DSpace at VNU: Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applicat...
Cell Tissue Bank DOI 10.1007/s10561-015-9541-6 ORIGINAL PAPER Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications Phuc Van Pham Nhat Chau Truong Phuong Thi-Bich Le Tung Dang-Xuan Tran Ngoc Bich Vu Khanh Hong-Thien Bui Ngoc Kim Phan Received: September 2015 / Accepted: 11 December 2015 Ó Springer Science+Business Media Dordrecht 2015 Abstract Umbilical cord (UC) is a rich source of rapidly proliferating mesenchymal stem cells (MSCs) that are easily cultured on a large-scale Clinical applications of UC–MSCs include graft-versus-host disease, and diabetes mellitus types and UC– MSCs should be isolated and proliferated according to good manufacturing practice (GMP) with animal component-free medium, quality assurance, and quality control for their use in clinical applications This study developed a GMP standard protocol for UCMSC isolation and culture UC blood and UC were collected from the same donors Blood vasculature was removed from UC UC blood was used as a source of activated platelet rich plasma (aPRP) Small fragments (1–2 mm2) of UC membrane and Wharton’s jelly were cut and cultured in DMEM/F12 medium containing % antibiotic–antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 °C in % CO2 The MSC properties of UC–MSCs at passage such as osteoblast, chondroblast and adipocyte differentiation, and markers including CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were confirmed UC–MSCs also were analyzed for karyotype, expression of tumorigenesis related genes, cell cycle, doubling time as well as in vivo tumor formation in NOD/SCID mice Control cells consisted of UC–MSCs cultured in DMEM/F12 plus % antibiotic–antimycotic, and 10 % fetal bovine serum (FBS) All UC-MSC (n = 30) samples were successfully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS UC–MSCs in these four groups exhibited similar marker profiles Moreover, the proliferation rates in medium with PRP, especially 7.5 and 10 %, were significantly quicker compared with 2.5 and % aPRP or 10 % FBS These cells maintained a normal karyotype for 15 sub-cultures, and differentiated into P Van Pham (&) Á N C Truong Á N B Vu Á N K Phan Laboratory of Stem Cell Research and Application, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam e-mail: pvphuc@hcmuns.edu.vn P T.-B Le Á T D.-X Tran Van Hanh Stem Cell Unit, Van Hanh Hospital, Ho Chi Minh City, Vietnam e-mail: drbphuong@gmail.com N C Truong e-mail: tcnhat@hcmus.edu.vn N B Vu e-mail: vbngoc@hcmus.edu.vn N K Phan e-mail: pvphuc@hcmus.edu.vn T D.-X Tran e-mail: drxuantung@gmail.com K H.-T Bui University Medical Center, University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam e-mail: khanhbui1969@yahoo.com 123 Cell Tissue Bank osteoblasts, chondroblasts, and adipocytes The analysis of pluripotent cell markers showed UC–MSCs maintained the expression of the oncogenes Nanog and Oct4 after long term culture but failed to transfer tumors in NOD/SCID mice Replacing FBS with aPRP in the culture medium for UC tissues allowed the successful isolation of UC–MSCs that satisfy the minimum standards for clinical applications Keywords Activated platelet rich plasma Á Clinical application of mesenchymal stem cells Á Umbilical cord Á Umbilical cord derived mesenchymal stem cells Á Good manufacturing practice Á UC–MSCs Introduction Although umbilical cord blood (UCB) and the umbilical cord (UC) were previously considered medical waste, they are a rich source of stem cells In UCB, at least different kinds of stem cells have been isolated including hematopoietic stem cells, mesenchymal stem cells (MSCs) and endothelial progenitor cells (Phuc et al 2012; Pineault and Abu-Khader 2015; Lee et al 2004; Mareschi et al 2001) The UC is also a source of MSCs (Romanov et al 2003; Kim et al 2004) Compared with other MSC sources, the advantages of UC–MSCs are non-invasive recovery, high abundance, and can be used off-the-shelf A recent study by Ding et al (Ding et al 2015) showed that UC–MSCs strongly expressed HLA-G, a nonclassical HLA allele with strong immune-inhibitory properties, which efficiently inhibited a lymphocyte reaction assay (Ding et al 2015) Therefore, UC– MSCs are promising candidates for the treatment of immune system and autoimmune diseases MSCs have been used for the treatment of diseases in both preclinical and clinical trials Pre-clinically, UC–MSCs have been used to treat myocardial infarction (Santos et al 2014), enhance hematopoiesis after cord blood transplantation (Wu et al 2013a, b), and traumatic brain injury (Peng et al 2015) Clinically, UC–MSCs have been transplanted for the treatment of refractory systemic lupus erythematosus nephritis (Woodworth and Furst 2014; Wang et al 2014; Gu et al 2014; Wang et al 2013; Sun et al 2010; Shi et al 2012), type diabetes mellitus (Kong et al 2014), sequelae of thoracolumbar spinal cord injury (Cheng et al 2014), autism (Lv et al 2013), hereditary 123 spinocerebellar ataxia (Jin et al 2013), spinocerebellar ataxia and multiple system atrophy-cerebellar type (Dongmei et al 2011), spinal cord injury (Liu et al 2013), primary biliary cirrhosis (Wang et al 2013), sequelae of traumatic brain injury (Wang et al 2013), acute-on-chronic liver failure (Shi et al 2012), decompensated liver cirrhosis (Zhang et al 2012; Chen et al 2012), stroke (Jiang et al 2013), and steroid-resistant severe acute graft-versus-host disease (Chen et al 2012) According to the ClinicalTrials.gov registry and results database of clinical studies of human participants Clinical trials [clinicaltrial.gov], there are about 96 clinical trials using UC–MSCs for the treatment of more than 10 different diseases, including type diabetes mellitus (NCT01219465, NCT01143168), type diabetes mellitus (NCT02302599), Hepatic Cirrhosis (NCT01224327, NCT01342250, NCT01220492), Ulcerative Colitis (NCT01221428), Hereditary Ataxia (NCT01360164), Diabetic Foot (NCT01216865), Cerebral Hemorrhage Sequela (NCT02283879), Duchenne Muscular Dystrophy (NCT02285673, NCT02235844), Articular Cartilage Defect (NCT02291926), Progressive Multiple Sclerosis and Neuromyelitis Optica (NCT0136 4246), Idiopathic Dilated Cardiomyopathy (NCT0121 9452), Acute Burn (NCT01443689), and Autism (NCT 02192749, NCT01343511) Previous studies have successfully isolated MSCs from UC (Buyl et al 2014; Mori et al 2015; Badraiq et al 2015) These studies confirmed that UC–MSCs expressed an MSC phenotype characterized by adherence to plastic disks with a fibroblast like shape, the expression of cell markers CD44, CD73, CD90, CD105, a lack of CD14, CD34, CD45 and HLA-DR expression; and differentiation into three kinds of mesenchymal cells including adipocytes, osteoblasts, and chondroblasts (Buyl et al 2014) The main concern of using UC–MSCs for clinical applications is that in vitro expansion is affected by the culture medium Production protocols of UC–MSCs for use under clinical conditions, it is essential to include sterility controls, analysis for viral markers, and genetic testing such as karyotyping However, most studies culture UC–MSCs in medium supplemented with FBS (Buyl et al 2014; Mori et al 2015; Iftimia-Mander et al 2013; Zhang et al 2012) Recent studies have suggested alternatives to medium containing FBS or allogenic serum, such as chemically defined conditions (Badraiq et al 2015) Some novel methods used human serum or platelet- Cell Tissue Bank rich plasma (PRP) to replace FBS Recent studies have used PRP from peripheral blood (Kocaoemer et al 2007; Bieback et al 2009; Jonsdottir-Buch et al 2013; Rauch et al 2011; Blande et al 2009) and UCB (Ding et al 2013; Shetty et al 2007; Ma et al 2012; Murphy et al 2012) to culture MSCs from bone marrow (Bieback et al 2009; Shetty et al 2007), UCB (Ding et al 2013; Baba et al 2013), or adipose tissue (Kocaoemer et al 2007; Escobedo-Lucea et al 2013) We previously isolated and proliferated MSCs from UCB using activated (a) PRP derived from the same source as the UCB Taken together, these studies indicate PRP might replace FBS for in vitro MSC expansion According to the European Medicines Agency and regulation No [EC] 1394/2007 of the European Commission, MSC are considered as medicinal products (Fekete et al 2012) and must be produced in compliance with GMP; therefore, cells should be produced with the highest standards of sterility, quality control, and documentation following a standard operating procedure In this study, we aimed to establish an UC-MSC isolation protocol using aPRP from the same source as the UC sample This protocol is GMP compliant and can be used for clinical applications Methods Human UC and UCB Both UCB and UC were collected from the same donor with informed consent of the mother The collection was performed in accordance with the ethical standards of the local ethics committee Preparation of cell culture medium Culture medium used in this study was DMEM/F12 (the basal medium, Gibco, Thermo Scientific, Cat no 11039-021) supplemented with 10 % FBS, % antibiotic–antimycotic as published previously (Buyl et al 2014) and DMEM/F12 (the basal medium, Gibco, Thermo Scientific, Cat no 11039-021) supplemented with activated platelet rich plasma (aPRP) prepared from the same donor as the UC sample (Fig 1) Briefly, after birth, UCB was collected into a tube containing CDP-A anticoagulation (Terumo, Japan) Both UCB and UC were transferred into the laboratory at a cool temperature Then UCB was used to prepare the aPRP as published previously (Pham et al 2014; Van Pham and Phan 2015) Blood samples were centrifuged at 2000 rpm for 15 The plasma was collected and centrifuged at 3500 rpm for 10 To prepare aPRP, a third of the plasma volume and the platelet pellet was collected and resuspended, and then 100 lL CaCl2 per mL of PRP was added to activate growth factor release The samples were then incubated at 37 °C for 30 or until the occurrence of clotting Cells were grown in culture medium with different concentrations of aPRP or 10 % FBS: DMEM/F12 supplemented with 2.5 % aPRP (Group I); DMEM/ F12 supplemented with 5.0 % aPRP (Group II); DMEM/F12 supplemented with 7.5 % aPRP (Group III), DMEM/F12 supplemented with 10 % aPRP (Group IV), and DMEM/F12 supplemented with 10 % FBS (Group V) DMEM/F12, FBS, CaCl2 and antibiotic–antimycotic were purchased from SigmaAldrich, St Louis, MO, USA Primary cell culture of UC derived MSCs After arteries and veins were removed, the remaining tissue and Wharton’s jelly, was cut into 0.5–1 cm3 pieces and suspended in culture medium The tissue was left undisturbed for days in a 37 °C humidified incubator with % CO2 to allow cells to migrate from the explants Culture medium was replaced every days The cells were passaged through a TrypLE solution when cells reached 80–90 % confluence (Invitrogen, Gibco) Immunophenotypic Analysis Primary antibodies against human antigen CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and histocompatibility antigen DR alpha chain (HLA-DR) were purchased from BD Biosciences (San Jose, CA) MSCs (5 105 cells) were resuspended in 500 lL phosphate-buffered saline (PBS; InvitrogenGibco) and cultured with fluorescein-isothiocyanate(FITC) or phycoerythrin- (PE) conjugated primary antibodies for 20 at room temperature (RT) FITCor PE-conjugated human IgGs were used as isotype controls at the same concentration as the specific primary antibodies The fluorescence intensity of the cells was evaluated by flow cytometry (FACScan; BD Biosciences) Data were analyzed using CELLQUEST software (BD Biosciences) 123 Cell Tissue Bank Fig Procedure of isolation and culture of umbilical cord derived mesenchymal stem cells Both umbilical cord and umbilical cord blood were collected from the same donor Umbilical cord was used to isolate MSCs, and umbilical cord blood was used to produce PRP as supplement of culture medium Secondary culture and doubling time assay This assay was performed as previously described (Pham et al 2014) ascorbate-2-phosphate (Sigma-Aldrich) The medium was changed every days Osteogenic differentiation was confirmed by the Alizarin Red staining Adipogenic induction Differentiation Assays Osteogenic induction Upon reaching 50 % confluency, cells were cultured for 14–21 days in LG-DMEM containing 10 % FBS, 0.1 lM dexamethasone (Sigma-Aldrich), 10 mM bglycerophosphate (Sigma-Aldrich), and 100 lM 123 Upon reaching 100 % confluency, cells were cultured for 14–21 days in LG-DMEM containing 10 % FBS, lM dexamethasone, 0.5 lM isobutyl methylxanthine (Sigma-Aldrich), 100 lM indomethacin (Sigma-Aldrich), and 10 lg/mL insulin (Sigma-Aldrich) Adipogenic differentiation was evaluated by the cellular accumulation of neutral Cell Tissue Bank lipid vacuoles that were stained with Oil Red O (Sigma-Aldrich) statistical analyses and performed with GraphPad Prism software, version 4.0 P values \ 0.05 were considered statistically significant Karyotyping assay Proliferating MSCs were treated with colcemid at a concentration of 0.10 lg/ml for h Primary cells were harvested, and then used for karyotyping as previously published Briefly, a single cell suspension was incubated in hypotonic solution for 30 at 37 °C, and then fixed at least times in Carnoy’s solution, which included an overnight fixative step The fixed cell suspension was dropped on wellprepared slides and stained according to the G-Banding protocol Sets of chromosomes were analyzed using Ikaros software (MetaSystems, Altlussheim, Germany) Tumorigenicity assay The tumorigenicity of UC–MSCs was examined in athymic nude mice All manipulations of mice were approved by the Local Ethics Committee of Stem Cell Research and Application, University of Science (Ho Chi Minh City, Vietnam) Each mouse was injected subcutaneously with 106 UC–MSCs (three mice per group) As a positive control, mice were injected with breast cancer cells at a different site Tumor formation in mice was followed up for months Gene expression assay At the 5th, 10th and 15th passage, UC–MSCs were collected and total RNA was extracted according to the standard protocol of easy-BlueTM Total RNA Extraction Kit (iNtRON Biotechnology, INC.) Total RNA was used for gene expression analysis immediately Tumor suppressor genes included p15, p53 and pten; oncogenes included Oct3/4, Nanog, Sox-2 The Gapdh gene was used as an internal control The PCR program was set up using the Realplex 2.2 software (Eppendorf, Germany) with a gradient Tm for different pairs of primers Statistical methods The results were expressed as the mean ± SD Oneway ANOVA and two-tailed tests were utilized for all Results Primary culture of UC For all groups, after days of culture, cells inside tissue fragments migrated and adhered to the surface of culture dishes Adherent cells exhibited an MSClike shape, similar to fibroblasts Most cultures were confluent after 14 days These cells were continuously cultured after sub-culturing and were considered as passage In the primary culture, there were some differences between groups including time for adherent cells to appear, and the quality of fragments for the growth of adherent cells All UC-MSC (n = 30) samples were successfully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS The results showed that in groups III and IV, cells appeared on day 3; while in other groups cells appeared on day Light microscopy observation indicated that cells in groups III and IV rapidly increased with many mitotic cells in the microscope field After primary culture, isolated cells were collected by TrypLE to obtain single cells that were secondarily cultured up to passage and used to evaluate stemness characteristics Proliferation of derived cells Cells of all groups at passage were cultured in an xCelligence plate to evaluate cell proliferation (Fig 2) The proliferation rate was different between groups Cells in group V (10 % FBS) slowly proliferated with a longer doubling time, and small slope value, whereas cells in groups I, II, III and IV rapidly increased, especially groups III and IV The doubling time of cells in groups III and IV were not significantly different, but were significantly different compared with groups I, II and V (P \ 0.05) By comparing slope values and doubling times analyzed by the xCelligence system between groups, the proliferation rates of cells in groups III and IV were nearly doubled compared with those in groups I, II and V 123 Cell Tissue Bank Fig Cell proliferation evaluated by the xCelligence system a Proliferation curve, b doubling time, c slope value Fig Mesenchymal stem cell marker expression Cells from all groups expressed specific mesenchymal stem cell markers, positive with CD13, CD44, CD73, CD90, CD105, CD29; negative with CD14, CD34, HLA-DR and CD45 123 Cell Tissue Bank Fig Differentiation potential of mesenchymal stem cell from umbilical cord tissues Cells grown from all samples showed mesenchymal stem cell (MSC) shapes (a–e, respectively groups I–V) Isolated cells successfully differentiated into adipocytes (f–k, respectively groups I–V), osteoblasts (l–p, respectively groups I–V) All figures were captured at 109 magnification Expression of mesenchymal stem cell markers 5.83 ± 0.85 % for group I, II, III, IV and V, respectively) All cells in all groups satisfied the MSC marker profiles They expressed CD13, CD29, CD44, CD73, CD90, CD105, but were negative for markers of other cells such as CD14 (monocytes), CD34 (hematopoietic stem cells), CD45 (leukocytes), and HLA-DR (mature leukocytes) (Fig 3) Cells at passage were used to evaluate the expression of MSC markers including CD13 (93.5 ± 3.21 %, 93.0 ± 4.53 %, 95.27 ± 2.12 %, 94.1 ± 1.99 %, 96.11 ± 3.88 % for group I, II, III, IV and V, respectively), CD14 (4.11 ± 1.18 %, 2.57 ± 1.03 %, 3.85 ± 1.05 %, 3.75 ± 1.02 %, 3.73 ± 0.42 % for group I, II, III, IV and V, respectively), CD34 (3.81 ± 2.05 %, 3.32 ± 1.32 %, 3.10 ± 1.15 %, 4.21 ± 0.92 %, 2.01 ± 0.22 % for group I, II, III, IV and V, respectively), CD44 (96.53 ± 2.13 %, 92.0 ± 1.21 %, 93.15 ± 2.92 %, 97.11 ± 2.89 %, 92.43 ± 1.78 % for group I, II, III, IV and V, respectively), CD73 (86.13 ± 2.09 %, 85.09 ± 4.82 %, 87.75 ± 4.22 %, 90.51 ± 3.01 %, 88.56 ± 4.18 % for group I, II, III, IV and V, respectively), CD90 (92.78 ± 2.83 %, 91.01 ± 1.90 %, 93.44 ± 2.12 %, 98.41 ± 2.19 %, 97.35 ± 2.18 % for group I, II, III, IV and V, respectively), CD105 (92.23 ± 3.64 %, 91.42 ± 2.09 %, 93.45 ± 1.22 %, 95.21 ± 1.56 %, 98.13 ± 1.55 % for group I, II, III, IV and V, respectively), and HLA-DR (5.61 ± 1.58 %, 4.45 ± 1.43 %, 4.15 ± 1.25 %, 4.24 ± 1.14 %, 4.14 ± 1.12 % for group I, II, III, IV and V, respectively), CD29 (91.93 ± 3.73 %, 92.56 ± 1.31 %, 91.55 ± 1.22 %, 93.41 ± 2.19 %, 95.13 ± 2.88 % for group I, II, III, IV and V, respectively) and CD45 (2.81 ± 1.08 %, 2.97 ± 0.93 %, 2.85 ± 1.15 %, 5.15 ± 1.52 %, Derived cells successfully differentiate into mesoderm lineage cells Next, MSC candidates were actively differentiated into three kinds of mesoderm, including adipocytes, and osteoblasts After 21 days culture in inducing medium, most derived cells had changed phenotype toward a differentiated cell including adipocytes that store lipid drops inside the cytoplasm, osteoblasts that elongate with extracellular matrix formation and stained positive with Alizarin red (Fig 4) UC–MSCs maintained stemness and proliferation during long time culture To evaluate the effects of aPRP on MSC stemness for long-term cultures compared with FBS, we compared MSCs in groups V and group IV after continuous subculture up to passage 15, by characterizing the expression of markers and differentiation potentials 123 Cell Tissue Bank 123 Cell Tissue Bank b Fig UC–MSCs in groups IV and V at passage 5th, 10th and 15th Cells maintained the shapes (a–c, d–f; respectively PRP 7.5 %, FBS 10 %), adipocyte differentiation (g–i, k–m, respectively PRP 7.5 %, FBS 10 %), osteoblast differentiation (n–p, q–s, respectively PRP 7.5 %, FBS 10 %) All figures were captured at 109 magnification MSCs from groups IV and V maintained stemness with the specific marker profiles of MSCs, and successful differentiation into adipocytes, osteoblasts, and chondrocytes However, the proliferation rate of cells at passage 15th was significantly decreased Fig Comparison of UC–MSCs at 5th and 15th passages between cultured in 7.5 % PRP and 10 % FBS a They did not change the expression of particular markers However, they were significantly different the cell proliferation, doubling time and slope value b Fig Karyotypes of mesenchymal stem cells were maintained stably up to 15 passages MSCs cultured 7.5 % PRP at passages 5th (a), 15th (c); 10 % FBS at passages 5th (b), 15th (d) 123 Cell Tissue Bank Fig The expression of some tumor suppressor genes and oncogenes in UC–MSCs after 5th, 10th and 15th passages The results showed that tumor suppressor genes (p15, p53 and pten) nearly did not changes the expression level during 5, 10 and 15th passages, while oncogenes (Oct3/4, Nanog and Sox-2) slightly increased after 10 and 15th passages but not significantly compared with those of passage MSCs in group V The results of xCelligence analysis of doubling time and slope values confirmed this observation (Figs 5, 6) 5th, 10th and 15th passages) (Fig 8) Importantly, MSCs at passage 15 did not cause tumors when transferred into NOD/SCID mice UC MSCs maintain chromosome stability in longtime culture Discussion MSCs in groups IV and V at passages and 15 were used to for karyotype analysis The karyotype of MSCs in both groups and at any passage maintained a normal karyotype that contained 2n = 46 chromosomes (Fig 7) UC MSCs maintain the expression of oncogenes and tumor suppressor genes and not form tumors in NOD/SCID mice To evaluate the safety of these MSCs for long-term culture, we compared the expression of oncogenes and tumor suppressor genes at passages 5th and 15th in MSCs from groups IV and V The cells changed oncogene expression after long-term culture but these changes were non-significant (Oct3/4: 15.00 ± 1.15; 14.33 ± 1.86; 18.0 ± 1.73 compared to GAPDH, respectively for 5th, 10th and 15th passages; Nanog: 39.00 ± 2.08; 41.33 ± 1.86; 44.67 ± 5.17 compared to GAPDH, respectively for 5th, 10th and 15th passages; and Sox-2: 12.00 ± 1.73; 12.67 ± 2.40; 16.00 ± 3.06 compared to GAPDH, respectively for 123 UC is a rich source of MSCs that have a huge potential for regenerative medicine In recent years, UC–MSCs have been used clinically to treat a number of diseases However, UC–MSCs are normally isolated and cultured in xenogenic FBS or allogenic human serum, which might increase risks of immunological reactions and viral transmission The current study developed a novel procedure to reduce these risks associated with MSC culture using xenogenic or allogenic serum In this study, we replaced xenogenic serum or allogenic serum with autologous serum in the form of aPRP prepared from UCB obtained from UC Using this method, UC–MSCs were not subject to xenogenic or allogenic immunological reactions, or contaminated by prions from cows or animal and human viruses More importantly, cells obtained from medium supplemented with aPRP exhibited an MSC phenotype that satisfied all minimal criteria of MSCs suggested by Dominici et al (Dominici et al 2006) MSCs proliferated in plastic flasks, exhibited a fibroblast-like shape, strongly expressed CD13, Cell Tissue Bank CD29, CD44, CD73, CD90 and CD105, and lacked the expression of CD14, CD34, CD45, and HLA-DR Finally, they successfully differentiated into cells belonging to the mesoderm, including adipocytes, and osteoblasts in vitro In this study, we also determined which concentrations of aPRP had an effect on UC-MSC proliferation Using 7.5 or 10 % aPRP, UC–MSCs rapidly proliferated compared with those cultured in 2.5 and % aPRP Of note, aPRP stimulated UC–MSCs stronger than FBS did In 10 % FBS, UC–MSCs proliferation was similar to those cultured with 2.5 and % aPRP These results were confirmed by doubling time and slope value analysis In previous studies, aPRP also was demonstrated to stimulate MSCs stronger than FBS did (Van Pham and Phan 2015) Indeed, aPRP contains high amounts of adhesion proteins such as fibrin, fibronectin, vitronectin, and thrombospondin (Ding et al 2013; Marx 2004) and contains several growth factors that stimulate cell proliferation, such as epidermal growth factor, acidic fibroblast growth factor, keratinocyte growth factor, vascular endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor, and basic fibroblast growth factor (Ding et al 2013; Murphy et al 2012; Lee et al 2011) Therefore, autologous aPRP removed risks related to allogenic and xenogenic materials as well as stimulating UC MSC proliferation to a greater degree than FBS Regenerative medicine requires a large number of UC–MSCs Therefore, we evaluated the effects of medium containing aPRP on long-term UC-MSC culture aPRP maintained the UC–MSCs phenotype better than FBS did during long-term culture After continuous subculture (15 times) of greater than 60 days, UC–MSCs in aPRP medium maintained a normal MSC phenotype with 46 chromosomes and their stemness was maintained with differentiation potentials as well as proliferation There were no significant changes in cell cycle, and they stably kept their doubling time In contrast, UC–MSCs cultured for 15 passages in FBS medium became uniform in shape, with significantly reduced population doublings, and cell cycle accumulation at G0/G1 These results confirmed previous observations when UC– MSCs were cultured in FBS medium (Otte et al 2013) The expression of both oncogenes and tumor suppressor genes in UC–MSCs cultured in aPRP was unchanged at passages and 15 Therefore, although aPRP stimulated UC-MSC proliferation it did not stimulate the over-expression of oncogenes similar to pluripotent stem cells, and inhibited tumor suppressor genes These might also explain why UC–MSCs cultured in both media supplemented with aPRP and FBS did not cause tumors in NOD/SCID mice Taken together, autologous aPRP can replace FBS for the culture and proliferation of UC–MSCs Regarding the quantity of aPRP required for MSC culture, we showed that the mean amount obtained from a sample of UCB was about 60–140 mL of blood (included anticoagulants), from which we successfully produced 30–70 mL of aPRP With a supplement concentration of 7.5 % aPRP, we could produce 400–900 mL of complete medium If UC– MSCs are cultured in T-75 cm2 flasks, then 40–90 flasks of UC–MSCs can be cultured to obtain 40–90 106 UC–MSCs This amount of UC–MSCs can be used directly for clinical transplantation or cell banking When banked UC–MSCs are cultured to proliferate after thawing, the recipient’s aPRP must be used In combination with our previously published study regarding the isolation and proliferation of UCBderived MSCs using autologous aPRP, we hope that all sources of MSCs including UCB- and UC–MSCs can now be isolated to satisfy clinical grades (Pham et al 2014; Van Pham and Phan 2015) Conclusion UC is a rich source of MSCs UC–MSCs can be isolated and cultured with xenogenic and allogeneic component-free medium In this study, we successfully established a GMP-compliant UC-MSC isolation protocol Autologous aPRP is used to replace FBS UC–MSCs can be isolated by expanding the culture in DMEM/F12 supplemented with 7.5 or 10 % aPRP and % antibiotic-mycotic The MSCs obtained from this protocol maintain the MSC phenotype, stemness, and differentiation potential The use of aPRP in this protocol induced a greater degree of proliferation compared with FBS and reduced MSC aging in vitro In particular, the isolated MSCs stably expressed oncogenes and tumor suppressor genes and did not form tumors at high doses when transferred into athymic nude mice Therefore, we hope this protocol will be suitable for the isolation of UC–MSCs for use in clinical applications 123 Cell Tissue Bank Acknowledgments This research was funded by Ministry of Science and Technology via project Grant No DTDL.2012-G/ 23 Authors’ contributions PVP conceived the study, performed PRP preparation, evaluated the effects of PRP on mesenchymal stem cell proliferation NBV primarily cultured mesenchymal stem cells from mononuclear cells; TDXT, PTBL collected umbilical cord blood, isolated mononuclear cells from umbilical cord blood; KHTB carried out the differentiation assays; NTC, NKP evaluated the, karyotype, and tumorigenecity of MSCs in mice model All authors read and approved the final manuscript Compliance with ethical standards Conflict of interests competing interests The authors declare that they have no References Baba K, Yamazaki Y, Ishiguro M, Kumazawa K, Aoyagi K, Ikemoto S, Takeda A, Uchinuma E (2013) Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: a preliminary study J Craniomaxillofac Surg 41:775–782 Badraiq H, Devito L, Ilic D (2015) Isolation and expansion of mesenchymal stromal/stem cells from umbilical cord under chemically defined conditions Methods Mol Biol 1283:65–71 Bieback K, Hecker A, Kocaomer A, Lannert H, Schallmoser K, Strunk D, Kluter H (2009) Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow Stem Cells 27:2331–2341 Blande IS, Bassaneze V, Lavini-Ramos C, Fae KC, Kalil J, Miyakawa AA, Schettert IT, Krieger JE (2009) Adipose tissue mesenchymal stem cell expansion in animal serumfree medium supplemented with autologous human platelet lysate Transfusion 49:2680–2685 Buyl K, Vanhaecke T, Desmae T, Lagneaux L, Rogiers V, Najar M, De Kock J (2014) Evaluation of a new standardized enzymatic isolation protocol for human umbilical cordderived stem cells Toxicol In Vitro 29:1254–1262 Chen GH, Yang T, Tian H, Qiao M, Liu HW, Fu CC, Miao M, Jin ZM, Tang XW, Han Y, He GS, Zhang XH, Ma X, Chen F, Hu XH, Xue SL, Wang Y, Qiu HY, Sun AN, Chen ZZ, Wu DP (2012) Clinical study of umbilical cord-derived mesenchymal stem cells for treatment of nineteen patients with steroid-resistant severe acute graft-versus-host disease Zhonghua Xue Ye Xue Za Zhi 33:303–306 Cheng H, Liu X, Hua R, Dai G, Wang X, Gao J, An Y (2014) Clinical observation of umbilical cord mesenchymal stem cell transplantation in treatment for sequelae of thoracolumbar spinal cord injury J Transl Med 12:253 Ding Y, Yang H, Feng JB, Qiu Y, Li DS, Zeng Y (2013) Human umbilical cord-derived MSC culture: the replacement of animal sera with human cord blood plasma In Vitro Cell Dev Biol Anim 49:771–777 123 Ding DC, Chou HL, Chang YH, Hung WT, Liu HW, Chu TY (2015) Characterization of HLA-G and related immunosuppressive effects in human umbilical cord stroma derived stem cells Cell Transplant doi:10.3727/09636 8915X688182 Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells The International Society for Cellular Therapy position statement Cytotherapy 8: 315–317 Dongmei H, Jing L, Mei X, Ling Z, Hongmin Y, Zhidong W, Li D, Zikuan G, Hengxiang W (2011) Clinical analysis of the treatment of spinocerebellar ataxia and multiple system atrophy-cerebellar type with umbilical cord mesenchymal stromal cells Cytotherapy 13:913–917 Escobedo-Lucea C, Bellver C, Gandia C, Sanz-Garcia A, Esteban FJ, Mirabet V, Forte G, Moreno I, Lezameta M, Ayuso-Sacido A, Garcia-Verdugo JM (2013) A xenogeneic-free protocol for isolation and expansion of human adipose stem cells for clinical uses PLoS One 8:e67870 Fekete N, Rojewski MT, Furst D, Kreja L, Ignatius A, Dausend J, Schrezenmeier H (2012) GMP-compliant isolation and large-scale expansion of bone marrow-derived MSC PLoS One 7:e43255 Gu F, Wang D, Zhang H, Feng X, Gilkeson GS, Shi S, Sun L (2014) Allogeneic mesenchymal stem cell transplantation for lupus nephritis patients refractory to conventional therapy Clin Rheumatol 33:1611–1619 Iftimia-Mander A, Hourd P, Dainty R, Thomas RJ (2013) Mesenchymal stem cell isolation from human umbilical cord tissue: understanding and minimizing variability in cell yield for process optimization Biopreserv Biobank 11:291–298 Jiang Y, Zhu W, Zhu J, Wu L, Xu G, Liu X (2013) Feasibility of delivering mesenchymal stem cells via catheter to the proximal end of the lesion artery in patients with stroke in the territory of the middle cerebral artery Cell Transplant 22:2291–2298 Jin JL, Liu Z, Lu ZJ, Guan DN, Wang C, Chen ZB, Zhang J, Zhang WY, Wu JY, Xu Y (2013) Safety and efficacy of umbilical cord mesenchymal stem cell therapy in hereditary spinocerebellar ataxia Curr Neurovasc Res 10:11–20 Jonsdottir-Buch SM, Lieder R, Sigurjonsson OE (2013) Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells PLoS One 8:e68984 Kim JW, Kim SY, Park SY, Kim YM, Kim JM, Lee MH, Ryu HM (2004) Mesenchymal progenitor cells in the human umbilical cord Ann Hematol 83:733–738 Kocaoemer A, Kern S, Kluter H, Bieback K (2007) Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue Stem Cells 25:1270–1278 Kong D, Zhuang X, Wang D, Qu H, Jiang Y, Li X, Wu W, Xiao J, Liu X, Liu J, Li A, Wang J, Dou A, Wang Y, Sun J, Lv H, Zhang G, Zhang X, Chen S, Ni Y, Zheng C (2014) Umbilical cord mesenchymal stem cell transfusion ameliorated hyperglycemia in patients with type diabetes mellitus Clin Lab 60:1969–1976 Cell Tissue Bank Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH (2004) Isolation of multipotent mesenchymal stem cells from umbilical cord blood Blood 103:1669–1675 Lee JY, Nam H, Park YJ, Lee SJ, Chung CP, Han SB, Lee G (2011) The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells In Vitro Cell Dev Biol Anim 47:157–164 Liu J, Han D, Wang Z, Xue M, Zhu L, Yan H, Zheng X, Guo Z, Wang H (2013) Clinical analysis of the treatment of spinal cord injury with umbilical cord mesenchymal stem cells Cytotherapy 15:185–191 Lv YT, Zhang Y, Liu M, Qiuwaxi JN, Ashwood P, Cho SC, Huan Y, Ge RC, Chen XW, Wang ZJ, Kim BJ, Hu X (2013) Transplantation of human cord blood mononuclear cells and umbilical cord-derived mesenchymal stem cells in autism J Transl Med 11:196 Ma HY, Yao L, Yu YQ, Li L, Ma L, Wei WJ, Lu XM, Du LL, Jin YN (2012) An effective and safe supplement for stem cells expansion ex vivo: cord blood serum Cell Transplant 21:857–869 Mareschi K, Biasin E, Piacibello W, Aglietta M, Madon E, Fagioli F (2001) Isolation of human mesenchymal stem cells: bone marrow versus umbilical cord blood Haematologica 86:1099–1100 Marx RE (2004) Platelet-rich plasma: evidence to support its use J Oral Maxillofac Surg 62:489–496 Mori Y, Ohshimo J, Shimazu T, He H, Takahashi A, Yamamoto Y, Tsunoda H, Tojo A, Nagamura-Inoue T (2015) Improved explant method to isolate umbilical cord-derived mesenchymal stem cells and their immunosuppressive properties Tissue Eng Part C Methods 21:367–372 Murphy MB, Blashki D, Buchanan RM, Yazdi IK, Ferrari M, Simmons PJ, Tasciotti E (2012) Adult and umbilical cord blood-derived platelet-rich plasma for mesenchymal stem cell proliferation, chemotaxis, and cryo-preservation Biomaterials 33:5308–5316 Otte A, Bucan V, Reimers K, Hass R (2013) Mesenchymal stem cells maintain long-term in vitro stemness during explant culture Tissue Eng Part C Methods 19:937–948 Peng W, Sun J, Sheng C, Wang Z, Wang Y, Zhang C, Fan R (2015) Systematic review and meta-analysis of efficacy of mesenchymal stem cells on locomotor recovery in animal models of traumatic brain injury Stem Cell Res Ther 6:47 Pham PV, Vu NB, Pham VM, Truong NH, Pham TL, Dang LT, Nguyen TT, Bui AN, Phan NK (2014) Good manufacturing practice-compliant isolation and culture of human umbilical cord blood-derived mesenchymal stem cells J Transl Med 12:56 Phuc PV, Ngoc VB, Lam DH, Tam NT, Viet PQ, Ngoc PK (2012) Isolation of three important types of stem cells from the same samples of banked umbilical cord blood Cell Tissue Bank 13:341–351 Pineault N, Abu-Khader A (2015) Advances in umbilical cord blood stem cell expansion and clinical translation Exp Hematol 43:498–513 Rauch C, Feifel E, Amann EM, Spotl HP, Schennach H, Pfaller W, Gstraunthaler G (2011) Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media ALTEX 28:305–316 Romanov YA, Svintsitskaya VA, Smirnov VN (2003) Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord Stem Cells 21:105–110 Santos Nascimento D, Mosqueira D, Sousa LM, Teixeira M, Filipe M, Resende TP, Araujo AF, Valente M, Almeida J, Martins JP, Santos JM, Barcia RN, Cruz P, Cruz H, Pinto-do OP (2014) Human umbilical cord tissue-derived mesenchymal stromal cells attenuate remodeling after myocardial infarction by proangiogenic, antiapoptotic, and endogenous cell-activation mechanisms Stem Cell Res Ther 5:5 Shetty P, Bharucha K, Tanavde V (2007) Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells Cell Biol Int 31:293–298 Shi D, Wang D, Li X, Zhang H, Che N, Lu Z, Sun L (2012a) Allogeneic transplantation of umbilical cord-derived mesenchymal stem cells for diffuse alveolar hemorrhage in systemic lupus erythematosus Clin Rheumatol 31:841–846 Shi M, Zhang Z, Xu R, Lin H, Fu J, Zou Z, Zhang A, Shi J, Chen L, Lv S, He W, Geng H, Jin L, Liu Z, Wang FS (2012b) Human mesenchymal stem cell transfusion is safe and improves liver function in acute-on-chronic liver failure patients Stem Cells Transl Med 1:725–731 Sun L, Wang D, Liang J, Zhang H, Feng X, Wang H, Hua B, Liu B, Ye S, Hu X, Xu W, Zeng X, Hou Y, Gilkeson GS, Silver RM, Lu L, Shi S (2010) Umbilical cord mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus Arthritis Rheum 62:2467–2475 Van Pham P, Phan NK (2015) Production of good manufacturing practice-grade human umbilical cord blood-derived mesenchymal stem cells for therapeutic use Methods Mol Biol 1283:73–85 Wang D, Zhang H, Liang J, Li X, Feng X, Wang H, Hua B, Liu B, Lu L, Gilkeson GS, Silver RM, Chen W, Shi S, Sun L (2013a) Allogeneic mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus: years of experience Cell Transplant 22:2267–2277 Wang L, Li J, Liu H, Li Y, Fu J, Sun Y, Xu R, Lin H, Wang S, Lv S, Chen L, Zou Z, Li B, Shi M, Zhang Z, Wang FS (2013b) Pilot study of umbilical cord-derived mesenchymal stem cell transfusion in patients with primary biliary cirrhosis J Gastroenterol Hepatol 28(Suppl 1):85–92 Wang S, Cheng H, Dai G, Wang X, Hua R, Liu X, Wang P, Chen G, Yue W, An Y (2013c) Umbilical cord mesenchymal stem cell transplantation significantly improves neurological function in patients with sequelae of traumatic brain injury Brain Res 1532:76–84 Wang D, Li J, Zhang Y, Zhang M, Chen J, Li X, Hu X, Jiang S, Shi S, Sun L (2014) Umbilical cord mesenchymal stem cell transplantation in active and refractory systemic lupus erythematosus: a multicenter clinical study Arthritis Res Ther 16:R79 Woodworth TG, Furst DE (2014) Safety and feasibility of umbilical cord mesenchymal stem cells in treatment-refractory systemic lupus erythematosus nephritis: time for a double-blind placebo-controlled trial to determine efficacy Arthritis Res Ther 16:113 Wu KH, Tsai C, Wu HP, Sieber M, Peng CT, Chao YH (2013a) Human application of ex vivo expanded umbilical cordderived mesenchymal stem cells: enhance hematopoiesis after cord blood transplantation Cell Transplant 22: 2041–2051 123 Cell Tissue Bank Wu KH, Sheu JN, Wu HP, Tsai C, Sieber M, Peng CT, Chao YH (2013b) Cotransplantation of umbilical cord-derived mesenchymal stem cells promote hematopoietic engraftment in cord blood transplantation: a pilot study Transplantation 95:773–777 Zhang Z, Lin H, Shi M, Xu R, Fu J, Lv J, Chen L, Lv S, Li Y, Yu S, Geng H, Jin L, Lau GK, Wang FS (2012a) Human 123 umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients J Gastroenterol Hepatol 27(Suppl 2):112–120 Zhang H, Zhang B, Tao Y, Cheng M, Hu J, Xu M, Chen H (2012b) Isolation and characterization of mesenchymal stem cells from whole human umbilical cord applying a single enzyme approach Cell Biochem Funct 30:643–649 ... medium for UC tissues allowed the successful isolation of UC–MSCs that satisfy the minimum standards for clinical applications Keywords Activated platelet rich plasma Á Clinical application of mesenchymal. .. CELLQUEST software (BD Biosciences) 123 Cell Tissue Bank Fig Procedure of isolation and culture of umbilical cord derived mesenchymal stem cells Both umbilical cord and umbilical cord blood were... rich source of stem cells In UCB, at least different kinds of stem cells have been isolated including hematopoietic stem cells, mesenchymal stem cells (MSCs) and endothelial progenitor cells (Phuc