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 consen...

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Datum:	2017
Hauptverfasser:	Zlatska, A.V., Rodnichenko, A.E., Gubar, O.S., Zubov, D.O., Novikova, S.N., Vasyliev, R.G.
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Sprache:	English
Veröffentlicht:	Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України 2017
Schriftenreihe:	Experimental Oncology
Schlagworte:	Original contributions
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Zitieren:	Endometrial stromal cells: isolation, expansion, morphological and functional properties / A.V. Zlatska, A.E. Rodnichenko, О.S. Gubar, D.О. Zubov, S.N. Novikova, R.G. Vasyliev // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 197–202. — Бібліогр.: 12 назв. — англ.

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spelling	irk-123456789-1385342018-06-20T03:05:16Z Endometrial stromal cells: isolation, expansion, morphological and functional properties Zlatska, A.V. Rodnichenko, A.E. Gubar, O.S. Zubov, D.O. Novikova, S.N. Vasyliev, R.G. Original contributions 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. 2017 Article Endometrial stromal cells: isolation, expansion, morphological and functional properties / A.V. Zlatska, A.E. Rodnichenko, О.S. Gubar, D.О. Zubov, S.N. Novikova, R.G. Vasyliev // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 197–202. — Бібліогр.: 12 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138534 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	English
topic	Original contributions Original contributions
spellingShingle	Original contributions Original contributions Zlatska, A.V. Rodnichenko, A.E. Gubar, O.S. Zubov, D.O. Novikova, S.N. Vasyliev, R.G. Endometrial stromal cells: isolation, expansion, morphological and functional properties Experimental Oncology
description	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.
format	Article
author	Zlatska, A.V. Rodnichenko, A.E. Gubar, O.S. Zubov, D.O. Novikova, S.N. Vasyliev, R.G.
author_facet	Zlatska, A.V. Rodnichenko, A.E. Gubar, O.S. Zubov, D.O. Novikova, S.N. Vasyliev, R.G.
author_sort	Zlatska, A.V.
title	Endometrial stromal cells: isolation, expansion, morphological and functional properties
title_short	Endometrial stromal cells: isolation, expansion, morphological and functional properties
title_full	Endometrial stromal cells: isolation, expansion, morphological and functional properties
title_fullStr	Endometrial stromal cells: isolation, expansion, morphological and functional properties
title_full_unstemmed	Endometrial stromal cells: isolation, expansion, morphological and functional properties
title_sort	endometrial stromal cells: isolation, expansion, morphological and functional properties
publisher	Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
publishDate	2017
topic_facet	Original contributions
url	http://dspace.nbuv.gov.ua/handle/123456789/138534
citation_txt	Endometrial stromal cells: isolation, expansion, morphological and functional properties / A.V. Zlatska, A.E. Rodnichenko, О.S. Gubar, D.О. Zubov, S.N. Novikova, R.G. Vasyliev // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 197–202. — Бібліогр.: 12 назв. — англ.
series	Experimental Oncology
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first_indexed	2025-07-10T05:59:51Z
last_indexed	2025-07-10T05:59:51Z
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fulltext	Experimental Oncology 39, 197–202, 2017 (September) 197 ENDOMETRIAL STROMAL CELLS: ISOLATION, EXPANSION, MORPHOLOGICAL AND FUNCTIONAL PROPERTIES A.V. Zlatska1, 2, , A.E. Rodnichenko1, 2, О.S. Gubar2, 3, D.О. Zubov1, 2, S.N. Novikova1, R.G. Vasyliev1, 2 1State Institute of Genetic and Regenerative Medicine of National Academy of Medical Sciences of Ukraine, Kyiv 04114, Ukraine 2Biotechnology Laboratory ilaya.regeneration, Medical Company ilaya®, Kyiv 03115, Ukraine 3Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv 03143, Ukraine 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 sta- bility 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. Key Words: endometrium, mesenchymal stromal cells, secretome, regenerative medicine. The development of cell biology and regenerative medicine methods open up new opportunities for the treatment of a number of pathological conditions, the study of their pathogenesis and the possibilities for correction. Mesenchymal stem/stromal cells (MSC), the ma- jor cell type for cell therapy, have been used in the clinic for approximately 15 years [1]. They are free of ethical concerns and have numerous sources, low immunogenicity and no risk of teratoma forma- tion. For this reasons the MSC are the most com- monly used stem cells in current clinical applications. Although MSC for cell therapy have been shown to be safe and effective, there are still challenges that need to be tackled before their wide application in the clinic. MSC exist in almost all tissues. They can be easily isolated from the bone marrow, adipose tissue, umbili- cal cord, dental pulp, and extra-embryonic tissues and can be successfully expanded in vitro [2–7]. The endometrium is a unique structure that is able to complete self-renewal over the month cycle, and undergoes these changes over 400 times during woman reproductive age [8]. A significant regenera- tive potential is due to the presence of stem cells in the endometrium, such as mesenchymal, epithelial and endothelial progenitor cells [9]. The endometrium is a hormone-sensitive structure. For this reason, it is necessary to use tissue-specific MSC for develop- ment of cell-based regenerative medicine approaches. Interest for MSC using in regenerative medicine related not only of their structural integration in dam- aged or diseased tissues and organs, but also for their trophic effect. So, the study of secretion profile of interleukins (IL), chemokines and growth factors of endometrial mesenchymal stromal cells (eMSC) is important task. Considering of above mention the endometrium is a promising object for MSC isolation and in vitro large-scale expansion for their further application in reproductology and regenerative medicine. MATERIALS AND METHODS Experiments were done in accordance with the bioethics and biological safety norms confirmed by the Medical Company ilaya® Bioethics Commission permission. 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. Submitted: August 08, 2017. Correspondence: E-mail: alenazlacka@gmail.com Tel.: +380978669174 Abbreviations used: eMSC — endometrial mesenchymal stromal cells; FACS — fluorescence-activated cell sorting; FGF-2 — basic fibroblast growth factor; GM-CSF — granulocyte-macrophage colony-stimulating factor; IFNγ — interferon gamma; IL-1ra — in- terleukin-1 receptor antagonist; IL-6 — interleukin-6; IL-8 — inter- leukin-8; IP-10 — interferon gamma-induced protein 10; ISCT — International Society for Cellular Therapy; MCP-1 — monocyte chemoattractant protein-1; MSC — mesenchymal stromal cells; VEGF — vascular endothelial growth factor. Exp Oncol 2017 39, 3, 197–202 198 Experimental Oncology 39, 197–202, 2017 (September) Cell isolation and culturing. Endometrial frag- ments were dissociated by enzymatic treatment for 1h in 0.1% collagenase IA and 0.1% pronase and 2% FBS. The cells were cultured in DMEM/F12 supplement- ed with 10% FBS, 2 mМ L-glutamine and 1 ng/ml basic fibroblast growth factor (FGF-2) in a multi-gas incubator at 5% CO2 and 5% O2. All reagents were from Sigma- Aldrich, USA. The P3 cells were used for the phenotype determination, directed adipogenic, osteogenic and chondrogenic differentiation assays, colony forming units test and karyotype stability. Flow cytometry. The cell phenotype was assessed by fluorescence-activated cell sorting (FACS) on the BD FACSAria flow cytometer (BD Pharmingen, BD Ho- rizon, USA). Staining with the monoclonal antibodies (PerCP-Cy5.5 mouse anti-human CD105, APC mouse anti-human CD73, FITC mouse anti-human CD90, PE- Cy5 mouse anti-human HLA-DR, APC mouse anti-human CD34, FITC mouse anti-human CD45, PE-CF592 mouse anti-human CD146, BV421 mouse anti-human CD166, PE mouse anti-human CD106, PerCP-Cy5.5 mouse anti- human CD140a, PE-CF592 mouse anti-human CD140b, APC mouse anti-human CD184, PE-CF592 mouse anti- human CD271, FITC mouse anti-human CD325) was performed according to the manufacturer’s instructions (BD Pharmingen, BD Horizon, USA). Fig. 1. Morphology of eMSC: 24 h in culture (a, b); 72 h in culture (c, d) ; cell culture at P3 (e, f). a, c — epiteloid-like morphology; b, d, e, f — fibroblast-like morphology. Phase-contrast microscopy. a, b, f — the bar = 50 μm; c, d, e — the bar = 100 μm Experimental Oncology 39, 197–202, 2017 (September) 199 The cell secretome was assessed by BioRad Multi- plex immunoassay kit according to manufacturer’s in- structions. Directed multilineage differentiation. Adipo- genic differentiation was performed in DMEM with high glucose content (4.5 g/l) (Sigma-Aldrich, USA) sup- plemented with 5% horse serum (PAA, Austria), 10% FBS, 1 μM dexamethasone, 200 μM indomethacine, 500 μM isobutylmethylxanthine and 5 μg/ml insulin (all from Sigma-Aldrich, USA). After 14 days the cells were fixed and stained with Oil Red O (Sigma-Aldrich, USA). Osteogenic differentiation was performed in DMEM with low glucose content (1 g/l) with 10% FBS, 100 nM dexamethasone, 10 mM β-glycerophosphate Fig. 2. Representative FACS graphics 200 Experimental Oncology 39, 197–202, 2017 (September) and 50 μg/ml ascorbate-2-phosphate (all from Sigma- Aldrich, USA). After 21 days the cells were fixed and stained with Alizarin Red S (Sigma-Aldrich, USA). Chondrogenic differentiation of cultures was carried out using the micromass culture method, 300,000 cells were centrifuged for 14 min at 2000 rpm to obtain a precipitate in 15-ml tubes (Nunc, USA). Further the cell pellets were cultured in chondrogenic induc- tive medium containing DMEM with 4.5 g/l glucose (PAA, Germany) with addition of 50 μg/ml ascorbate- 2-phosphate (Sigma-Aldrich, USA), 40 μg/ml L-proline (Sigma-Aldrich, USA), 100 μg/ml sodium pyruvate (Sig- ma-Aldrich, USA), 10 ng/ml rhTGF-β3 (Sigma, USA), 10-7 M dexamethasone (Sigma-Aldrich, USA), 1% ITS containing 6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 ng/ml selenic acid (Gibco, USA), 1.25 mg/ml bo- vine serum albumin (Sigma-Aldrich, USA). Colony forming units test. 100 cells were seeded per 100 mM cell culture dish and cultured for two weeks in standard culture medium with 20% FBS with- out medium change. The colonies were fixed with cold 96% ethanol and stained by Romanowsky. GTG-banding was performed as decribed previ- ously [10]. The population doubling time was calculated at http://www.doubling-time.com/compute.php [11]. RESULTS AND DISCUSSION eMSC primary population morphology. We have obtained five eMSC cultures from five donors 34.0 ± 3.3 years old. One of the characteristics of the MSC Fig. 4. Multilineage differentiation of eMSC. a — adipogenic differentiation. Red Oil O (red) and Romanowsky (violet) stain. Light microscopy. The bar = 50 μm; b — osteogenic differentiation. Alizarin Red S (red) stain. Light microscopy. The bar = 200 μm. c, d — chondrogenic differentiation. Light microscopy. The bar = 200 μm. c — Alcian Blue stain (blue), d — Goldner stain (black) Fig. 3. eMSC 3D-spheroid formation. Phase-contrast microscopy. The bar = 200 μm (a); the bar = 10 μm (b); the bar = 50 μm (c) Experimental Oncology 39, 197–202, 2017 (September) 201 is their ability to adhere to plastic under standard cul- ture conditions. After 24 h in culture the cells began to adhere to the plastic. The resulting cell population had a heterogeneous morphology (Fig. 1, a, b). After 48 h of culture, part of the cells began to mi- grate from non-dissociated conglomerates, forming colonies of small round cells with epithelial-like morphology. There were also single cells with typical fibroblast-like morphology. Further these cells with fibroblast-like morphology actively multiplied and formed colonies, whereas in epithelial-like colonies part of the cells died after 72 h in culture (Fig. 1, c, d). At first two passages, population of eMSC re- mained heterogeneous and consisted of fibroblast- like and epithelial-like cells which is consistent with previous reports [8, 9]. However, at P3 the cell population became rather homogeneous: the majority of cells had fibroblast-like morphology, actively proliferated and formed a mono- layer (Fig. 1, e, f). eMSC immunophenotype. The results of the cell immunophenotyping at P3 are presented in Table 1 with representative histograms at Fig. 2. eMSC demonstrated high levels of MSC markers expression, e.g. CD73, CD90, CD105, CD90; and did not express markers of hemato- poietic progenitor cells, e.g. CD34, CD45, HLA-DR. Posi- tive expression of CD146+CD166+CD140a+CD140b+ was also studied. eMSC did not express CD106, CD184, CD271, CD325. Table 1. Flow cytometry results of eMSC surface antigen expression (data presented as a percentage of parent population from 5 donors) Antigens № 1 № 2 № 3 № 4 № 5 Mean ± SD CD105 93.2 93.7 95.3 95 95.4 94.5 ± 1.0 CD73 95.4 98.9 99.2 99.1 97.9 98.1 ± 1.6 CD90 84.0 95.9 94.7 93.1 93.3 92.2 ± 4.7 CD34 0.1 0.3 0.3 0.2 0.2 0.2 ± 0.1 CD45 0.2 0.3 0.3 0.1 0.3 0.2 ± 0.9 HLA-DR 0.7 0.3 0.5 0.8 0.3 0.5 ± 0.2 CD140a 67.5 66.8 61.2 67.0 86.7 69.8 ± 9.7 CD140b 96.9 92.7 94.8 92.9 97.1 94.9 ± 2.1 CD146 85.2 78.6 94.7 75.1 83.1 83.4 ± 7.5 CD166 87.9 77.1 88.6 82.1 79.3 83.0 ± 5.1 CD106 0.3 0.6 0.3 0.2 0.3 0.35 ± 0.2 CD184 0.9 1.5 1.7 1.8 2.1 1.6 ± 0.4 CD271 6.4 4.2 5.1 5.3 6.9 5.58 ± 1.1 CD325 0.1 0.2 0.1 0.15 0.2 0.14 ± 0.1 Colony forming units efficiency and 3D spher- oid formation of eMSC. The colony-forming units assay was also performed at P3. All cultures showed 35.7 ± 2.3% colony forming efficiency. Moreover, when seeded at high density and cultured for a long time without splitting, all eMSC cell cultures demonstrated a tendency to 3D spheroid formation (Fig. 3). Directed mult i l ineage dif ferentiat ion of eMSC. In accordance with the minimal criteria of the International Society for Cellular Therapy (ISCT) [12], multipotency is a mandatory property of any MSC and is determined by their ability for directed in vitro differen- tiation into mesenchymal derived cell types (adipocytes, osteoblasts and chondrocytes). All eMSC cultures successfully underwent adipo- genic differentiation (Fig. 4, a). However, it was non- canonic (comparing to bone marrow MSC where fat vacuoles are significantly larger and evenly distributed throughout the cytoplasm of the cell) as there were plen- ty tiny lipid vacuoles located in the perinuclear region. The eMSC cell culture also showed positive osteogenic differentiation with the production of Ca2+-mineralized extracellular matrix beginning from the day 14 of induction and with a high-density matrix at day 21 (Fig. 4, b). The chondrogenic differentiation was also successful with a dense chondroid formation at day 21 of induction (Fig. 4, c, d). eMSC secretome. After 48 h incubation period in se- rum-free medium eMSC secreted anti-inflammatory IL- 1ra (74.6 ± 9.5 pg/ml), as well as IL-6 (29.8 ± 8.3 pg/ml), IL-8 (138.5 ± 33.3 pg/ml) and interferon gamma (IFNγ) (55.9 ± 3.8 pg/ml), angiogenic factors, vascular endo- thelial growth factor (VEGF) (92.2 ± 19.8 pg/ml), granu- locyte-macrophage colony-stimulating factor (GM-CSF) (133.2 ± 5.1 pg/ml) and FGF-2 (17.8 ± 4.3 pg/ml), che- mokines interferon gamma-induced protein 10 (IP-10) (39.9 ± 3.3 pg/ml) and monocyte chemoattractant protein-1 (MCP-1) (41.1 ± 6.7 pg/ml) (Table 2). Karyotype stability and cell doubling time of eMSC. Cell doubling time at P3 was in average 29.6 ± 1.3 h for all five cell cultures. Moreover, eMSC maintained stable karyotype during long-term culturing up to P8 (Fig. 5). Altogether these data indicate a high proliferation rate and genetic stability of eMSC. Fig. 5. Representative eMSC karyotype at P8: metaphase plate (a), karyogram (b). Normal female 46,XX karyotype 202 Experimental Oncology 39, 197–202, 2017 (September) Table 2. Levels of cytokines and growth factors production of eMSC Cytokines IL-1ra, pg/ml 74.6 ± 9.5 IL-6, pg/ml 29.8 ± 8.3 IL-8, pg/ml 138.5 ± 33.3 IFNγ, pg/ml 55.9 ± 3.8 Growth factors VEGF, pg/ml 92.2 ± 19.8 GM-CSF, pg/ml 133.2 ± 5.1 FGF-2, pg/ml 17.8 ± 4.3 Chemokines IP-10, pg/ml 39.9 ± 3.3 MCP-1, pg/ml 41.1 ± 6.7 Thus, obtained eMSC meet minimal ISCT criteria for MSCs, such as adherence to plastic in standard culture conditions, expression of typical phenotype markers and ability for the directed differentiation in vitro. eMSC are stable during long-term culturing. They also produce a range of cytokines, chemokines and growth factors. Proliferative potential, karyotype stability and secretome profile make them a perspec- tive object for the use for the regenerative medicine application. REFERENCES 1. Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2008; 2: 313–9. 2. Gnecchi M1, Melo LG. Bone marrow-derived mes- enchymal stem cells: isolation, expansion, characterization, viral transduction, and production of conditioned medium. Methods Mol Biol 2009; 482: 281–94. 3. Dhanasekaran M, Indumathi S, Poojitha R, et al. Plas- ticity and banking potential of cultured adipose tissue derived mesenchymal stem cells. Cell Tissue Bank 2013; 14: 303–15. 4. Romanov YA, Svintsitskaya VA, Smirnov VN. Search- ing for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells 2003; 21: 105–10. 5. Karaöz E, Doğan BN, Aksoy A, et al. Isolation and in vitro characterisation of dental pulp stem cells from natal teeth. Histochem Cell Biol 2010; 133: 95–112. 6. Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004; 22: 1338–45. 7. Fukuchi Y, Nakajima H, Sugiyama D, et al. Human placenta-derived cells have mesenchymal stem/progenitor cell potential. Stem Cells 2004; 22: 649–58. 8. Gargett C. Stem cells in gynaecology. Austral New Zealand J Obstet Gynaecol 2004; 44: 380–6. 9. Gargett C. Identification and characterization of hu- man endometrial stem/progenitor cells. Austral New Zealand J Obstet Gynaecol 2006; 46: 250–3. 10. Fayazi M, Salehnia M, Ziaei S. The effect of stem cell factor on proliferation of human endometrial CD146+ cells. Int J Reprod Biomed 2016; 14: 437–42. 11. Roth V. 2006 Doubling Time Computing. Available from: http://www.doubling-time.com/compute.php. 12. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. Int Soc Cell Ther Cytother 2006; 8: 315–7. Copyright © Experimental Oncology, 2017

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

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