimm and Red-Horse, Vascular endothelial cell development and diversity, Nat Rev Cardiol, 2022
Based on the Annotation excel file, SMC Inner Muscle Cells should be FOXF2+, MORN5+, but this cluster is not
T-cell contamination needs to be deleted from this object.
Seems to be mostly T cells with CD2, CD3, CD69?
I'm not sure that this subclusterization of glial cells is correct. I coudn't find any marker that clearly separates each of the subyptes, and the markers we had from the Annotation grup do not follow this clusterization. Which granularity would you like to reach at this stage? I might suggest having just one cluster of Glia (NRXN1, S100B) truly reliable
Clearly a lymphoid tissue FRC cluster. CD24+ CCL19/CCL21+ CLU+ As CXCL13/CXCL14/FDCSP are also on the list, this is likely a T cell zone / B cell zone mixed cluster
Overclustered, this cluster does not add more information. Could be merged with S2b fibs (they express NRG1).
this cluster also expressed NRG1, demonstrated to be crypt top (validated in https://doi.org/10.1038/s41467-023-40156-6 by ISH).
F3+ subepithelial fibroblasts. See Kinchen et al. 2018.
Clearly LYVE-1 lymphatic endothelial cells (LEC), also the "Lacteal" label makes sense (LYVE1hi, PROX1hi KDR+...) https://www.nature.com/articles/nrgastro.2017.79 But please delete "vascular" as this is indicative that is NOT a lymphatic cell, but a blood endothelial cell (BEC).
This is for sure a glial cell. PLP1, Sox10, L1CAM. I am not sure this is purely a submucosal glial cell. Check out the expression of all the MHCII-related genes and there are also some interferon response genes. Could this be more a "reactive glia" = activated cluster?
Specific expression of ADARB2 and KCNQ3, but I'm not sure about them being angiogenic pericytes. Another good marker of angiogenic pericytes is HIGD1B, but its expression is not limited to this cluster
ESAM+CD36+, ACTA2lower than the other pericytes. Absence of endothelial cell markers (e.g. PECAM1) and fibroblast markers (e.g. PDPN)
Epithelial cells
Definitely containing some enterocytes. CLDN1, CALB2, PDZK1IP1
OPN4 and BMP8A seem to differentiate well this cluster. But are they markers of "Pericytes secretory"? Expression seems quite sparse, is this cluster worthy?
I think this label is in line with the Elmentaite et al paper (https://www.nature.com/articles/s41586-021-03852-1). However, given that some of the main DEG are e.g. TNFSF11 and CXCL14, I think one could also label them MRC = marginal reticular cells. Especially TNSF11 is a hallmark of MRC from secondary lymphoid tissues such as Peyers patches and isolated lymphoid follicles. (https://www.nature.com/articles/s41590-021-00894-5); (10.1084/jem.20250471)
For real myofibroblasts, I would expect MYH11, ACTA2, ACTG2 etc. expression = genes associated with muscle function. These fibroblasts are CTHRC1 and FAP positive and are most likely small amounts of pro-fibrotic inflammation-associated fibroblasts as described previously. --> Call them "Inflammation-associated fibroblasts" (IAF)? E.g. Bauer-Rowe et al. https://www.sciencedirect.com/science/article/abs/pii/S0092867425010189 Zhang et al. https://www.jci.org/articles/view/179472 Review by Brügger/Basler https://www.cell.com/trends/cell-biology/fulltext/S0962-8924(23)00048-X?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS096289242300048X%3Fshowall%3Dtrue
Delete
ANPEP+DPP4, FABP1 clear epithelial cells. Remove from this object as there is a separate epithelial lineage object?
Delete.
Surely containing plasma cells (MZB1, JCHAIN, CD79A), but also other lymphocytes (likely incl. T cells IL7R, CD69hi). As this is a stromal cell object, they cluster together. Delete this cluster from this object?
Very few cells, located in the UMAP with the S1 fibroblasts... Why are they glia muscularis propria and not just glia?