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. 2022 Jun 20;24(7):1038–1048. doi: 10.1038/s41556-022-00931-x

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 3 — a. Representative FACS gating scheme of LT-HSCs within GPRC5C^pos/neg population from CD34⁺ BM donors. Frequency of LT-HSCs within GPRC5C^pos/neg cells from CD34⁺ BM donors. n = 7 biological replicates. b. Experimental design to assess the in vivo reconstitution ability of BM GPRC5C^pos/neg-HSCs. c. Lineage distribution of GPRC5C^pos/neg-HSCs. Data presented refer to the distribution of myeloid (CD33), B-lymphoid (CD19) and immature surface phenotype (CD34). n = 9 biological replicates. d. Percentage of determined colony types derived from single BM GPRC5C^pos/neg -LT-HSCs following culture under differentiating conditions for three weeks. n = 2 biological replicates. e. Experimental design to characterize GPRC5C in human CB. f. FACS analysis of cell cycle from CB GPRC5C^pos/neg-HSCs. n = 2 biological replicates. g. SiC division assay of GPRC5C^pos/neg-HSCs from CB donors quantified after 48 hours in vitro culture. n = 2 biological replicates. h. qPCR of CDK6 expression from CB GPRC5C^pos/neg-HSCs. Normalized to housekeeping gene GAPDH and GPRC5C^pos-HSCs. n = 9 biological replicates. i. Frequency of LT-HSCs within GPRC5C^pos/neg cells from CD34⁺ CB donors. n = 6 biological replicates. j. Assessment of reconstitution dynamics. Engraftment was assessed by percentage of human CD45 cells in the PB. n = 6 biological replicates. k. Lineage contribution of CB GPRC5C^pos/neg-HSCs. Data presented refer to the distribution of myeloid (CD33), B-lymphoid (CD19), and immature surface phenotype (CD34). n = 6 biological replicates. l. Table showing the number of cells injected per dose, number of mice per dose, and the number of engrafted mice per dose. These data were used to calculate the HSC frequency seen in Fig. 2i. All data presented as mean ± SD. Statistical significance was determined using two-tailed t-test (a, h, i, j) or two-way ANOVA (c and k) and at least two independent experiments were performed. ns, not significant. n indicates number of replicates. Source numerical data are available in source data.

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