Department of Cell Regulatory Mechanisms

Exploring the potential of Cell therapy

Senior investigator

Olena Deryabina

Study of the umbilical cord MSC: isolation, expansion, characterization and development of the approaches for their therapeutic applications

A technology of MSC isolation from human umbilical cord Wharton jelly with maximal preservation of their native characteristics has been developed. The necessity of individual approach for the cells obtaining from umbilical cord was confirmed. The optimal culturing conditions, in particular, considering an influence of gas mixture were determined. It has been shown that lowered oxygen concentration and nitrogen substitution with argon had positive effect on the umbilical cord MSC during their cultivation. The morphological characteristics as well as surface markers expression were studied. MSC from umbilical cord were shown to be heterogeneous at isolation (0 passage), but already at the first passage they obtained spindle-shaped morphology, which is typical for MSC. They were CD73, CD90, CD105 positive and CD34, CD45 negative, which is characteristic of MSC.

The characteristics of human umbilical cord MSCs: A - chondrogenic differentiation was detected by Alcian blue staining; B - osteogenic differentiation was detected by Alizarin red S staining; C - adipogenic differentiation was detected by Oil Red O staining; D – expression of MSC surface markers from human umbilical cord.

After the second ex vivo passage, gradual change of the MSC properties occurred (cell morphology, surface markers expression, doubling time, etc.), as well as the cases of spontaneous differentiation to adipo- and chondrocytes began. These findings indicate that cultures at early passages should have preferences for use.

A – Gene Therapy Correction of Streptozotocin-induced Diabetic Hyperglycemia in Landras Swine, B – Gene Therapy for Experimental Hypercholesterolemia in Rabbits

In recent years, the use of transplanted cells producing active factors has been proven to be an emergent technology. However, the transplantation of allogenic and xenogenic cell material results in the immune rejection of cell transplants. Cell microencapsulation is a promising tool to prevent an attack of immune system even in case of xenogenic transplants. Microcapsules, prepared from semipermeable membranes, ensure transmission of target proteins, on the one hand, and defend the cells from attacks of immune system – on the other. It makes possible to avoid the use of long-term therapies of modulating and/or immunosuppressive agents, which have potentially severe side-effects. Usage of encapsulated cells, producing growth factors, cytokines, hormones and other therapeutic proteins, is at present a very promising way of delivery of therapeutic material into an organism. The Department takes part in this field of research. The genetically engineered cells are a source of therapeutic proteins. Transgenic mammalian cell lines producing some important recombinant human cytokines (LIF, FGF2, IL10) have been obtained by us via a nonviral gene transfer technique. The recombinant protein secretion into the cultural medium was shown. When the cells producing cytokines were encapsulated in alginate microcapsules, the production of the recombinant cytokines was continuing and the cytokine molecules are secreted from the microcapsules into the cultural medium (Fig. 3). The therapeutic effects of the cytokines, produced by genetically modified eukaryotic cells, are planned to be tested on animal models when the microcapsules will be transplanted to animals suffering from some disordes caused by the lack of tested cytokines.

Senior investigator

Nadiia Shuvalova

Optimizing the oxygen concentration for cultivation of mesenchymal stem cells from umbilical cord

It is known that the cultivation of MSCs, obtained from different sources under standard CO₂ incubator conditions, is accompanied by aging of the culture and loss of its regenerative potential. Numerous studies have shown that the level of oxygen in the culture environment modulates the production of reactive oxygen species, and the conditions of mild hypoxia prevent the loss of regenerative potential of MSCs. The cytoprotective effect of inert gases (argon,in particular) on cultures of neural origin, is also described, but there is no information about the biological effects of inert gases on stem cell cultures.

In the department, the systems for creating gas mixtures with low oxygen content, based on nitrogen and argon, were developed. The studies on impact of mild hypoxia on various aspects of obtaining and cultivating MSC Wharton's jelly (WJ-MSC) were performed. We found that during five passages, after seven days of cultivation, the number of WJ-MSC, in gas mixtures, containing 3% oxygen, based on nitrogen and argon, was higher than in the control CO₂ incubator conditions, even considering the background decrease in proliferative activity after the third passage. Cultivation of MSCs in gas mixtures containing 3% O₂ also reduced the heterogeneity of MSC culture, and lowered the number of cells with the senescent phenotype, compared to cultivation under standard conditions of CO₂ incubator. For the first time, the difference in the biological effects of nitrogen- and argon-based gas mixtures was shown. Although both had a positive effect on the preservation of the native characteristics of the culture, the nitrogen-based mixture in all variants proved to be more effective.

Also, for the first time the method of optimization of non-viral transfection of MSCs in the conditions of physiological oxygen concentrations was developed in the department. It was shown that in gas mixtures, containing 3% oxygen, based on nitrogen and argon, the percentage of WJ-MSC expressing the marker protein eGFP after transfection, was significantly higher than in the control group: on average, the number of eGFP-positive cells was 2,58-time increase in nitrogen-based mixture, and 1,37 in argon-based mixture.

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collaboration with O.S.Kolomiychenko Institute of Otolaryngology of NAMS of Ukraine

A study of therapeutic effects of MSCs transplantation in the treatment of viral lesions on the model of progressive atrophic rhinitis caused by infection

The studies were performed in collaboration with SI “O.S.Kolomiychenko Institute of Otolaryngology of NAMS of Ukraine”. The study revealed that the administration of MSCs induced the regeneration of previously completely destructed nasal mucous tissues in a model of progressive atrophic rhinitis.

The effects of transplantation of human umbilical cord MSCs on the morphological characteristics of the nasal mucosa (NM) in mice with experimental atrophic rhinitis (Staining H&E, х200):

А, В – NM in mice of the experimental group, after the transplantation of umbilical cord MSCs 1(A) and 2 (B) months after transplantation;
C- normal NM in mice of the negative control group;
D- NM in mice with experimentally induced atrophic rhinitis.

collaboration with Romodanov Neurosurgery Institute of NAMS of Ukraine

A study of therapeutic effects of MSCs transplantation for the treatment of multiple sclerosis

The possibility of using MSCs to treat multiple sclerosis was demonstrated in the experimental model (experimental allergic encephalomyelitis). The studies were performed in collaboration with SI “Romodanov Neurosurgery Institute of NAMS of Ukraine”. The data obtained revealed that the transplantation of MSCs led to the complete disappearance of clinical symptoms and the restoration of injured neural cells.

Average clinical scores over time. Clinical scores of experimental rats after the human MSCs transplantation, or administration of the recombinant interleukin-10, and after their combined use was compared to untreated animals with the disease.

Morphological changes in the structure of the nerve root of the spinal cord in the lumbar region. Thionine staining, x800.

collaboration with Laboratory of Cell Receptors Immunology, Palladin Institute of Biochemistry NAS Ukraine

A study of therapeutic effects of MSCs transplantation for the treatment of Alzheimer’s disease

The Department of Cell Regulatory mechanisms in collaboration with Laboratory of Cell Receptors Immunology, Palladin Institute of Biochemistry NAS Ukraine, and State Institute of Genetic and Regenerative Medicine, NAMS Ukraine, performed the studies on the therapeutic effects of MSC transplantation for treatment of Alzheimer disease.

Previously, Laboratory of Cell Receptors Immunology, Palladin Institute of Biochemistry NAS Ukraine, showed that bacterial lipopolysaccharide (LPS) injection leads to the AD-like symptoms in mice. Our work in collaboration demonstrated, that transplantation of human Wharton jelly MSC (WJ-MSC), simultaneously with LPS injection, prevented α7 nicotinic acetylcholine receptors decrease, amyloid beta peptide accumulation in brain, as well as episodic memory decline. An injection of both WJ- MSCs and WJ-MSC-derived conditioned medium, 3 weeks after LPS injection, when memory decline was already observed in mice, both increased α4, α9 and β2 nAChR subunits in their brains and brain mitochondria. WJ-MSCs also up-regulated β4 subunits, decreased the level of Aβ and restored memory of LPS-treated mice and significantly improved mitochondria sustainability to Ca2+. It was demonstrated that WJ-MSCs supported memory of LPS-treated mice for at least 3 weeks, though the effect of a single injection of conditioned medium disappeared after 2 weeks.

Another set of experiments demonstrated that after hMSC (α7+ cells) transplantation to α7−/− mice, α7-specific signals could be detected on day 14 in the brain. Both injection of hMSCs and hMSCs-conditioned medium (though no α7- signal was detected after it) improved episodic memory of α7−/−mice and decreased cytochrome c release from their brain mitochondria under the effect of Ca2+. It was demonstrated that either MSCs or conditioned media increased IL-6 level in the brain, which coincided with the improvement of episodic memory.

The effect of either hMSCs or mMSCs on α7 nAChR (A) or Aβ (1–42) (B) levels in the brain, memory impairment (C) and cyt c release from the brain mitochondria (Mch) under the effect of Ca2+ (D). Ctrl – samples of non-treated mice. Each column (A–C) or point (D) corresponds to M ± SD of data for separate mice in each group (n = 8). **p < 0.005; ***p < 0.0005 compared to Ctrl. Designations of columns are similar in (A) and (B).

Senior investigator

Svitlana Rymar

Study of restorative potential of MSCs transplantation in the therapy of liver cirrhosis

Mesenchymal stem cells are the most promising regenerative medicine tool for treating various diseases, including liver disease, although the exact mechanism of their therapeutic action remains unclear. It was found that MSCs are captured by the lungs after systemic transplantation, quickly disappear, and are not detected at the site of injury but at the same time exhibit an obvious therapeutic effect. Comparison of the MSC efficiency depending on the route of their administration may shed light on the mechanisms involved in implementing MSC therapeutic potential. In this work, we compared the therapeutic effects of human umbilical cord MSCs (hUC-MSCs) administered systemically and intraperitoneally in the form of MSCs encapsulated in alginate capsules in a CCl₄ - induced model of liver cirrhosis in rats. Our study showed that both treatments resulted in liver recovery. MSC transplantation by two different routes led to a decrease in collagen deposition, the disappearance of the fibrous area by the 13th week, and the normalization of the morphometric parameters of liver parenchyma cells (Fig.1 ).

Histological analysis of liver tissue sections after hUC-MSC transplantation to rats with CCl₄-induced liver cirrhosis. Van Giezon staining and H&E staining of rat liver sections 8 and 13 weeks after MSCs transplantation: I group - stained liver sections after transplanted MSCs (Van Giezon staining); II group - stained liver sections after transplantation of encapsulated MSCs in alginate capsules (Van Giezon staining); III group - stained liver sections after transplanted MSCs (H&E staining). A - negative control group (normal); B- sham control group with induced liver cirrhosis; C. - regeneration of the liver parenchyma of the groups that received intravenous and intraperitoneal injections of MSCs after 8 weeks; D. - regeneration of the liver parenchyma of the groups that received intravenous and intraperitoneal injections of MSCs after 13 weeks. Scale bar: A – D - 100μm (Van Giezon staining), A - D - 400μm (H&E staining).

In addition, the expression of some genes (EGF, alpha SMA, GFAP) which is activated in liver injury, decreased to the level observed in negative control animals. However, a detailed study of liver recovery in dynamics showed that encapsulated MSCs led to faster normalization in several parameters of the liver tissue. Our results showed that human umbilical cord MSCs effectively exhibit their therapeutic properties when using both transplantation methods. However, intraperitoneal administration of encapsulated MSCs accelerated the process of liver regeneration.

The expression of EGF (A) and α-SMA (B) genes: 0 point - 13 weeks after intraperitoneal administration of CCl₄ to rats; 3, 8 and 13 weeks - after intravenous administration of MSCs and intraperitoneal administration of encapsulated MSCs to rats with liver cirrhosis. Results are expressed as mean±SEM. P values were calculated by ANOVA with Tukey’s test.

^* p<0.01 – negative control vs. sham control

^# p<0.01 – sham control vs. MSC administration

^## p<0.01 – sham control vs. encapsulated MSC administration

^### p<0.01 – MSC administration vs. encapsulated MSC administration

The expression of GFAP (A) and eNOS (B) genes: 0 point - 13 weeks after intraperitoneal administration of CCl₄ to rats; 3, 8 and 13 weeks - after intravenous administration of MSCs and intraperitoneal administration of encapsulated MSCs to rats with liver cirrhosis. Results are expressed as mean±SEM. P values were calculated by ANOVA with Tukey’s test.

* p<0.01 – negative control vs. sham control

# p<0.01 – sham control vs. MSC administration

## p<0.01 – sham control vs. encapsulated MSC administration

collaboration with L.V.Gromashevsky Institute of Epidemiology and Infectious Diseases of NAMS of Ukraine

Study of the possibilities of using the therapeutic potential of MSCs in the treatment of viral injury using influenza A virus-induced pneumonia model

The studies were performed in collaboration with SI “L.V. Gromashevsky Institute of Epidemiology and Infectious Diseases of NAMS of Ukraine” and Bogomolets National Medical University. The data obtained revealed differences in effects obtained on the different stages of viral disease. If MSCs were administrated during the acute phase of the disease, a unique interacting, the highly dynamic triple system would be created, namely organism-virus-MSC. Properties of the system were studied on the model of severe influenza A-induced pneumonia in mice. An animal mortality rate in the control group inoculated with the virus only was 100 %. The development and course of the pathological process in the group were typical of severe influenza A-induced pneumonia (Fig.A).

On Day 5 of the experiment, there was diffuse exudative serofibrinous inflammation in the lungs of the animals with the hemorrhagic component (1). Lymphocytes, macrophages, and desquamated alveolocytes were found in the lumens of alveoli (2). Serohemorrhagic exudate (b).Staining- H&E, х200

In experimental groups, a dose of MSCs administered had little effect on disease progression and survival. The administration routes had a more significant impact on these parameters. And the development and course of the pathological process in such animals differed significantly from that in the viral control group (Fig. B).

Influenza A virus + MSCs (1x106 cells/ administered intravenously)

On Day 7 of the experiment, there was significant diffuse lymphoid-macrophage infiltration, desquamation of alveolocytes in the parenchyma of the lungs of the dead animals, in the lumens of bronchi, alveoli and between alveolar septa (1). Blood vessels were overfilled with blood and had RBC hemolysis areas (2).

Staining- H&E, х200

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