microRNA Sequencing of CD34+ Sorted Adipose Stem Cells Undergoing Endotheliogenesis

Authors

Shahensha Shaik, Bioengineering Laboratory, Department of Mechanical Engineering and Louisiana State University, Baton Rouge, Louisiana, USA.
Elizabeth Martin, Department of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, Louisiana, USA.
Daniel Hayes, Department of Biomedical Engineering, Pennsylvania State University, University Park, Pennsylvania, USA.
Jeffrey Gimble, La Cell, LLC and Obatala Sciences, Inc., New Orleans, Louisiana, USA.
Ram Devireddy, Bioengineering Laboratory, Department of Mechanical Engineering and Louisiana State University, Baton Rouge, Louisiana, USA.

Document Type

Article

Publication Date

3-1-2021

Abstract

While several microRNAs (miRNAs) that regulate the endotheliogenesis and further promote angiogenesis have been identified in various cancers, the identification of miRNAs that can drive the differentiation of adipose derived stromal/stem cells (ASCs) into the endothelial lineage has been largely unexplored. In this study, CD34+ ASCs sorted using magnetic bead separation were induced to differentiate along the endothelial pathway. miRNA sequencing of ASCs at day 3, 9, and 14 of endothelial differentiation was performed on Ion Proton sequencing system. The data obtained by this high-throughput method were aligned to the human genome HG38, and the differentially expressed miRNAs during endothelial differentiation at various time points (day 3, 9, and 14) were identified. The gene targets of the identified miRNAs were obtained through miRWalk database. The network-pathway analysis of miRNAs and their targets was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tools to determine the potential candidate miRNAs that promote endothelial differentiation. Based on these analyses, six upregulated miRNAs (miR-181a-5p, miR-330-5p, miR-335-3p, miR-15b-5p, miR-99a-5p, and miR-199a-5p) and six downregulated miRNAs (miR-145-5p, miR-155-5p, miR-193a-3p, miR-125a-5p, miR-221-5p, and miR-222-3p) were chosen for further studies. In vitro evaluation of these miRNAs to induce endothelial differentiation when transfected into CD34+ sorted ASCs was studied using Von Willebrand Factor (VWF) staining and quantitative real time-polymerase chain reaction (qRT-PCR). Our results suggest that miRNAs: 335-5p, 330-5p, 181a-5p and anti-miRNAs: 125a-5p, 145-5p can likely induce endothelial differentiation in CD34+ sorted ASCs. Further studies are clearly required to elucidate the specific mechanisms on how miRNAs or anti-miRNAs identified through bioinformatics approach can induce the endotheliogenesis in ASCs.

Publication Source (Journal or Book title)

Stem cells and development

First Page

265

Last Page

288

Recommended Citation

Shaik, S., Martin, E., Hayes, D., Gimble, J., & Devireddy, R. (2021). microRNA Sequencing of CD34+ Sorted Adipose Stem Cells Undergoing Endotheliogenesis. Stem cells and development, 30 (5), 265-288. https://doi.org/10.1089/scd.2020.0173