Supporting data on combined transcriptomic and phosphoproteomic analysis of BMP4 signaling in human embryonic stem cells

Human embryonic stem cells exhibit great potential as a therapeutic tool in regenerative medicine due to their self-renewal and trilineage differentiation capacity. Maintaining this unique cellular state has been shown to rely primarily on the Activin A / TGFβ signaling pathway. While most conventional culture media are supplemented with TGFβ, in the current study we utilize a modified version of the commercially available mTeSR1, substituting TGFβ for Activin A in order to preserve pluripotency. (1) Cells cultured in ActA-mTesR express pluripotency factors NANOG, OCT4 and SOX2 at comparable levels with cells cultured in TGFβ-mTeSR. (2) ActA-mTeSR cultured cells retain a physiological karyotype. (3) Cells in ActA-mTeSR maintain their trilineage differentiation capacity as shown in the teratoma formation assay. This system can be used to dissect the role of Activin A, downstream effectors and signaling cascades in human embryonic stem cell responses.

(3) Cells in ActA-mTeSR maintain their trilineage differentiation capacity as shown in the teratoma formation assay. This system can be used to dissect the role of Activin A, downstream effectors and signaling cascades in human embryonic stem cell responses.
© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) Table   Subject Cell Biology Specific subject area Human Embryonic Stem Cells Type of data Graph, Figure  How data were acquired Image capture (Leica TCS SP8 and SP5 confocal microscopes, LASAF software) Western blotting (LI-COR Odyssey imaging system, image studio software) Data analysis (GraphPad Prism version 6.0) qRT-PCR (Light Cycler) Teratoma formation assay (H&E staining) Data format Raw, Analyzed Parameters for data collection H1 cells were cultured in TGF β or Activin A-based mTeSR1 for several passages were tested for expression of pluripotency markers, karyotypic abnormalities as well as for in vitro and in vivo differentiation capacity. Description of data collection Pluripotency assessment was performed by indirect immunofluorescence of H1 colonies with antibodies against NANOG, OCT4 and SOX2. In vitro differentiation potential was assessed by subjecting H1 cells to three separate protocols for mesoderm, endoderm and ectoderm differentiation. Karyotypic analysis was performed by colchicine-induced mitotic arrest and Giemsa staining. Assessing in vivo differentiation potential required the formation of teratomas in NOD/SCID mice and the subsequent H&E staining. Data

Value of the Data
• These data provide an Activin A dependent culture system substituting TGF β in mTeSR1 and capable of maintaining the pluripotency of hESCs. • The culture system developed is a versatile tool for studying signaling pathways in hESCs.
• This system allows for complete control over the growth factors and compounds included in a standard hESC culture and can therefore be employed to develop novel pluripotency maintenance or differentiation protocols.

Data Description
To develop an mTeSR-dependent human embryonic stem cell (hESC) culture method replacing TGF β with Activin A, H1 cells were cultured in mTeSR without select factors supplemented with LiCl, GABA, pipecolic acid and FGF2 at the concentrations stated in the manufacturer's protocol [2] . Media was also supplemented with 0.6 ng/ml TGF β (TGF β-mTeSR) or 0.5 ng/ml Activin A (ActA-mTeSR). After adapting cells in ActA-mTeSR for 10 passages, localization of the pluripotency factors NANOG, OCT4 and SOX2 was assessed by immunofluorescence ( Fig. 1 A), protein levels of NANOG and OCT4 were quantified by western blotting ( Fig. 1 B) and the karyotypic profile was inspected by Giemsa staining ( Fig. 1 C). Subsequently, ActA-mTeSR-adapted cells were assessed for their in vitro trilineage differentiation capacity. For mesodermal specification, H1 cells were induced with Activin A and BMP4. BRACHYURY levels were analyzed by Western blotting ( Fig. 2 A). For ectodermal differentiation, H1 cells were subjected to an embryoid body formation assay and stained for PAX6 ( Fig. 2 B). For endodermal differentiation, H1 cells were induced with Activin A in a serum-containing medium and transcription levels of SOX17 were evaluated by qPCR ( Fig. 2 C).
Last, ActA-mTeSR cultured cells were assessed for their capacity to form in vivo teratomas.
Cells cultured in regular TGF β-mTeSR were used as a positive control. Teratoma size differences between the two cell groups were not scored for, as this methodology was applied in a qualitative manner as a pluripotency test ( Fig. 2 D). Haematoxylin and eosin staining was applied to visualize the different cell types that emerged ( Fig. 2 E). Activin A was first implicated in pluripotency when its precursor was found in the secretome of mouse embryonic fibroblasts (MEFs) which served as feeders for hESC cultures [3] . Since then, several Activin A-dependent systems have been developed for the undifferentiated propagation of hESCs bypassing the need for MEFs or MEF secreted factors [ 4 , 5 ]. Our study provides an additional versatile Activin A-dependent medium which allows for the study of pluripotency and differentiation in a chemically defined background.
On day 10 cells were fixed with 4% PFA.

Teratoma formation assay
Sub-confluent H1 cells cultured in ActA-or TGF β-mTeSR were manually dissected with an insulin needle and digested with 1 mg/ml dispase (Gibco; 17,105-041). The cell clumps were spun down for 3 min at 100 g and resuspended in 150 μl matrigel. The mixture was injected through a 25 G 7/8 needle (BD Biosciences; 305,124) subcutaneously into the hind legs of immunodeficient NOD/SCID mice. Prior to injection, the matrigel, tips and syringes were stored on ice to avoid matrix polymerization. Mice were maintained under pathogen free conditions, at the animal house of the Biomedical Research Foundation (Academy of Athens, Greece). 10 weeks later, animals were sacrificed and the resulting teratomas were fixed, embedded in paraffin, dissected and stained with haematoxylin/eosin.

Ethics Statement
All animal experimentation was performed in accordance with directive 2010/63/EU and the Amsterdam protocol on animal protection and welfare. Permission for the use of H1 embryonic stem cells was obtained from the Steering Committee for the UK Stem Cell Bank and Use of Human Stem Cells, MRC, UK.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.
for the Strategic Development on the Research and Technological Sector", funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020 ) and co-financed by Greece and the European Union (European Regional Development Fund).