Angiogenic properties of aged adipose derived mesenchymal stem cells after hypoxic conditioning

Background: Mesenchymal stem cells derived from adipose tissue (ADSC) are multipotent stem cells, originated from the vascular-stromal compartment of fat tissue. ADSC are used as an alternative cell source for many different cell therapies, however in ischemic cardiovascular diseases the therapeutic benefit was modest. One of the reasons could be the use of autologous aged ADSC, which recently were found to have impaired functions. We therefore analysed the effects of age on age markers and angiogenic properties of ADSC. Hypoxic conditioning was investigated as a form of angiogenic stimulation. Methods: ADSC were harvested from young (1-3 month), adult (12 month) and aged (18-24 month) mice and cultured under normoxic (20%) and hypoxic (1%) conditions for 48 h. Differences in proliferation, apoptosis and telomere length were assessed in addition to angiogenic properties of ADSC. Results: Proliferation potential and telomere length were decreased in aged ADSC compared to young ADSC. Frequency of apoptotic cells was higher in aged ADSC. Gene expression of pro-angiogenic factors including vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and hepatic growth factor (HGF) were down-regulated with age, which could be restored by hypoxia. Transforming growth factor (TGF-b) increased in the old ADSC but was reduced by hypoxia. Expression of anti-angiogenic factors including thrombospondin-1 (TBS1) and plasminogen activator inhibitor-1 (PAI-1) did increase in old ADSC, but could be reduced by hypoxic stimulation. Endostatin (ENDS) was the highest in aged ADSC and was also down-regulated by hypoxia. We noted higher gene expression of proteases system factors like urokinase-type plasminogen activator receptor (uPAR), matrix metalloproteinases (MMP2 and MMP9) and PAI-1 in aged ADSC compared to young ADSC, but they decreased in old ADSC. Tube formation on matrigel was higher in the presence of conditioned medium from young ADSC in comparison to aged ADSC. Conclusions: ADSC isolated from older animals show changes, including impaired proliferation and angiogenic stimulation. Angiogenic gene expression can be partially be improved by hypoxic preconditioning, however the effect is age-dependent. This supports the hypothesis that autologous ADSC from aged subjects might have an impaired therapeutic potential

Angiogenic properties of aged adipose derived mesenchymal stem cells after hypoxic conditioning

Background: Mesenchymal stem cells derived from adipose tissue (ADSC) are multipotent stem cells, originated from the vascular-stromal compartment of fat tissue. ADSC are used as an alternative cell source for many different cell therapies, however in ischemic cardiovascular diseases the therapeutic benefit was modest. One of the reasons could be the use of autologous aged ADSC, which recently were found to have impaired functions. We therefore analysed the effects of age on age markers and angiogenic properties of ADSC. Hypoxic conditioning was investigated as a form of angiogenic stimulation.Methods: ADSC were harvested from young (1-3 month), adult (12 month) and aged (18-24 month) mice and cultured under normoxic (20%) and hypoxic (1%) conditions for 48 h. Differences in proliferation, apoptosis and telomere length were assessed in addition to angiogenic properties of ADSC.Results: Proliferation potential and telomere length were decreased in aged ADSC compared to young ADSC. Frequency of apoptotic cells was higher in aged ADSC. Gene expression of pro-angiogenic factors including vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and hepatic growth factor (HGF) were down-regulated with age, which could be restored by hypoxia. Transforming growth factor (TGF-β) increased in the old ADSC but was reduced by hypoxia.Expression of anti-angiogenic factors including thrombospondin-1 (TBS1) and plasminogen activator inhibitor-1 (PAI-1) did increase in old ADSC, but could be reduced by hypoxic stimulation. Endostatin (ENDS) was the highest in aged ADSC and was also down-regulated by hypoxia. We noted higher gene expression of proteases system factors like urokinase-type plasminogen activator receptor (uPAR), matrix metalloproteinases (MMP2 and MMP9) and PAI-1 in aged ADSC compared to young ADSC, but they decreased in old ADSC. Tube formation on matrigel was higher in the presence of conditioned medium from young ADSC in comparison to aged ADSC.Conclusions: ADSC isolated from older animals show changes, including impaired proliferation and angiogenic stimulation. Angiogenic gene expression can be partially be improved by hypoxic preconditioning, however the effect is age-dependent. This supports the hypothesis that autologous ADSC from aged subjects might have an impaired therapeutic potential. © 2011 Efimenko et al; licensee BioMed Central Ltd

NEUROPSYCHOLOGY OF COGNITIVE DYSFUNCTIONS IN SENILE DEMENTIA WITH LOBAR SYMPTOMS PREVALENCE

Цель: изучить нейропсихологические аспекты когнитивных нарушений у пациентов с преобладанием лобной симптоматики в сравнении с когнитивными нарушениями у пациентов сенильной деменцией иной структуры.Материалы и методы. Обследовано 48 человек, страдающих сенильной деменцией. Основную группу с преобладанием лобной симптоматики составили 24 больных, из них 12 (50%) женщины, средний возраст – 84±12 лет. Контрольную группу с иной структурой сенильной деменции также составили 24 больных, из них 12 (50%) женщины, средний возраст – 83±13 лет.Исследование проводилось с разрешения этического комитета ФГБОУ ВО ЮУГМУ. Каждый участник (его законный представитель) подписывал стандартную форму добровольного согласия для участия в медицинском исследовании.Использовались Батарея тестов для оценки лобной дисфункции (БЛД); Краткая шкала оценки психического статуса (MMSE); Монреальская шкала оценки когнитивных функций (MоСA); Схема нейропсихологического обследования Е.Д. Хомской: пробы на исследование праксиса, речи, слухо- и зрительно-моторного гнозиса.Непараметрическое сравнение результатов проводилось при помощи критерия Манна-Уитни, параметрическое – при помощи углового преобразования Фишера (УПФ) в среде статистической программы SPSS v.20.Результаты. Констатирующий эксперимент раскрыл характер нейропсихологических паттернов когнитивных нарушений, свойственных больным сенильной деменцией с преобладанием лобной симптоматики в клинической картине расстройства. Выявлены сравнительные особенности влияния лобной симптоматики на характер когнитивных нарушений в структуре сенильной деменции. Сформированы предпосылки для модернизации существующих программ реабилитации данной категории больных.Заключение. Таким образом, выявлены предпосылки для динамической оценки эффективности лечебно-реабилитационных мероприятий у больных сенильной деменцией с преобладанием лобной симптоматики.Background: comparative cognitive dysfunctions in lobar and non-lobar senile dementia patients (constitute experiment).Materials & Methods: 48 senile dementia patients, equal 24 in lobar (84±12 mean age) and non-lobar (83±13 mean age) groups, 50% female. Ethic committee granted. MMSE, MoCa, Lobar dysfunction battery (LDB), E. Homskaya probes (praxis, speech, audio- and visual motor gnosis) were used. Mann-Whitney and Fisher criteria for comparison in SPSS v. 20 were used.Results: constitute experiment presents neuropsychological patterns of cognitive dysfunctions in senile dementia patients with lobar symptoms prevalence. Comparative measures of lobar induction on cognitive dysfunctions are discussed.Conclusion: thus, even if a non-statistically meaning year of life is The Factor of senile dementia (and we’ll try to equalize it in process of further investigations), the basis for dynamic research of efficacy of treatment and rehabilitation of this patients is grounded

“Cell-Free Therapeutics” from Components Secreted by Mesenchymal Stromal Cells as a Novel Class of Biopharmaceuticals

Regenerative medicine is a fast growing multidisciplinary field aiming at the regeneration or replacement of damaged cells, tissues, or organs. Adult multipotent mesenchymal stromal cells (MSCs) are often used as a principal therapeutic tool in this field. Along with differentiation potency, MSCs secrete a wide spectrum of paracrine factors and extracellular vesicles participating in tissue repair and regeneration. Thus, for injuries that require trophic stimulation, cell survival support, and/or resident stem cells activation to be restored, one can apply MSC-conditioned medium, a combination of products and extracellular vesicles in cell culture growth medium, secreted by MSC. It could mediate most of beneficial regenerative effects of MSC without possible side effects of using MSC themselves. However, before the clinical application of this promising biopharmaceutical, several issues such as manufacturing protocols, quality control, and others must be addressed. Subsequently, we highlight the questions considering donor material variability, manufacturing, cell culture medium and auxiliary components selection, and potency tests development

Abstract P-41: Contribution of Matrix-bound Vesicles Produced by Mesenchymal Stromal Cells in the Differentiation of Multipotent Stem Cells in vitro

Background: According to the current view on the extracellular matrix (ECM) composition and functions, it includes not only structural proteins and components of cell adhesion, but also various deposited components, including enzymes involved in ECM remodeling, growth factors, and matrix-bound vesicles (MBV). MBV can presumably participate in the formation of a specific microenvironment for stem cells and regulate their differentiation. However, the contribution of MBV to these processes remains poorly understood. In our work, we evaluated the effects of MBV within native ECM produced by mesenchymal stromal cells (MSCs) cultured in cell sheet on multipotent stem cell differentiation. Methods: We isolated MBV from decellularized MSC-produced ECM by treatment with the following enzymes: collagenase, hyaluronidase, or trypsin, and centrifugation on 1000 kDa filters. The nanostructure and relative size in each sample were observed using TEM. The particle size and concentration were also studied with NTA. In addition, the obtained MBV were examined for the presence of key exosome markers using Western blot. Then we investigated the effect of MBV on the formation of capillary-like structures by endothelial cells (in vitro model of angiogenesis) as well as on the differentiation of primary MSCs isolated from human adipose tissue in the adipogenic, osteogenic, and chondrogenic directions. Results: As a result of comparative analysis of isolation protocols, it was shown that all MBV samples had the characteristics of extracellular vesicles (EV), but differed in size and representation of exosomal markers. The MBV isolated from ECM did not stimulate the formation of capillary-like structures by endothelial cells, in contrast to EV secreted by MSCs to the conditioned medium, but maintained the viability of the endothelium. Isolated MBV stimulated osteogenic and adipogenic differentiation of MSCs similar to secreted EV. On the other hand, preincubation of MSCs with MBV leads to reorganization of cell monolayer to spheroid-like structures during chondrogenic differentiation. Conclusion: Here, we developed the protocol of isolation of MBV from ECM that have distinguished characteristics and functional activity

Extracellular matrix-induced signaling pathways in mesenchymal stem/stromal cells

Abstract The extracellular matrix (ECM) is a crucial component of the stem cell microenvironment, or stem-cell niches, and contributes to the regulation of cell behavior and fate. Accumulating evidence indicates that different types of stem cells possess a large variety of molecules responsible for interactions with the ECM, mediating specific epigenetic rearrangements and corresponding changes in transcriptome profile. Signals from the ECM are crucial at all stages of ontogenesis, including embryonic and postnatal development, as well as tissue renewal and repair. The ECM could regulate stem cell transition from a quiescent state to readiness to perceive the signals of differentiation induction (competence) and the transition between different stages of differentiation (commitment). Currently, to unveil the complex networks of cellular signaling from the ECM, multiple approaches including screening methods, the analysis of the cell matrixome, and the creation of predictive networks of protein–protein interactions based on experimental data are used. In this review, we consider the existing evidence regarded the contribution of ECM-induced intracellular signaling pathways into the regulation of stem cell differentiation focusing on mesenchymal stem/stromal cells (MSCs) as well-studied type of postnatal stem cells totally depended on signals from ECM. Furthermore, we propose a system biology-based approach for the prediction of ECM-mediated signal transduction pathways in target cells. Video Abstrac

The Power of Gene Technologies: 1001 Ways to Create a Cell Model

Modern society faces many biomedical challenges that require urgent solutions. Two of the most important include the elucidation of mechanisms of socially significant diseases and the development of prospective drug treatments for these diseases. Experimental cell models are a convenient tool for addressing many of these problems. The power of cell models is further enhanced when combined with gene technologies, which allows the examination of even more subtle changes within the structure of the genome and permits testing of proteins in a native environment. The list and possibilities of these recently emerging technologies are truly colossal, which requires a rethink of a number of approaches for obtaining experimental cell models. In this review, we analyze the possibilities and limitations of promising gene technologies for obtaining cell models, and also give recommendations on the development and creation of relevant models. In our opinion, this review will be useful for novice cell biologists, as it provides some reference points in the rapidly growing universe of gene and cell technologies

Novel Potency Assay for MSC Secretome-Based Treatment of Idiopathic Male Infertility Employed Leydig Cells and Revealed Vascular Endothelial Growth Factor as a Promising Potency Marker

Idiopathic male infertility is a highly prevalent diagnosis in developed countries with no specific treatment options. Although empirical medical treatment is widely used to restore male fertility, its efficacy remains limited and inconclusively proven. Therefore, the development of novel therapeutic approaches in this field is a high-priority task. Since the failure of testicular microenvironment components might be involved in the pathogenesis of idiopathic male infertility, application of mesenchymal stromal cells (MSCs) as well as the MSC secretome is worth considering. Previously, we showed that the intratesticular injection of MSCs or the MSC secretome led to the recovery of spermatogenesis at least through replenishing the testicular microenvironment and its maintenance by MSC-secreted paracrine factors. However, the clinical use of such products has been limited to single trials to date. This may be due to the lack of relevant potency tests reflecting mechanisms of action of the MSC secretome in male infertility models. Based on the presumptive MSC secretome mode of action on the testicular microenvironment, we suggest a novel approach to test the potential efficacy of the MSC secretome for idiopathic male infertility treatment. It represents a potency assay based on evaluation of testosterone production by isolated Leydig cells. We demonstrated that the MSC secretome stimulated testosterone secretion by Leydig cells in vitro. We then hypothesized that among the major factors of the MSC secretome, vascular endothelial growth factor (VEGF) could be responsible for the observed effects, which we confirmed by the revealed correlation between the extent of stimulated testosterone production and VEGF concentration in the MSC secretome. The pilot results obtained from the doxorubicin-induced male infertility murine model also indicate the important impact of VEGF in the MSC secretome’s regenerative effects. Utilizing VEGF as a surrogate factor, a novel approach to study the potency of MSC secretome-based products for idiopathic male infertility treatment is suggested. Further validation is required for its implementation into the biopharmaceutical manufacturing process