Introduction:

Therapeutic application of umbilical cord blood mononuclear cells (UCB-MCs) has been actively studied for at least during the last 30 years. Currently, UCB-MCs are extensively being investigated in the regeneration of the central nervous system (CNS) and the treatment of human neurodegenerative disorders. Translational studies dedicated to the application of UCB-MCs have revealed their capacity for stimulation of neurogenesis in the aged brain and promising potency for being therapeutic agents for treating such diseases as Alzheimer`s disease, amyotrophic lateral sclerosis (ALS), ischemic stroke, traumatic brain injury, Parkinson`s disease etc. Still the exact mechanism providing therapeutic effect remains unclear. Several studies modulating neurodegeneration have demonstrated immunomodulating and pro-inflammatory activity of UCB-MCs. Moreover, the unique feature of UCB-MCs, which underlies in the optional concordance of HLA or immunosuppression during transplantation, has been shown. At the same time, the main challenge in the application of UCB-MCs is the limited amount of available cells from a single donor. Therefore, the development of methods increasing therapeutic potency is an important task when using UCB-MC. In this regard, in our opinion, one of the options for increasing the therapeutic potential of cells can be ex vivo genetic modification of the transplanted UCB-MC. This approach is beneficial to get cells with the desired therapeutic properties. At the same time, little is known about the effect of genetic modification and over-expression of therapeutic genes on the UCB-MC transcriptome. In this regard in our present study, we evaluated the transcriptomic landscape of gene engineered UCB-MC.

Methods:

UCB-MCs were separated using Ficoll density gradient and modified recombinant adenoviruses (Ad-EGFP or Ad-VEGF165, MOI 10). Total RNA from all test samples was extracted using Trizol reagent. The quality and concentration of the isolated RNA samples were evaluated using Agilent Bioanalyzer 2100. Total mRNA from genetically modified and non-treated cells was sequenced Sequencing-By-Synthesis (SBS) technology on Illumina NextSeq 500 platform in 2×75 bp mode. After quality control, reads were aligned to human reference transcriptome GRCh38 using Kallisto pseudoaligner. Differentially expressed transcripts and genes were calculated with R package "sleuth".

Results:

We comprehensively profiled the whole-transcriptome landscape of human genetically modified UCB-MC. Totally 12 cDNA libraries, obtained from 6 individual donors, were analyzed. Transcriptomic analysis has revealed 2,4-2,8×106 of paired reads. A total of 10164 genes in the RNA-seq data were detected and analyzed. UCB-MCs were shown to express a broad range of pro- and anti-inflammatory cytokines, chemokines, growth factors, and metalloproteinases. The principal component analysis (PCA) of the RNA-seq data showed that samples representing different biological conditions do not differ from each other and are grouped according to the source of their receipt (isolation) (Fig.1A). Genetic modification and expression of transgenes did not lead to a global shift in the transcriptome profile of UCB-MC. At the same time, the recombinant genes EGFP (log2FC = 7.15, q <0.05) and VEGF (log2FC = 4.41, q <0.05), as expected, showed increased expression compared to NTC. We performed Gene Ontology (GO) analysis of the expressed genes. The results demonstrated that most genes associated with biological processes were related to metabolism. In the category of cellular components, most detected genes were associated with cellular membrane and nucleus, while in molecular function category - protein binding (Fig.1B).

Conclusion:

It has been shown that genetic modification and expression of UCB-MC transgenes did not affect the global transcriptome profile. Transcriptome profiling can be useful in the creation and testing of personalized gene cell products that meet biological safety and efficacy criteria. This work was supported by the RFBR grant №18-44-160029 and subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities 0671-2020-0058.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.