The First Experience of Application the Umbilical Cord-Derived Human Autologous Mesenchymal Stromal Cells for the Rehabilitation Therapy of Premature Infants with Extremely Low Body Weight and Hypoxic-Ischemic Encephalopathy.

Two clinical cases of the use of cell therapy with umbilical cord-derived autologous mesenchymal stromal cells in the rehabilitation therapy of extremely premature newborns (27-28 weeks gestation, body weights 900 and 870 g, respectively) with hypoxic-ischemic encephalopathy are described. The girls were born by caesarean section; the 1-min Apgar score was 6 points. After resuscitation including artificial ventilation, stabilization of the condition was achieved against the background of the development of hypoxic-ischemic encephalopathy. Rehabilitation therapy included administration of umbilical cord-derived mesenchymal stromal cells harvested at birth. The cells were injected in a dose of 1.6-7 million/kg body weight at the age of 3, 6, 12 months (the first patient) and 3, 6, 9, 15 months (the second patient). Psychoneurological developmental delay was scored using the Hellbrügge scale. Cell therapy induced no significant adverse reactions and improved the psychomotor development of children.

Dichotomy of Local Th1- and Systemic Th2/Th3-Dependent Types of Immune Response in Rheumatoid Arthritis.

Biomarkers of activity of rheumatoid arthritis were analyzed in the blood serum and synovial fluid of affected joints in 84 patients. Significant differences between the serum and synovial fluid levels were revealed for 10 of 17 analyzed biomarkers; the levels of IgM and proinflammatory cytokines TNFα, IL-1ß, IFNγ, and IL-6 were higher in the synovial fluid. The concentration of IL-10 in the synovial fluid was also elevated. In the peripheral blood, the content of antinuclear antibodies, circulating immune complexes, and cytokines IL-4 and TGF-ß was elevated. These findings attest to the development of local Th1 type immune response in affected joints paralleled by compensatory elevation of immunosuppressive cytokine IL-10 and systemic Th2/Th3 type immune response (judging from peripheral blood parameters) in patients with rheumatoid arthritis.

The Use of Autologous Mesenchymal Stem Cells for Cell Therapy of Patients with Amyotrophic Lateral Sclerosis in Belarus.

We studied a new method of treatment of amyotrophic lateral sclerosis with autologous mesenchymal stem cells. Autologous mesenchymal stem cells were injected intravenously (intact cells) or via lumbar puncture (cells committed to neuronal differentiation). Evaluation of the results of cell therapy after 12-month follow-up revealed slowing down of the disease progression in 10 patients in comparison with the control group consisting of 15 patients. The cell therapy was safe for the patients.

Effect of platelet releasate on osteogenic differentiation of human mesenchymal bone marrow stem cells.

We studied the effect of platelet releasate on osteogenic differentiation of human mesenchymal BM stem cells. Histological staining and reverse transcription PCR analysis showed that addition of platelet releasate to osteogenic differentiation medium stimulated the formation of calcium ossificates and alkaline phosphatase and increased expression of osteopontin and alkaline phosphatase genes.

Non-viral engineering of skin precursor-derived Schwann cells for enhanced NT-3 production in adherent and microcarrier culture.

Genetic engineering of stem cells and their derivatives has the potential to enhance their regenerative capabilities. Here, dendrimer- and lipofection-based approaches were used for non-viral neurotrophin-3 (NT-3) over-expression in Schwann cells differentiated from skin precursors (SKP-SCs). A variety of dendrimers were first tested for transfection efficiency on HEK 293T cells, with PAMAMNH2 G4 found most effective and used subsequently for SKP-SCs transfection. Plasmid-based expression resulted in increased NT-3 release from SKP-SCs in both adherent and microcarrier-based culture. In a proof-of-concept study, the microcarrier/SKP-SCs were implanted into the injured nerve, and transfected cells were shown to detach, integrate into the nerve tissue and associate with regenerating axons. Virus-free systems for transient neurotrophin expression are a feasible and biologically safe option to increase the therapeutic value of stem cells and stem cell-derived cells in nerve repair strategies. Further work to develop bioprocesses for expansion of SKP-SCs on microcarriers in bioreactors is still needed.

[Highly efficient transfer and stable expression of two genes upon lentivirus transduction of mesenchymal stem cells from human bone marrow].

The efficiency of human bone marrow (BM) mesenchymal stem cell (MSC) transduction with a bicistronic lentivirus vector was estimated, and the stability of transgene expression in genetically modified MSCs was determined. First-passage BM MSCs were capable of efficient transduction with the bicistronic lentivirus vector. The transduction efficiency depended on the multiplicity of infection (MOI), being 64 +/- 6.5 and 88.6 +/- 2.9% at MOI 10 and 20, respectively. The lentivirus transduction efficiency proved independent on the number of passages of a BM MSC culture, and expression of the egfp and dsRed1 transgenes in genetically modified MSCs remained stable for one month of culturing. A comparison showed that the level of egfp and dsRed1 transgene expression was preserved upon hepatogenic differentiation in vitro. The results provide a basis for further development of multigenic modification of human BM MSCs for research and/or therapeutic purposes.

Neurons and stromal stem cells as targets for polycation-mediated transfection.

Expression of transgenes in neurons and stromal/mesenchymal stem cells (MSC) can greatly enhance their therapeutic potential. In transfection experiments, we studied properties of linear and branched (dendrimers) polycations as transgene delivery vehicles. Linear polyethyleneimine transfected neurons, but was ineffective in MSC. Polyamidoamine dendrimers showed greater transfection efficiency and mean GFP fluorescence intensity compared to phosphorus dendrimers of the same (4th) generation. Expression of neurotrophic factor BDNF in MSC transfected with polyamidoamine dendrimers was also by more than 10 times higher.

Hepatogenic potential of human bone marrow and umbilical cord blood mesenchymal stem cells.

Conditions of human BM and umbilical cord blood MSC in vitro differentiation in the hepatogenic direction were studied. Changes in cell morphology, phenotype, acquisition of the capacity to produce albumin and accumulate glycogen, express cytokeratin, alkaline phosphatase, and albumin mRNA indicated that BM and umbilical cord blood MSC differentiated in vitro into immature hepatocyte-like cells.

Neurogenic induction of human mesenchymal stem cells in fibrin 3D matrix.

We studied the potential of neurogenic induction of human bone marrow mesenchymal stem cells in fibrin-based 3D matrix. The best results were obtained after incubation of mesenchymal stem cells in fibrin 3D matrix in the presence of neurogenic medium containing EGF and bFGF growth factors. Under these conditions, most cells formed numerous branching processes and expressed neural cell marker ßIII-tubulin. Optimal combination of 3D matrix and neurogenic factors of the medium can provide new insight into neurogenic potential of mesenchymal stem cells, which can be used for the therapy of neural traumas and neurodegenerative diseases.

Fourth generation phosphorus-containing dendrimers: prospective drug and gene delivery carrier.

Research concerning new targeting delivery systems for pharmacologically active molecules and genetic material is of great importance. The aim of the present study was to investigate the potential of fourth generation (P4) cationic phosphorus-containing dendrimers to bind fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS), anti-neoplastic drug cisplatin, anti-HIV siRNA siP24 and its capability to deliver green fluorescent protein gene (pGFP) into cells. The interaction between P4 and ANS (as the model drug) was investigated. The binding constant and the number of binding centers per one molecule of P4 were determined. In addition, the dendriplex between P4 and anti-HIV siRNA siP24 was characterized using circular dichroism, fluorescence polarization and zeta-potential methods; the average hydrodynamic diameter of the dendriplex was calculated using zeta-size measurements. The efficiency of transfection of pGFP using P4 was determined in HEK293 cells and human mesenchymal stem cells, and the cytotoxicity of the P4-pGFP dendriplex was studied. Furthermore, enhancement of the toxic action of the anti-neoplastic drug cisplatin by P4 dendrimers was estimated. Based on the results, the fourth generation cationic phosphorus-containing dendrimers seem to be a good drug and gene delivery carrier candidate.

[Neurogenic differentiation of mesenchymal stem cells: a transgenic approach].

Mesenchymal stem cells (MSC), alongside with "traditional" osteogenic, chondrogenic and adipogenic differentiation potentials, are considered by many researches as capable of giving rise to neurogenic lineage as well. We overview transgenic approaches to the study of neurogenic differentiation of MSC, including expression of neurotrophic factors, signalling molecules and other transgenes with neurogenic properties.

[Gene therapy based on human mesenchymal stem cells: strategies and methods].

Major technologies of transient and stable transgene expression in hMSC are reviewed. Properties and efficiencies of recombinant lentiviruses, adenoviruses, AAV and baculoviruses used for hMSC transduction are compared. The aims oftransgenesis include directed differentiation of hMSC, function improvement, correction of pathology factors and proliferatrion control, as well as many basic research challenges.

[Use of LAK-cells and systemic chemotherapy with hyperthermia in the management of chemo-resistant tumors].

Tentative results of LAK-cell and whole-body hyperthermia (WBH) were evaluated in 19 children with advanced chemorefractory tumors. LAK-cells were obtained by extracorporeal incubation of peripheral blood lymphocytes: a germ-cell rhabdomyosarcoma was detected in 4, Askin's tumor--2--2, renal cell carcinoma--2 and miscellaneous--7. Autologous LAK-cells were infused twice: on completion of WBH as body temperature fell to as low as (+) 40 deg. C and on day after WBH. The latter was well tolerated. Complete or partial response to thermochemobiotherapy was reported in 8 patients. Overall 5-year survival was 43% (median follow-up--12.6 months).

Plasticity of human mesenchymal stem cell phenotype and expression profile under neurogenic conditions.

Human bone marrow MSC cultured in neurogenic medium containing EGF and FGFbeta demonstrated alteration of the phenotype and expression of neuronal precursor/early neuron markers nestin and NSE. Signals of expression of neuronal and oligodendroglial markers MAP-2, dm-20, and MBP were detected after prolongation of incubation in neurogenic medium to 2 weeks. Cells with neuronal morphology were immunopositive to early neuronal marker beta-III-tubulin. Replacement of neurogenic medium for alpha-MEM with 10% fetal calf serum induced reversion of the phenotype to that typical for human MSC. This indicates high plasticity of the phenotype and expression profile of neuronal markers in MSC cultured under neurogenic conditions or possibility of dedifferentiation of MSC reaching the stage of neuronal precursors/early neurons.

In vitro growth of human umbilical blood mesenchymal stem cells and their differentiation into chondrocytes and osteoblasts.

Conditions for culturing and differentiation of human umbilical blood mononuclear cells in vitro were studied. The growth of mesenchymal stem cells was attained in 31 of 54 (57.4%) umbilical blood samples and morphological and immunophenotypical authenticity of these cells was confirmed. Stimulatory effects of 20% AB(IV) human serum and transforming growth factor-beta (TGF-beta) on the growth of mesenchymal stem cells were demonstrated. Osteogenic cells formed in the presence of differentiation factors ascorbic acid, dexamethasone, and beta-glycerophosphate, while chondrogenic cells developed in the presence of dexamethasone, ascorbic acid, and TGF-beta. Differentiation of mesenchymal stem cells was confirmed by histochemical and molecular genetic tests.

Viral vectors for stable transduction of human mesenchymal stem cells: systems based on adeno-associated viruses and lentiviruses.

We compared the efficiency of transduction with lentivectors based on HIV-1 and adeno-associated viruses serotype 2 and stability of transgene expression in human mesenchymal bone marrow stem cells.

Study of expression and functional activity of P-GP membrane glycoprotein in children with acute leukemia.

We studied expression and functional activity of tumor cell P-gp in children with various leukemia variants and analyzed its prognostic role in acute lymphoblastic leukemia. Functional activity of P-gp increased in acute myeloid leukemia, relapses of acute lymphoblastic leukemia (in comparison with primary disease), and in a group of patients in whom no remission was attained. The survival of patients with acute lymphoblastic leukemia was lower in cases with increased expression and function of P-gp.

[Effect of interleukin-2 on apoptosis and function of human lymphocytes in in-vitro cytostatics culture].

In vitro experiments were conducted to determine the impact of interleukin-2 (IL-2) on apoptosis and function of cytostatic-cultured lymphoid (mononuclear) cells (MNC) of peripheral blood from healthy subjects and children with cancer. Neither slight or any effect on vepesid-16 or carboplatin--cultured MNC apoptosis, nor any phytohemagglutinin--induced proliferation was found. By contrast, in carboplatin- cultured MNC from healthy subjects, IL-2 significantly potentiated their toxicity for tumor cells by producing interferon-gamma. It was concluded that IL-2 predominantly supported MNC functional activity rather than inhibited lymphoid MNC apoptosis in in vitro culture with cytostatic drugs.

Rearrangements of IgH, TCRD and TCRG genes as clonality marker of childhood acute lymphoblastic leukemia.

AIM: Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient-specific targets for clonality studies and monitoring of acute lymphoblastic leukemia (ALL). THE AIMS of the study were to select the optimum panel of primers, evaluate incidence of particular types of monoclonal and oligoclonal gene rearrangements and observe alteration of rearrangement profile between diagnosis of ALL and subsequent relapse(s). METHODS: We used polymerase chain reaction (PCR) for amplification of junctional region of rearranged IgH, TCRD and TCRG genes in combination with heteroduplex analysis in polyacrylamide gel. RESULTS: TCRD gene rearrangements were detected in 64%, TCRG - in 45%, and IgH - in 79% of B-precursor ALL patients. For patients with T-ALL, TCRD gene rearrangements were found in 47%, TCRG gene - in 66%, and IgH - in 19% of cases. Evaluation of biallelic and oligoclonal rearrangements was performed in the study. The highest incidence of oligoclonal rearrangements - 25% was shown for IgH gene in patients with B-precursor ALL. Seven pair cases of patients with de novo leukemia and relapses were analyzed and revealed subclonal deviation in rearrangements of IgH or TCR genes during disease evolution. CONCLUSION: We propose a panel of 13 types of rearrangements (primer pairs) sufficient for tumor cell clonality detection in 96% of patients with ALL. Applications of PCR-based analysis of rearranged IgH, TCRD and TCRG genes for discrimination of mono- and oligoclonality and identification of the origin of relapse were demonstrated.

[Detection of rearranged immunoglobulin and T-cell receptor genes as a method for characterization of tumor cell clonality in acute lymphoblast leukemia in children]. / Vyiavlenie rearrazhirovki genov immunoglobulinov i T-kletochnogo retseptora kak metod kharakteristiki klonal'nosti opukholevykh kletok pri ostrykh limfoleikozakh u detei.

Selection of primers for polymerase chain reaction (PCR) as used in detection of the reconstructed genes of immunoglobulins and T-cells receptor in tumor cells in children with acute lymphatic leukemia is described in the paper. PCR potentialities and limitations in characterizing the monoclonality observed in primary acute lymphatic leukemia are demonstrated. Cross linkages of the heavy chain of immunoglobulins and of T-cell receptor were found between T- and B-linear acute lymphatic leukemia forms, but not between acute lymphatic leukemia forms and myeloleukemia in children.