Multilineage potential research of Beijing duck amniotic mesenchymal stem cells

Multilineage potential research of Beijing duck amniotic mesenchymal stem cells Cell Tissue Bank https://doi.org/10.1007/s10561-018-9701-6 Multilineage potential research of Beijing duck amniotic mesenchymal stem cells . . . . . Caiyun Ma Kunfu Wang Hongda Ji Hongliang Wang Liangcai Guo . . . Zhiyong Wang Han Ren Xishuai Wang Weijun Guan Received: 7 September 2017 / Accepted: 14 May 2018 The Author(s) 2018 Abstract Amnion, which is usually discarded as CD105, CD166, Vimentin and Fibronection), while medical waste, is considered as abundant sources for the expression of CD34 and CD45 were undetectable. mesenchymal stem cells. In human and veterinary Additionally, AMSCs also expressed the pluripotent medicine, the multipotency of mesenchymal stem marker gene OCT4. Particularly, when appropriately cells derived from amnion (AMSCs) together with induced, AMSCs could be induced to trans-differen- their plasticity, self-renewal, low immunogenicity and tiate into adipocytes, osteoblasts, chondrocytes and nontumorigenicity characteristics make AMSCs a neurocytes in vitro. Together, these results demon- promising candidate cell for cell-based therapies and strated that the isolated AMSCs maintained their tissue engineering. However, up till now, the multi- stemness and proliferation in vitro, which may be potential characteristics and therapeutic potential of useful for future cell therapy in regenerative medicine. AMSCs on preclinical studies remain uncertain. In this work, we successfully obtained AMSCs from Beijing Keywords Beijing duck  Amniotic mesenchymal duck embryos in vitro, and also attempted to detect stem cells  Multiply differentiation  Biological their biological characteristics. The isolated AMSCs characteristics were phenotypically identified, the growth kinetics and karyotype were tested. Also, the cells were positive for MSCs-related markers (CD29, CD71, Introduction Caiyun Ma and Kunfu Wang have contributed equally to this To date, stem cell research has meant a tremendous work. advance for cell therapy and tissue engineering (Alizadeh et al. 2016; Bai et al. 2016; Gao et al. C. Ma  H. Ji  H. Wang  X. Wang  W. Guan (&) Institute of Animal Science, Chinese Academy of 2016; Guo et al. 2017; Zhang et al. 2016). Embryonic Agricultural Sciences, Beijing 100193, China stem cells and adult type stem cells are current sources e-mail: wj_guan301@126.com of stem cells(Gurel Pekozer et al. 2018; Kariminekoo et al. 2016; Mohammadian et al. 2016; Momenzadeh K. Wang College of Wildlife Resources, Northeast Forestry et al. 2017). However, given the ethical and technical University, Harbin, China problems, the use of embryonic stem cells may have obvious drawbacks, such as limited availability, L. Guo  Z. Wang  H. Ren complicated culture system and tumorigenicity (Blum Mudanjiang Normal University, Mudanjiang 157011, and Benvenisty 2008; Gruen and Grabel 2006). China 123 Cell Tissue Bank Reagents and experimental animals Acquisition of adult stem cells from bone marrow (BM-MSCs) is involved in invasive surgical manip- All the reagents were purchased from Sigma (Sigma- ulation, the number and self-renewal ability of BM- MSCs significantly decreases with donor age (Gother- Aldrich, St, Louis, MO, USA), unless stated other- wise. 14-day old Beijing duck embryos were provided strom et al. 2005). Expanding on this research, amnion (Bilic et al. 2004), amniotic fluid (Gao et al. 2014), by Poultry Experimental Base of Chinese Academy of Agricultural Sciences, Beijing, China. placental tissue(Gekas et al. 2010), umbilical cord blood (Kim et al. 2017) and the Wharton’s Jelly (Taghizadeh et al. 2011) which are rich in stem cells Cell isolation and culture have captured the attention of researchers. The amnion is filled with fluid composed of Initially, the amniotic membrane tissues were exposed and mechanically peeled off from 10 Beijing duck basement layer, compact layer, fibroblastic layer and spongy layer, which is a source of important mes- embryos under sterile conditions. After rinsed well (6 times) with phosphate-buffered saline (PBS), trans- enchymal stem cells with pluripotential characteristics (Cai et al. 2010). Intensive research efforts have been parent amnion layer were cut into small pieces and incubated for 5 min in 0.125% (w/v) trypsin/EDTA reported that the AMSCs are derived from the spongy layer, which, in cell based therapies, have advantage solution to remove epithelial amniotic cells (AECs). After that, membrane fragment were transfered into a over adult type stem cells, such as a higher in vitro expansion potential, telomerase activity, immunolog- clean culture dish and subsequently submitted to 0.1% ical tolerance (Roubelakis et al. 2012). Importantly, collagenase II treatment at 37 C for 20 min. Single- convenient procurement without ethical conflict cell suspensions were extracted by filtration through a makes AMSCs a promising candidate cell for regen- 74 lm cell strainer. The pellets were resuspended with basal DMEM/F12 medium supplemented with 10% erative medicine. Although isolation and characterization of AMSCs fetal bovine serum (FBS), 10 ng/mL basic fibroblast growth factor (bFGF), 1%(v/v) GlutaMAX, and 1% from humans, rats and livestock have been reported, little literature has been done on the avian. Similar to (w/v) non-essential amino acids (NEA) after centrifu- gation at room temperature. After counted, 1 9 10 mammalian development, the avian embryos play a crucial role in developmental and cell biology. Addi- cells/cm were seeded in 60-mm-diameter culture tionally, the avian eggs characterized by small body dishes and incubated at 37 Cina 5% CO atmo- size, ease of manipulation and a low maintenance cost sphere. After 24 h post-seeding, non-adherent cells may serve as significant model system for stem cell were removed from the plate by refreshing medium. research (Li et al. 2011). Notably, our present study When reached 80–90% confluency, attached AMSCs aimed to isolate AMSCs from 14-day old Beijing duck were subcultured with 0.125% trypsin–EDTA and purified AMSCs were harvested after 4–5 passages. embryos and examine their biological characteristics with regard to growth kinetics, karyotype, Growth kinetics and karyotype assay immunophenotype, specific mesenchymal markers and differentiation potential. To evaluate cell proliferative ability, AMSCs at P4, P10, and P18 were subjected to growth kinetics analysis. After detached with 0.125% trypsin/EDTA, Experimental section cells were seeded into 24-well plates at 1 9 10 cells/ Ethics statement well. Subsequently, the cells from three random wells were counted each day for 8 days. Growth curves were All animal experiments were approved and performed drawn in accordance with mean cell numbers and the population doubling time (PDT) was calculated based in accordance with the guidelines established by the Institutional Animal Care and Use Committee at on the formula PDT = (t - t ) lg2/(lgN - lgN ), t : 0 t 0 0 starting time of culture; t: termination time of culture; Chinese Academy of Agriculture Sciences (GB14925- 2010). N : initial cell number of culture; N : ultimate cell 0 t number of culture. The chromosomal profiles of 123 Cell Tissue Bank AMSCs were investigated and analyzed according to cDNA synthesis using 5 9 All-In-One MasterMix standard methods. Briefly, AMSCs at P4 were treated with AccuRT Genomic DNA Removal Kit (abm). with 10 mg/mL colcemid for 2 h. Subsequently, the Specific primers were designed by NCBI primer-blast cells were centrifuged, fixed and stained. And then and details of primers were list in Table 1. PCR chromosome numbers were observed under an oil amplification program included: an initial denatura- immersion objective. tion at 94 C for 3 min, followed by 30 cycles at 94 C for 30 s, annealing for 30 s, an extension at 72 C for Immunofluorescence 30 s and a final extension for 10 min at 72 C. AMSCs at P4 which were grown to confluence in Multiple differentiations potential 6-well plates were prepared and fixed at room temperature for immunofluorescence analysis. After Induction of osteogenic/adipogenic/chondrogenic soaked (15 min) with 4% paraformaldehyde solution, differentiation AMSCs were processed by permeabilizing the mem- brane with 0.25% Triton X-100 for 15 min, followed For the assessment of the mesodermal differentiation by 10% goat serum in PBS and blocked for 1 h at room potential, AMSCs at P4 were targeted for adipogenic, temperature. The direct antibodies used were FITC osteogenic, and chondrogenic differentiation as pre- viously reported (Ma et al. 2017). When reached goat anti-rabbit CD29 (1:100; BIOSS), FITC goat anti-rabbit CD166 (1:100; BIOSS), FITC goat anti- 50–60% confluence, cells were cultured in adipogenic rabbit CD71 (1:100; BIOSS), FITC goat anti-rabbit (10% FBS, 1 mM dexamethasone, 0.5 mM IBMX, CD105 (1:100; BIOSS) and FITC goat anti-rabbit 10 lg/mL insulin, 200 lM indomethacin), the osteo- OCT4 (1:100; BIOSS). For nuclear staining, the cells genic (10% FBS, 0.1 mM dexamethasone, 10 mM b- were incubated with 1 lg/mL DAPI in the dark for glycerophosphate, 0.05 mM ascorbate), or the chon- 15 min. Fluorescence images were acquired by Nikon drogenic (10 lM dexamethasone, 1% Insulin-Trans- TE-2000-E confocal microscope equipped with Nikon ferrin-Selenium, 50 lg/mL L-proline, 1% sodium ZE-1-C1 3.70 digital camera system. pyruvate, 50 lg/mL vitamin C and 10 ng/mL TGF- b3) differentiation medium. The differentiation med- Immunofluorescence characterization ium were changed twice weekly. After 2 weeks of induction, the differentiated AMSCs were detected by For the assessment of immunophenotyping, culture- RT-PCR and visualized by Oil Red O, Alizarin Red expanded AMSCs in logarithmic phase were subjected and Alcian Blue staining, respectively. to flow cytometry analysis. Place the cells into FACS tubes and add precooling 70% ethanol. Incubate Induction of neuronal differentiation overnight at 4 C. The next day, the cells were transferred into a clean FACS tubes with 0.25% Triton To access neuronal differentiation, AMSCs at P4 was X-100 solution in PBS and incubated 15 min at room initially cultured in 10% FBS-DMEM/F-12 in the temperature. After that, 10% BSA (bovine serum presence of 20 ng/mL EGF, 40 ng/mL bFGF, 2% B27 albumin) in PBS were used to block nonspecific and 2 mM L-glutamine. After 6 days of pre-induction, binding. AMSCs were subsequently stained with the 10 ng/mL GDNF, 50 lg/mL vitamin C and 1% N2 following polyclonal antibodies, respectively: CD29- inducing factors were added. After 14 days, the cells FITC, CD166-FITC, CD71-FITC, CD105-FITC, and were detected by immunocytochemical staining and OCT4-FITC. RT-PCR analysis. RNA extraction and RT-PCR Following the manufacturer’s instructions, total RNA (2 lg) that was extracted using Trizol reagent (Invit- rogen) from AMSCs at P8 (90% confluence) or induced differentiated cells served as a template for 123 Cell Tissue Bank Table 1 Primer sequences Gene Primers Products (bp) used for RT-PCR 0 0 CD29 F:5 -CAGAGAGCAACGCAGAGGTT-3 226 0 0 R:5 -ATTGTCACCACCACTTGGCT-3 0 0 CD71 F:5 -GAACCGGTACCTTGAGTGGG-3 415 0 0 R:5 -GCCAGTCCTGAGCCATTTCT-3 0 0 CD166 F:5 -AGGCAAAGCTAATAGTGGGCA-3 209 0 0 R:5 -TCTGGAATGATGACTGACGCA-3 0 0 Vimentin F:5 -GACCAGCTCACCAACGACAA-3 395 0 0 R:5 -GCAGCAACGCTTTCGTACTG-3 0 0 Fibronectin F:5 -CCTCCAACTTCCATCGTGCT-3 320 0 0 R:5 -TCTGGGTGGTACCGGATTCT-3 0 0 PPAR-c F:5 -GCATCGACCAGCTAAACCCT-3 259 0 0 R:5 -TGACATCGCTGGAAAATGCG-3 0 0 LPL F:5 -TTTTCCTTACGGACGCCTGC-3 369 0 0 R:5 -GTGAGCACCCAGACTGTACC-3 0 0 ATF4 F:5 -CCCAGACTCCTACCTGGGAA-3 239 0 0 R:5 -CTGCCCTCTTCTTCTGTCGG-3 0 0 COL1A2 F:5 -GGAATAGCTAGCCACCGACC-3 421 0 0 R:5 -CTCACCGGGAACACCTTGAA-3 0 0 ACAN F:5 -AGTGGCAGCTAATGTGGTCT-3 547 0 0 R:5 -AGCTTGCTCCACTTGATCCG-3 0 0 VIM F:5 -ACGAAAGCGTTGCTGCTAAG-3 218 0 0 R:5 -CTCCATTTCACGCATCTGGC-3 0 0 MAP2 F:5 -ATCAATGGAGAGCTGTCGGC-3 221 0 0 R:5 -GCTCCAGTTTGCTCAGAAGC-3 0 0 GAPDH F:5 -GAGGAGCTGCCCAGAACATT-3 426 0 0 R:5 -GGTCTGCATGCTTGGCTTAC-3 0 0 CD34 F:5 -CTCAACGAGTCCAACACCTG-3 338 0 0 R:5 -CCAGAAGTGACCAAAGCAGTC-3 0 0 CD45 F:5 -CTCACCACACGCACTCTCAC-3 350 Results subculture 4–5 passages (Fig. 1a). After the highest number of passages (P18), the appearance of most Morphological characterization and karyotype cells gradually changed and displayed senescent signs analysis of AMSCs (Fig. 1a). Population growth kinetics of the cells from the low, middle and high passages (P4, P10 and P18) AMSCs were successfully isolated from amnion emerged obvious ‘‘S’’ shapes (Fig. 1b), and PDTs of tissues of 14-day-old Beijing duck embryos and were AMSCs cultures were 34.4, 35.9 and 39.6 h, respec- expanded until passage 18. Approximately 24 h after tively. The chromosome number of duck AMSCs is the initial primary culture, a few cells were observed to 2n = 78, as shown in Fig. 1c. And there were no adhere to petri dishes (Fig. 1a). The cells at passage 0 obvious difference about diploid rates of the cell with then began to proliferate and became confluent after 2n = 78 among different passages. These results 7 days. The initial growth of the cells was mixed with verified the AMSCs cell line we isolated was repro- the AECs. According to different tolerances to trypsin, ducibly diploid. a homogenous monolayer of spindle-shaped fibrob- last-like AMSCs was obtained after successive 123 Cell Tissue Bank Fig. 1 Characterization of duck AMSCs in vitro. a Morpho- with cell density in the Y-axis. c Karyotype analysis of duck logical characteristics of primary and sub-cultured AMSCs AMSCs. Chromosomes at metaphase (left) and karyotype (bar = 50 lm). b Growth kinetics of AMSCs at P4, P10 and P18 (right) Characterization of cells mesenchymal cell genes assessed by RT-PCR exper- iments were also consistent with the immunofluores- The results of immunofluorescence staining showed cence results above, but the expression of CD34 and that culture-expanded cells characteristically CD45 were obviously undetectable (Fig. 2b). expressed pluripotent stem cell marker OCT4. And, the expression of MSC markers CD29, CD166, CD71 Multipotential capacity of AMSCs and CD105 were also positive, as presented in Fig. 2a. Moreover, more than 90% of viable MSC population Adipogenic differentiation isolated from amnion were positive for the MSC markers as assessed by flow cytometry analysis Adipocyte-inducing medium (AID) was prepared to (Fig. 2c). Additionally, expression levels of induce AMSCs to trans-differentiate into adipocytes. 123 Cell Tissue Bank Fig. 2 Detection of surface markers in AMSCs. a The AMSCs and CD45 were not detected. c AMSCs at P4 were colabeled could express pluripotent marker gene OCT4 and MSC- with surface antigens (CD29, CD166, CD71, CD105, OCT4), associated markers by immunofluorescence stain (bar = 50 and the positive rates were all above 99% by flow cytometry lm), DAPI, Blue. b mRNA expression levels of AMSCs analysis markers were detected by RT-PCR, but the expression of CD34 7 days after differentiation, we could observe signif- were positively expressed by RT-PCR analysis icant morphological changes from fibroblast-like to (Fig. 3b). oblate with the formation of oil droplet in the AMSCs. As the induction time progressed, the number of larger Osteogenic differentiation droplets gradually increased. By day 14, the differen- tiated cells were visualized by 0.3% Oil red O staining Alizarin Red staining was performed to evidence (Fig. 3a). And the genes peroxisome proliferator- osteogenic differentiation of the AMSCs. Following activated receptor-gamma (PPAR-c) and lipoprotein induction in osteogenic differentiation medium (OID) lipase (LPL), which are involved in adipogenesis, for 7 days, the induced cells was transformed from 123 Cell Tissue Bank Fig. 3 Adipocyte, osteoblast differentiation of AMSCs (bar = PPAR-c and LPL. c The differentiated cells cultured in OID for 50 lm). a Numerous Lipid droplets, apparent in cytoplasm of 14 days were monitored using Alizarin Red staining. d RT-PCR induced cells, were positive for Oil Red staining. b RT-PCR was assays revealed osteoblast specific genes ATF4 and COL1A2 used to examine the expression of adipocyte marker genes were expressed in the differentiated osteoblast spindle shape into polygonal in appearance, followed Chondrogenic differentiation by noticeable accumulation of mineralization. And under the continued effects of osteogenic supplements Subsequent to chondrogenic differentiation in medium (CID) for 7 days, most cells interconnected to gener- on cells, the calcium deposits nodules increased and were revealed with Alizarin Red (Fig. 3c). At 14 days ate cluster-like aggregation. With prolonged induc- post-induction, the differentiated AMSCs seeded in tion, the cells changed shape with significantly the well expressed the collagen type I alpha 2 chain increased nucleoplasmic ratio and colonies. At day (COL1A2) and activating transcription factor 4 14, Alcian Blue staining was used to assess chondro- (ATF4) genes related to osteogenesis by RT-PCR genesis (Fig. 4a). To further confirm chondrogenic analysis (Fig. 3d). differentiation, RT-PCR amplification of 123 Cell Tissue Bank Fig. 4 Chondrocyte and neurocyte differentiation of AMSCs c The differentiated cells cultured in NID for 14 days expressed (bar = 50 lm). a The differentiated chondrocyte was visualized MAP2 by immunocytochemistry. d RT-PCR detection of the by Alcian Blue staining. b The expression of chondrocyte- neuronal marker MAP2 expression specific genes ACAN and VIM were analyzed by RT-PCR. chondrocyte-specific genes ACAN and VIM were immunofluorescence. After incubation in neural-in- performed with RNA isolated from induced AMSCs ducing medium (NID) for 14 days, the induced cells (Fig. 4b). gradually progressed toward neuron-like morphology with elongated cell bodies, long branches and axon of Neural differentiation neurons (Fig. 4c). And then neural cells derived from AMSCs were identified by RT-PCR (Fig. 4d). The The neuronal differentiation study focused on the results showed that differentiated cells expressed expression of neuronal-specific markers (MAP2) by 123 Cell Tissue Bank neural lineage specific gene MAP2, which were in pluripotent markers Nanog, TRA-1-60, TRA-1-81 accordance with the immunofluorescence results. and STRO-1, but lack expression for HLA-A, HLA-B, and surface molecules (Diaz-Prado et al. 2011; Kastrinaki et al. 2008; Mrugala et al. 2009). Discussion In this present work, AMSCs could be differenti- ated in vitro into adipocytes, osteocytes, chondrocytes, Amniotic membrane is a nearly transparent avascular and neurogenic cells by culturing them in specific membrane without nerves and is composed of AECs induction media which contains a cocktail of inducing and AMSCs. The major role of amniotic membrane is mediators. Dexamethasone, IBMX and insulin could to protect the fetus and provide supplemental nutrients promote adipogenic differentiation of AMSCs. How- during development. Amniotic membrane, known as ever, their exact regulatory mechanisms on adipoge- bio-material, was shown to block proteinase activity nesis remained to be analyzed. Also, b- and promote the wound repair process (Kim et al. glycerophosphate combined with dexamethasone and 2000). It has been reported that amniotic membrane ascorbate was effective in converting AMSCs into could express indispensable growth factors that is osteogenic-specific gene-expressing cells(Le Pape critical for reducing inflammation and fibrosis (Tseng et al. 2018; Zhang et al. 2016). Treatment with et al. 1999). More importantly, the isolated amniotic chondrogenic supplements led to chondrogenesis of AMSCs, and in combination with serum-free medium membrane stem cells could eliminate the concern of teratoma formation in vivo after transplantation, they may reduce apoptosis from the AMSCs (Ibrahim additional properties of noninvasive isolation, multi- et al. 2015). Currently, neurotrophic factors GDNF, potency, anti-inflammatory, minimal ethical problem under appropriate conditions, in vitro, was added to make them promising tools or appropriate sources for enhance the trans-differentiation process of AMSCs clinical treatment, such as corneal tissue (Shimmura (Yang et al. 2014). and Tsubota 2002), treatment scaffolds of corneal Taken together, all these findings show that iso- transplantation (Dua and Azuara-Blanco 1999), lated AMSCs retained self-renewal ability and multi- Parkinson’s disease (Kakishita et al. 2003), spinal potentiality in vitro. However, its preclinical applica- cord injury (Gao et al. 2016), and brain infarction bility still remains controversial in cell-based thera- (Sakuragawa et al. 1997). pies and tissue engineering in vivo, both securely and Interestingly, we successfully harvested heteroge- technically. Further detailed studies are required to neous population of AMSCs from amniotic membrane define the specific surface markers for AMSCs of 14-day old Beijing duck embryos and attempted to characterization and to investigate molecular mecha- investigate their biological characteristics in vitro. In nisms for AMSCs differentiation, which would per- the experimental set-up, enzymatic digestion was haps facilitate more effective therapies of AMSCs in employed to isolate and purify AMSCs. The cells in regenerative medicine. culture were expanded at least 18 passages in vitro. And, evaluation of karyotyping and growth curves demonstrated that culture-expanded AMSCs main- Conclusions tained renewal activity and genetic stability. In terms of the maintenance of stemness of stem cells, gene AMSCs were isolated from Beijing duck embryos. The self-renewal ability and differentiation potential expression was significant. Similar to other sources of MSCs, AMSCs possessed MSC characteristics and of the isolated AMSCs was evaluated in vitro. Our characteristically expressed a set of MSC markers, findings provide a platform for the establishment of a such as CD29, CD71, CD105, CD166 and OCT4, but Beijing duck AMSCs bank. These results have impli- negativity for CD45 and CD34. Expanding on this cations for the potential application of AMSCs as a research, the pluripotency of AMSCs was monitored stem-cell source for regenerative medical therapies. through the expression of OCT4 which is the marker Acknowledgements This research was supported by the of non-differentiation stage (Corradetti et al. 2013). In National Natural Science Foundation of China (31472099, addition, several lines of evidence suggest that 31672404), the project National Infrastructure of Animal cultured AMSCs also positively express the 123 Cell Tissue Bank Germplasm Resources (2016 year), the project of Agricultural Gekas C, Rhodes KE, Van Handel B, Chhabra A, Ueno M, Science and Technology Innovation Program (cxgc-ias-01), and Mikkola HK (2010) Hematopoietic stem cell development the domestic animal germplasm resources sharing construction in the placenta. Int J Dev Biol 54:1089–1098 (Y2017LM21). Gotherstrom C, West A, Liden J, Uzunel M, Lahesmaa R, Le Blanc K (2005) Difference in gene expression between human fetal liver and adult bone marrow mesenchymal Compliance with ethical standards stem cells. Haematologica 90:1017–1026 Gruen L, Grabel L (2006) Concise review: scientific and ethical Conflict of interest The authors declare that they have no roadblocks to human embryonic stem cell therapy. Stem conflict of interest. Cells 24:2162–2169 Guo DL, Wang ZG, Xiong LK, Pan LY, Zhu Q, Yuan YF, Liu Open Access This article is distributed under the terms of the ZS (2017) Hepatogenic differentiation from human adi- Creative Commons Attribution 4.0 International License (http:// pose-derived stem cells and application for mouse acute creativecommons.org/licenses/by/4.0/), which permits unre- liver injury. Artif Cells Nanomed Biotechnol 45:224–232 stricted use, distribution, and reproduction in any medium, Gurel Pekozer G, Ramazanoglu M, Schlegel KA, Kok FN, provided you give appropriate credit to the original Torun Kose G (2018) Role of STRO-1 sorting of porcine author(s) and the source, provide a link to the Creative Com- dental germ stem cells in dental stem cell-mediated bone mons license, and indicate if changes were made. tissue engineering. 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Multilineage potential research of Beijing duck amniotic mesenchymal stem cells

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Abstract

Cell Tissue Bank https://doi.org/10.1007/s10561-018-9701-6 Multilineage potential research of Beijing duck amniotic mesenchymal stem cells . . . . . Caiyun Ma Kunfu Wang Hongda Ji Hongliang Wang Liangcai Guo . . . Zhiyong Wang Han Ren Xishuai Wang Weijun Guan Received: 7 September 2017 / Accepted: 14 May 2018 The Author(s) 2018 Abstract Amnion, which is usually discarded as CD105, CD166, Vimentin and Fibronection), while medical waste, is considered as abundant sources for the expression of CD34 and CD45 were undetectable. mesenchymal stem cells. In human and veterinary Additionally, AMSCs also expressed the pluripotent medicine, the multipotency of mesenchymal stem marker gene OCT4. Particularly, when appropriately cells derived from amnion (AMSCs) together with induced, AMSCs could be induced to trans-differen- their plasticity, self-renewal, low immunogenicity and tiate into adipocytes, osteoblasts, chondrocytes and nontumorigenicity characteristics make AMSCs a neurocytes in vitro. Together, these results demon- promising candidate cell for cell-based therapies and strated that the isolated AMSCs maintained their tissue engineering. However, up till now, the multi- stemness and proliferation in vitro, which may be potential characteristics and therapeutic potential of useful for future cell therapy in regenerative medicine. AMSCs on preclinical studies remain uncertain. In this work, we successfully obtained AMSCs from Beijing Keywords Beijing duck  Amniotic mesenchymal duck embryos in vitro, and also attempted to detect stem cells  Multiply differentiation  Biological their biological characteristics. The isolated AMSCs characteristics were phenotypically identified, the growth kinetics and karyotype were tested. Also, the cells were positive for MSCs-related markers (CD29, CD71, Introduction Caiyun Ma and Kunfu Wang have contributed equally to this To date, stem cell research has meant a tremendous work. advance for cell therapy and tissue engineering (Alizadeh et al. 2016; Bai et al. 2016; Gao et al. C. Ma  H. Ji  H. Wang  X. Wang  W. Guan (&) Institute of Animal Science, Chinese Academy of 2016; Guo et al. 2017; Zhang et al. 2016). Embryonic Agricultural Sciences, Beijing 100193, China stem cells and adult type stem cells are current sources e-mail: wj_guan301@126.com of stem cells(Gurel Pekozer et al. 2018; Kariminekoo et al. 2016; Mohammadian et al. 2016; Momenzadeh K. Wang College of Wildlife Resources, Northeast Forestry et al. 2017). However, given the ethical and technical University, Harbin, China problems, the use of embryonic stem cells may have obvious drawbacks, such as limited availability, L. Guo  Z. Wang  H. Ren complicated culture system and tumorigenicity (Blum Mudanjiang Normal University, Mudanjiang 157011, and Benvenisty 2008; Gruen and Grabel 2006). China 123 Cell Tissue Bank Reagents and experimental animals Acquisition of adult stem cells from bone marrow (BM-MSCs) is involved in invasive surgical manip- All the reagents were purchased from Sigma (Sigma- ulation, the number and self-renewal ability of BM- MSCs significantly decreases with donor age (Gother- Aldrich, St, Louis, MO, USA), unless stated other- wise. 14-day old Beijing duck embryos were provided strom et al. 2005). Expanding on this research, amnion (Bilic et al. 2004), amniotic fluid (Gao et al. 2014), by Poultry Experimental Base of Chinese Academy of Agricultural Sciences, Beijing, China. placental tissue(Gekas et al. 2010), umbilical cord blood (Kim et al. 2017) and the Wharton’s Jelly (Taghizadeh et al. 2011) which are rich in stem cells Cell isolation and culture have captured the attention of researchers. The amnion is filled with fluid composed of Initially, the amniotic membrane tissues were exposed and mechanically peeled off from 10 Beijing duck basement layer, compact layer, fibroblastic layer and spongy layer, which is a source of important mes- embryos under sterile conditions. After rinsed well (6 times) with phosphate-buffered saline (PBS), trans- enchymal stem cells with pluripotential characteristics (Cai et al. 2010). Intensive research efforts have been parent amnion layer were cut into small pieces and incubated for 5 min in 0.125% (w/v) trypsin/EDTA reported that the AMSCs are derived from the spongy layer, which, in cell based therapies, have advantage solution to remove epithelial amniotic cells (AECs). After that, membrane fragment were transfered into a over adult type stem cells, such as a higher in vitro expansion potential, telomerase activity, immunolog- clean culture dish and subsequently submitted to 0.1% ical tolerance (Roubelakis et al. 2012). Importantly, collagenase II treatment at 37 C for 20 min. Single- convenient procurement without ethical conflict cell suspensions were extracted by filtration through a makes AMSCs a promising candidate cell for regen- 74 lm cell strainer. The pellets were resuspended with basal DMEM/F12 medium supplemented with 10% erative medicine. Although isolation and characterization of AMSCs fetal bovine serum (FBS), 10 ng/mL basic fibroblast growth factor (bFGF), 1%(v/v) GlutaMAX, and 1% from humans, rats and livestock have been reported, little literature has been done on the avian. Similar to (w/v) non-essential amino acids (NEA) after centrifu- gation at room temperature. After counted, 1 9 10 mammalian development, the avian embryos play a crucial role in developmental and cell biology. Addi- cells/cm were seeded in 60-mm-diameter culture tionally, the avian eggs characterized by small body dishes and incubated at 37 Cina 5% CO atmo- size, ease of manipulation and a low maintenance cost sphere. After 24 h post-seeding, non-adherent cells may serve as significant model system for stem cell were removed from the plate by refreshing medium. research (Li et al. 2011). Notably, our present study When reached 80–90% confluency, attached AMSCs aimed to isolate AMSCs from 14-day old Beijing duck were subcultured with 0.125% trypsin–EDTA and purified AMSCs were harvested after 4–5 passages. embryos and examine their biological characteristics with regard to growth kinetics, karyotype, Growth kinetics and karyotype assay immunophenotype, specific mesenchymal markers and differentiation potential. To evaluate cell proliferative ability, AMSCs at P4, P10, and P18 were subjected to growth kinetics analysis. After detached with 0.125% trypsin/EDTA, Experimental section cells were seeded into 24-well plates at 1 9 10 cells/ Ethics statement well. Subsequently, the cells from three random wells were counted each day for 8 days. Growth curves were All animal experiments were approved and performed drawn in accordance with mean cell numbers and the population doubling time (PDT) was calculated based in accordance with the guidelines established by the Institutional Animal Care and Use Committee at on the formula PDT = (t - t ) lg2/(lgN - lgN ), t : 0 t 0 0 starting time of culture; t: termination time of culture; Chinese Academy of Agriculture Sciences (GB14925- 2010). N : initial cell number of culture; N : ultimate cell 0 t number of culture. The chromosomal profiles of 123 Cell Tissue Bank AMSCs were investigated and analyzed according to cDNA synthesis using 5 9 All-In-One MasterMix standard methods. Briefly, AMSCs at P4 were treated with AccuRT Genomic DNA Removal Kit (abm). with 10 mg/mL colcemid for 2 h. Subsequently, the Specific primers were designed by NCBI primer-blast cells were centrifuged, fixed and stained. And then and details of primers were list in Table 1. PCR chromosome numbers were observed under an oil amplification program included: an initial denatura- immersion objective. tion at 94 C for 3 min, followed by 30 cycles at 94 C for 30 s, annealing for 30 s, an extension at 72 C for Immunofluorescence 30 s and a final extension for 10 min at 72 C. AMSCs at P4 which were grown to confluence in Multiple differentiations potential 6-well plates were prepared and fixed at room temperature for immunofluorescence analysis. After Induction of osteogenic/adipogenic/chondrogenic soaked (15 min) with 4% paraformaldehyde solution, differentiation AMSCs were processed by permeabilizing the mem- brane with 0.25% Triton X-100 for 15 min, followed For the assessment of the mesodermal differentiation by 10% goat serum in PBS and blocked for 1 h at room potential, AMSCs at P4 were targeted for adipogenic, temperature. The direct antibodies used were FITC osteogenic, and chondrogenic differentiation as pre- viously reported (Ma et al. 2017). When reached goat anti-rabbit CD29 (1:100; BIOSS), FITC goat anti-rabbit CD166 (1:100; BIOSS), FITC goat anti- 50–60% confluence, cells were cultured in adipogenic rabbit CD71 (1:100; BIOSS), FITC goat anti-rabbit (10% FBS, 1 mM dexamethasone, 0.5 mM IBMX, CD105 (1:100; BIOSS) and FITC goat anti-rabbit 10 lg/mL insulin, 200 lM indomethacin), the osteo- OCT4 (1:100; BIOSS). For nuclear staining, the cells genic (10% FBS, 0.1 mM dexamethasone, 10 mM b- were incubated with 1 lg/mL DAPI in the dark for glycerophosphate, 0.05 mM ascorbate), or the chon- 15 min. Fluorescence images were acquired by Nikon drogenic (10 lM dexamethasone, 1% Insulin-Trans- TE-2000-E confocal microscope equipped with Nikon ferrin-Selenium, 50 lg/mL L-proline, 1% sodium ZE-1-C1 3.70 digital camera system. pyruvate, 50 lg/mL vitamin C and 10 ng/mL TGF- b3) differentiation medium. The differentiation med- Immunofluorescence characterization ium were changed twice weekly. After 2 weeks of induction, the differentiated AMSCs were detected by For the assessment of immunophenotyping, culture- RT-PCR and visualized by Oil Red O, Alizarin Red expanded AMSCs in logarithmic phase were subjected and Alcian Blue staining, respectively. to flow cytometry analysis. Place the cells into FACS tubes and add precooling 70% ethanol. Incubate Induction of neuronal differentiation overnight at 4 C. The next day, the cells were transferred into a clean FACS tubes with 0.25% Triton To access neuronal differentiation, AMSCs at P4 was X-100 solution in PBS and incubated 15 min at room initially cultured in 10% FBS-DMEM/F-12 in the temperature. After that, 10% BSA (bovine serum presence of 20 ng/mL EGF, 40 ng/mL bFGF, 2% B27 albumin) in PBS were used to block nonspecific and 2 mM L-glutamine. After 6 days of pre-induction, binding. AMSCs were subsequently stained with the 10 ng/mL GDNF, 50 lg/mL vitamin C and 1% N2 following polyclonal antibodies, respectively: CD29- inducing factors were added. After 14 days, the cells FITC, CD166-FITC, CD71-FITC, CD105-FITC, and were detected by immunocytochemical staining and OCT4-FITC. RT-PCR analysis. RNA extraction and RT-PCR Following the manufacturer’s instructions, total RNA (2 lg) that was extracted using Trizol reagent (Invit- rogen) from AMSCs at P8 (90% confluence) or induced differentiated cells served as a template for 123 Cell Tissue Bank Table 1 Primer sequences Gene Primers Products (bp) used for RT-PCR 0 0 CD29 F:5 -CAGAGAGCAACGCAGAGGTT-3 226 0 0 R:5 -ATTGTCACCACCACTTGGCT-3 0 0 CD71 F:5 -GAACCGGTACCTTGAGTGGG-3 415 0 0 R:5 -GCCAGTCCTGAGCCATTTCT-3 0 0 CD166 F:5 -AGGCAAAGCTAATAGTGGGCA-3 209 0 0 R:5 -TCTGGAATGATGACTGACGCA-3 0 0 Vimentin F:5 -GACCAGCTCACCAACGACAA-3 395 0 0 R:5 -GCAGCAACGCTTTCGTACTG-3 0 0 Fibronectin F:5 -CCTCCAACTTCCATCGTGCT-3 320 0 0 R:5 -TCTGGGTGGTACCGGATTCT-3 0 0 PPAR-c F:5 -GCATCGACCAGCTAAACCCT-3 259 0 0 R:5 -TGACATCGCTGGAAAATGCG-3 0 0 LPL F:5 -TTTTCCTTACGGACGCCTGC-3 369 0 0 R:5 -GTGAGCACCCAGACTGTACC-3 0 0 ATF4 F:5 -CCCAGACTCCTACCTGGGAA-3 239 0 0 R:5 -CTGCCCTCTTCTTCTGTCGG-3 0 0 COL1A2 F:5 -GGAATAGCTAGCCACCGACC-3 421 0 0 R:5 -CTCACCGGGAACACCTTGAA-3 0 0 ACAN F:5 -AGTGGCAGCTAATGTGGTCT-3 547 0 0 R:5 -AGCTTGCTCCACTTGATCCG-3 0 0 VIM F:5 -ACGAAAGCGTTGCTGCTAAG-3 218 0 0 R:5 -CTCCATTTCACGCATCTGGC-3 0 0 MAP2 F:5 -ATCAATGGAGAGCTGTCGGC-3 221 0 0 R:5 -GCTCCAGTTTGCTCAGAAGC-3 0 0 GAPDH F:5 -GAGGAGCTGCCCAGAACATT-3 426 0 0 R:5 -GGTCTGCATGCTTGGCTTAC-3 0 0 CD34 F:5 -CTCAACGAGTCCAACACCTG-3 338 0 0 R:5 -CCAGAAGTGACCAAAGCAGTC-3 0 0 CD45 F:5 -CTCACCACACGCACTCTCAC-3 350 Results subculture 4–5 passages (Fig. 1a). After the highest number of passages (P18), the appearance of most Morphological characterization and karyotype cells gradually changed and displayed senescent signs analysis of AMSCs (Fig. 1a). Population growth kinetics of the cells from the low, middle and high passages (P4, P10 and P18) AMSCs were successfully isolated from amnion emerged obvious ‘‘S’’ shapes (Fig. 1b), and PDTs of tissues of 14-day-old Beijing duck embryos and were AMSCs cultures were 34.4, 35.9 and 39.6 h, respec- expanded until passage 18. Approximately 24 h after tively. The chromosome number of duck AMSCs is the initial primary culture, a few cells were observed to 2n = 78, as shown in Fig. 1c. And there were no adhere to petri dishes (Fig. 1a). The cells at passage 0 obvious difference about diploid rates of the cell with then began to proliferate and became confluent after 2n = 78 among different passages. These results 7 days. The initial growth of the cells was mixed with verified the AMSCs cell line we isolated was repro- the AECs. According to different tolerances to trypsin, ducibly diploid. a homogenous monolayer of spindle-shaped fibrob- last-like AMSCs was obtained after successive 123 Cell Tissue Bank Fig. 1 Characterization of duck AMSCs in vitro. a Morpho- with cell density in the Y-axis. c Karyotype analysis of duck logical characteristics of primary and sub-cultured AMSCs AMSCs. Chromosomes at metaphase (left) and karyotype (bar = 50 lm). b Growth kinetics of AMSCs at P4, P10 and P18 (right) Characterization of cells mesenchymal cell genes assessed by RT-PCR exper- iments were also consistent with the immunofluores- The results of immunofluorescence staining showed cence results above, but the expression of CD34 and that culture-expanded cells characteristically CD45 were obviously undetectable (Fig. 2b). expressed pluripotent stem cell marker OCT4. And, the expression of MSC markers CD29, CD166, CD71 Multipotential capacity of AMSCs and CD105 were also positive, as presented in Fig. 2a. Moreover, more than 90% of viable MSC population Adipogenic differentiation isolated from amnion were positive for the MSC markers as assessed by flow cytometry analysis Adipocyte-inducing medium (AID) was prepared to (Fig. 2c). Additionally, expression levels of induce AMSCs to trans-differentiate into adipocytes. 123 Cell Tissue Bank Fig. 2 Detection of surface markers in AMSCs. a The AMSCs and CD45 were not detected. c AMSCs at P4 were colabeled could express pluripotent marker gene OCT4 and MSC- with surface antigens (CD29, CD166, CD71, CD105, OCT4), associated markers by immunofluorescence stain (bar = 50 and the positive rates were all above 99% by flow cytometry lm), DAPI, Blue. b mRNA expression levels of AMSCs analysis markers were detected by RT-PCR, but the expression of CD34 7 days after differentiation, we could observe signif- were positively expressed by RT-PCR analysis icant morphological changes from fibroblast-like to (Fig. 3b). oblate with the formation of oil droplet in the AMSCs. As the induction time progressed, the number of larger Osteogenic differentiation droplets gradually increased. By day 14, the differen- tiated cells were visualized by 0.3% Oil red O staining Alizarin Red staining was performed to evidence (Fig. 3a). And the genes peroxisome proliferator- osteogenic differentiation of the AMSCs. Following activated receptor-gamma (PPAR-c) and lipoprotein induction in osteogenic differentiation medium (OID) lipase (LPL), which are involved in adipogenesis, for 7 days, the induced cells was transformed from 123 Cell Tissue Bank Fig. 3 Adipocyte, osteoblast differentiation of AMSCs (bar = PPAR-c and LPL. c The differentiated cells cultured in OID for 50 lm). a Numerous Lipid droplets, apparent in cytoplasm of 14 days were monitored using Alizarin Red staining. d RT-PCR induced cells, were positive for Oil Red staining. b RT-PCR was assays revealed osteoblast specific genes ATF4 and COL1A2 used to examine the expression of adipocyte marker genes were expressed in the differentiated osteoblast spindle shape into polygonal in appearance, followed Chondrogenic differentiation by noticeable accumulation of mineralization. And under the continued effects of osteogenic supplements Subsequent to chondrogenic differentiation in medium (CID) for 7 days, most cells interconnected to gener- on cells, the calcium deposits nodules increased and were revealed with Alizarin Red (Fig. 3c). At 14 days ate cluster-like aggregation. With prolonged induc- post-induction, the differentiated AMSCs seeded in tion, the cells changed shape with significantly the well expressed the collagen type I alpha 2 chain increased nucleoplasmic ratio and colonies. At day (COL1A2) and activating transcription factor 4 14, Alcian Blue staining was used to assess chondro- (ATF4) genes related to osteogenesis by RT-PCR genesis (Fig. 4a). To further confirm chondrogenic analysis (Fig. 3d). differentiation, RT-PCR amplification of 123 Cell Tissue Bank Fig. 4 Chondrocyte and neurocyte differentiation of AMSCs c The differentiated cells cultured in NID for 14 days expressed (bar = 50 lm). a The differentiated chondrocyte was visualized MAP2 by immunocytochemistry. d RT-PCR detection of the by Alcian Blue staining. b The expression of chondrocyte- neuronal marker MAP2 expression specific genes ACAN and VIM were analyzed by RT-PCR. chondrocyte-specific genes ACAN and VIM were immunofluorescence. After incubation in neural-in- performed with RNA isolated from induced AMSCs ducing medium (NID) for 14 days, the induced cells (Fig. 4b). gradually progressed toward neuron-like morphology with elongated cell bodies, long branches and axon of Neural differentiation neurons (Fig. 4c). And then neural cells derived from AMSCs were identified by RT-PCR (Fig. 4d). The The neuronal differentiation study focused on the results showed that differentiated cells expressed expression of neuronal-specific markers (MAP2) by 123 Cell Tissue Bank neural lineage specific gene MAP2, which were in pluripotent markers Nanog, TRA-1-60, TRA-1-81 accordance with the immunofluorescence results. and STRO-1, but lack expression for HLA-A, HLA-B, and surface molecules (Diaz-Prado et al. 2011; Kastrinaki et al. 2008; Mrugala et al. 2009). Discussion In this present work, AMSCs could be differenti- ated in vitro into adipocytes, osteocytes, chondrocytes, Amniotic membrane is a nearly transparent avascular and neurogenic cells by culturing them in specific membrane without nerves and is composed of AECs induction media which contains a cocktail of inducing and AMSCs. The major role of amniotic membrane is mediators. Dexamethasone, IBMX and insulin could to protect the fetus and provide supplemental nutrients promote adipogenic differentiation of AMSCs. How- during development. Amniotic membrane, known as ever, their exact regulatory mechanisms on adipoge- bio-material, was shown to block proteinase activity nesis remained to be analyzed. Also, b- and promote the wound repair process (Kim et al. glycerophosphate combined with dexamethasone and 2000). It has been reported that amniotic membrane ascorbate was effective in converting AMSCs into could express indispensable growth factors that is osteogenic-specific gene-expressing cells(Le Pape critical for reducing inflammation and fibrosis (Tseng et al. 2018; Zhang et al. 2016). Treatment with et al. 1999). More importantly, the isolated amniotic chondrogenic supplements led to chondrogenesis of AMSCs, and in combination with serum-free medium membrane stem cells could eliminate the concern of teratoma formation in vivo after transplantation, they may reduce apoptosis from the AMSCs (Ibrahim additional properties of noninvasive isolation, multi- et al. 2015). Currently, neurotrophic factors GDNF, potency, anti-inflammatory, minimal ethical problem under appropriate conditions, in vitro, was added to make them promising tools or appropriate sources for enhance the trans-differentiation process of AMSCs clinical treatment, such as corneal tissue (Shimmura (Yang et al. 2014). and Tsubota 2002), treatment scaffolds of corneal Taken together, all these findings show that iso- transplantation (Dua and Azuara-Blanco 1999), lated AMSCs retained self-renewal ability and multi- Parkinson’s disease (Kakishita et al. 2003), spinal potentiality in vitro. However, its preclinical applica- cord injury (Gao et al. 2016), and brain infarction bility still remains controversial in cell-based thera- (Sakuragawa et al. 1997). pies and tissue engineering in vivo, both securely and Interestingly, we successfully harvested heteroge- technically. Further detailed studies are required to neous population of AMSCs from amniotic membrane define the specific surface markers for AMSCs of 14-day old Beijing duck embryos and attempted to characterization and to investigate molecular mecha- investigate their biological characteristics in vitro. In nisms for AMSCs differentiation, which would per- the experimental set-up, enzymatic digestion was haps facilitate more effective therapies of AMSCs in employed to isolate and purify AMSCs. The cells in regenerative medicine. culture were expanded at least 18 passages in vitro. And, evaluation of karyotyping and growth curves demonstrated that culture-expanded AMSCs main- Conclusions tained renewal activity and genetic stability. In terms of the maintenance of stemness of stem cells, gene AMSCs were isolated from Beijing duck embryos. The self-renewal ability and differentiation potential expression was significant. Similar to other sources of MSCs, AMSCs possessed MSC characteristics and of the isolated AMSCs was evaluated in vitro. Our characteristically expressed a set of MSC markers, findings provide a platform for the establishment of a such as CD29, CD71, CD105, CD166 and OCT4, but Beijing duck AMSCs bank. These results have impli- negativity for CD45 and CD34. Expanding on this cations for the potential application of AMSCs as a research, the pluripotency of AMSCs was monitored stem-cell source for regenerative medical therapies. through the expression of OCT4 which is the marker Acknowledgements This research was supported by the of non-differentiation stage (Corradetti et al. 2013). In National Natural Science Foundation of China (31472099, addition, several lines of evidence suggest that 31672404), the project National Infrastructure of Animal cultured AMSCs also positively express the 123 Cell Tissue Bank Germplasm Resources (2016 year), the project of Agricultural Gekas C, Rhodes KE, Van Handel B, Chhabra A, Ueno M, Science and Technology Innovation Program (cxgc-ias-01), and Mikkola HK (2010) Hematopoietic stem cell development the domestic animal germplasm resources sharing construction in the placenta. Int J Dev Biol 54:1089–1098 (Y2017LM21). Gotherstrom C, West A, Liden J, Uzunel M, Lahesmaa R, Le Blanc K (2005) Difference in gene expression between human fetal liver and adult bone marrow mesenchymal Compliance with ethical standards stem cells. Haematologica 90:1017–1026 Gruen L, Grabel L (2006) Concise review: scientific and ethical Conflict of interest The authors declare that they have no roadblocks to human embryonic stem cell therapy. Stem conflict of interest. Cells 24:2162–2169 Guo DL, Wang ZG, Xiong LK, Pan LY, Zhu Q, Yuan YF, Liu Open Access This article is distributed under the terms of the ZS (2017) Hepatogenic differentiation from human adi- Creative Commons Attribution 4.0 International License (http:// pose-derived stem cells and application for mouse acute creativecommons.org/licenses/by/4.0/), which permits unre- liver injury. Artif Cells Nanomed Biotechnol 45:224–232 stricted use, distribution, and reproduction in any medium, Gurel Pekozer G, Ramazanoglu M, Schlegel KA, Kok FN, provided you give appropriate credit to the original Torun Kose G (2018) Role of STRO-1 sorting of porcine author(s) and the source, provide a link to the Creative Com- dental germ stem cells in dental stem cell-mediated bone mons license, and indicate if changes were made. tissue engineering. 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Journal

Cell and Tissue BankingSpringer Journals

Published: Jun 1, 2018

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