Postnatal extra-embryonic tissues as a source of multiple cell types for regenerative medicine applications

Aim: We aimed to isolate and characterize the cell types which could be obtained from postnatal extra-embryonic tissues. Materials and Methods: Fresh tissues (no more than 12 h after delivery) were used for enzymatic or explants methods of cell isolation. Obtained cultures were further maintained at 5% oxygen. At P3 cell phenotype was assessed by fluorescence-activated cell sorting, population doubling time was calculated and the multilineage differentiation assay was performed. Results: We have isolated multiple cell types from postnatal tissues. Namely, placental mesenchymal stromal cells from placenta chorionic disc, chorionic membrane mesenchymal stromal cells (ChM-MSC) from free chorionic membrane, umbilical cord MSC (UC-MSC) from whole umbilical cord, human umbilical vein endothelial cells (HUVEC) from umbilical vein, amniotic epithelial cells (AEC) and amniotic MSC (AMSC) from amniotic membrane. All isolated cell types displayed high proliferation rate together with the typical MSC phenotype: CD73⁺CD90⁺CD105⁺CD146⁺CD166⁺CD34⁻CD45⁻HLA⁻DR⁻. HUVEC constitutively expressed key markers CD31 and CD309. Most MSC and AEC were capable of osteogenic and adipogenic differentiation. Conclusion: We have shown that a wide variety of cell types can be easily isolated from extra-embryonic tissues and expanded ex vivo for regenerative medicine applications. These cells possess typical MSC properties and can be considered an alternative for adult MSC obtained from bone marrow or fat, especially for allogeneic use

Endometrial stromal cells: isolation, expansion, morphological and functional properties

Aim: We aimed to study biological properties of human endometrial stromal cells in vitro. Materials and Methods: The endometrium samples (n = 5) were obtained by biopsy at the first phase of the menstrual cycle from women with endometrial hypoplasia. In all cases, a voluntary written informed consent was obtained from the patients. Endometrial fragments were dissociated by enzymatic treatment. The cells were cultured in DMEM/F12 supplemented with 10% FBS, 2 mМ L-glutamine and 1 ng/ml FGF-2 in a multi-gas incubator at 5% CO₂ and 5% O₂. At P3 the cells were subjected to immunophenotyping, multilineage differentiation, karyotype stability and colony forming efficiency. The cell secretome was assessed by BioRad Multiplex immunoassay kit. Results: Primary population of endometrial cells was heterogeneous and contained cells with fibroblast-like and epithelial-like morphology, but at P3 the majority of cell population had fibroblast-like morphology. The cells possessed typical for MSCs phenotype CD90⁺CD105⁺CD73⁺CD34⁻CD45⁻HLA⁻DR⁻. The cells also expressed CD140a, CD140b, CD146, and CD166 antigents; and were negative for CD106, CD184, CD271, and CD325. Cell doubling time was 29.6 ± 1.3 h. The cells were capable of directed osteogenic, adipogenic and chondrogenic differentiation. The cells showed 35.7% colony forming efficiency and a tendency to 3D spheroid formation. The GTG-banding assay confirmed the stability of eMSC karyotype during long-term culturing (up to P8). After 48 h incubation period in serum-free medium eMSC secreted anti-inflammatory IL-1ra, as well as IL-6, IL-8 and IFNγ, angiogenic factors VEGF, GM-CSF and FGF-2, chemokines IP-10 and MCP-1. Conclusion: Thus, cultured endometrial stromal cells meet minimal ISCT criteria for MSC. Proliferative potential, karyotype stability, multilineage plasticity and secretome profile make eMSC an attractive object for the regenerative medicine use

Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications

Aim: The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from human hair follicle for their further clinical use. Materials and Methods: Adult neural crest-derived multipotent stem cells were obtained from human hair follicle by explant method and were expanded at large-scale up to a clinically significant number. The resulted cell cultures were examined by flow cytometry and immunocytochemical analysis. Their clonogenic potential, ability to self-renewal and directed multilineage differentiation were also investigated. Results: Cell cultures were obtained from explants of adult human hair follicles. Resulted cells according to morphological, phenotypic and functional criteria satisfied the definition of neural crest-derived multipotent stem cells. They had the phenotype Sox2⁺Sox10⁺Nestin⁺CD73⁺CD90⁺CD105⁺CD140a⁺CD 140b⁺CD146⁺CD166⁺CD271⁺CD349⁺ CD34⁻CD45⁻CD56⁻HLA⁻DR⁻, showed high clonogenic potential, ability to self-renewal and directed differentiation into the main derivatives of the neural crest: neurons, Schwann cells, adipocytes and osteoblasts. Conclusion: The possibility of a large-scale expansion of adult neural crest-derived multipotent stem cells up to 40–200·106 cells from minimal number of hair follicles with retention of their phenotype and functional properties are the significant step towards their translation into the clinical practice

Tissue-engineered bone for treatment of combat related limb injuries

Aim: Based on our preliminary positive clinical results with use of cultured bone marrow-derived multipotent mesenchymal stem/stromal cells in traumatology, our aim was to develop living three-dimensional tissue-engineered bone equivalent transplantation technology for restoration of critical sized bone defects caused by combat related high energy trauma. Materials and Methods: To fabricate bone equivalent we used devitalized allogeneic bone scaffolds (blocks and chips) seeded with cultured autologous cells: bone marrow-derived multipotent mesenchymal stem/stromal cells in mix with periosteal progenitor cells and endothelial progenitor cells. Quality/identity of cell cultures was assured by donor and cell culture infection screening (immunofluorescence assay, polymerase chain reaction), flow cytometry (cell phenotype), karyotyping (GTG banding), functional assays (colony forming units analysis, multilineage differentiation assay). Bone defect treatment with bone equivalent application was fully completed in 39 combat-injured with 42 defects. New bone formation was assessed by the radiographic examination. Results: Casualties were included in a treatment program an average of 10.1 months after injury, provided the ineffectiveness of conventional surgery methods. All cell type cultures had a normal karyotype and appropriate phenotype, differentiation potential and functional properties, ~30% colony forming units frequency and hadn’t any signs of cell senescence. The fluorescein diacetate/propidium iodide combined staining and histology analysis of graft samples before transplantation showed their regular seeding with viable cells. Pathomorphological analysis of bone equivalent specimens 3–6 months post-op revealed the active remodeling processes and immature bone tissue formation. Bone defect restoration was observed 5–6 months post-op. Conclusion: The developed biotechnology of living three-dimensional tissue-engineered bone equivalent transplantation with overall effectiveness 90.4% allows restoring the bone integrity, forming new bone tissue in a site of bone defect, and significantly reducing the rehabilitation period of a patient

Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions

Aim. The work is aimed at obtaining the culture of neural crest-derived multipotent stem cells (NC-MSCs) in new culture conditions and to investigate their biological properties. Methods. NC-MSCs were grown from the explants of the bulge region of whisker follicle of adult mice. The cell cultures were examined by the following methods: sphere-forming assay, directed multilineage differentiation, CFU assay, immunocytochemistry, flow cytometry, RT-PCR. Results. The obtained NC-MSCs expressed the typical neural crest markers (nestin, Sox10 and Sox2) and were differentiated into adipocytes, osteoblasts and Schwann cells. Under our original growing conditions, the culture of NC-MSCs at the third passage had the following parameters: 66.8 % nestin+, 3.1 % ALDH brigth and 33.3 % clonogenic cells. The NC-MSCs growth rate depended on plating density. EGF and bFGF demonstrated a dose-dependent mitogenic action on NC-MSCs. Conclusions. The proposed approach permits the NC-MSC expansion with the maintenance of their main functional properties. Further optimization of the culture conditions will be based on the use of growth factors and low plating density.Мета. Отримати культуру мультипотентних стовбурових клітин – похідних нервового гребеня (МСК-ПНГ) за нових умов культивування та дослідити їхні біологічні властивості. Методи. МСК-ПНГ отримували з експлантів бульбарного району волосяного фолікула вібриса дорослих мишей. Культури клітин вивчали з використанням наступних методів: дослідження на здатність до сфероутворення, направлене мультилінійне диференціювання, тест на колонієутворювальну здатність, імуноцитохімія, проточна цитометрія, ЗТ-ПЛР. Результати. Отримані МСК-ПНГ експресували характерні для нервового гребеня маркери (нестин, Sox10 и Sox2) та диференціювалися в адипоцити, остеобласти та Шванівські клітини. У запропонованих нами умовах культура МСК-ПНГ на третьому пасажі містила: 66,8 % нестину+, 3,1 % ALDHbrigth и 33,3 % колонієутворювальних клітин. Встановлено, що швидкість росту МСК-ПНГ залежить від щільності посіву. Виявлено дозозалежну мітогенну дію EGF і bFGF на МСК-ПНГ. Висновки. Розроблено новий підхід, що дозволяє нарощувати МСК-ПНГ із збереженням їхніх основних функціональних властивостей. Подальша оптимізація умов культивування буде заснована на використанні факторів росту і низької щільності посіву.Цель. Получить культуру мультипотентных стволовых клеток – производных нервного гребня (МСК-ПНГ) в новых культуральных условиях и исследовать их биологические свойства. Методы. МСК-ПНГ получали из эксплантов бульбарного района волосяного фолликула вибрисса взрослых мышей. Культуры клеток изучали следующими методами: исследование на способность к сферогенезу, направленная мультилинейная дифференциация, тест на колониеобразующую способность, иммуноцитохимия, проточная цитометрия, ОТ-ПЦР. Результаты. Полученные МСК-ПНГ экспрессировали характерные маркеры нервного гребня (нестин, Sox10 и Sox2) и дифференцировались в адипоциты, остеобласты и Шванновские клетки. В предложенных нами условиях культура МСК-ПНГ на третьем пассаже содержала: 66,8 % нестина+, 3,1 % ALDHbrigth и 33,3 % колониеобразующих клеток. Определено, что скорость роста МСК-ПНГ зависит от плотности посева. Показано дозозависимое митогенное действие EGF и bFGF на МСК-ПНГ. Выводы. Предложен подход, позволяющий наращивать МСК-ПНГ с сохранением их основных функциональных свойств. Дальнейшая оптимизация условий культивирования будет основана на использовании факторов роста и низкой плотности посева

POSTNATAL EXTRA-EMBRYONIC TISSUES AS A SOURCE OF MULTIPLE CELL TYPES FOR REGENERATIVE MEDICINE APPLICATIONS

Aim: We aimed to isolate and characterize the cell types which could be obtained from postnatal extra-embryonic tissues. Materials and Methods: Fresh tissues (no more than 12 h after delivery) were used for enzymatic or explants methods of cell isolation. Obtained cultures were further maintained at 5% oxygen. At P3 cell phenotype was assessed by fluorescence-activated cell sorting, population doubling time was calculated and the multilineage differentiation assay was performed. Results: We have isolated multiple cell types from postnatal tissues. Namely, placental mesenchymal stromal cells from placenta chorionic disc, chorionic membrane mesenchymal stromal cells (ChM-MSC) from free chorionic membrane, umbilical cord MSC (UC-MSC) from whole umbilical cord, human umbilical vein endothelial cells (HUVEC) from umbilical vein, amniotic epithelial cells (AEC) and amniotic MSC (AMSC) from amniotic membrane. All isolated cell types displayed high proliferation rate together with the typical MSC phenotype: CD73⁺CD90⁺CD105⁺CD146⁺CD166⁺CD34⁻CD45⁻HLA⁻DR⁻. HUVEC constitutively expressed key markers CD31 and CD309. Most MSC and AEC were capable of osteogenic and adipogenic differentiation. Conclusion: We have shown that a wide variety of cell types can be easily isolated from extra-embryonic tissues and expanded ex vivo for regenerative medicine applications. These cells possess typical MSC properties and can be considered an alternative for adult MSC obtained from bone marrow or fat, especially for allogeneic use

ISOLATION OF MULTIPOTENT MESENCHIMAL STROMAL CELLS FROM MINIMAL HUMAN ENDOMETRIUM BIOPSY

The aim of the research was establishing a cell culture from a minimal human endometrial biopsy and assessment its conformity with the criteria for multipotent mesenchymal stromal cells. It was shown that cells in the culture possess adhesion to plastic, have characteristic fibroblast-like morphology, express CD73+CD90+CD105+, and are negative for hematopoietic markers (CD34-CD45-HLA-DR-), have the ability to directed adipogenic, osteogenic and chondrogenic differentiation. Due to these properties, the cell population isolated from the minimal endometrial biopsy can be attributed to multipotent mesenchymal stromal cells

ИЗМЕНЕНИЯ МЕТАБОЛИЧЕСКОЙ АКТИВНОСТИ НОРМАЛЬНЫХ И РАКОВЫХ КЛЕТКОК ЧЕЛОВЕКА IN VITRO ПРИ РАЗНОЙ КОНЦЕНТРАЦИИ ДЕЙТЕРИЯ В КУЛЬТУРАЛЬНОЙ СРЕДЕ

In current study we have shown the in vitro normal and cancer cells in deuterated growth medium demonstrated decrease in metabolic activity. In contrast, in deuterium-depleted medium there was an increase metabolic activity.В настоящем исследовании мы показали, что нормальные и раковые клетки in vitro в дейтерированной ростовой среде демонстрируют снижение метаболической активности, а в обедненной дейтерием среде повышение

TISSUE-ENGINEERED BONE FOR TREATMENT OF COMBAT RELATED LIMB INJURIES

Aim: Based on our preliminary positive clinical results with use of cultured bone marrow-derived multipotent mesenchymal stem/stromal cells in traumatology, our aim was to develop living three-dimensional tissue-engineered bone equivalent transplantation technology for restoration of critical sized bone defects caused by combat related high energy trauma. Materials and Methods: To fabricate bone equivalent we used devitalized allogeneic bone scaffolds (blocks and chips) seeded with cultured autologous cells: bone marrow-derived multipotent mesenchymal stem/stromal cells in mix with periosteal progenitor cells and endothelial progenitor cells. Quality/identity of cell cultures was assured by donor and cell culture infection screening (immunofluorescence assay, polymerase chain reaction), flow cytometry (cell phenotype), karyotyping (GTG banding), functional assays (colony forming units analysis, multilineage differentiation assay). Bone defect treatment with bone equivalent application was fully completed in 39 combat-injured with 42 defects. New bone formation was assessed by the radiographic examination. Results: Casualties were included in a treatment program an average of 10.1 months after injury, provided the ineffectiveness of conventional surgery methods. All cell type cultures had a normal karyotype and appropriate phenotype, differentiation potential and functional properties, ~30% colony forming units frequency and hadn’t any signs of cell senescence. The fluorescein diacetate/propidium iodide combined staining and histology analysis of graft samples before transplantation showed their regular seeding with viable cells. Pathomorphological analysis of bone equivalent specimens 3–6 months post-op revealed the active remodeling processes and immature bone tissue formation. Bone defect restoration was observed 5–6 months post-op. Conclusion: The developed biotechnology of living three-dimensional tissue-engineered bone equivalent transplantation with overall effectiveness 90.4% allows restoring the bone integrity, forming new bone tissue in a site of bone defect, and significantly reducing the rehabilitation period of a patient

LARGE-SCALE EXPANSION AND CHARACTERIZATION OF HUMAN ADULT NEURAL CREST-DERIVED MULTIPOTENT STEM CELLS FROM HAIR FOLLICLE FOR REGENERATIVE MEDICINE APPLICATIONS

Aim: The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from human hair follicle for their further clinical use. Materials and Methods: Adult neural crest-derived multipotent stem cells were obtained from human hair follicle by explant method and were expanded at large-scale up to a clinically significant number. The resulted cell cultures were examined by flow cytometry and immunocytochemical analysis. Their clonogenic potential, ability to self-renewal and directed multilineage differentiation were also investigated. Results: Cell cultures were obtained from explants of adult human hair follicles. Resulted cells according to morphological, phenotypic and functional criteria satisfied the definition of neural crest-derived multipotent stem cells. They had the phenotype Sox2⁺Sox10⁺Nestin⁺CD73⁺CD90⁺CD105⁺CD140a⁺CD 140b⁺CD146⁺CD166⁺CD271⁺CD349⁺ CD34⁻CD45⁻CD56⁻HLA⁻DR⁻, showed high clonogenic potential, ability to self-renewal and directed differentiation into the main derivatives of the neural crest: neurons, Schwann cells, adipocytes and osteoblasts. Conclusion: The possibility of a large-scale expansion of adult neural crest-derived multipotent stem cells up to 40–200·106 cells from minimal number of hair follicles with retention of their phenotype and functional properties are the significant step towards their translation into the clinical practice