Umbilical Cord Blood-Derived Cells Can Reconstruct Hematopoiesis in an Aplastic Anemia Animal Model

Abstract

Objectives: To explore the efficacy and the mechanism of the umbilical cord-derived cells combined with cyclosporine A (CsA) in treating aplastic anemia (AA) in mice.

Methods: Immune-mediated AA model mice were treated with CsA + UC mesenchymal stem cells (UC-MSC), CsA + umbilical cord blood regulatory T cells (UCB-T_reg), UC-MSC, UCB-T_reg, CsA alone, or blank control, respectively (n = 9 mice/group). CsA and the cell infusion was administered on d0. Routine peripheral blood testing was performed once weekly; bone marrow colony culture, bone marrow cell flow cytometry, peripheral blood T cell subsets, and serum inflammatory cytokines tests were performed on d14. Transcriptome sequencing was performed for cells from CsA + UC-MSC, CsA + UCB-T_reg, and CsA groups to detect the possible related genes. Gene function cluster and signal pathway enrichment analysis were also performed.

Results: Blank control mice died due to pancytopenia within 21 days, whereas mice in other groups survived for >28 days. On d14, the CsA + UC-MSC and CsA + UCB-T_reg groups had higher white blood cell (WBC) counts than the other groups (p < 0.05), along with higher burst-forming unit (BFU) and colony-forming unit-granulocyte, macrophage (CFU-GM) counts (p < 0.01). The CsA + UC-MSC group had the highest BFU count (p < 0.01). The CsA + UC-MSC and CsA + UCB-T_reg groups exhibited the highest bone marrow CD34⁺ cell proportion (9.68% ± 1.35% and 8.17% ± 0.53%, respectively; p < 0.01). Tumor necrosis factor (TNF)-α and interleukin (IL)-2 levels in the CsA + UC-MSC group (p < 0.05) and TNF-α, interleukin-2, and interferon (INF)-γ levels in the CsA + UC-T_reg group (p < 0.01) were lower than those in the CsA group. Compared with CsA treatment, CsA + UC-MSC significantly downregulated the histone methylation pathway (p < 0.05), whereas CsA + UCB-T_reg significantly upregulated energy metabolism processes (p < 0.05). Treatment with CsA + UC-MSC upregulated superoxide dismutase activity compared with CsA + UCB-T_reg treatment.

Conclusions: Adding UC-MSC or UCB-T_reg to CsA markedly enhanced the reconstruction of hematopoiesis in AA mice, with UC-MSC eliciting greater efficiency than UCB-T_reg. Accordingly, the addition of these cells could further improve immune abnormalities.

Figure 1

Induction of AA murine model and hematopoiesis recovery in mice with different treatments. (a–c) White blood cell (WBC), hemoglobin (HGB), and platelet (PLT) levels in mice subjected to different treatments at different time points (represented as the means and SD).  ^∗,  ^∗∗, and  ^∗∗∗ indicate a significant difference among all groups (p < 0.05, p < 0.01, and p < 0.001, respectively). (d–f) WBC, HGB, and PLT levels in mice subjected to different treatments at D14 (represented as means ± SD).  ^∗,  ^∗∗, and  ^∗∗∗ indicate a significant difference between two groups (p < 0.05, p < 0.01, and p < 0.001, respectively).

Figure 2

Colony numbers in different groups (n = 3; on 2 × 10⁴ bone marrow nucleated cells). (a) Burst-forming unit (BFU) colony numbers in different groups. (b) Colony-forming unit-granulocyte, macrophage (CFU-GM) colony numbers in different groups. (c) Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) colony numbers in different groups. Data are presented as means ± standard deviation (SD).  ^∗,  ^∗∗, and  ^∗∗∗ indicate a significant difference between two groups (p  < 0.05, p  < 0.01, and p  < 0.001, respectively). CsA, cyclosporine A; MSC, mesenchymal stem cells; UC, umbilical cord; UCB, umbilical cord blood; and T_reg, regulatory T cells.

Figure 3

CD34⁺ bone marrow nucleated cells analysis in different treatment groups. (a) CD34⁺cell population ratio of mice in each treatment group. (b) Detection of bone marrow CD34⁺cell by flow cytometry.  ^∗,  ^∗∗, and  ^∗∗∗ indicate a significant difference between two groups (p  < 0.05, p  < 0.01, and p  < 0.001, respectively). NS indicates no significant difference between the two groups. ▲ indicates a significant difference between this group and any other group (all meet the requirements of p  < 0.01). CsA, cyclosporine A; MSC, mesenchymal stem cells; UC, umbilical cord; UCB, umbilical cord blood; and T_reg, regulatory T cells.

Figure 4

Changes in T cell subsets in different treatment groups. (a) The radio of CD4⁺/CD8⁺ in each treatment group. (b) The ratio of T_reg/CD4⁺ in each treatment group. (c) Detection of CD4⁺/CD8⁺ T cell subsets and T_reg (CD25⁺ and Foxp3⁺) subsets in CD4⁺ T cell subsets in different treatment groups by flow cytometry.  ^∗,  ^∗∗, and  ^∗∗∗ indicate a significant difference between two groups (p  < 0.05, p  < 0.01, and p  < 0.001, respectively). CsA, cyclosporine A; MSC, mesenchymal stem cells; UC, umbilical cord; UCB, umbilical cord blood; and T_reg, regulatory T cells.

Figure 5

Serum levels of inflammatory factors in different treatment groups. (a) INF-γ levels in different treatment groups. (b) TNF-α levels in different treatment groups. (c) IL-2 levels in different treatment groups.  ^∗,  ^∗∗, and  ^∗∗∗ indicate a significant difference between the two groups (p < 0.05, p  < 0.01, and p  < 0.001, respectively). CsA, cyclosporine A; MSC, mesenchymal stem cells; UC, umbilical cord; UCB, umbilical cord blood; T_reg, regulatory T cells; IFN-γ, interferon-γ; IL-2, interleukin-2; and TNF-α, tumor necrosis factor-α.

Figure 6

PCA plot and heatmap of significantly altered genes between two kinds of infusion treatments and CsA monotherapy. (a) PCA plot of all the groups. (b) Heatmap of significantly altered genes between the CsA + UC-MSC and CsA group. (c) Heatmap of significantly altered genes between the CsA + UCB-T_reg and CsA group. CsA, cyclosporine A; MSC, mesenchymal stem cells; UC, umbilical cord; UCB, umbilical cord blood; T_reg, regulatory T cells; and PCA, principal component analysis.

Figure 7

Transcriptome sequence analysis between two kinds of infusion treatments and CsA monotherapy. (a) GO analysis of differential genes between the CsA + UC-MSC and CsA group. (b) KEGG analysis of differential genes between the CsA + UC-MSC and CsA group. (c) GO analysis of differential genes between the CsA + UCB-T_reg and CsA group. (d) KEGG analysis of differential genes between CsA+UCB-T_reg and CsA group. CsA, cyclosporine A; MSC, mesenchymal stem cells; UC, umbilical cord; UCB, umbilical cord blood; T_reg, regulatory T cells; GO, gene ontology; and KEGG, Kyoto Encyclopedia of Genes and Genomes.