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. 2024 Jul 3;27(8):110445.
doi: 10.1016/j.isci.2024.110445. eCollection 2024 Aug 16.

Decoding human bone marrow hematopoietic stem and progenitor cells from fetal to birth

Xiaowei Xie  1   2 , Fanglin Gou  3 , Zhaofeng Zheng  1   2 , Yawen Zhang  1   2 , Yingchi Zhang  1   2 , Fang Dong  1   2 , Tao Cheng  1   2 , Hui Cheng  1   2
Affiliations

Decoding human bone marrow hematopoietic stem and progenitor cells from fetal to birth

Xiaowei Xie et al. iScience. .

Abstract

Bone marrow (BM) is the dominant site of hematopoiesis after 20 post-conception weeks (PCWs), but the intricacies of hematopoietic development in fetal BM up to birth and its involvement in malignancies remain unknown. Here, we compared the single-cell transcriptomic profile of BM hematopoietic stem and progenitor cells (HSPCs) at the early (12-14 PCW), middle (19-22 PCW) second trimester, and the neonatal stage. The stemness of hematopoietic stem cell and multipotent progenitor (HSC/MPP) is established at the middle second trimester, then maintained until birth. Furthermore, differentiation potentials toward three lineages are enhanced after the middle second trimester for birth, accompanied by the upregulation of aerobic metabolism. Notably, decreased stemness in HSCs/MPPs and higher interferon signals in progenitors at the early second trimester rendered the HSPCs more proximal to leukemogenesis. Collectively, our work elucidated the dynamics of fetal hematopoiesis in preparation for birth, offering valuable insights into the pathological processes underlying leukemia.

Keywords: Haematology; Molecular biology; Transcriptomics.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Single-cell transcriptomic profile of human bone-marrow-derived HSPCs from the fetal to birth (A) A schematic diagram showing the experimental design. eST, early stage of second trimester; mST, middle stage of second trimester. (B) UMAP displaying the distribution of 14,286 single cells. Colors indicate 19 cell clusters determined by Seurat. (C) The number of differentially expressed genes (DEGs) between two adjacent stages for 11 cell clusters, which contain enough single cells among three developmental stages. Red and blue colors represent the number of upregulated and downregulated genes, respectively. (D) Circos plot displaying the overlap in upregulated genes among 11 cell clusters in the middle second trimester (eST versus mST). Red band represents the common genes, whereas orange band represents the specific genes for each cell cluster. Purple lines indicate overlapping genes between two cell clusters. Heatmap presents the enriched GO terms for the upregulated genes in the corresponding clusters. (E) Circos plot displaying the overlap in upregulated genes among 11 cell clusters at neonatal stage (mST versus neonate). Heatmap presents the enriched GO terms for the upregulated genes in the corresponding clusters.
Figure 2
Figure 2
Enhanced expressions of HSC/MPP stemness signature genes in the middle second trimester (A) Relative expression of intrinsic apoptotic signaling pathway by p53 class mediator in the HSC/MPPs from fetal to birth. Red line is implemented by loess algorithm. (B) GSEA of gene set associated with somatic stem cell population maintenance between the HSC/MPPs from the early (eST) and middle second trimester (mST). (C) Percentages of HSC/MPPs in different cell-cycle phases for three developmental stages. The percentages of HSC/MPPs in G1 phase: eST versus mST, p value = 0.03; mST versus neonates, p value = 0.003 by chi-squared tests. (D) UMAP visualization of the distribution of 1,406 HSC/MPPs by Monocle3. Colors indicate three developmental stages. (E) Relative expression of HSC/MPP signature genes in the HSC/MPP from fetal to birth. Red line is implemented by loess algorithm. (F) Bubble plots displaying the relative expression of individual HSC/MPP signature gene represented by MLLT3, MEIS1, etc., among three developmental stages. (G) Scatterplots present the activated transcription factors in the HSC/MPP from different developmental stages. Red and larger dots highlight the transcription factors associated with HSC stemness. Top, eST versus mST; bottom, mST versus neonate. Adjusted p value <0.05. (H) Cytoscape showing the stemness-related transcription factor regulatory network activated in the HSC/MPP at middle second trimester (mST versus eST and neonate, adjusted p value <0.05). Each edge represents a transcription factor-target gene pair, whereas nodes represent transcription factors and genes. The thickness of edge represents the target weight evaluated by SCENIC. Red and larger dots highlight the transcription factors associated with HSC stemness. Gold dots emphasize the genes associated with HSC stemness. (I) Representative images of clones generated from Lin−CD43+CD34+ HSPCs derived from the bone marrow at eST and mST stages. (J) One thousand five hundred Lin−CD43+CD34+ fetal BM HSPCs at eST and mST stages were sorted and cultured in MethoCult GF H4435 for 2 weeks (n = 3 biologically independent samples in each group, n = 4 technical replicates of each sample). BFU-E, CFU-GM, and CFU-GEMM were calculated. BFU-E: burst-forming unit-erythroid; CFU-GM: colony-forming unit-granulocyte, macrophage; CFU-GEMM: colony-forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte. Error bars represent the mean ± SD. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; unpaired two-tailed Student’s t test. (K) The percentage of each lineage output analyzed by flow cytometry compared between eST and mST groups (eST = 195, mST = 377), based on single-cell liquid culture of HSC (Lin−CD43+CD34+CD38lowCD45RA−CD90+). Mean ± SD; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; unpaired two-tailed Student’s t test.
Figure 3
Figure 3
Single-cell transcriptomic landscape of human fetal-liver-derived HSPCs (A) UMAP displaying the distribution of 2,487 HSPCs from fetal liver. Colors indicate four cell clusters determined by Seurat. (B) Relative expression of intrinsic apoptotic signaling pathway by p53 class mediator in the HSC/MPPs from fetal liver. Red line is implemented by loess algorithm. (C) Relative expression of fetal-liver-derived HSC/MPP signature genes in the HSC/MPPs from fetal liver. Red line is implemented by loess algorithm. (D) Relative expression of fetal-liver-derived CLP, GMP, and MEP signature genes in the HSC/MPP and corresponding CLP (left), GMP (middle), and MEP (right) from the early and middle second trimester of fetal liver. (E) Bar plots displaying the normalized enrichment score (NES) (mST versus eST) evaluated by GSEA for fetal-liver-derived CLP, GMP, and MEP signature genes in the HSC/MPP and corresponding CLP (left), GMP (middle), and MEP (right) cell clusters of fetal liver. (F–H) Proportions of NK-containing colonies (eST = 47, mST = 136) (F), myeloid-containing colonies (eST = 185, mST = 355) (G), and erythroid-containing colonies (eST = 45, mST = 58) (H), generated by the HSCs respectively from eST and mST. n = 6–7 biologically independent samples in each stage. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; unpaired two-tailed Student’s t test. M, myeloid; E, erythroid; MK, megakaryocyte; NK, natural killer. (I) Boxplots showing the relative expression of cell migration and HSC migration in the HSC/MPP from the early and middle second trimester of fetal liver. Wilcoxon test: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. (J) Boxplots showing the relative expression of cell migration in the CLP, GMP, and MEP from the early and middle second trimester of fetal liver. Wilcoxon test: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. (K) Bubble plots displaying the relative expression of individual migration gene in the HSPC from the early and middle second trimester of fetal liver.
Figure 4
Figure 4
Enhanced lineage differentiation potentials of progenitors at neonatal stage (A) Relative expression of oxidative phosphorylation in the HSC/MPP, CLP, GMP, and MEP from the bone marrow at the middle second trimester and neonatal stages. (B) Bubble plots displaying the relative expression of individual oxidative phosphorylation gene in the HSC/MPP, CLP, GMP, and MEP from the bone marrow at the middle second trimester and neonatal stages. (C) Relative expression of CLP, GMP, and MEP signature genes in the HSC/MPP and corresponding CLP (left), GMP (middle), and MEP (right) from the bone marrow at the middle second trimester and neonatal stages. (D) Bar plots displaying the NES (neonate versus mST) evaluated by GSEA for the CLP, GMP, and MEP signature genes in the HSC/MPP and corresponding CLP (left), GMP (middle), and MEP (right) cell clusters. (E) Relative expression of individual lineage differentiation gene in the HSC/MPP along with CLP (top), GMP (middle), and MEP (bottom) at the middle second trimester and neonate. Wilcoxon test: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. (F) Heatmap showing the activated transcription factors associated with lineage differentiation in the CLP, GMP, and MEP at the middle second trimester (blue) and neonate (red) (neonate versus mST, adjusted p value <0.05).
Figure 5
Figure 5
Similar transcriptomic features of HSC/MPPs in the early second trimester and in ALL (A) UMAP displaying the distribution of 14,007 bone-marrow-derived HSPCs from fetus, neonate, adult, and leukemia patients. Colors indicate seven cell clusters determined by Seurat. (B) The lineage differentiation potential of HSC/MPP toward CLP, GMP, and MEP from three developmental stages and leukemia patients. (C) Clustering of HSC/MPP from three developmental stages and leukemia patients based on the expression levels of HSC/MPP signature genes, by the “complete” method from pheatmap package, which could be reproduced by the “centroid” method. (D) Volcano plot displaying the differentially expressed genes between the HSC/MPP from the early second trimester adding leukemia and the remaining stages. Red and green dots respectively indicate the upregulated and downregulated genes in the early second trimester adding leukemia. (E) Boxplots showing the relative expression of HSC/MPP signature genes in the HSC/MPP from fetus, neonate, adult, and leukemia patients. Wilcoxon test: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. (F) Heatmap showing the expression profile of leukemia genes across the HSC/MPP from three developmental stages and leukemia patients. (G) Boxplots showing the relative expression of interferon alpha production (top) and interferon beta production (bottom) in the HSC/MPP, CLP, GMP, and MEP from three developmental stages. Wilcoxon test: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001.
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