Abstract

   The function and heterogeneity of neutrophils in neonatal umbilical
   cord blood (UCB) have not been characterized. In this study, we
   analyzed the neutrophils in UCB and healthy adults using single-cell
   RNA sequencing analysis for the first time. We found that neutrophils
   divided into six subpopulations (G2, G3, G4, G5a, G5b, and G5c) with
   different marker genes and different functions under homeostasis.
   Compared with healthy adults, neutrophils of UCB were more naïve and
   have more obvious degranulation and activation functions. Moreover, we
   found significant differences in the amount and function of G5b cells
   between healthy adults and UCB. The amount of G5b group in UCB was
   lower, but it has more degranulation, secretion and activation
   functions. In addition, we noted a new subset of G5c labeled by CD52,
   which almost did not exist in UCB. Besides, its differential genes were
   enriched in terms such as protein synthesis and mRNA transcription.
   Furthermore, uncharacteristic transcription factors ZNF-276, ZNF-319
   and ZNF-354A were identified in our study. In summary, we first
   examined the heterogeneity and functional diversity of neutrophils in
   UCB, and these data provided new insights into the mechanism of
   neutrophil-mediated diseases of neonates and the wider use of
   neutrophils in UCB.

   Keywords: umbilical cord blood, neonate, neutrophils, single cell,
   transcriptome

Background

   The neutrophil is a major component of the innate immune system and are
   the first cells recruited to injured or infected sites. In the presence
   of abnormal neutrophil numbers or quality, it might disrupt the immune
   system’s homeostasis environment and contribute to the development or
   progression of disease. Neutropenia or neutrophil dysfunction would put
   patients at high risk for fatal infection ([41]1). With the in-depth
   study of neutrophils, researchers have proved that neutrophil
   dysfunction developed many diseases, such as malignant tumors,
   autoimmune diseases and severe infections ([42]2, [43]3). Granulocyte
   transfusion as one of the main treatment methods has a long history in
   treating patients with neutropenia or neutrophilic dysfunction, and can
   be used to prevent and treat infection ([44]4). In addition, in recent
   years, with the emergence of immunotherapy, neutrophilic adoptive
   therapy also began to be used in the treatment of refractory infectious
   diseases and malignant tumors, and good therapeutic effect was achieved
   ([45]5).

   It was increasingly recognized that UCB, previously considered medical
   waste, was actually a valuable source of therapeutic cells ([46]6). The
   cells of UCB were easier to collect than that of healthy adults, and
   their unique properties made them especially appropriate for cell
   therapy. The unique properties of these cells included their naive
   nature ([47]7, [48]8), the high proportion of stem and progenitor cells
   ([49]7, [50]9), and non-hematopoietic cells with therapeutic potential
   ([51]8). But neutrophils in UCB, the most numerous of the immune cells,
   were rarely mentioned. There were no clear differences between
   UCB-derived neutrophils and healthy adults’ neutrophils in terms of how
   they function.

   With the help of single-cell RNA sequencing(scRNA-seq), an important
   method for characterizing immune cells and their function, we
   preliminary explored the differences between neutrophils of UCB and
   adults. We provided the first reference map of neutrophils subsets in
   UCB, and the functional difference of neutrophils in UCB were
   explained. The results of our study lay the theoretical groundwork for
   the further study of UCB neutrophils.

Result

   To study the functional changes of neutrophils in UCB, we tested
   neutrophils with scRNA-seq in healthy adults and UCB. Neutrophils
   samples for sequencing were collected from peripheral blood of 25
   healthy adults and UCB of 40 healthy neonates of full-term natural
   delivery ([52] Table 1 ). Among them, 5 UCB samples and 5 peripheral
   blood samples from healthy adults were used for the detection of
   seRNA-seq. The magnetic bead separation was used for the separation of
   neutrophils.

Table 1.

   Clinical data of UCB (including maternity and neonate) and healthy
   adults submitted for examination.
   Characteristics Maternity Neonate Healthy adult
   Number n=40 n=40 n=25
   Sex, female (%) 100% 45% 44%
   Age (year) 26 (21-34) N/A 25 (22-35)
   Gestational age(week) N/A 39 (38-41) N/A
    Delivery mode,
   spontaneous labor (%) N/A 100% N/A
   Intrapartum fever (%) 0 N/A N/A
   GBS Colonization (%) 0 N/A N/A
     *  PIH (%)

   0 N/A N/A
   Gestational diabetes (%) 0 N/A N/A
   Birth weight (g) N/A 3447.29 ± 570.45 N/A
   1min Apgar score N/A 10 N/A
   5min Apgar score N/A 10 N/A
   10min Apgar score N/A 10 N/A
   WBC count (10^9/L) 7.49± 1.23 N/A N/A
     *  Neutrophil count (10^9/L)

   5.24 ± 1.01 N/A N/A
   [53]Open in a new tab

   Data presented as median (age range and birth weight); mean (standard
   deviation) or number (percentage); GBS, group B β hemolytic
   streptococcus; PIH, Pregnancy induced hypertension; WBC, white blood
   cell; N/A, not applicable.

Conservative classification of neutrophils in UCB

   The 45905 high-quality cells were obtained through rigorous quality
   control ([54] Supplementary Figure 1A ). We identified 13 major cell
   populations by graph-based clustering ([55] Figure 1A ). With the known
   genetic markers (HBB, HBA1, and HBA2) of red blood cell (RBC) ([56]10),
   groups 12 were considered RBC ([57] Supplementary Figure 1B ).
   Furthermore, 10 and 11 clusters were also characterized by a high
   percent of mitochondrial unique molecular identifier (UMI) count and
   low UMI count per cell ([58] Supplementary Figure 1C ). Therefore, in
   further analysis we discarded groups 10,11 and 12. FCGR3B and S100A8
   are considered as specific marker genes of neutrophil ([59]11), and
   CD10 encoded by MME is considered to be a surface marker of mature
   neutrophils ([60]12). For these reasons, we identified groups 0-9 as
   neutrophils, and subgroups 6 and 9 as immature neutrophils ([61]
   Figure 1B ). The specific marker genes Kit and CD34 of
   granulocyte-monocyte progenitors (GMP) ([62]13) are not expressed on
   neutrophils ([63] Supplementary Figure 1D ). As a result, no GMP was
   found in the neutrophils we collected. In addition, the correlation
   analysis based on cell subpopulation differential genes and cell
   cycle-related genes ([64] Figure 1C and [65]Supplementary Table-Sheet 1
   ) suggested that 9 cluster could have a proliferative function in the
   neutrophil subsets. According to the study of Xie et al ([66]14),
   neutrophils have been classified into eight subsets (G0-G5c) in bone
   marrow, tissue and circulation. Using the genetic markers present in
   the references ([67] Supplementary Table-Sheet 2 ), we conducted