The study was conducted in accordance with the Karolinska Institutet Ethics Committee to derive and culture hESCs, with the Ethics Committee of Pirkanmaa Hospital District to culture, characterize and differentiate hESCs derived at Karolinska Institutet (R05051), to derive, culture, characterize and differentiate new hESC lines at Regea (R05116) and with the permission of the National Authority for Medicolegal Affairs to do research with human embryos (Dnro 1426/32/300/05). Donated embryos were received from Tampere University Hospital in vitro fertilization (IVF) clinic. An informed consent form was signed by both partners after receiving an oral and written description of the study. The donors did not receive financial compensation. Animal experiments were performed at the animal facilities of Karolinska University Hospital or at Turku Center for Disease Modeling and Department of Physiology, Institute of Biomedicine, University of Turku in accordance with the approval of the institution's Ethics Committee. Adipose stem cells (ASCs) were isolated from adipose tissue samples collected from female donors undergoing elective surgical procedures at the Department of Plastic Surgery, Tampere University Hospital with the permission of the Ethics Committee of Pirkanmaa Hospital District. Human iPS cells were derived and characterized at the University of Helsinki, with the permission of the Ethics Committee of the University of Helsinki.
The hESC lines HS237, HS346, and HS401 used in the optimization of the xeno-free formulation RegES were initially derived and characterized at the Karolinska Institutet, Stockholm, Sweden 
. These cell lines were derived and cultured using conventional hES medium containing KO-DMEM basal medium supplemented with 20% KO-SR, 2 mM Glutamax, 0.1 mM β-mercaptoethanol (all from Invitrogen, Carlsbad, CA), 0.1 mM MEM non-essential amino acids (NEAA), 1% penicillin-streptomycin (both from Cambrex Bio Science, Walkersville, MD) and 8 ng ml/ml recombinant human bFGF (R&D Systems Inc., Minneapolis, MN). Human iPSC lines FiPS 5-7 and FiPS 6-14 were derived and characterized at the University of Helsinki. Briefly, human cDNAs of Oct4
, were amplified by direct PCR from hESC cDNA, and cloned into pMXs retroviral vector (Addgene Inc., Cambridge, MA). 293-GPG packaging cells were transfected with each pMXs-cDNA vector separately using Fugene HD (Roche Diagnostics GmbH, Mannheim, Germany). Next day, fresh MEF media was added. Viral supernatant was collected 3 times at days 4, 5, and 6 post-transfection, combined and then filtered. The human neonatal foreskin fibroblast line CCCD-1112Sk (ATCC) was plated on gelatin-coated petri dishes and incubated overnight. Cells were then infected with an equally mixed combination of fresh virus-containing supernatant twice at 24 h intervals. At day 3, infected cells were harvested and re-seeded on a Mitomycin C-treated MEF layer. The following day, the medium was changed to hES medium. After 20 to 30 days post-transduction, ES-like colonies were picked, expanded, and characterized as previously described for hESCs 
. Transgene expression was studied using RT-PCR to confirm transgene silencing. FiPS 5-7 was generated using Oct4, Sox2, Nanog, and Lin28 encoding retroviruses, while FiPS 6-14 was generated using the same factors plus to Klf4.
Human ESCs and iPSCs were gradually adapted to the RegES medium (Table S1
20, and 100
0) during the first four passages. Commercially available hFFs (CRL-2429, ATCC, Manassas, VA) cultured in IMDM with L-glutamine and 25 mM HEPES (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin (Cambrex Bio Science) were used for hESCs and iPSCs culture. Confluent monolayers of hFFs were mitotically inactivated by irradiation (40 Gy) and plated at a density of 3.8×104
. The growth of hESCs and iPSCs was monitored microscopically and culture media were changed daily. The hESC and iPSC cultures were mechanically passaged every 6 to 7 days to new mitotically inactivated feeder cells. Single-cell enzymatic passaging was performed for hESCs as described previously 
using TrypLE Select (Invitrogen) and an 8 to 12 day splitting interval. The hESCs were frozen using RegES medium supplemented with 10% DMSO (Sigma-Aldrich, St. Louis, MO). Xeno-free commercially available HEScGRO medium (Millipore Corporation, Billerica, MA) without any supplementation was compared to RegES medium for the culture of hESCs.
ASCs were isolated from adipose tissue samples collected from female donors undergoing elective surgical procedures in the Department of Plastic Surgery, Tampere University Hospital. Isolation of ASCs from adipose tissue samples was carried out as described previously 
. Briefly, the adipose tissue was minced manually into small fragments and digested with 1.5 mg/mL collagenase type I (Life Technologies, Paisley, UK) in a shaking water bath at 37°C. To separate the adipose stem cells from the surrounding tissue the digested tissue was centrifuged and filtered in sequential steps. The isolated cells were expanded in DMEM/F-12 1
1, supplemented with 1% GlutaMAX, 1% antibiotics/antimycotic (a/a; 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B) (all from Invitrogen) and 10% alloHS (PAA Laboratories GmbH, Pasching, Austria), (HS medium). For testing defined, xeno-free conditions, ASCs were transferred to RegES culture medium in flasks with pre-coated CELLstart (Invitrogen). For detachment of ASCs TrypLE Select (Invitrogen) was used.
Derivation of hESC lines
Surplus poor quality embryos that could not be used for infertility treatment were obtained as donations from couples undergoing in vitro
fertilization treatment at Tampere University Hospital, Tampere, Finland. The mechanical derivation of the cell lines from blastocyst-stage embryos with a hardly visible inner cell mass was performed using a specially made flexible metal needle (Hunter Scientific, Essex UK) and surgical knife as described previously 
. For successful derivation of the 06/015 cell line, we used 10 surplus embryos (arrested around day 3–4) without blastocyst development. Derivation of the cell line Regea 06/015 was performed by removing the zona pellucida and plating the remaining cells on the top of hFF cells using a flexible needle. Human ESC line Regea 06/040 was derived using hES medium and hESC lines Regea 06/015, Regea 07/046, and Regea 08/013 were derived using RegES medium. Human ESC line Regea 06/015 was derived on feeder cells (C-12300 PromoCell GmbH, Heidelberg, Germany) cultured in IMDM with L-glutamine and 25 mM HEPES (Invitrogen) supplemented with 10% human serum (Sigma-Aldrich), 0.1 mM NEAA (Cambrex Bio Science), and 0.1 mM penicillin-streptomycin (Cambrex Bio Science).
Immunocytochemistry was performed as previously described 
. The primary antibodies used for hESCs and iPSCs were specific for Nanog, Oct4 (both from R&D Systems), SSEA-4 and Sox2 (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and TRA-1-60 (Millipore). The cells were incubated with primary antibody solution overnight at 4°C. The cells were probed with Alexa Fluor 568 or Alexa Fluor 488 (both from Invitrogen) secondary antibodies for 1 h in the dark at room temperature. The differentiated cardiomyocytes were incubated overnight at 4°C with anti-cardiac troponin T (Abcam, Cambridge, UK) and anti-ventricular myosin heavy chain (Chemicon). Alexa Fluor 488 or 568 (Invitrogen) conjugated anti-goat and anti-mouse antibodies were used as secondary antibodies. Differentiated neural cells were immunostained after 9 weeks of differentiation. For immunocytochemical staining neurospheres were plated on human laminin (Sigma Aldrich) coated wells in the absence of bFGF and after growing three days the cells were fixed. The fixed neuronal cells were incubated overnight at 4°C with polyclonal rabbit anti- MAP-2 (Chemicon) for neuronal cells and polyclonal sheep anti-GFAP (R&D Systems) for astrocytes. Alexa Fluor 488 and 568 conjugated anti-rabbit or anti-sheep (Invitrogen) were used as secondary antibodies. Human ESCs labeled only with secondary antibodies, were used as negative controls and no fluorescense in these samples were detected. After incubation, the cells were mounted in Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA). The labeled cells were photographed with an Olympus IX51 phase contrast microscope with fluorescence optics and Olympus DP30BW camera (Olympus Corporation, Tokyo, Japan).
Alkaline phosphatase staining
The in vitro
osteogenic differentiation capacity was determined 14 days after the initiation of differentiation by alkaline phosphatase staining (ALP) as described previously 
. The cell cultures were fixed with a 4% paraformaldehyde (PFA) solution and stained with the leukocyte alkaline phosphatase kit according to the Sigma Procedure No. 86 (#86R-1KT). The ALP staining was confirmed by a purple staining visualized by microscopy.
Adipogenesis Oil red O staining
The adipogenic differentiation was confirmed with Oil red O staining as described previously 
. Cell cultures were maintained for 14 days and subsequently fixed with 4% PFA, pretreated with 60% isopropanol and stained with a 0.5% Oil red O solution in 60% isopropanol. After fixation and staining, the wells were rinsed with distilled water and visualized by microscopy. Adipocytes were identified as cells with red-stained lipid vesicles.
Chondrogenesis Alcian blue staining
The chondrogenic differentiation cultures were maintained for 14 days and were subsequently fixed with 4% PFA, and stained with 1% Alcian blue stain. Cells stained with Alcian blue verified the presence of sulphated proteoglycans within the chondrogenic cell pellet.
Human ESCs and iPSCs cultured in hES and RegES media were analyzed by flow cytometry using antibodies against SSEA-4-PE (R&D Systems) and TRA-1-81-FITC (BD Pharmingen, San Diego, CA). Human ESC samples cultured in hES and RegES media were from 7 day old colonies, iPSC samples cultured in hES medium from 6 day old colonies and samples cultured in RegES medium from 7 day old (5–7) or 8 day old colonies (6–14). Both undifferentiated and differentiated cells present in the culture dish were included in the analyses. Alexa Fluor 488 (Invitrogen) and R-phycoerythrin-conjugated (Caltag-Medsystems Ltd, Buckingham, UK) secondary antibodies were used as isotype controls. ASCs cultured in HS and RegES media (n
4, passages 2–4) were analyzed by flow cytometry using monoclonal antibodies against CD90-APC (BD Biosciences), CD45-FITC (Miltenyi Biotech, Bergisch Gladbach, Germany), CD34-APC, HLA-ABC-PE, HLA-DR-PE (all from Immunotools GmbH Friesoythe, Germany), and CD105-PE (R&D Systems). The samples were analysed by flow cytometry (FACSAria®, BDBiosciences). The acquisition was set for 10 000 events per sample. The data were analyzed using FACSDiva Software version 4.1.2 (BD Biosciences).
RNA isolation and reverse transcription
Total RNA was isolated using an RNeasy mini plus kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The concentration and quality of isolated RNA was determined using a ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Complementary DNA (cDNA) was synthesized from 50 ng of total RNA using a Sensiscript Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions.
Both undifferentiated and differentiated hESCs and iPSCs in the culture plate were included in the quantitative RT-PCR analyses. Quantitative RT-PCR was performed for hESCs and iPSCs with Applied Biosystems Gene Expression Assays: GAPDH
(Hs99999905_m1; 122-bp product), DNMT3B
(Hs01003405_m1; 80-bp product), TDGF1
(Hs02339496_g1; 102-bp product), Nanog
(Hs2387400_g1; 109-bp product), GDF3
(Hs00220998_m1; 65-bp product), GABRB3
(Hs01115771_m1; 72-bp product), and Oct4
(Hs00999632_g1; 77-bp product). All samples and the no-template controls were analyzed in triplicate. Quantitative RT-PCR was performed with an Applied Biosystems 7300 Real-Time PCR system using the following conditions: 40 cycles of 50°C, 2 min; 95°C, 10 min; and 95°C, 15 s; followed by 60°C, 1 min. The data were analyzed with a 7300 System SDS Software (Applied Biosystems). The cycle threshold (Ct) values were determined for every reaction. Relative quantification was calculated using the 2−ΔΔCt
. All data were normalized to the expression of the gene GAPDH
whose expression does not change under the experimental conditions. The data are presented as mean fold-change. Neural cells:
RNA isolation and reverse transcription of RT-PCR samples (≥10 spheres/sample) was performed as described above. The primer set for RT-PCR contained Oct4
for undifferentiated hESC; α-fetoprotein
for endodermal lineage; brachyury/T
for mesodermal lineage; Musashi
, and PAX6
for neuroectodermal cells; MAP-2
for neuronal cells; GFAP
for astrocytes, and Olig1
for oligodentrocyte precursor cells. Data were normalized to the expression of the gene GAPDH
whose expression does not change under the experimental conditions. Primer sequences are shown in supplementary Table S2
. The RT-PCR reactions were performed in an Eppendorf Mastercycler using 35 PCR cycles with an initializing step at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 5 min. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing 0.4 µg/ml ethidium bromide (Sigma-Aldrich).
Cell proliferation assay
Cell proliferation of the hESC lines was determined using a colorimetric immunoassay (Roche Diagnostics) based on the measurement of bromodeoxyuridine (BrdU) incorporation during DNA synthesis. The assay was performed according to the manufacturer's instructions. The hESC colonies at day 5 were labeled with BrdU-labeling solution overnight at 37°C. The hESCs (105 cells/well) were added to a 96-well plate, with 8 replicates per cell line. Cells that were not labeled with BrdU were used as a background control. The absorbance of the samples was measured at 450 nm using a Viktor 1429 Multilabel Counter (PerkinElmer-Wallace, Norton, OH).
The cell viability and proliferation activity of the ASCs was assessed using PreMix WST-1 Cell Proliferation Assay System (Takara Bio Inc., Shiga, Japan). The ASCs (n
7 donor cell samples, with 4 replicates per cell line) were seeded on a 48-well plate at a density of 4500 cells/cm2
, and the proliferation was determined at 1, 4, 7 and 11 days. Briefly, the cell culture medium was removed and DPBS and PreMix WST-1 were added 10
1. The well plate was incubated for 4 hours in 37°C and the cell proliferation activity was measured using a Viktor 1429 Multilabel Counter at 450 nm.
Karyotype analysis was performed by G-banding technique in Medix Laboratories Inc, Helsinki, Finland. In brief, the hESCs were treated with colchicine for 2 hours, and cells in metaphase were analyzed using conventional light microscopy (Olympus, BX 50). Karyotypes were determined using IKAROS-software designed for chromosome analysis (MetaSystems GmbH, Altlussheim, Germany). A total of 20 cells in metaphase were analyzed for each cell line.
Analysis of pluripotency in vitro
The embryoid bodies (EBs) formed by mechanically dissecting the hESC and iPSC colonies were cultured without feeder cells in RegES or hES medium without bFGF for 4 weeks before RNA isolation. The media was changed every 2 to 3 days. RNA isolation and reverse transcription from EBs was performed as described above. The expression of markers characteristic of ectoderm (PAX6
, DNA Technologies Inc. Gaithersburg, MD), endoderm (α-fetoprotein
, DNA Technologies Inc.), and mesoderm (α-cardiac actin
, Proligo, Sigma-Aldrich and T
, DNA Technologies Inc.) development in EBs was determined using RT-PCR primers. β-actin
(DNA Technologies Inc.) whose expression does not change under the experimental conditions was used as a control. Primer sequences are shown in supplementary Table S2
. Negative controls contained sterilized water instead of the cDNA template. The RT-PCR reactions were performed in an Eppendorf Mastercycler as follows: denaturation at 95°C for 3 min and 40 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 5 min. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing 0.4 µg/ml ethidium bromide (Sigma-Aldrich) and 50 bp DNA standard (MassRulerTM DNA Ladder Mix, Fermentas).
Analysis of pluripotency in vivo
Animal experiments with hESC lines Regea 06/015, Regea 06/040 and Regea 07/046 were performed at the infection-free animal facility of Karolinska University Hospital and experiments with hESC line Regea 08/013 at the animal facility of Turku Center for Disease Modeling and Department of Physiology, Institute of Biomedicine, University of Turku. Human ESCs were harvested from the culture plates using dispase or TrypleSelect and mechanical treatment. Five colonies (103
hESC) were washed twice in PBS and subsequently implanted beneath the testicular capsule of a young (6–8 week-old) severe combined immunodeficiency (scid)/beige male mouse (C.B.-17/GbmsTac-scid-bgDF N7, M&B, Ry, Denmark) or in the case of Regea 08/013 male Nude mouse (Hsd;Athymic Nude-Foxn1nu
, Harlan). Three animals were used for cell line. Teratoma growth was determined by palpation once per week, and the mice were killed by cervical dislocation 8 to 9 weeks after implantation. The in vivo
pluripotency of the hESC lines Regea 06/015, Regea 06/040 and Regea 07/046 was analyzed as previously described 
. Immunohistochemical studies of Regea 08013 cell line teratomas were done in paraffin sections. The dewaxed and rehydrated sections were treated in pressure cooker in 1 mM EDTA, pH 8, (Desmin and HNF3β) or 10 mM sodium citrate buffer, pH 6, (NCAM) for 5 minutes to reveal antigenic sites, cooled at room temperature (RT) for 2 hours, and rinsed in phosphate buffer saline (PBS). After rinsing with PBS the sections were incubated with primary antibodies (rabbit anti-desmin, sc-14026, mouse anti-HNF3β, sc-101060, purchased from Santa Cruz Biotechnology, or rabbit anti-NCAM, AB5032, purchased from Chemicon International. Primary antibodies were diluted (1
3000, and 1
800 respectively) in PBS containing 0.05% Tween® and incubated overnight at +4 degrees. The next day, after several rinses with PBS, endogenous peroxidase was inactivated with 1% hydrogen peroxide in PBS for 20 minutes. After rinsing several times with PBS, the sections were incubated with secondary antibodies (HRP conjugated Dako Envision™+ Systems) for 30 minutes (RT). Slides were rinsed with PBS. Color was developed with diaminobenzidine substrate (Dako Envision™+ Systems). Sections were slightly counterstained with Mayer's hematoxylin, dehydrated, and mounted.
Differentiation of hESCs to cardiomyocytes and neural cells
Cardiomyocyte differentiation was carried out as described previously 
by co-culturing Regea 08/013 hESCs, maintained in RegES (p46) and in hES (p52) media, with mouse visceral endodermal-like (END-2) cells, which were a kind gift from Prof. Mummery, Humbrecht Institute, Utrecht, Netherlands. Briefly, undifferentiated hESC colonies were dissected mechanically into aggregates containing a few hundred cells and placed on the top of plated END-2 cells in hES culture medium without KO-SR and bFGF. Differentiating cell colonies were monitored by microscopy daily and the medium was changed after 5 days of culturing. After 12 days the 10% KO-SR (Invitrogen) was added to the medium. Differentiation was performed in 12-well plates, 15 hESC colony pieces in a well. The beating areas were dissociated by collagenase II treatment. The cells were plated on 0.1% gelatin coated cell culture plates in a medium containing 7.5% FBS.
Neural differentiation was performed as previously described 
. Briefly, hESC (Regea 08/013) colonies maintained in RegES (p53) and in hES (p58) media, were mechanically dissociated into small clusters containing ~3000 cells. Clusters were transferred into 6-well ultra low attachment plates (Nunc, Thermo Fisher Scientific, Rochester, NY) and cultured as floating aggregates, e.g. neurospheres, for up to 20 weeks in neural differentiation medium consisting of 1
1 DMEM/F-12:Neurobasal media supplemented with 2 mM Glutamax, 1xB27, 1xN2 (all from Invitrogen), 25 U/ml penicillin-streptomycin (Lonza Group Ltd), and 20 ng/ml bFGF (R&D Systems). Medium was changed 3 times/week and the spheres were mechanically passaged weekly.
Differentiation of ASCs to adipogenic, osteogenic and chondrogenic lineages
ASCs (passage 3) cultured in RegES medium were analyzed for their capacity to differentiate toward the adipogenic, osteogenic, and chondrogenic lineages with ASCs cultured in medium containing HS as reference. Briefly, osteogenic differentiation analyses were performed on ASCs seeded onto a 12-well plate at a density of 2.5×103 cells/cm2 in RegES media supplemented with 150 µM L-ascorbic acid 2-phosphate (Sigma), 10 mM β-glycerophosphate (Sigma) and 100 nM dexamethasone (Sigma) in culture vessels were pre-coated with CELLstart. The control cultures were maintained in RegES media without supplements in pre-coated culture vessels. The cultures were maintained 14 days and osteogenic differentiation was detected by alkaline phosphatase activity staining.
Adipogenic differentiation was induced by culturing ASCs for two weeks in adipogenic media with an initial seeding density of 2×104 cells/cm2 in culture vessels were pre-coated with CELLstart. For the adipogenic induction, RegES media supplemented with 33 µM biotin (Sigma), 1 µM dexamethasone, 100 nM insulin (Invitrogen) and 17 µM pantothenate (Fluka, Buchs, Switzerland) was used while the control cultures were maintained in RegES without adipogenic supplements. The cultures were analyzed using an Oil red O stain as an indicator of intracellular lipid accumulation.
The chondrogenic differentiation capacity analysis was assessed by micromass culture technique 
. Briefly, 1×105
cells were seeded onto a 24-well culture plate in a 10 µl volume, and were let adhere for 3 h in a cell incubator prior to addition of the chondrogenic RegES media containing 1% ITS+1 (Sigma), 50 µM L-ascorbic acid 2-phosphate, 55 µM sodium pyruvate (Invitrogen), L-proline 23 µM (Sigma). The control cultures were maintained in RegES without chondrogenic supplements. The chondrogenic induction cultures and control cultures were performed without CELLstart pre-coating. The cultures were maintained for 14 days prior to analysis. Chondrogenesis was confirmed using Alcian blue stain.
ANOVA by the methodology of mixed models was used for the proliferation data analysis of ASCs between the different culture media compositions within each separate experiment setup to account for the correlation of multiple measurements within donors. Mann-Whitney U test was applied in the surface marker expression of ASCs to compare the different culture conditions. The surface marker expression of ASCs was analyzed using Wilcoxon signed-rank statistics, which was used for pairwise comparison between the different culturing conditions. The statistical analyses were performed at the significance level p<0.05. The analyses were performed using SPSS software version 13 (SPSS Inc., Chicago, IL, USA).