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

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-Treg), UC-MSC, UCB-Treg, 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-Treg, 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-Treg 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-Treg 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-Treg 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-Treg significantly upregulated energy metabolism processes (p < 0.05). Treatment with CsA + UC-MSC upregulated superoxide dismutase activity compared with CsA + UCB-Treg treatment. Conclusions Adding UC-MSC or UCB-Treg to CsA markedly enhanced the reconstruction of hematopoiesis in AA mice, with UC-MSC eliciting greater efficiency than UCB-Treg. Accordingly, the addition of these cells could further improve immune abnormalities.


Introduction
Aplastic anemia (AA) is characterized by low bone marrow (BM) hyperplasia and pancytopenia.Acquired AA can be further divided into secondary and idiopathic AA based on the presence of clear secondary factors [1].Abnormalities in hematopoietic stem cells, hematopoietic microenvironment, and immune system have been observed in idiopathic AA [2,3,4].BM-derived mesenchymal stem cells (MSCs) are well known to participate in hematopoietic support and immune regulation, both of which are reduced in patients with idiopathic AA [5].Likewise, patients with AA reportedly exhibit a reduced number of regulatory T cells (T regs ), although functional defects in these cells were enhanced [6,7].Immunosuppressive therapy (IST) is reportedly effective in 70%-80% of patients with AA who are ineligible to undergo transplantation, although novel treatment options need to be explored for patients who fail to benefit from IST [1].Preclinical and clinical studies have confirmed the safety and efficacy of infusion therapy with MSCs or T regs derived from the BM or other tissues in patients with AA [8,9,10,11,12].
Umbilical cord blood (UCB) is typically collected from the umbilical cord (UC) and placenta of newborns.Owing to the abundance of hematopoietic stem cells and immature immune cells, UCB can be employed in transplantation and transfusion therapy for hematological malignancies and BM failure diseases [13,14,15,16].In patients with AA, cord blood infusion therapy can hasten the onset of immunosuppressive therapy and improve the long-term prognosis [14].Importantly, there have been considerable advancements in technologies applied to isolate and culture human UCderived MSCs (UC-MSC) and human UCB-T reg from fresh UC and UCB [17,18].UC-MSCs exhibit stronger hematopoietic support and immune regulation function than BMderived MSCs, and UC-MSC infusion may elicit robust efficacy, while UCB-T reg may be more suitable for infusion therapy [19,20,21].MSCs were found to induce CD4 + T cells to enhance CD25 and forkhead box protein P3 (Foxp3) expression under coculture conditions in vitro.UC-MSC infusion may play a therapeutic role by predominantly increasing the number of T reg in patients with autoimmune diseases [19,22].Although the application of UC-MSC therapy in patients with AA has been reported, to the best of our knowledge, investigations on treatment with UCB-T reg or comparisons between UC-MSCs and UCB-T reg are lacking.
In the current study, in the context of cyclosporine A (CsA) therapy, we examined the effects of UC-MSC and UCB-T reg infusion in an immune-mediated AA mouse model.We attempted to illustrate the differences between UC-MSC and UCB-T reg infusions in terms of their ability to induce hematopoietic stimulation, immune-regulation effects, and gene transcription to lay a foundation for further advancing cell infusion therapy.

Induction of AA Murine Model and Umbilical Cell
Preparation.A murine AA model was established as described previously [9,23,24].Lymph node cells from C57BL/6 (male, 49-72 days old, SPF grade) donors were homogenized, washed, and filtered.In total, 54 C57BL/6 mice (male, 49-72 days old, SPF grade) were subjected to sublethal irradiation (5 Gy Co-60 total body irradiation) followed by the administration of a single dose of 5 × 10 6 lymph node cell infusion through the lateral tail vein within 4 hr postirradiation.The AA model mice were randomly assigned to six groups and administered different treatments, as shown in Table 1.Human UC-MSCs and human UCB-T reg were provided by Shandong Cord Blood Bank, China.UC-MSC or UCB-T reg infusion was administered within 24 hr after AA model induction.Table 1 presents the therapeutic drugs and cells used in the study.All experimental operations on mice were conducted in accordance with the Declaration of Helsinki and were approved by the ethics committee of Peking Union Medical College Hospital, Beijing, China.

Survival Status and Routine Blood
Monitoring.The survival status of mice in each group was observed daily, starting from d0.Routine blood tests were conducted on days 0, 7, 14, and 28 by collecting blood from the intraorbital venous plexus of the mice.

Flow Cytometry.
Peripheral blood was collected from the intraorbital venous plexus, and BM cells were extracted from the dissected tibia and femur of each mouse.The BM cell suspension was repeatedly pipetted to disperse cells and then filtered through a 200-mesh screen.The suspension was centrifuged at 500 × g for 5 min at room temperature, and the cells were resuspended in phosphate-buffered saline for counting.After processing with red blood cell lysate, peripheral blood lymphocytes and BM cells were centrifuged at 4°C.Monoclonal antibodies were added and incubated according to the instructions provided with the antibodies.Monoclonal antibodies against murine CD4-FITC, CD8-perCP-Cy5.5, CD25-APC, Foxp3-PE, and CD34-APC were procured from BD Biosciences.Stained and unstained cells as blank control were analyzed using a FACSC auto II flow cytometer (BD Biosciences).

BM Colony
Assay.BM cells were collected and filtered as previously described and plated at a density of 2.0 × 10 4 /plate in methylcellulose-based medium with recombinant cytokines (including erythropoietin (EPO)) for mouse cells (MethoCult™ GF M03434, STEMCELL Technology Inc., Canada).The colonies were maintained at 37°C in a 5% CO 2 incubator for 12 days.The number of BM colonies was counted using an inverted microscope.

Combination Therapy Enhanced the Recovery of
Peripheral Blood Cell Count.Based on the preliminary results, untreated mice (group C2) revealed a significant simultaneous decline in peripheral blood ternary sorts on d7 and d14, and all untreated mice died within 21 days postradiation.
As shown in Figures 1(a), 1(b), and 1(c), the number of blood cells in all mice was the lowest on d7 after model generation, gradually recovering thereafter.Considering each group, the white blood cell (WBC) count did not differ at d0 and d7 but differed significantly at d14 and d28 (d14, p <0:001 and d28, p <0:05).The hemoglobin level differed significantly between groups only on d14 (p <0:001) but not on d0, d7, and d28.There were no differences in platelet levels in each group at d0, although a significant difference was observed at d7 and d14 (d7, p <0:01 and d28, p <0:001), and the difference disappeared at d28.There were no significant differences in baseline blood cell counts in the above groups.On d14, the CsA + UC-MSC group exhibited significantly better recovery in all three blood cells than the UC-MSC, CsA, and blank control groups (all p <0:001).Likewise, the CsA + UCB-T reg group had significantly better hematopoiesis recovery than the UCB-T reg , CsA, and blank control group (Figures 1(d), 1(e), and 1(f)).On d28, there were no significant differences in blood cell counts between the different treatment groups; however, the CsA + UC-MSC and CsA + UCB-T reg groups exhibited higher WBC counts than the other groups (p <0:05).Accordingly, the UC-MSC infusion therapy was superior to the UCB-T reg infusion therapy in terms of the speed of recovery of the tertiary blood system with or without CsA treatment (Figures 1(a), 1(b), 1(c), 1(d), 1(e), and 1(f)).

Combination
Therapy Elicited Superior Recovery of the BM Colony.Untreated mice (group C2) showed a significant decline in the BM nucleated cells (BMNC) colony cultures at d14 (p <0:01).All treated groups had significantly improved BM colony counts than the control group (p <0:01), as shown in Figure 2. The CsA + UC-MSC and CsA + UCB-T reg groups had higher burst-forming unit (BFU) and colony-forming unit-granulocyte, macrophage (CFU-GM) colony counts than the CsA, UC-MSC, or UCB-T reg groups (p <0:01); the CsA + UC-MSC group exhibited a better recovery in the BFU colony count than the CsA + UCB-T reg group (p <0:01).CFU-GM and colony-forming unitgranulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) numbers were higher in the CsA + UC-MSC than those in the CsA + UCB-T reg group, although the difference was not significant.

Combination Therapy
Enhanced the Numbers of BM CD34 + Cells.On d14, the proportion of CD34 + cells in the BM significantly decreased in blank control mice when compared with those in normal mice (p <0:01).Conversely, all treated mice had significantly increased proportions of BM CD34 + cells (p <0:01) when compared with the blank control, as shown in Figure 3. Combined treatment groups, that is, the CsA + UC-MSC and CsA + UCB-T reg groups, had a higher number of CD34 + cells than the CsA monotherapy group (CsA + UC-MSC vs. CsA, 9.68% AE 1.35% vs. 2.89% AE 0.60%, p <0:01 and CsA + UCB-T reg vs. CsA, 8.17% AE 0.53% vs 2.89% AE 0.60%, p <0:01).No significant difference was detected between the CsA + UC-MSC and CsA + UCB-T reg groups (p ¼ 0:073), and the CsA + UC-MSC group exhibited similar findings to the normal group in terms of CD34 + BMNC analysis (p ¼ 0:121).

Combined Cell and Drug Therapies Restored the T Cell
Balance in AA Mice.T cell subsets were detected in normal mice and untreated, CsA + UC-MSC, CsA + UCB-T reg , and CsA single treatment groups.As shown in Figures 4(a), 4(b), and 4(c), compared with normal mice, AA mice showed extensive infiltration of CD8 + T cells in peripheral blood,

Transcriptome Analysis of Mechanisms Underlying Enhanced Hematopoiesis following Combination Treatment.
Given that the treatment with CsA + UC-MSC and CsA + UCB-T reg exhibited better hematopoiesis reconstruction ability than CsA monotherapy, we next aimed to explore the possible mechanism.Accordingly, peripheral blood was collected from mice in the CsA + UC-MSC, CsA + UCB-T reg , and CsA groups for transcriptome sequencing.Compared with the CsA group, the CsA + UC-MSC group had 28 genes significantly up-and 113 genes downregulated, and the difference was mainly focused on the downregulation of genes related to histone methylation, including Kmt2a, Kmt2c, Kdm4a, and Arid4a; in the CsA + UCB-T reg group, we identified 252 and 87 upregulated and downregulated genes, respectively, with the difference mainly observed in the upregulation of energy metabolism, including tricarboxylic acid cycle, oxidative phosphorylation and glycolytic process (Figure 6).Compared with the CsA + UCB-T reg group, the CsA + UC-MSC group exhibited 420 and 407 upregulated and downregulated genes, respectively, with the difference primarily focused on the upregulation of superoxide dismutase (SOD) activity and oxidative phosphorylation pathway in the CsA + UC-MSC group.

Discussion
The pathogenesis of idiopathic AA has long been summarized by a "seeds, soil, worms" theory [4].Recently, BM-MSCs and T reg in patients with AA were shown to exhibit reduced number and loss of function [6,7].BM-MSCs are an important constituent of the BM hematopoietic microenvironment, supporting hematopoiesis and functioning as an immune regulator by interacting with immune cells or secreting inflammatory cytokines [25].T reg cells are a group of T lymphocytes characterized by the coexpression of CD4, CD25, and Foxp3, capable of inhibiting the proliferation, activation, and killing of effector T cells by directly contacting or secreting cytokines.Impaired T reg function has been reported in patients with AA, which could not be attributed to the attack by effector T cells [7].
Owing to the close relationship between AA pathogenesis and the dysfunction of MSC and T reg , the application of MSC or T reg cells to treat AA has been examined in a few reports, either in humans or in animal models, eliciting promising results [5,8,25,26,27].Cord blood UC-MSC and UCB-T reg were easier to obtain and exerted stronger immune regulation ability than MSC or T reg cells derived from other tissues.T reg cells play the most crucial role in immune regulation and may be the key component for cord blood transfusion.However, the separation of T reg can be complicated and unstable when compared with MSC separation; therefore, it is important to identify which cell is more efficient in vivo, as well as the possible mechanism [19,20,21].However, only a few studies have focused on UC-MSC infusion therapy for patients with AA or in AA animal models.To date, no study has explored the potential of UCB-T reg infusion for treating AA [28].
In the current study, we further compared the effects of UC-MSC or UCB-T reg addition to CsA in our AA mouse model.To the best of our knowledge, this is the first study to apply UCB-T reg infusion and CsA + UCB-T reg therapy to treat AA model mice.Furthermore, this study is the first to comprehensively compare the efficacy of UCB-T reg therapy with UC-MSC therapy in AA model mice, with or without an immunosuppressant.
Our AA model mimicked patients with AA-not only did mice exhibit BM failure, but showed abnormal immunity, as observed in patients with AA.These defects can be partly corrected with CsA alone, as reported previously [29].Likewise, rescue with UC-MSC or UCB-T reg alone was also found to be efficient, as evidenced by the avoidance of early deaths and restoration of hematopoiesis, which was similar to CsA monotherapy.Next, we added UC-MSC or UCB-T reg to CsA, which elicited better recovery in blood cell count, BMNC colony cultures, and the proportion of BM CD34 + cells than CsA or MSC or T reg cells alone.Simultaneously, the addition of MSC or T reg to CsA could further reduce the inflammatory factors when compared with CsA monotherapy, although a significant improvement in the CD4 + /CD8 + T cell ratio was not observed.Upon comparing the treatment with CsA + UC-MSC and CsA + UCB-T reg , we observed that CsA + UC-MSC seemed to recover the blood cell count 8 Stem Cells International better than CsA + UCB-T reg on d14 but not on d28.Conversely, the CsA + UCB-T reg group showed a greater reduction in inflammatory factor levels.To the best of our knowledge, this effect has not previously been reported in the literature.
Transcriptome sequencing of differently treated mice revealed that CsA + UC-MSC treatment could significantly alter histone methylation and the complement and coagulation cascade reaction pathways, whereas the treatment with CsA + UCB-T reg upregulated energy metabolism-related  The most enriched GO terms Stem Cells International processes, including glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation, compared with CsA alone.Moreover, treatment with CsA + UC-MSC and CsA + UCB-T reg showed differences in the histone methylation process, energy metabolism-related processes, and SOD activity (Figure 7).
Histone methylation refers to the process of altering the interaction between histone and chromatin by adding or removing methyl groups to histone lysine or arginine residues, which could regulate the replication, expression, damage repair, and other functions of chromatin [30,31].Histone methyltransferases and demethylases play key roles in the histone methylation process and can be classified into multiple families according to the functional residues and structural characteristics.Downregulation of KMT2a and KMT2c (lysine methyltransferase 2a/2c) and upregulation of KDM4a (lysine demethylase 4a) were detected in the CsA + UC-MSC group, which eventually decreased the histone methylation.Although these genes have not been identified in AA, upregulated expression of KMT2a and KMT2c and downregulated KDM4a expression, compared with normal control, have been reported in patients with autoimmune dermatosis [32].Agger et al. [33] found that the hematopoietic function was significantly reduced in a KDM4a-gene-knockdown mouse model, while another study reported that hypermethylation of the H3K36 site was related to the enhancement of MSC adipogenic differentiation tendency, which could be suppressed by KDM4a expression [34].These data confirm that the KDM4a gene is crucial for long-term hematopoiesis.Thus, the addition of UC-MSC to CsA could alter histone methylation-related enzyme genes and may affect hematopoiesis and immune regulation.The complement and coagulation cascade pathways are another pathway with significant enrichment of differential expression genes.Single-cell sequencing and proteomic and metabonomic analyses of CD8 + T cells from patients with AA have revealed that the complement and coagulation cascade pathway is significantly activated in these patients [35].
The addition of UCB-T reg to CsA significantly enriched genes in the energy metabolism process, primarily related to glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation.Alterations in the energy metabolism mode of hematopoietic stem cells (preferring anaerobic glycolysis or aerobic respiration) may underlie the pathogenesis of several kinds of hemopathy [36,37,38], and the unique changes after UCB-T reg addition may be mediated via these pathways.
CsA + UC-MSC significantly enriched the SOD activityrelated pathway when compared with CsA + UCB-T reg .SOD is an important antioxidant enzyme that can protect cells from reactive oxygen species (ROS)-mediated damage.ROS can cause oxidative damage to hematopoietic stem cells and osteoblasts, increase fat production in BM, and induce hematopoietic failure by directly attacking hematopoietic stem cells and inhibiting the hematopoietic microenvironment.ROS played a central role in the onset of AA in the chemotherapy-induced AA animal model, and the expression of SOD in this animal model was significantly reduced [39,40].CsA + UC-MSC significantly upregulated the SOD activity-related pathway, which may further improve the therapeutic effects.

Conclusions
In summary, the addition of UC-MSC or UCB-T reg to CsA could further improve the efficacy of CsA in treating an AA mouse model.The treatment with CsA + UC-MSC elicited superior hematologic improvements, whereas CsA + UCB-T reg was advantageous in immune regulation.The addition of different cord blood components can function through different pathways.In future investigations, we plan to explore the safety and efficacy of treatment with UCB-derived cells combined with CsA in humanized AA animal models, closely followed by early clinical trials.Collectively, these results suggest the potential for improving the treatment of AA in humans using our approach.12 Stem Cells International

FIGURE 7 :
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.

TABLE 1 :
Treatment regimens for AA mice in different groups.
Stem Cells Internationaland the proportion of CD4 + /CD8 + T cells and that of T reg / CD4 + T cells rapidly decreased.Changes in T cell subsets were consistent with the immunological features observed in human AA.Following the administration of various treatments, the ratio of CD4 + /CD8 + T cells was significantly increased (CsA + UC-MSC group, 1.27 AE 0.05; CsA + UCB-T reg group, 1.18 AE 0.14; (p <0:05).The levels of INF-γ, TNF-α, and IL-2 were reduced in the CsA + UC-MSC and CsA + UCB-T reg groups when compared with those in the untreated group (all p <0:05).Treatment with CsA reduced levels of TNF-α and IL-2 (all p <0:05), whereas those of INF-γ showed no significant difference (p >0:05).The levels of INF-γ and IL-2 were further reduced in the CsA + UCB-T reg group when compared with those in the CsA + UC-MSC group (p <0:05).Following the CsA + UCB-T reg treatment, TNF-α and IL-2 reached levels comparable with those in the normal control group, showing no significant differences.Similarly, after CsA + UC-MSC treatment, TNF-α levels aligned with those of the normal control group, exhibiting no notable variance (all p >0:05).