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 w...

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Дата:	2014
Автори:	Vasyliev, R.G., Rodnichenko, A.E., Zubov, D.A., Rymar, S.Y., Gubar, O.S., Labunets, I.F., Novikova, S.N.
Формат:	Стаття
Мова:	English
Опубліковано:	Інститут молекулярної біології і генетики НАН України 2014
Назва видання:	Вiopolymers and Cell
Теми:	Molecular and Cell Biotechnologies
Онлайн доступ:	http://dspace.nbuv.gov.ua/handle/123456789/154583
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Цитувати:	Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions / R.G. Vasyliev, A.E. Rodnichenko, D.A. Zubov, S.Y. Rymar, O.S. Gubar, I.F. Labunets, S.N. Novikova // Вiopolymers and Cell. — 2014. — Т. 30, № 6. — С. 469-476. — Бібліогр.: 21 назв. — англ.

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spelling	irk-123456789-1545832019-06-16T01:32:21Z Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions Vasyliev, R.G. Rodnichenko, A.E. Zubov, D.A. Rymar, S.Y. Gubar, O.S. Labunets, I.F. Novikova, S.N. Molecular and Cell Biotechnologies 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. 2014 Article Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions / R.G. Vasyliev, A.E. Rodnichenko, D.A. Zubov, S.Y. Rymar, O.S. Gubar, I.F. Labunets, S.N. Novikova // Вiopolymers and Cell. — 2014. — Т. 30, № 6. — С. 469-476. — Бібліогр.: 21 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008C2 http://dspace.nbuv.gov.ua/handle/123456789/154583 576.524 + 576.35 : 577.12 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	English
topic	Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies
spellingShingle	Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies Vasyliev, R.G. Rodnichenko, A.E. Zubov, D.A. Rymar, S.Y. Gubar, O.S. Labunets, I.F. Novikova, S.N. Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions Вiopolymers and Cell
description	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.
format	Article
author	Vasyliev, R.G. Rodnichenko, A.E. Zubov, D.A. Rymar, S.Y. Gubar, O.S. Labunets, I.F. Novikova, S.N.
author_facet	Vasyliev, R.G. Rodnichenko, A.E. Zubov, D.A. Rymar, S.Y. Gubar, O.S. Labunets, I.F. Novikova, S.N.
author_sort	Vasyliev, R.G.
title	Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions
title_short	Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions
title_full	Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions
title_fullStr	Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions
title_full_unstemmed	Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions
title_sort	biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions
publisher	Інститут молекулярної біології і генетики НАН України
publishDate	2014
topic_facet	Molecular and Cell Biotechnologies
url	http://dspace.nbuv.gov.ua/handle/123456789/154583
citation_txt	Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded in new culture conditions / R.G. Vasyliev, A.E. Rodnichenko, D.A. Zubov, S.Y. Rymar, O.S. Gubar, I.F. Labunets, S.N. Novikova // Вiopolymers and Cell. — 2014. — Т. 30, № 6. — С. 469-476. — Бібліогр.: 21 назв. — англ.
series	Вiopolymers and Cell
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fulltext	UDC 576.524 + 576.35 : 577.12 Biological properties of neural crest-derived multipotent stem cells from the bulge region of whisker follicle expanded under new culture conditions R. G. Vasyliev1, 2, A. E. Rodnichenko1, 2, D. A. Zubov1, 2, S. Y. Rymar3, O. S. Gubar3, I. F. Labunets1, S. N. Novikova1 1State Institute of Genetic and Regenerative Medicine, National Academy of Medical Science of Ukraine 67, Vyshhorodska Str., Kyiv, Ukraine, 04114 2Biotechnology laboratory ilaya.regeneration, Medical company ilaya® 9, I. Kramskoho Str., Kyiv, Ukraine, 03115 3Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 rvasiliev@ukr.net Aim. The work is aimed to obtain the culture of neural crest-derived multipotent stem cells (NC-MSCs) under new culture conditions and to investigate their biological properties. Methods. NC-MSCs were grown from the ex- plants of the bulge region of whisker follicle of adult mice. The cell cultures were examined by the following me- thods: sphere-forming assay, directed multilineage differentiation, CFU assay, immunocytochemistry, flow cy- tometry, 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. Keywords: neural crest, multipotent stem cells, colony-forming units, plating density, growth factors. Introduction. In the recent 15 years the neural crest- derived multipotent stem cells (NC-MSCs) have been isolated from various tissues and organs of adult mam- mals (for review see [1]). The given cell type is of great interest for the basic research in cell biology as well as for the practical application in the regenerative me- dicine. This is explained by the NC-MSCs capacity for self-renewal and differentiation into multiple cell types such as neurons, Schwann cells, corneal stromal (kera- tocytes) and endothelial cells, melanocytes, osteo- blasts, etc. NC-MSCs exist in various adult mammalian tis- sues as a minor cell population. For their practical use in regenerative medicine, reliable and reproducible me- thods are needed for their expansion. The majority of NC-MSCs culturing protocols use a combination of the chick embryo extract (CEE) and fetal bovine serum (FBS) as growth supplements [2]. The CEE and FBS have an undefined, complicated and variable composi- tion. As a commercial product the CEE is now very rarely produced by few companies and predominantly as a protein-free ultrafiltrate unable to maintain NC- MSCs expansion. The CEE production in laboratory is a complicated and labor-consuming process. Thus a creation of the GMP-compliant xeno-free media for the NC-MSCs culturing for clinical application is very ac- tual. For the human cell culture a successful replace- ment of FBS by human blood derivatives (e. g. platelet 469 ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 6. P. 469–476 doi: http://dx.doi.org/10.7124/bc.0008C2 � Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014 lysate) was shown [3]. Hence the development of CEE- free growth media for NC-MSCs is a great challenge. The second aspect of the NC-MSCs culture is a search for the markers of stemness. The clonal analysis performed by us and others has shown that at early cul- ture stages the cells are heterogenic by their clonogenic potential [2, 4]. In other words, not all of the cells in cul- ture are stem cells. A clonal analysis is the gold standard for determination of the amount of stem/progenitor cells in culture or tissue samples. However, this is a ti- me-consuming process allowing only a retrospective assessment. Nestin is thought to be one of the most popular mar- kers of neural stem/progenitor cells and NC-MSCs [2, 5]. It is the class VI intermediate filament protein with a cytoplasmic localization and thus it is technically unsui- table for the isolation of live positive cells. Currently the role of nestin as a marker of stem cells and the idea of its exclusive expression in the neural and neural crest cells are re-estimated [6]. Nowadays there are no phe- notypic markers that allow identifying and isolating NC-MSCs from the committed cells with a limited pro- liferative potential (progenitor cells). Search for the markers suitable for the prospective evaluation and se- lection of NC-MSCs is very actual. A perspective non- immunological method is the identification of the stem/ progenitor cells based on a high activity of aldehyde dehydrogenase (ALDH). High ALDH expression has been reported for normal and cancer stem/precursor cells of various lineages including hematopoietic, mam- mary, endothelial, mesenchymal, and neural (for review see [7]). The ALDH activity for NC-MSCs has not been estimated so far. Our aims were to obtain the NC-MSCs culture from the bulge region of the whisker follicle of adult mice in the new CEE-free culture conditions, (b) to confirm the NC-MSCs identity, (c) to investigate functional signs and markers of stemness in culture (clonogenicity, num- ber of nestin+ and ALDHbrigth cells), (d) to assess effects of plating density and growth factors on the NC-MSCs proliferation. Materials and methods. Our experiments were done in accordance with the bioethics and biological safety norms and confirmed by the conclusion of the Bioethical Committee of the State Institute of Genetic and Regenerative Medicine NAMS of Ukraine. Five cell cultures obtained from males of FVB strain 4–6- month old mice were used. Isolation and culturing of NC-MSCs from the bulge region (BR) of the whisker follicle (WF). NC-MSCs we- re isolated using an explants technique from the BR of the WF according to Sieber-Blum et al. [2]. Briefly, the specimens of BR were obtained via microdissection of WF under stereomicroscope. Afterwards each three ex- plants were placed in the collagen-coated 35 mm Petri dish and cultured in the multigas incubator CB210 («BINDER», Germany) at 37 oC, saturated humidity in the artificial atmosphere containing 90 % of N2, 5 % of O2 and 5 % of CO2 in the following growth medium: DMEM:F12 («Sigma») supplemented with 10 % fetal bovine serum (FBS) («Sigma»), 1% vitamins MEM («Sigma»), 2 mM glutamine («Sigma»), 2 % B27 («Gibco», USA) and 2 % insulin, transferrin, selenium (ITS) supplements («BD», USA). On day 11 the explants were discarded and the cells were subcultured into T25 flask. All culture dishes were coated with type I colla- gen. When subcultured, the cells were dissociated with 0.05 % trypsin in 0.53 mM Na2-EDTA («Sigma»). In order to check the NC-MSC phenotype and con- tamination by other cell types, a part of the explants was plated on a cover glass. The cells of primary culture were stained on day 11 as described below (Immunofluores- cent staining section). For the rest of experiments we used the 3rd passage cells. Sphere-forming assay. The cells were dissociated and washed twice by phosphate buffered saline (PBS, pH 7.4) with centrifugation (200 g, 5 min) to get rid of the serum residues and then plated in the concentration 104 cells/ml in the 35 mm Petri dish for suspension cell culture in the serum-free medium containing: DMEM: F12, 2 % B27 supplement, 2 mÌ glutamine, 20 ng/ml basic fibroblast growth factor (bFGF, FGF-2) («Sig- ma», USA ), 40 ng/ml epidermal growth factor (EGF) («Sigma»). The growth factors were added each 48 h. Half of the medium was changed once a week. The cells were cultured for 14–21 days. Directed multilineage differentiation. Adipogenic differentiation was performed in the following medium: DMEM with high glucose content (4.5 g/l) («Sigma») with 5 % horse serum («PAA», Austria), 10 % FBS, 1 µM dexamethasone, 200 µM indomethacine, 500 µM isobutylmethylxanthine and 5 µg/ml insulin («Sigma»). 470 VASYLIEV R. G. ET AL. After 14 days the cells were fixed and stained. To de- tect adipogenic differentiation, the cells were stained with Oil Red O («Sigma»). Osteogenic differentiation was performed in the following medium: DMEM with low glucose content (1 g/l) with 10 % FBS, 100 nM dexamethasone, 10 mM �-glycerophosphate and 50 µg/ml ascorbate-2-phos- phate («Sigma»). After 21 days the cells were fixed and stained. To detect osteogenic differentiation, the cells were stained with Alizarin Red S («Sigma»). Differentiation into glial cells (Schwann cells) was done in Neuronal Medium («PAA») with 10 % mouse brain extract, 1 % B27 («Gibco»), 2 µM retinoid acid and 2 µM forskolin («Sigma»). After 14 days the cells were fixed and stained. Colony-forming units (CFU) assay. Two hundred cells of each cell culture were seeded in triplicate on the 100 mm Petri dishes in the growth medium (FBS con- centration was increased to 20 %). The cells were grown during 14 days. The colony-forming efficiency (CFE) was estimated according to the standard for- mula: CFE = (number of colonies formed/number of cells plated) �100 % [8]. To assess the types of CFU, the cultures were stained with Romanovsky-Gimsa («Mac- rochim», Ukraine). Flow cytometry. The number of nestin+ and ALDHbrigth cells was measured on the flow cytofluorimeter-sorter BD FACSAria («BD»). Staining by monoclonal anti- nestin (PE) antibodies was performed according to the manufacturer’s instructions («BD»). ALDHbrigth cells were identified using the ALDEFLUORTM kit («STEM CELL Technologies», Canada) according to the manu- facturer’s instructions. RT-PCR analysis. Cells were harvested by trypsini- zation and washed by PBS. Total cellular mRNA was isolated by the column-based kit «NucleoSpin RNA» («Macherey-Nagel», Germany) according to the manu- facturer’s instructions. cDNA were synthesized with the RevertAid First Strand cDNA Synthesis Kit using oli- go(dT) primer («Thermo Scientific», USA) according to the manufacturer’s instructions. For the detection of Sox10 and Sox2 mRNA ex- pression we used the following primers: Sox10 – F: 5'-GACTGGACGCTGGTGCCCATGCCC-3', R: 5'- CCGTTCTTCCGCCGCCGAGGTTG-3'; Sox2 – F: 5'-TCACAACAATCGCGGCGG-3', R: 5'-GTTCATGTGCGCGTAGCTGT-3'. To evaluate the quality of purified RNA, a control RT-PCR was performed using the GAPDH primers from the kit («Thermo Scientific»). For the DNA conta- mination control we used the samples without reverse transcriptase. Immunofluorescent staining of cells. NC-MSCs we- re fixed with Cytofix reagent («Sigma») for 30 min at room temperature, washed twice with PBS («Sigma»), then permeabilized with 0.1 % Triton X-100 («Sigma») for 10 min, washed twice with PBS, saturated with 4 % bovine serum albumin (BSA) («Sigma») for 30 min and stained with FITC-conjugated anti-pancytokeratin mou- se antibodies (1:50, «Sigma») and primary rabbit anti- nestin antibodies (1:100, «Sigma») for 1 h at room tem- perature. The cells were washed three times with PBS and incubated with secondary goat anti-rabbit Alexa 594-conjugated antibodies (1:2000, «Abcam», UK) for 1 h and washed two more times with PBS. Glial diffe- rentiation was detected with primary mouse anti-S-100 (1:500, «Abcam») and secondary goat anti-mouse fluo- rescein isothiocyanate (FITC)-conjugated antibodies (1:2000, «Abcam»). Cell nuclei were contrasted with Hoechst 33342 (2 µg/ml) or propidium iodide (PI, 2 µg/ml) for 10 min after the incubation with the secondary antibodies. Then the cells were washed two times with PBS and mounted in Mowiol («Sigma»). Estimation of plating density effect on growth rate. Cells were plated into T25 flasks (in triplicate) with pla- ting density 10, 50, 100, 1000 and 5000 cells/cm2 and cultured for 7 days (change of the medium was done every 48 h). Thereafter the cells were calculated. The cell population doubling number (PDN) and the popula- tion doubling time (PDT) were estimated according to the standard formula: PDN = 3.32 � lg(Xk/X0) and PDT = = T/3.32 � lg(Xk/X0), where Xk – number of expanded cells; X0 – number of plated cells; T – cell culture time [8]. Assessment of growth factors effect on proliferation. The cells were seeded on 6-well plates (in triplicate) with plating density 1000 cells/cm2 and cultured for 7 days in the standard growth medium (control) or in the growth medium supplemented with EGF (5, 10, 20 and 50 ng/ml) or bFGF (1, 2, 5 and 10 ng/ml). The medium 471 BIOLOGICAL PROPERTIES OF NEURAL CREST-DERIVED MULTIPOTENT STEM CELLS 472 VASYLIEV R. G. ET AL. �200 �500 �1000 bp Sox2 � GAPDH � Sox10 � Sox 10 Sox2 M RT- GAPDH GAPDH Culture 1 Culture 2 Sox 10 Sox2 A B C D E F HG Fig. 1. Morphology, phenotype and functional properties of NC-MSCs: A – an explant of BR WF with migrating NC-MSCs, 11 days of primary cultu- re, phase contrast, scale 200 µm; B – the morphology of NC-MSCs, phase contrast, scale 50 µm; C – NC-MSCs are nestin + (red) cytokeratin – (green), scale 20 µm; D – RT-PCR analysis of the expression of NC markers in two NC-MSC cultures (M – 100 bp DNA ladder; RT- – control without reverse transcriptase); E – NC-MSCs form spheres in serum-free conditions, pha- se contrast, scale 20 µm; F – adipo- genic differentiation of NC-MSCs, Oil Red stain, scale 200 µm; G – osteo- genic differentiation of NC-MSC, Ali- zarin Red stain, scale 200 µm; H – dif- ferentiation of NC-MSC into Schwann cells, anti-S-100 stain (green), fluo- rescent microscopy, scale 20 µm A B Fig. 2. ALDH bright cell detection in the 3 rd passage culture of NC-MSCs: A – control (cells treated with the inhi- bitor of ALDH; B – tested cells. P1 – population of ALDH bright cells A B C D Fig. 3. Clonal heterogeneity of NC- MSCs: A – type I CFU; B – type II CFU; C – type III CFU (colony cen- ter with high cell density and 3D struc- tures formation (spheroids)); D – type III CFU (colony periphery with acti- vely migrating fibroblast-like cells). Romanowsky-Giemsa stain, scale 200 µm was changed every 48 h. The cell number was calcula- ted on days 3, 5 and 7. Cell counting and viability assay. The cell number and viability were estimated after staining with 0.4 % trypan blue. The living and dead cells were counted using the Goryaev chamber. The cell viability (CV) was estimated according to the standard formula: CV = (number of viable cells/ total cell number) � 100 % [8]. Microscopy. Inverted fluorescent microscope Axio Observer A1 equipped with digital camera AxioCam ERc 5s and ZEN 2012 («Carl Zeiss», Germany) soft- ware was used. Statistics. The data are presented as means and stan- dard deviation (M ± s). Statistical significance was esti- mated using the Student’s t-criterion. Results and discussion. Under proposed culture con- ditions cell migration from the BR explants and their ac- tive proliferation began on day 4. It was previously shown that the culture from BR of WF besides NCSC may also contain other cell types such as epidermal stem cells and keratinocytes [2, 9]. To check the purity and homoge- neity of the obtained cultures we analyzed cell morpho- logy (fibroblast-like for NCSC vs epithelial-like for ke- ratinocytes) and expression of the specific markers (nes- tin for NCSC and cytokeratin for keratinocytes). All cells in primary culture had a characteristic fibroblast-like morphology and were nestin+cytokeratin– (Fig. 1, A, B, C). They also expressed transcription factors Sox10 (one of the central regulators of neural crest development, which maintains neural crest cell identity) [10] and Sox2 (a marker of early neuroepithelial cells, factor of pluri- potency and stemness, reexpressed in adult NC-MSCs) [11] (Fig. 1, D). Thus the cells expressed main pheno- typic markers of the neural crest-derived cells. Next, we expanded the cells up to 3rd passage and studied their functional properties. Importantly, the 3rd passage allowed obtaining a sufficient number of cells for most experimental and potential clinical applica- tions. When cultured in serum-free conditions the 3rd passage NC-MSCs formed floating spheres like neuro- spheres formed by neural stem cells (Fig. 1, E). The cells also possessed the capacity for a directed differentia- tion into adipocytes, osteoblasts and glial cells (Fig. 1, F, G, H). Thus the cells expanded in our culture condi- tions corresponded to the NC-MSCs criteria [1]. The study on functional and phenotypic markers of stemness in 3rd passage cultures revealed that at a given time point the number of nestin+ cells was 66.82 ± 5.91 % and the CFE was 33.33 ± 3.57 % (Table). Besides, a mi- nor subpopulation of ALDHbright cells (3.06 ± 1.13 %) was present in the NC-MSCs culture (Fig. 2, Table). An analysis of the colonies formed in the CFU assay showed that the 3rd passage NC-MSCs were hetero- genic in their proliferative potential. Depending on the cell number, all CFUs could be divided into three ty- pes: type I CFU consisted of 50–100 cells; type II CFU consisted of 100–300 cells; and type III CFU (about 1000 cells) (Fig. 3, Table). Earlier we showed that only type III CFUs were able to form secondary clonal colo- nies [4, 12]. Noteworthy, the subcloning of type III CFU resulted in the appearance of all the three types of colonies again [4, 12]. Thus, type III CFUs possess the ability for self-renewal. Previously we had also shown that the clonal cultures from type III CFUs maintain the ability for directed multilineage differentiation into adi- pocytes, osteoblasts, neurons and Schwann cells [13]. Therefore, the type III CFUs in vitro manifest the main functional signs of stem cells, i. e. self-renewal and mul- 473 BIOLOGICAL PROPERTIES OF NEURAL CREST-DERIVED MULTIPOTENT STEM CELLS Cell culture, N Nestin+ cells, % ALDHbright cells, % Colony-forming efficiency, % Type I colony- forming units (CFU), % of plated cells Type II CFU, % of plated cells Type III CFU, % of plated cells 1 69.8 3.5 35.8 12.0 20.2 3.6 2 67.6 3.1 31.7 10.2 18.5 3.0 3 74.6 4.7 38.0 12.0 20.8 5.2 4 59.8 1.9 29.0 9.4 17.8 1.8 5 62.3 2.1 32.2 8.7 21.3 2.2 M ± s 66.82 ± 5.91 3.06 ± 1.13 33.33 ± 3.57 10.43 ± 1.53 19.73 ± 1.51 3.12 ± 1.33 Parameters of NC-MSC cultures at the 3 rd passage tilineage differentiation and hence their founder cells are the NC-MSCs per se. For further optimization of the NC-MSC culture conditions we studied the effects of plating density and growth factors (bFGF and EGF) on the cell growth rate. NC-MSCs show strong dependence of growth rate on plating density (Fig. 4). The highest growth rate was ob- served at the lowest plating density (10 cells per cm2): the population doubling number was almost three times bigger whereas the population doubling time was three times less than that of the cells with high plating density (5000 cells per cm2). We found that in given conditions the optimal cell plating density for NC-MSCs is 50–100 cells per cm2 that allows fast and steady expansion. As for the growth factors effect, both bFGF and EGF produced a dose-dependent mitogenic effect (Fig. 5). Notably, the bFGF effect was more pronounced and ap- peared at lower concentrations: 1 ng/ml of bFGF was enough to increase the cell number three times compa- ring to the control. A higher bFGF concentration (from 2 to 10 ng/ml) gave similar curves and about 4-fold in- crease in the cell number. EGF had less striking effect and at the concentration of 10 ng/ml only doubled the NC-MSCs number comparing to the control. Studies on NC-MSCs and development of culture methods for their expansion are an actual and complex task. It was believed for a long time that NC-MSCs have the transitory nature similar to neural crest and exist only in the narrow time span during embryonic develop- ment. In recent 15 years this concept has changed in view of the isolation of cells possessing NC-MSC properties from many tissues of adult mammals. However, their study is very challenging. Firstly, NC-MSCs exist in adult organism tissues in the form of minor population and currently there are no phenotypic markers for their discrimination from the cells of mesenchymal origin. Secondly, several of the neural crest-derived differentiated cell types, such as me- lanocytes and Schwann cells, possess the capacity for dedifferentiation and subsequent multilineage differen- 474 VASYLIEV R. G. ET AL. 0 10 20 30 40 50 60 70 80 90 100 10 50 100 1000 5000 Plating density, cells/cm2 P o p u la ti o n d o u b li n g ti m e, h 0 1 2 3 4 5 6 7 8 9 10 10 50 100 1000 5000 Plating density, cells/cm2 P o p u la ti o n d o u b li n g n u m b er A B Fig. 4. Effects of plating density on NC-MSC growth rate: A – popula- tion doubling number; B – popula- tion doubling time. p < 0.05 * * * * * * * * * * * * * * 0 50 100 150 200 250 300 350 400 450 0 3 5 7 days C el l n u m b er � 1 0 3 * * * * * * * * * * * * * * * 0 100 200 300 400 500 600 700 0 3 5 7 days C el l n u m b er � 1 0 3 A B 1 2 3 4 5 1 2 3 4 5 Fig. 5. Effect of the growth factors on NC-MSC proliferation. Growth curve of NC-MSCs cultured with different concentration of EGF (A: 1 – 50 ng/ml; 2 – 20 ng/ml; 3 – 10 ng/ml; 4 – 5 ng/ml; 5 – control) and bFGF (B: 1 – 10 ng/ml; 2 – 5 ng/ml; 3 – 2 ng/ml; 4 – 1 ng/ml; 5 – control). *p < 0.05 tiation into other cell types [14, 15]. As has been already mentioned, the phenotypic markers of NC-MSCs allow- ing their distinction from the neural crest-derived diffe- rentiated cells are lacking. The engineering of special transgenic mice strains allowed solving the problem of isolation of neural crest- derived cells from an adult organism for the investiga- tion of their biological properties [16]. It was shown that BR of WF is the convenient source for the isolation of NC-MSCs and a significant part of cells in the prima- ry culture appears to be stem cells [2]. According to our results, the culture of neural crest-derived cells has the hierarchic organization and is composed of three cell subpopulations: a) stem cells with a large proliferative potential that are capable of self-renewal (type III CFU); b) progenitor cells with a moderate proliferative potential that are incapable of self-renewal (type II CFU); c) differentiated cells with limited proliferation ca- pacity (type I CFU). Such hierarchic organization of the cells in culture was reported by other authors for the NC-MSCs obtained from different tissues and organs [2, 17]. As we demonstrated the number of nestin+ cells greatly exceeds the number of type III CFU. Thus nes- tin is rather the marker of a proliferative state than the stem-like state of neural crest-derived cells. We were first to demonstrate the existence of ALDHbright cells in the culture of neural crest-derived cells. Interestingly, the number of ALDHbright cells in the culture is comparable with the number of type III CFU. Further investigations are needed to separate the ALDHbright and ALDHdim subpopulations and compare their proliferative and clonogenic potential. Similar to other cell types [7] the activity of ALDH may appear to be the marker of stem cells in the population of neural crest-derived cells. According to our data, the growth rate of NC- MSCs depends on plating density. Such dependence was first demonstrated for the bone marrow mesen- chymal stem cells (BMMSCs) from different species [18]. Apparently, this phenomenon is the consequence of a hierarchic organization of the BMMSCs in culture [18]. Later these data were used to work out the effec- tive protocols for the BMMSCs expansion for clinical applications, when the time necessary for receiving the therapeutic cell doses is crucial [19]. The use of a serum-based medium with hormonal supplements and hypoxia allows an effective expansion of NC-MSCs from BR of WF. Further search for the growth factors promoting NC-MSCs self-renewal is ne- cessary to increase the number of type III CFU in cul- ture. The effect of growth factors on the fetal neural crest stem cells has been thoroughly studied [20]. However, the adult NC-MSCs are prone to change their respon- siveness to the certain of them [21]. For this reason, in the case of adult NC-MSCs the impact of growth fac- tors on proliferation and differentiation should be deter- mined anew [21]. EGF and bFGF were empirically used in the serum-free growth medium composition for the assessing of NC-MSC sphere-forming capacity [16]. We demonstrated that EGF and bFGF possess potent mitogenic action when used in the serum-based growth medium. In our future investigations we plan to explore the effects of the given growth factors on the NC-MSC clonogenic capacity and the frequency of the appea- rance of different CFU types. Conclusions. Our findings open new aspects for the NC-MSC biology and can promote translation of the ex- perimental therapeutic methods based on NC-MSCs into the clinical practice of regenerative medicine. Á³îëîã³÷í³ âëàñòèâîñò³ ìóëüòèïîòåíòíèõ ñòîâáóðîâèõ êë³òèí – ïîõ³äíèõ íåðâîâîãî ãðåáåíÿ ç áóëüáàðíîãî ðàéîíó âîëîñÿíîãî ôîë³êóëà â³áðèñà ïðè ¿õíüîìó íàðîùóâàíí³ çà íîâèõ óìîâ êóëüòèâóâàííÿ Ð. Ã. Âàñèëüºâ, À. ª. Ðîäí³÷åíêî, Ä. Î. Çóáîâ, Ñ. Þ. Ðèìàð, Î. Ñ. Ãóáàð, ². Ô. Ëàáóíåöü, Ñ. Ì. Íîâ³êîâà Ðåçþìå Ìåòà. Îòðèìàòè êóëüòóðó ìóëüòèïîòåíòíèõ ñòîâáóðîâèõ êë³- òèí – ïîõ³äíèõ íåðâîâîãî ãðåáåíÿ (ÌÑÊ-ÏÍÃ) çà íîâèõ óìîâ êóëü- òèâóâàííÿ òà äîñë³äèòè ¿õí³ á³îëîã³÷í³ âëàñòèâîñò³. Ìåòîäè. ÌÑÊ-ÏÍÃ îòðèìóâàëè ç åêñïëàíò³â áóëüáàðíîãî ðàéîíó âîëîñÿ- íîãî ôîë³êóëà â³áðèñà äîðîñëèõ ìèøåé. Êóëüòóðè êë³òèí âèâ÷àëè ç âèêîðèñòàííÿì íàñòóïíèõ ìåòîä³â: äîñë³äæåííÿ íà çäàòí³ñòü äî ñôåðîóòâîðåííÿ, íàïðàâëåíå ìóëüòèë³í³éíå äèôåðåíö³þâàííÿ, òåñò íà êîëîí³ºóòâîðþâàëüíó çäàòí³ñòü, ³ìóíîöèòîõ³ì³ÿ, ïðî- òî÷íà öèòîìåòð³ÿ, ÇÒ-ÏËÐ. Ðåçóëüòàòè. Îòðèìàí³ ÌÑÊ-ÏÍÃ åêñïðåñóâàëè õàðàêòåðí³ äëÿ íåðâîâîãî ãðåáåíÿ ìàðêåðè (íåñòèí, Sox10 è Sox2) òà äèôåðåíö³þâàëèñÿ â àäèïîöèòè, îñòåîáëàñòè òà Øâàí³âñüê³ êë³òèíè. Ó çàïðîïîíîâàíèõ íàìè óìîâàõ êóëüòó- ðà ÌÑÊ-ÏÍÃ íà òðåòüîìó ïàñàæ³ ì³ñòèëà: 66,8 % íåñòèíó + , 3,1 % ALDH brigth è 33,3 % êîëîí³ºóòâîðþâàëüíèõ êë³òèí. Âñòàíîâëåíî, ùî øâèäê³ñòü ðîñòó ÌÑÊ-ÏÍÃ çàëåæèòü â³ä ù³ëüíîñò³ ïîñ³âó. Âèÿâëåíî äîçîçàëåæíó ì³òîãåííó ä³þ EGF ³ bFGF íà ÌÑÊ-ÏÍÃ. 475 BIOLOGICAL PROPERTIES OF NEURAL CREST-DERIVED MULTIPOTENT STEM CELLS Âèñíîâêè. Ðîçðîáëåíî íîâèé ï³äõ³ä, ùî äîçâîëÿº íàðîùóâàòè ÌÑÊ-ÏÍÃ ³ç çáåðåæåííÿì ¿õí³õ îñíîâíèõ ôóíêö³îíàëüíèõ âëàñòè- âîñòåé. Ïîäàëüøà îïòèì³çàö³ÿ óìîâ êóëüòèâóâàííÿ áóäå çàñíîâà- íà íà âèêîðèñòàíí³ ôàêòîð³â ðîñòó ³ íèçüêî¿ ù³ëüíîñò³ ïîñ³âó. Êëþ÷îâ³ ñëîâà: íåðâîâèé ãðåá³íü, ìóëüòèïîòåíòí³ ñòîâáóðîâ³ êë³òèíè, êîëîí³ºóòâîðþâàëüí³ îäèíèö³, ù³ëüí³ñòü ïîñ³âó, ôàêòî- ðè ðîñòó. Áèîëîãè÷åñêèå ñâîéñòâà ìóëüòèïîòåíòíûõ ñòâîëîâûõ êëåòîê – ïðîèçâîäíûõ íåðâíîãî ãðåáíÿ èç áóëüáàðíîãî ðàéîíà âîëîñÿíîãî ôîëëèêóëà âèáðèññà, ðàçìíîæåííûõ â íîâûõ óñëîâèÿõ êóëüòèâèðîâàíèÿ Ð. Ã. Âàñèëüåâ, À. Å. Ðîäíè÷åíêî, Ä. À. Çóáîâ, Ñ. Å. Ðûìàðü, Î. Ñ. Ãóáàðü, È. Ô. Ëàáóíåö, Ñ. Í. Íîâèêîâà Ðåçþìå Öåëü. Ïîëó÷èòü êóëüòóðó ìóëüòèïîòåíòíûõ ñòâîëîâûõ êëåòîê – ïðîèçâîäíûõ íåðâíîãî ãðåáíÿ (ÌÑÊ-ÏÍÃ) â íîâûõ êóëüòóðàëü- íûõ óñëîâèÿõ è èññëåäîâàòü èõ áèîëîãè÷åñêèå ñâîéñòâà. Ìåòîäû. ÌÑÊ-ÏÍÃ ïîëó÷àëè èç ýêñïëàíòîâ áóëüáàðíîãî ðàéîíà âîëîñÿíî- ãî ôîëëèêóëà âèáðèññà âçðîñëûõ ìûøåé. Êóëüòóðû êëåòîê èçó÷à- ëè ñëåäóþùèìè ìåòîäàìè: èññëåäîâàíèå íà ñïîñîáíîñòü ê ñôåðî- ãåíåçó, íàïðàâëåííàÿ ìóëüòèëèíåéíàÿ äèôôåðåíöèàöèÿ, òåñò íà êîëîíèåîáðàçóþùóþ ñïîñîáíîñòü, èììóíîöèòîõèìèÿ, ïðîòî÷- íàÿ öèòîìåòðèÿ, ÎÒ-ÏÖÐ. Ðåçóëüòàòû. Ïîëó÷åííûå ÌÑÊ-ÏÍÃ ýêñïðåññèðîâàëè õàðàêòåðíûå ìàðêåðû íåðâíîãî ãðåáíÿ (íåñòèí, Sox10 è Sox2) è äèôôåðåíöèðîâàëèñü â àäèïîöèòû, îñòåîáëàñòû è Øâàííîâñêèå êëåòêè. Â ïðåäëîæåííûõ íàìè óñëîâèÿõ êóëüòóðà ÌÑÊ-ÏÍÃ íà òðåòüåì ïàññàæå ñîäåðæàëà: 66,8 % íåñòèíà + , 3,1 % ALDH brigth è 33,3 % êîëîíèåîáðàçóþùèõ êëåòîê. Îïðåäåëåíî, ÷òî ñêîðîñòü ðîñòà ÌÑÊ-ÏÍÃ çàâèñèò îò ïëîòíîñòè ïîñåâà. Ïîêàçàíî äîçîçàâèñèìîå ìèòîãåííîå äåéñòâèå EGF è bFGF íà ÌÑÊ-ÏÍÃ. Âûâîäû. Ïðåäëîæåí ïîäõîä, ïîçâîëÿþùèé íàðàùè- âàòü ÌÑÊ-ÏÍÃ ñ ñîõðàíåíèåì èõ îñíîâíûõ ôóíêöèîíàëüíûõ ñâîéñòâ. Äàëüíåéøàÿ îïòèìèçàöèÿ óñëîâèé êóëüòèâèðîâàíèÿ áó- äåò îñíîâàíà íà èñïîëüçîâàíèè ôàêòîðîâ ðîñòà è íèçêîé ïëîò- íîñòè ïîñåâà. 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Received 30.09.14 476 VASYLIEV R. G. ET AL.

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

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