Deciphering Transcriptome Alterations in Bone Marrow Hematopoiesis at Single-Cell Resolution in Prolonged Thrombocytopenia after Umbilical Cord Blood Transplantation

Background: Prolonged isolated thrombocytopenia (PIT) is a frequent complication after allogeneic hematopoietic stem cell transplantation, especially after umbilical cord blood transplantation (UCBT), with an incidence ranging from 5% to 48.6%, significantly increasing non-relapse mortality after transplantation. Currently, there is no systematic study on PIT, and the pathogenesis of PIT after UCBT remains unclear. Thus, we performed a single-cell transcriptome profiling of bone marrow mononuclear cells to explore the molecular mechanism of PIT.

Method: We included 8 patients who diagnosed with AML and underwent UCBT, from whom a total of 10 bone marrow specimens were collected. They were divided into two groups based on platelet engraftment status at +60 days post-UCBT: the PIT group (4 samples at +30 days, and 2 samples at +60 days) and the non-PIT group (4 samples at +30 days). We applied single-cell transcriptome sequencing to detect and integrate sequencing results from 10 bone marrow single-nucleus cell samples.

Results:

After quality control, a total of 258,067 cells were used for downstream analysis. We defined 29 cellular subtypes based on shared characteristic genes. The UMAP plot showed that HSC, megakaryocyte-erythroid progenitors (MEP)/megakaryocyte progenitors (MKP), and red blood cells in the PIT groups were significantly reduced compared to the non-PIT group. Using the Monocle2 method to reconstruct the differentiation trajectory of hematopoietic stem/progenitor cell groups, both groups of patients had similar differentiation trajectories, but the number of HSC and MEP/MKP was sparser in the PIT group at the initial stage.

We also evaluated the transcriptome changes in HSCs and MEP/MKP in the PIT group. Differential gene expression analysis results showed that immune response-related genes were upregulated in HSCs and MEP/MKP in the PIT group, while a series of genes related to cell cycle and proliferation were downregulated. In the MEP/MKP, genes related to mitochondria and energy metabolism were upregulated, while platelet function-related genes (GP9) were downregulated. Enrichment analysis results showed that the oxidative phosphorylation pathway and reactive oxygen species signaling pathway were upregulated, while the hematopoietic regulation signaling pathway was downregulated in the MEP/MKP cell group in the PIT group. In the bone marrow of PIT patients at +60 days compared to +30 days, both HSCs and MEP/MKP showed upregulation of the IL-17 signaling pathway, with downregulation of important biological functions such as DNA replication and protein processing in HSCs. This suggests the crucial role of immune response in the pathogenesis of PIT.

We further observed that, compared to the non-PIT group at +30 days post-UCBT, the proportion of CD4⁺Th1 cells in bone marrow decreased, while the proportion of CD4⁺Treg cells slightly increased and the proportion of CD8⁺TEFF cells significantly increased in the PIT group. Within the PIT group, at +60 days post-UCBT, the proportions of CD4⁺TEM, CD4⁺Treg, CD8⁺TEFF, and CD8⁺TEM cells in the bone marrow were significantly higher compared to +30 days. Enrichment analysis revealed significant upregulation of Th17 differentiation and/or IL-17 signaling pathways in the CD4⁺TEM/CD8⁺TEM/CD8⁺TEFF subsets at +60 days post-UCBT in the bone marrow of PIT patients, as well as in the CD8⁺TEFF subset at +30 days post-UCBT. These findings suggest that immune cells in the bone marrow of PIT patients are in a state of dysregulation, and overactivation of the IL-17 signaling pathway may be associated with the occurrence of PIT.

We used the CellPhoneDB method to analyze the interaction between cell subgroups in each group. Overall cell interactions were reduced in the PIT group, suggesting that the bone marrow cell niche of PIT patients is damaged, potentially leading to disruption of hematopoietic function.

Conclusions:

Through single-cell sequencing, we confirmed changes in the quantity and transcriptome of HSCs and MEPs/MKPs in PIT patients and found that the IL-17 signaling pathway may be involved in the pathogenesis of PIT.

Keywords:

Unrelated umbilical cord blood transplantation, Prolonged isolated thrombocytopenia, single-cell transcriptome sequencing