211 - Identification of miRNAs by sequencing of Cord blood plasma (CB) and Placental derived Small Extracellular Vesicles (sEVs) from Full-Term Healthy Pregnancies

Publication Number: 211.5466

Said Omar, Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University. Regional Neonatal Intensive Care Unit, University of Michigan Health-Sparrow, East Lansing, MI, United States; Ranga Prasanth Thiruvenkataramani, Michigan State University College of Human Medicine, Lansing, MI, United States; Amal Abdul-Hafez, Michigan State University, East Lansing, MI, United States; Ahmed Abdelgawad, University of Delaware, Newark, DE, United States; Mona Batish, University of Delaware, Newark, DE, United States; Sherif Abd El Fattah. Ibrahim, University of Eastern Finland, KUOPIO, Pohjois-Savo, Finland; Burra V. Madhukar, Michigan State University College of Human Medicine, E. Lansing, MI, United States

Background: sEVs are small membrane-enclosed vesicles released from cells, carrying an abundance of nucleic acids especially small noncoding RNAs such as miRNAs. These miRNAs play a major role in the regulation of diverse biological processes. Umbilical CB and conditioned medium of cultured human Wharton Jelly (WJ) and placenta (Pl) mesenchymal stem cells (MSCs) are abundant source of sEVs and may have a therapeutic effect in preventing hyperoxia induced acute lung injury and development of Bronchopulmonary Dysplasia (BPD). Previously we have reported the characteristics of sEVs isolated from CB, WJ and Pl and their role in prevention of hyperoxia induced acute lung injury. However, miRNA cargo of these placental sEVs has not been previously reported.

Objective: To examine differential expression profiles of miRNAs extracted from sEVs of CB, WJ MSCs and Pl MSCs isolated from the placenta of healthy full-term pregnancies and to elucidate the molecular mechanisms underlying biological functions, therapeutic effects and biomarker potentials of these sEVs.

Design/Methods: Placentas and CB were collected after IRB approval and obtaining maternal informed consent. sEVs with a size range of 50-200nm were isolated and concentrated from CB, WJ MSCs and Pl MSCs using ultrafiltration. Total RNA from these sEVs was extracted using Qiagen miRNeasy Plasma kit. Libraries were prepared using the SMARTer smRNA-seq kit for Illumina and miRNA was sequenced using Illumina NovaSeq6000 sequencer.

Results: miRNA sequencing was performed on 7 pooled samples of CB, PL, WJ sEVs. Each library had between 8-13M reads that passed QC with 95% of the bases having quality scores of 30 or better. Hierarchical clustering analysis of miRNAs with altered expression between the three groups is seen in Fig 1. miRNA expression profiles of sEVs identified 50 miRNA that are common in all groups. Unique miRNAs (21 in CB, 95 in PL and 28 in WJ sEVs) were also identified for each group. The common 50 miRNA fall in the regeneration pathway. Other significant biological processes related to immunity, regeneration and inflammation were identified in the gene ontology (GO) analysis of miRNAs that are common between CB, PL, and WJ sEVs and miRNAs unique to each of CB, PL, and WJ sEVs as seen in Fig 2 and table.

Conclusion(s): We have identified differentially expressed miRNAs in CB, PL and WJ sEVs and allocated them to key biological processes and pathways. These data will contribute to a better understanding of the properties of placental derived sEVs from healthy pregnancies and will provide novel resources for using these sEVs for therapeutic applications.

Fig 1. Hierarchical clustering analysis of miRNAs of CB and Placental sEV
Hierarchical clustering analysis of miRNAs with altered expression between the three groups (p < 0.05, fold change > 2). Red strip, high relative expression; blue strip, low relative expression; white strip, no change in gene expression. Color intensity reflects the degree of expression increase or decrease. CB: sEV isolated from Cord blood plasma; WJ: sEV isolated from Wharton Jelly derived MSCs; PL: sEV isolated from Placenta MSCs.

Fig 2. miRNA expression profiles in sEV isolated from umbilical cord blood plasma (CB), Pl, and WJ MSCs
miRNA expression profiles in sEVs isolated from umbilical cord blood plasma (CB), Pl MSCS sEVs and WJ MSCS sEVs. (A) Venn diagram representation of unique and overlapping miRNAs between CB, Pl and WJ. (B-E) Gene ontology analysis related to inflammation and regeneration of miRNAs that are common between CB, PL, and WJ, miRNAs unique to CB, miRNAs unique to PL, and miRNAs unique to WJ.

Table: Gene ontology (GO) IDs for miRNAs Sequencing of CB, PL and WJ sEVs
IDs of Gene ontology analysis related to miRNAs that are common between CB, PL, and WJ, miRNAs unique to CB, miRNAs unique to PL, and miRNAs unique