Differential expression of genes influencing mitotic processes in cord blood mononuclear cells after a pre-conceptional micronutrient-based randomized controlled trial: Pune Rural Intervention in Young Adolescents (PRIYA)

In the Pune Maternal Nutrition Study, low maternal vitamin B12 and high folate concentrations during pregnancy predicted adiposity and insulin resistance (diabesity) in the children. Based on these findings, we designed an RCT (PRIYA) to provide these nutrients pre-conceptionally to reduce diabesity in the offspring. The interventions included: 1) vitamin B12+multi-micronutrients and protein supplement (B12+MMN), 2) vitamin B12 alone, and 3) placebo. A total of 149 women became pregnant and delivered in the trial. We report results of RNA sequencing in the cord-blood mononuclear cells in 88 deliveries. Gene expression analysis revealed 20 differentially expressed genes (FDR <0.1, a discovery threshold) between the B12+MMN and placebo groups. The enriched biological processes included: chromosome segregation, nuclear division and organelle fission. A cell cycle checkpoint gene Claspin (CLSPN) was expressed less in the B12+MMN group but proliferation markers did not differ. No difference was observed between the B12 alone and placebo groups. The B-complex vitamins are essential for nucleotide synthesis and generation of S-Adenosyl Methionine, a universal cellular methyl donor and thereby may contribute to the regulation of gene expression. Vitamins A and D, and zinc in MMN may also influence gene expression. We propose that the multi-micronutrient intervention influenced rate of progression of specific phases of cell cycle without affecting proliferation. Vitamin B12 alone was not sufficient. We highlight a novel molecular mechanism for the effect of multi-micronutrients during fetal development, and a plausible mechanism for intergenerational nutritional programming. Follow up studies will help understand their implications on the future phenotype.

Umbilical cord blood-derived microglia-like cells to model COVID-19 exposure

Microglia, the resident brain immune cells, play a critical role in normal brain development, and are impacted by the intrauterine environment, including maternal immune activation and inflammatory exposures. The COVID-19 pandemic presents a potential developmental immune challenge to the fetal brain, in the setting of maternal SARS-CoV-2 infection with its attendant potential for cytokine production and, in severe cases, cytokine storming. There is currently no biomarker or model for in utero microglial priming and function that might aid in identifying the neonates and children most vulnerable to neurodevelopmental morbidity, as microglia remain inaccessible in fetal life and after birth. This study aimed to generate patient-derived microglial-like cell models unique to each neonate from reprogrammed umbilical cord blood mononuclear cells, adapting and extending a novel methodology previously validated for adult peripheral blood mononuclear cells. We demonstrate that umbilical cord blood mononuclear cells can be used to create microglial-like cell models morphologically and functionally similar to microglia observed in vivo. We illustrate the application of this approach by generating microglia from cells exposed and unexposed to maternal SARS-CoV-2 infection. Our ability to create personalized neonatal models of fetal brain immune programming enables non-invasive insights into fetal brain development and potential childhood neurodevelopmental vulnerabilities for a range of maternal exposures, including COVID-19.

Co-transplantation of Hypoxia Pretreated Human Adipose Derived Mesenchymal Stem Cells and Cord Blood Mononuclear Cells to Treat Rats with Acute Myocardial Infarction

BackgroundHuman adipose derived mesenchymal stem cells (ASCs) are ideal candidates for the treatment of acute myocardial infarction (AMI), due to their favorable availability and regenerative potential. However, in vivo studies showed that ASCs are not resilient at the infarcted area, for a shortage of blood and oxygen supply. Hypoxic pretreatment was proven to be an effective way to enhance cell survival in ischemic atmosphere. Moreover, co-transplantation of stem cells was another promising strategy to improve cardiac function after transplantation. So, we hypothesized that hypoxic pretreated ASCs combined with proangiogenic cord blood mononuclear cells (CBMNCs) would promote treatment efficacy after co-transplantation.MethodsASCs extracted from male volunteer were preconditioned in hypoxic condition (HP-ASC) for 24h, and total RNA were extracted after that. Gene expressions were compared between HP-ASC and ASC. Then, we transplanted stem cells to female Wistar rats which divided into different groups: (1) HP-ASCs group (n=10, 1x106ASCs); (2) HP-ASCs + CBMNCs group (n=10, 0.5×106 ASCs+0.5×106 CBMNCs); (3) CBMNCs group (n=10, 1×106 ASCs); (4) Control group (n=10, 40μL PBS); (5) Sham group (n=10). Echocardiogram was performed before (0d) and after (30d) after cell transplantation. Hearts were harvested at 30d to analyze the infarct size, myocardium apoptosis, stem cells viability and angiogenesis. ResultsIn vitro study showed that HP-ASCs had a wide range of paracrine function, with the incretion growth factors and their receptors, which would support the cell survivals. In addition, HP-ASCs also gained potentials in hypoxic adaptation (increased expression of HO-1 and SDF-1), as well as homing and immigrating abilities (CXCR4, ICAM-1 and ICAM-2). In vivo studies showed that, 30 days after transplantation in AMI rats, the HP-ASCs group had a better improvement in cardiac function; reduction of the infarct size; and decrease of ASCs death than the other groups (HP-ASCs > HP-ASCs + CBMNCs ≧ CBMNCs > PBS) (p<0.05). However, the combined group of HP-ASCs and CBMNCs had more significant angiogenesis than the other groups (HP-ASCs + CBMNCs > CBMNCs > HP-ASCs > PBS) (p <0.05).ConclusionsHP-ASCs alone had a greater potential in improving cardiac function in AMI rats. However, the combination of HP-ASCs and CBMNCs had a better result in angiogenesis.

Transcriptomic Landscape of Umbilical Cord Blood Mononuclear Cells after Genetic Modification

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 &lt;0.05) and VEGF (log2FC = 4.41, q &lt;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.

A Simple, Safe and Effective Approach for Personalized Precision Ex Vivo Gene Therapy Based on Autoinfusion of Gene-Modified Leucoconcentrate (GML) Prepared from Routine Unit of Patient's Peripheral Blood

Nowadays gene and cell therapy become the basic methods in regenerative medicine. However only few gene and cell products are currently approved for clinical usage. Biosafety problems, complexity of cell and gene technologies and high cost of manufacturing are the main reasons for the slow introduction of such approaches in practical medicine. Treatment of hereditary diseases of the immune system based on the correction of the mutant gene by delivering functional recombinant gene into WBC is the first successfully employed in the clinical practice approach of cell-mediated or ex vivo gene therapy. Earlier we have reported the strategy of the cell-mediated gene therapy based on umbilical cord blood mononuclear cells transduced with adenoviral vectors carrying recombinant genes encoding neurotrophic factors for treatment neurodegenerative diseases, neurotrauma and stroke. Significant disadvantage of this method is the usage of the umbilical cord blood mononuclear cells as a cell carrier for the therapeutic genes. Considering immunodeficiency treatment and our own data we developed a new approach of recombinant gene delivery for personalized ex vivo gene therapy. The method is based on autoinfusion of patient's WBC transduced with recombinant therapeutic genes for correction of certain pathological conditions. In the present study for the first time the human gene-modified leucoconcentrate (GML) producing recombinant reporter gene encoding green fluorescent protein (GFP) was obtained without culturing WBC in vitro. The routine unit of peripheral blood (450 ml) was collected into the plastic blood bag and the leucocyte- and platelet-rich concentrates (50 ml) were obtained by standard method using Macopress Smart (Macopharma, France). Afterwards the equal volume of hydroxyethyl starch 6% was added into the plastic blood bag which was centrifuged (DP-2065 R PLUS, Centrifugal Presvac RV; Presvac, Buenos Aires, Argentina) at 350 rpm for 10 min at 10°C. The obtained supernatant was transferred into the new plastic blood bag using manual plasma extractor FK-01 (Leadcore, Russia) and 200 ml of saline was added into the bag which was centrifuged at 1300 rpm for 10 min at 10°C and the supernatant was expressed out of the bag so that the remaining solution in the bag (30 ml) contained leucoconcentrate (WBC - 45.56 ± 23.93 × 106/ml and RBC - 1.76 ± 3.33 × 109/ml). Transduction of WBC with chimeric adenoviral vector (Ad5/35) carrying GFP gene was performed in the plastic bag with MOI 5 according to the count of WBC in the leucoconcentrate. After transduction for 12 hours, 200 ml of saline was added to the bag with leucoconcentrate, the mixture was centrifuged at 1000 rpm for 10 min at 10°C and the supernatant was squeezed out of the bag. The remained in the bag solution (30 ml) was considered as gene-modified leucoconcentrate carrying GFP gen (WBC - 22.63 ± 8.90 × 106/ml and RBC - 1.77 ± 1.21 × 109/ml). For in vitro study of GFP gene expression the samples of GML-GFP were cultivated for 60 hours after GML-GFP preparation. Fluorescent microscopy in the cytoplasm of the transduced WBC showed specific intensive green fluorescence. Flow cytometry analysis demonstrated that 2.5% of WBC from the GML-GFP efficiently expressed GFP. Thus leucoconcentrate after 72 h of transduction with Ad5/35-GFP with MOI 5 resulted in 2.5% of the GFP-positive cells. Thus the results of this study represent a simple, safe and effective approach for preparation of GML for personalized ex vivo gene therapy aimed at temporary production of the specific recombinant biologically active molecules for pathogenetic therapy of the varied nosological form, such as trauma, ischemic, degenerative, autoimmune, infection and other diseases. This study was supported by the grant of Russian Science Foundation 19-75-10030. Disclosures No relevant conflicts of interest to declare.

Umbilical cord blood derived microglia-like cells to model COVID-19 exposure

Microglia, the resident brain immune cells, play a critical role in normal brain development, and are impacted by the intrauterine environment, including maternal immune activation and inflammatory exposures. The COVID-19 pandemic presents a potential developmental immune challenge to the fetal brain, in the setting of maternal SARS-CoV-2 infection with its attendant potential for cytokine production and, in severe cases, cytokine storming. There is currently no biomarker or model for in utero microglial priming and function that might aid in identifying the neonates and children most vulnerable to neurodevelopmental morbidity, as microglia remain inaccessible in fetal life and after birth. This study aimed to generate patient-derived microglial-like cell models unique to each neonate from reprogrammed umbilical cord blood mononuclear cells, adapting and extending a novel methodology previously validated for adult peripheral blood mononuclear cells. We demonstrate that umbilical cord blood mononuclear cells can be used to create microglial-like cell models morphologically and functionally similar to microglia observed in vivo. We illustrate the application of this approach by generating microglia from cells exposed and unexposed to maternal SARS-CoV-2 infection. Our ability to create personalized neonatal models of fetal brain immune programming enables non-invasive insights into fetal brain development and potential childhood neurodevelopmental vulnerabilities for a range of maternal exposures, including COVID-19.