**1. Introduction**

The current options for ischemic stroke treatment are extremely limited and are aimed at restoring blood flow in the ischemic area by intravenous infusion of recombinant tissue plasminogen activator and/or physical removal of the clots [1]. To date, the main fundamental and clinical interest is focused on developing a neuroprotective treatment of the penumbra region within the therapeutic window. The strategy of cell-, gene-, and gene-cell therapy for neuroprotection in stroke treatment has been proven by numerous experiments in animal models [2–4]. Besides brain-specific cell types, umbilical cord blood (UCB) is widely used for neuroprotection in the central nervous system (CNS) for different pathological conditions [5]. UCB cells are considered a valuable source of stem cells, growth and neurotrophic factors for cell therapy. The mononuclear fraction of UCB contains populations of different immature cells that are capable of differentiating into many cell types [6] and, thus, represent an alternative to embryonic stem cells for transplantation to patients with post-ischemic, post-traumatic and degenerative diseases [7,8]. To date, the following have been discovered in UCB: Hematopoietic stem cells (HSCs), endothelial progenitor cells, mesenchymal stem cells (MSCs), unrestricted somatic stem cells (USSCs), and side population cells (SP) [9–12].

Due to the immaturity of the immune system of a new-born, the use of UCB cells for cell therapy does not require matching of genes relating to HLA (Human Leucocyte Antigens) human tissue compatibility, as evidenced by the absence of an acute or chronic form of the disease "graft-versus-host" (graft versus host disease) [13,14]. In addition, with UCB cell transplantation, tumor transformation of cells in the recipient's body is practically prevented [15].

Another attractive reason for using UCB cells for cell therapy is their ability to produce various biologically active molecules, such as proteins which are antioxidant, angiogenic, neurotrophic, and growth factors [16–20]. Thus, transplantation of UCB cells can be aimed at replacing dead cells and at preventing the further death of surviving cells due to secreted biologically active molecules. Enhancement of the positive effects of UCB cells on tissue regeneration after their genetic modification is a relatively new and promising gene-cell approach in cell therapy to stimulate post-traumatic or post-ischemic brain injury [21,22]. Gene-modified UCB cells may provide addressed delivery of therapeutic genes and supply the expression of the recombinant molecules at the site of regeneration.

In our previous studies, we showed the positive effect of gene-modified umbilical cord blood mononuclear cells (UCB-MC), simultaneously producing three recombinant molecules—vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (NCAM)—in animal models of amyotrophic lateral sclerosis [23], spinal cord injury [24], and stroke [25]. The rationale of using a combination of two neurotrophic factors with cell adhesion molecules is based on the well-known neuroprotective effects of VEGF and GDNF [26,27], with the expression of NCAM increasing the homing and survivability of UCB-MC at the brain injury site [28] supporting local production of the therapeutic molecules. In the model of middle cerebral artery occlusion (MCAO) in rats, we demonstrated that intrathecal injection of genetically-engineered UCB-MC over-expressing VEGF, GDNF, and NCAM, four hours after MCAO results in a reduction of infarct volume, the positive reaction of neuroglial cells and an increase in synaptic protein expression. Thus, ex vivo gene modification may enhance the naïve neuroprotective properties of UCB-MC.

The development of treatment under the threat of stroke is of particular interest. Preventive therapy may highly reduce the consequences of a stroke-induced brain injury. In the present study, we suggest the approach of preventive cell-mediated gene therapy for stroke. The efficacy of gene-engineered UCB-MC overexpressing recombinant molecules-stimulants of neuroregeneration VEGF, GDNF, and NCAM, administered intrathecally 3 days before MCAO in rats, was investigated using morphometric and immunofluorescent methods.

#### **2. Results**

#### *2.1. Molecular Analysis of Gene-Modified UCBC*

The efficiency of UCB-MC transduction by Ad5-GFP adenoviral vector was confirmed after 72 h of UCB-MC+Ad5-GFP cultivation using fluorescent microscopy. In the cytoplasm of UCB-MC+Ad5-GFP, a specific green glow was detected (Figure 1A). By the flow cytometry method, it was established that

the percentage of GFP-positive human UCB-MC at multiplicity of infection (MOI) equal to 10 reaches 29.5% (Figure 1B).

**Figure 1.** Molecular analysis of gene modified umbilical cord blood mononuclear cells (UCB-MC). (**A**,**B**)—Production of green fluorescent protein (GFP) in UCB-MC, 72 h after transduction with Ad5-GFP (MOI = 10). (**A**)—fluorescent microscopy shows a specific green glow in UCB-MC+Ad5-GFP. Cell nuclei are stained with Hoechst 33342 (blue glow). Scale bar = 200 μm. (**B**)—flow cytometry analysis demonstrates that 29.5% of UCB-MC effectively produce reporter GFP. (**C**)—Quantitative analysis of mRNA *vegf165*, *gdnf*, and *ncam1* levels in intact (naïve) UCB-MC and genetically-modified UCB-MC, 72 h after transduction with three adenoviral vectors simultaneously carrying the therapeutic genes (Ad5-VEGF, Ad5-GDNF, and Ad5-NCAM) or with Ad5-GFP (UCBC + Ad5-GFP), with a MOI = 10. Data from two independent experiments are represented as an average value of ± SE, \*—*p* < 0.05. (**D**)—Radial comparative diagram of cytokine, chemokines and growth factors in supernatant obtained 72 h after incubation of gene modified UCB-MC (UCB-MC+Ad5- vascular endothelial growth factor (VEGF)- glial cell-derived neurotrophic factor (GDNF)- neural cell adhesion molecule (NCAM) and UCB-MC+Ad5-GFP) and intact UCB-MC.

Evaluation of mRNA levels of transgenes (*vegf165*, *gdnf*, and *ncam1*) in genetically-modified UCB-MC was performed by RT-PCR, 72 h after incubation of UCB-MC+Ad5-VEGF-GDNF-NCAM. The molecular analysis revealed that the content of mRNA *vegf165* increased 141.8 ± 8.24 times, *gdnf* mRNA 167.51 ± 6.85 times and *ncam1* mRNA 122.9 ± 13.5 times compared with intact (naïve) UCB-MC (Figure 1C).

Multiplex analysis of cytokines, chemokines and growth factors in supernatants harvested after the cultivation of naïve UCB-MC identified a wide range of pro- and anti-inflammatory cytokines, chemokines and growth factors (Figure 1D). However, the concentrations of Flt-3L, IL12-p70, IL-15, IL-17a, IL-9, and TNFb were lower than the detection level. Genetic modification of human UCB-MC by recombinant adenovirus Ad5-GFP does not affect the secretory profile of modified cells in relation to the investigated factors when compared to the naïve cells. UCB-MC simultaneously transduced with Ad5-VEGF, Ad5-GDNF, and Ad5-NCAM also preserves the qualitative and quantitative profile of expression of the investigated factors. However, as expected, in comparison with the naïve and UCB-MC+Ad5-GFP, we observed a 200-fold increase of VEGF level. The presented results correlate with PCR-RT data above and confirm the efficiency of UCB-MC transduction and its ability to synthesize and secrete recombinant molecules, as with the example of VEGF. In addition, the results support that adenoviral vector does not affect the production of the studied biologically active molecules by human UCB-MC.
