Single-cell Transcriptomic Landscape Research On Nucleated Cells In Umbilical Cord Blood

Umbilical cord blood(UCB)has been used for more than a decade since it was used for bone marrow reconstruction and is now used for cancer treatment.Umbilical cord blood is an excellent source of hematopoietic progenitor cells and immune cells,and also has the advantage of convenient collection and non-invasiveness.The immune cells in cord blood are virtually free from external stimulant and infection,thus they are in a relatively more na?ve stage comparing to the peripheral blood.Such immunological immaturity is the key to alleviate the severity of GVHD by decreasing the alloreactive potential of lymphocytes.Immune cells in cord blood also play a role in the process of anti-virus and other microbial infections.Although UCB is now widely used for important clinical applications,we know surprisingly little about its cellular and molecular characteristics.With the development of biotechnologies and the continuous exploration on cord blood,umbilical cord blood cells have shown extraordinary heterogeneity,and these heterogeneities also significantly affect the clinical applications.The single-cell transcriptome sequencing technology becomes a powerful tool for exploring the heterogeneity of cord blood nucleated cells and inferring their functions.In this study,nucleated cells were screened from two cord blood samples,and single-cell transcriptome sequencing of cord blood nucleated cells was performed by 10x Genomics'Chromium System and BGISEQ-500 sequencer.To determine the unique cell subpopulations and the specific state of gene expression in UCB,we utilized the single-cell transcriptome dataset of peripheral blood(PB)cells for comparison.This dataset includes two independently generated libraries(PB1 and PB2),containing total of 11948 single-cell profiles of peripheral blood mononuclear cells(PBMC)measuring 1069 genes per cell on average,which are at comparable level with those of the UCB data.Strict multi-dimensional quality control and filtration were performed after combining the single cell data of cord blood and peripheral blood.Then a variety of methods were tried to correct and evaluate the batch effect,and the batch-removal method which is most suitable for this study was determined.Next,the single cell data was visualized and defined by clustering and dimensionality reduction.Finally,a more in-depth study of nucleated red blood cells,hematopoietic stem cells and cytotoxic cells in cord blood was conducted.Here,we first report a single-cell transcriptome landscape of 7852 and 9785 nucleated cells from the two UCB samples(UCB1 and UCB2).The average number of genes in nucleated cells is 1270 and 1460,respectively,covering 12 main cell types including nucleated red blood cells,T lymphocytes,and hematopoietic stem cells.Pseudotime analysis speculated the dual-polarity features of linear changes in nucleated red blood cell maturation,demonstrating changes in cell morphology,reprogramming of expression profiles and potential transcriptome activation differences during maturation of cord blood nucleated red blood cells.It suggests that cord blood may be an important place where nucleated red blood cells mature.A new subpopulation of progenitor cells present in cord blood was first identified.This group of progenitor cells was CD34 negative but very similar to the overall expression profile of CD34 positive progenitor cells.They simultaneously expresse the marker genes of granulocyte-monocyte progenitor cells(GMP),mast cells and basophils.Combined with the results of pseudotime analysis,we hypothesized that these cells represent the intermediates before the bifurcation during basophil and mast cell differentiation and termed them umbilical intermediate bi-potent cells(uIBC).Single cell data revealed the heterogeneity of cytotoxic lymphocyte including NK cells,NKT cells,and CTL in cord blood and peripheral blood.Five novel cytotoxic cell subsets in cord blood and peripheral blood were redefined by granzyme K,granzyme B,and cell lineage markers,which GZMB~+NKT cells only presented in cord blood.GO analysis revealed that GZMB~+NKT cells play an important role in innate immunity in cord blood.Differential gene integration analysis and co-expression analysis revealed core gene modules that are ubiquitously associated with GZMB and GZMK cytotoxicity,respectively.Taken together,our data provides the first single-cell transcriptome references for UCB,which could be used as a standard dataset for comparative analysis.We expect that this dataset will prove useful in uncovering the novel molecular signatures that define the cellular heterogeneity in UCB and provide markers for targeted enrichment of certain cell types of interest to researchers in multiple fields.The study of cytotoxic cell subsets provides important clues for improving the efficacy of cord blood cells in the treatment of tumors and resisting microbial infections.Our dataset is a rich resource to formulate hypothesis of signaling pathway activation,transcription control and other mechanistic studies in the field of functional immunology at single cell level.