Exposure of Cord Blood Hematopoietic Stem Cells to Bone Marrow Mesenchimal Cells-Derived Microvesicles Induces Cell Survival and Inhibition of Differentiation
Authors: De Luca Luciana , Trino Stefania , Simeon Vittorio , Laurenzana Ilaria , Raimondo Stefania , Podestà Marina , Santodirocco Michele , Di Mauro Lazzaro , La Rocca Francesco , Caivano Antonella , Morano Annalisa , Del Vecchio Luigi , Frassoni Francesco , Cilloni Daniela , Musto Pellegrino 
Published: October 2019

Abstract
Introduction
Hematopoietic stem cells (HSC) are used in transplantation for the treatment of several hematological or non-hematological diseases. HSC fate is bound to different factors and, among these, the cross-talk with the bone marrow microenvironment represents an important regulatory mechanism of aging, self-renewal, stemness and/or differentiation. Cells communicate by both direct interaction and through the secretion of soluble factor and extracellular vesicles (EVs). EVs are cell-derived vesicles enclosed by a lipid bilayer and enriched in phosphatidylserine, cytoplasmic protein, mRNA, miRNA and DNA. Recent studies suggest that the release of EVs is a highly regulated and important process for surface membrane traffic and horizontal transfer of RNAs, protein and DNA, present in different kind of cells, including tumor cells. Bone marrow mesenchimal stem cells (BM-MSC) are a component of hematopoietic microenvironment and support hematopoiesis by the constitutive ability to secrete soluble factor and EVs.
On these basis, we decided to study the interaction between the umbilical cord blood (UCBs) stem cells and the BM-MSCs mediated by EVs. The overarching goal of this project was to find out if and how the EVs can influence the fate of UCBs in transplantation.
Methods
Primary BM-MSC were cultured in DMEM containing 10% of fetal bovine serum (FBS). EVs were isolated from supernatant of BM-MSC by ultracentrifugation at 100.000 x g for 70 min at 4 ºC. After characterization by FACS analysis, EVs were stocked at -80 ºC and partially used for RNA extraction using TRIZOL reagent. EVs smallRNA was sequenced with HiScan SQ Illumina. UCBs CD34+ cells were isolated by Miltenyi Biotec separation kit and were cultured in DMEM containing 10% of FBS with or without BM-MSC EVs. After 24h of co-culture, viability by trypan blue count, apoptosis by Propide Iodure and Annexin V test and cell differentiation by FACS analysis of different markers (CD45, CD34, CD33, CD19 and CD38) were evaluated. RNA was extracted from the same samples and a gene expression profile experiment was performed using Illumina array. All genomic data were analyzed by Ingenuity Pathways Analysis software (IPA).
Results
EVs isolated from BM-MSC were positive for MSC markers like CD29 (61%), CD90 (72%), CD73 (63%), CD105 (35%), CD146 (32%), CD44 (70%) and for the exosome antigen CD81 (40%); they were negative, instead, for the hematopoietic marker CD45. The sequencing of smallRNA-EVs identified 87 miRNAs in BM-MSC EVs. Analysis of gene expression profile of UCBs CD34+ cells treated with EVs revealed 103 up-regulated and 100 down-regulated genes (with padj < 0.05 and logFC=0.7), when compared to UCBs CD34+ cells not treated with EVs. Interestingly, analyzing together the sequencing of EVs and the gene expression data of UCBs CD34+ cells treated with EVs, we found a direct correlation between EVs miRNAs and down-regulated genes, identifying at least one target gene for each EVs miRNA (e.g., miR-3168/LYZ, miR-27b-3p/ZFP36, miR21-5p/ANXA1). These results indicate that the EVs RNA content modifies the genes profile of receiving cells. Gene ontology analysis, using IPA, of the miRNA targeted genes identified different down-regulated biological function, like cell death and cellular development (e.g., CDKN1B, CEBPA, ANXA1, MPL). To confirm these data, we evaluated viability and apoptosis of UCBs treated with EVs respect to the UCBs control culture, and we observed an increase of 50% of cell viability and a reduction of 42% of apoptosis (p<0.05). Instead, a decrease of cellular maturation was confirmed by FACS analysis evaluating different hematological markers. The expression of CD19+, CD33+ and CD38+ resulted to be lower in UCBs cells treated with EVs, respectively by 45% (p<0.01), 13% (p<0.05) and 3% (p<0.01), when compared to control.
Furthermore, using IPA, we found that some up-regulated genes (e.g., IL6, CSF2, CCL3) are under the control of miRNA targeted genes (e.g., ZFP36/miR-27b-3p). The analysis of these genes identified some biological function up-regulated in cell after EVs treatment, the most important one being chemotaxis of the cells.
Conclusion
This study indicates, for the first time, the existence of a cross-talk between MSC and UCBs mediated by EVs. Moreover, it identifies a gene profile modification of UCBs cells after miRNA EVs treatment, providing new insights for cord blood transplantation.

Disclosures
No relevant conflicts of interest to declare.