A television apparatus designed for analysis of the microcirculation (at magnifications of × 30–160) was used to investigate the density of the microvascular network in the pia mater of the rat sensorimotor cortex after intracerebral transplantation of mesenchymal stem cells or (as controls) stem cell cultivation medium or physiological saline. The results showed that intracerebral transplantation did not alter the density of the microvascular network in the pia mater of the ipsilateral hemisphere, while there were no changes in either hemisphere in the control group. Transplantation of mesenchymal stem cells led to increases in the density of the microvascular network of the pia mater of the contralateral hemisphere by a factor of 1.8 as compared with control animals; the number of arterioles in this zone was 2.5 times greater than that in intact rats.

We studied the effect of intracerebral transplantation of bone marrow mesenchymal stem cells on microcirculation (density of microvascular network and reactivity of arterioles) in the pia mater of 2-3-month-old rats. It was found that after transplantation of mesenchymal stem cells, the density of pial microcirculatory network in the contralateral hemisphere significantly increased (by 1.7 times; p <0.05) in comparison with both intact animals and controls. The number of arterioles in the studied area increased most markedly (by ~2.5 times; p <0.05) in comparison with other groups. Intracerebral transplantation of mesenchymal stem cells or conditioned culture medium (α-MEM) had no effect on reactivity of pial arterioles.

Study aim - to elucidate possibilities of the use of precision administration of mononuclear bone marrow cells (MBMC) for the treatment of myocardial ischemia and heart failure. "Intramyocardial Multiple Precision Administration of Mononuclear Bone Marrow Cells in the Treatment of Myocardial Ischemia" was a double blind randomized placebo controlled study in which we included patients more or equal 6 months after Q-wave myocardial infarction with systolic myocardial dysfunction (ejection fraction <35%), not requiring myocardial revascularization, receiving stable optimal medical therapy for more or equal 8 weeks, and with implanted cardioverter-defibrillator. Transplantation of MBMC was guided by fluoroscopy and tridimensional NOGA XP Cardiac Navigation System. For assessment of efficacy of the method we used surrogate end points: decrease of number of fixed perfusion defects according to SPECT data and improvement of regional myocardial contractility according to data of echocardiography. Results of dynamic observation of the first experience of MBMC administration are presented in this paper.

This study aimed to investigate the effect of bone marrow- and adipose tissue-derived mesenchymal stem cell (BM-MSC and AD-MSC respectively) transplantation on left ventricular function and infarct area (IA) in the rat model of ischaemic heart failure. In anaesthetized Wistar rats, the left coronary artery (LCA) was occluded for 40 min with subsequent reperfusion for 7 days. Seven days following surgery, the animals with LCA occlusion/reperfusion were randomized into three groups: (i) Controls received intramyocardial injection of vehicle at three different locations within the peri-infarct zone, (ii) BM-MSC: cells were injected in the same way as in previous group (10(6) ), (iii) AD-MSC: using the same protocol as used in the BM-MSC group. In addition there was also a sham-treated group that had no injection. Two weeks following MSC transplantation, the hearts were isolated and perfused according to the Langendorff method followed by 30-min global ischaemia and 90-min reperfusion. After this IA was determined histologically. During Langendorff perfusion initial and postischaemic LV functions were the same in all groups although LV pressure at the 10th minute of reperfusion was higher in the AD-MSC group compared to controls. However, LV pressure during 30-min global ischaemia was significantly higher in BM-MSC as compared to controls and AD-MSC. The sham treated animals showed the same results as those seen with BM-MSC. Thus, BM-MSC transplantation, in contrast to transplantation of AD-MSC, resulted in better preservation of the LV ability to contract during ischaemia. Furthermore, IA was significantly smaller in BM-MSC group as compared to the controls and the AD-MSC groups. Thus this study has demonstrated that treatment with BM-MSC both ameliorates LV function and reduces histological scar size.

To assess safety and tolerability of treatment with autologic multipotent mesenchymal stem cells (MSC) in multiple sclerosis (MS), we have obtained autologic red bone marrow-derived MSC from 8 patients. Proliferation, immunophenotype and caryotype of MSC, their sterility, the absence of hemopoetic cells, chromosomal aberrations and signs of aging were controlled during the cell growth. The inverse injection of MSC in patient's blood was conducted in accordance to the elaborated protocol in a short intravenous infusion in dose 2.0 x 10(6)/kg of body mass once in 30 days. The duration of treatment was from 4 to 8 months. The efficacy of treatment was assessed after 4, 8 and 12 months. All patients tolerated repeated intravenous infusions of autologic MSC well with no significant side-effects as in the early as well in the remote periods of treatment. The distinct positive effect was seen in some cases 2 months after the beginning of treatment. The improvement of 0.5 point on EDSS was seen in 5/8 patients after 4 months. After 12 months, the improvement of 0.5-1 point on EDSS was seen in 6/8, stabilization in 1/8, progression in 1/8. These results revealed the safety of the elaborated protocol of treatment and the moderate clinical efficacy of treatment in non-curable patients or those with poor response to treatment that suggested continuing the study and enrollment of new patients.

Bone marrow (BM) and subcutaneous adipose tissue (Ad) are both considered being prospective sources of MSC for therapeutic applications. However, functional properties and therapeutic efficacy of MSC derived from different tissues of the same patient are still poorly investigated. In our study, BM-MSC and F-MSC cultures from 43 adult donors were evaluated in successive passages for immunophenotype, secretion of VEGF, SDF1, MCP1, IL6 and TGFβ1, frequency of colony-forming units (CFU-F), frequency of adipo- and osteo-progenitors (CFU-Ad, CFU-Ost), and for onset of in vitro replicative senescence. We have demonstrated that at early passages (P2-P4 or up to 14-15 in vitro population doublings) BM- and Ad- derived MSC cultures are comparable in such important characteristics as proliferation rate (population doubling time: 3,4±0,2% in BM-MSC, 3±0,3 % in F-MSC), clonogenity (CFU-F frequency: 32±5% in BM-MSC, 31±5% in F-MSC), differentiation potential (CFU-Ad frequency: 10,4±2% in BM-MSC, 13±3% in F-MSC; CFU-Ost frequency: 18,5±5,5% in BM-MSC, 18±5% in F-MSC), but differ significantly in abundance of CD146+ fraction within the sample (25±5% in BM-MSC, 7±3 % in F-MSC) and in a level of VEGF, SDF-1, MCP1 and TGFβ1 secretion. We have also demonstrated that BM-MSC enter senescence after P3-4 while most of F-MSC did not show senescence features up to P6-8. Together, these data demonstrate that specific properties of MSC from different sources should be always taken into account, when developing and optimizing the specific protocols for MSC expansion and evaluation for each particular clinical application.

We studied the effect of intracerebral transplantation of mesenchymal stem cells on the density of pial arterioles in rat brain cortex. It was shown that intracerebral transplantation although causes damage to about half the area of the ipsilateral pia mater, does not change the density of the microvascular network neither in border region of the injured tissue, nor in the contralateral hemisphere. Intracerebral transplantation of mesenchymal stem cells promoted arteriogenesis in the pia mater of the contralateral hemisphere: density of arterioles in this area was significantly (by about 2.5 times higher) than in other experimental animals.

The effects of the allogeneous demineralized bone transplants, seeded with bone marrow-derived multipotent mesenchymal stromal cells CMSCsJ, on the healing of the damaged articular cartilage and the subchondral bone were studied. It was shown that the use of allogeneous as well as autologous MSCs on the demineralized bone transplants and also on the transplants combined with type I collagen gel promote the remodeling of the regenerating tissue and the recovery of the histotypic cartilaginous and osseous structures in comparison with the articular surface regeneration without any therapy or after the procedure of the mosaic chondroplasty. Nevertheless, the positive effects of cell therapy were not clinically prominent, which means most probably in that case that the generally accepted experimental model is not adequate in full measure, but the approach used is promising.

Abstract Abstract 3856 Cell therapy with MSCs appears to be a promising method for treatment of different disorders. Bone marrow and fat tissue are both considered as a prospective source of MSCs for therapeutic applications. However, the differences in functional properties of specific populations of MSC derived from these two tissues of the same patient are still poorly investigated. Patients and methods: 23 cardiovasular patients and 4 healthy donors were involved in this study. All patients were enrolled in programme funded by EU FP7. MSC cultures from BM (BM-MSC) and subcutaneous adipose (F-MSC) of the same patient were evaluated in successive passages for immunophenotype (FACS analysis), frequency of colony-forming units (CFU) in MSC population, frequency of adipo- and osteo-progenitors (CFU-Ad, CFU-Ost) in the same population and for dynamic of changes in these properties with successive passage. CFU, CFU-Ad and CFU-Ost were studied by limiting dilution assay followed by induction of adipo- or osteo- differentiation as described (Mitchell et al. Stem Cells 2006) with some modifications. Cell suspension was serially diluted two folds across the 8 columns of 96-well plates, resulting in columns containing from 50 to 0,39 cells per well. After 10 days of culture the number of positive and negative wells was determined for each cell concentration and CFU frequency was calculated. Then plates were induced to undergo adipogenesis and osteogenesis and CFU-Ad was determined by Oil Red staining and CFU-Ost by Alizarin Red staining after 14 and 21 days respectively. All calculations were performed as for CFU. Results. Significant difference was observed in immunophenotype of MSC derived from different tissues of the same donor: while both BM- and F-MSC were positive for stromal cell-associated markers CD105, CD90 and CD73 and were negative for hematopoietic lineage cells markers CD34, CD19, CD14, CD45, the population of CD146+ cells was more abundant in BM-MSC than in F-MSC at passage 1 (39,6%± 8,3% vs 5,6 %± 1,8%; p<0,005), this population of cells further declined in successive passages by passage 5 (17,7%± 6,9% in BM-MSC vs 1,5%± 0,65% in F-MSC; p<0,01); (Figure 1). To further characterize MSC derived from BM and F we studied the frequency of CFU at passages P1-P4, and CFU-Ad and CFU-Ost at passages 2 and 4. Results are shown in Tables 1 and 2. These data support the idea that in vitro expanded BM- and F- derived MSC differ in their properties. While the frequency of CFU in BM derived MSC population declined by as much as 4-fold by passage 4, there was 2-fold increase in frequency of CFU in Ad-MSC over the same period of culturing and this frequency remain unchanged up to passage 7 (data not shown). These data are consistent with our observation that cultures of BM-MSC showed first signs of senescence (senescence-associated β-galactosidase activity) as early as at passage 4,3±0.5 while the first signs of senescence in Ad-MSC cultures were seen at passage 6,67±0,66 (p<0,02). We assume that early decline in CFU frequency in BM-MSC in vitro is because of cell senescence. Importantly, the difference in CFU-Ad and CFU-Ost was discovered: frequency of both declined significantly in BM-MSC by passage 4, while frequency of Ad- and Ost- lineage progenitors remain unchanged in F-MSC cultures. Conclusion: For a first time a broad study was performed to compare MSC derived from BM and adipose tissues for immunophenotype, self-renewal and differentiation potential. We have found that BM-MSC and F-MSC differ significantly in all these characteristics. Differences in BM- and F-MSC could be explained by their ontogeny and/or different microenvironment in “parent” tissue. These variations could affect their efficacy in different therapeutic applications. Disclosures: No relevant conflicts of interest to declare.

The study of the dynamic of morphological changes in the brain after ischemic stroke is very important for the preclinical trial of mesenchymal stem cell (MSC) therapy for this widespread disease. Experiments were carried out in inbred Wistar-Kyoto rats. MSCs were isolated, expanded in culture, and labeled with the vital fluorescent dye PKH-26. Animals were subjected to middle cerebral artery occlusion (MCAO), followed by an injection of 5 × 10 6 rat MSCs into the tail vein on the day of MCAO. Control group animals received PBS injection (negative control). Animals were sacrificed at 1, 2, 3, and 5 days and 1, 2, 4, and 6 weeks after the operation. MSCs were revealed in the brain on the third day after transplantation as being distributed around brain vessels both in the ipsilateral and contralateral hemispheres. This pattern of distribution remained unchanged throughout six weeks of observation. It was demonstrated that the inflammation process and scar formation in the cell therapy group were progressing at a rate 25–30% faster than in the control group. MSC transplantation stimulated endogenous stem cell proliferation in the subependimal zone of lateral ventricles (subventricular zone). In addition, MSC injection caused a neuroprotecting effect; most penumbra neurons retained their structure in cell therapy group, whereas in control group, animal penumbra neurons died or showed signs of serious damage.