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Activin A and BMP4 Signaling Expands Potency of Mouse Embryonic Stem Cells in Serum-Free Media.

ABSTRACT: Inhibitors of Mek1/2 and Gsk3β, known as 2i, and, together with leukemia inhibitory factor, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency (2i/L-ESCs). However, recent reports show that prolonged Mek1/2 suppression impairs developmental potential of ESCs, and is rescued by serum (S/L-ESCs). Here, we show that culturing ESCs in Activin A and BMP4, and in the absence of MEK1/2 inhibitor (ABC/L medium), establishes advanced stem cells derived from ESCs (esASCs). We demonstrate that esASCs contributed to germline lineages, full-term chimeras and generated esASC-derived mice by tetraploid complementation. We show that, in contrast to 2i/L-ESCs, esASCs display distinct molecular signatures and a stable hypermethylated epigenome, which is reversible and similar to serum-cultured ESCs. Importantly, we also derived novel ASCs (blASCs) from blastocysts in ABC/L medium. Our results provide insights into the derivation of novel ESCs with DNA hypermethylation from blastocysts in chemically defined medium.

SUBMITTER: Wu B

PROVIDER: S-EPMC7013251 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Activin A and BMP4 Signaling Expands Potency of Mouse Embryonic Stem Cells in Serum Free Media

Project description:Inhibitors of Mek1/2 and Gsk3b, known as 2i, and together with leukemia inhibitory factor, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency (2i/L-ESCs). However, recent reports show prolonged Mek1/2 suppression impairs the developmental potential of ESCs, and is rescued by serum (S/L-ESCs). In spite of the unclear roles of growth factors in serum, the coculture system has remained the gold standard. Here we show that culturing ESCs in Activin A, BMP4 and in the absence of MEK1/2 inhibitor (ABC/L medium) establishes stable ESCs, named advanced pluripotent stem cells derived from ESCs (esASCs). We demonstrate that esASCs contributed to germline lineages, full term chimeras and generated esASCs-derived mice by tetraploid complementation. We show that in contrast to 2i/L-ESCs, these esASCs display distinct molecular signatures and stable hypermethylated epigenome which is reversible and similar to S/L-ESCs. Importantly, we also derived novel ASCs (blASCs) from blastocysts in ABC/L medium. These blASCs are persisting at an intermediate state between naïve and primed state of pluripotency and stable hypermethylated epigenome. Our results provide insights into the derivation of novel ESCs with DNA hypermethylation from blastocysts in a chemically defined medium.

2020-01-07 | GSE119985 | GEO

Activin A and BMP4 Signaling Expands Potency of Mouse Embryonic Stem Cells in Serum Free Media

Project description:Activin A and BMP4 Signaling Expands Potency of Mouse Embryonic Stem Cells in Serum Free Media

| PRJNA491488 | ENA

Activin A and BMP4 Signaling for Efficient Cardiac Differentiation of H7 and H9 Human Embryonic Stem Cells.

Project description:The generation of cardiomyocytes from human embryonic stem cells (hESC) enables a variety of potential therapeutic and diagnostic applications. However, progress is challenged by the low efficiency of cardiomyocyte differentiation. Recently, Kattman et al., 2011 showed that individual hESC lines required proper balance of the Activin A and BMP4 signaling for efficient cardiac differentiation, presenting their differentiation protocols for several human and mouse ESC lines. However, two of the most utilized hESC lines, H7 and H9, were not included. Therefore, we set out to verify the published methodology for highly efficient cardiac specification and investigate the cardiac differentiation in the H7 and H9 ESC lines. Our studies examined a range of time points for the initial culture of hESC as embryoid bodies (EB) prior to transfer to monolayer culture, as well as, concentrations of Activin A and BMP4 in the medium formulations. The results highlight an efficient protocol for reproducibly generating cultures with approximately 50% cardiomyocytes from H7 and H9 ESC lines.

| S-EPMC3908295 | biostudies-literature

Establishment of Pig Embryonic Stem Cells using Simplified Serum-free media and Adaptation of Feeder-free conditions

Project description:Most porcine embryonic stem cells (pESC) derived from in vitro blastocysts Still require complex media compositions and feeder layers which make difficulty to adapt their broad and routine use. We derived pESC using simplified culture media (FIW) consisting of small molecules FGF2, IWR-1, and WH-4-023 in serum-free medium. We established several pESC-FIW lines capable of single cell passaging with short cell doubling time (about 12 hours). These cells expressed pluripotency markers, OCT4, SOX2, and NANOG, as well as cell surface markers, SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed stable proliferation rate and normal karyotype even after long-term culture over 50 passages. Established pESC showed formative characteristics based on the negative expression for the naive marker KLF4 and primed marker T, whereas strong expression for the formative marker OTX2 with high levels of related genes. Transcriptome analysis showed that pESC-FIW had similar cellular identities to reported pESC maintained in complex media formulations and had characteristics of gastrulating epiblast cells. In addition, pESC-FIW could be maintained for multiple passages in a feeder-free condition on fibronectin coated plate using mTeSR™, a commercial medium for feeder-free culture. These results indicate that the canonical WNT (IWR-1) inhibition and the SRC inhibition (WH-4-023) are sufficient to establish pESC capable of single cell passaging and feeder-free expansion under the serum-free condition. Easy to maintain-pESC can be a tool for gene editing application useful for agriculture and biomedicine, and lineage commitment studies.

2024-08-07 | GSE240454 | GEO

Comparative analysis of mesenchymal stem cells cultivated in serum free media.

Project description:Stem cells are attractive candidates for the regeneration of tissue and organ. Mesenchymal stem cells (MSCs) have been extensively investigated for their potential applications in regenerative medicine and cell therapy. For developing effective stem cell therapy, the mass production of consistent quality cells is required. The cell culture medium is the most critical aspect of the mass production of qualified stem cells. Classically, fetal bovine serum (FBS) has been used as a culture supplement for MSCs. Due to the undefined and heterologous composition of animal origin components in FBS, efforts to replace animal-derived components with non-animal-derived substances led to safe serum free media (SFM). Adipose derived mesenchymal stem cells (ADSCs) cultivated in SFM provided a more stable population doubling time (PDT) to later passage and more cells in a shorter time compared to FBS containing media. ADSCs cultivated in SFM had lower cellular senescence, lower immunogenicity, and higher genetic stability than ADSCs cultivated in FBS containing media. Differential expression analysis of mRNAs and proteins showed that the expression of genes related with apoptosis, immune response, and inflammatory response were significantly up-regulated in ADSCs cultivated in FBS containing media. ADSCs cultivated in SFM showed similar therapeutic efficacy in an acute pancreatitis mouse model to ADSCs cultivated in FBS containing media. Consideration of clinical trials, not only pre-clinical trial, suggests that cultivation of MSCs using SFM might offer more safe cell therapeutics as well as repeated administration due to low immunogenicity.

| S-EPMC9124186 | biostudies-literature

Activin/Nodal/TGF-β Pathway Inhibitor Accelerates BMP4-Induced Cochlear Gap Junction Formation During <i>in vitro</i> Differentiation of Embryonic Stem Cells.

Project description:Mutations in gap junction beta-2 (GJB2), the gene that encodes connexin 26 (CX26), are the most frequent cause of hereditary deafness worldwide. We recently developed an in vitro model of GJB2-related deafness (induced CX26 gap junction-forming cells; iCX26GJCs) from mouse induced pluripotent stem cells (iPSCs) by using Bone morphogenetic protein 4 (BMP4) signaling-based floating cultures (serum-free culture of embryoid body-like aggregates with quick aggregation cultures; hereafter, SFEBq cultures) and adherent cultures. However, to use these cells as a disease model platform for high-throughput drug screening or regenerative therapy, cell yields must be substantially increased. In addition to BMP4, other factors may also induce CX26 gap junction formation. In the SFEBq cultures, the combination of BMP4 and the Activin/Nodal/TGF-β pathway inhibitor SB431542 (SB) resulted in greater production of isolatable CX26-expressing cell mass (CX26+ vesicles) and higher Gjb2 mRNA levels than BMP4 treatment alone, suggesting that SB may promote BMP4-mediated production of CX26+ vesicles in a dose-dependent manner, thereby increasing the yield of highly purified iCX26GJCs. This is the first study to demonstrate that SB accelerates BMP4-induced iCX26GJC differentiation during stem cell floating culture. By controlling the concentration of SB supplementation in combination with CX26+ vesicle purification, large-scale production of highly purified iCX26GJCs suitable for high-throughput drug screening or regenerative therapy for GJB2-related deafness may be possible.

| S-EPMC8097046 | biostudies-literature

Maintenance of undifferentiated mouse embryonic stem cells in suspension by the serum- and feeder-free defined culture condition.

Project description:The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified. DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products.

| S-EPMC2559871 | biostudies-literature

Bio-instructive hydrogel expands the paracrine potency of mesenchymal stem cells.

Project description:The therapeutic efficacy of clinically applied mesenchymal stromal cells (MSCs) is limited due to their injection into harshin vivoenvironments, resulting in the significant loss of their secretory function upon transplantation. A potential strategy for preserving their full therapeutic potential is encapsulation of MSCs in a specialized protective microenvironment, for example hydrogels. However, commonly used injectable hydrogels for cell delivery fail to provide the bio-instructive cues needed to sustain and stimulate cellular therapeutic functions. Here we introduce a customizable collagen I-hyaluronic acid (COL-HA)-based hydrogel platform for the encapsulation of MSCs. Cells encapsulated within COL-HA showed a significant expansion of their secretory profile compared to MSCs cultured in standard (2D) cell culture dishes or encapsulated in other hydrogels. Functionalization of the COL-HA backbone with thiol-modified glycoproteins such as laminin led to further changes in the paracrine profile of MSCs. In depth profiling of more than 250 proteins revealed an expanded secretion profile of proangiogenic, neuroprotective and immunomodulatory paracrine factors in COL-HA-encapsulated MSCs with a predicted augmented pro-angiogenic potential. This was confirmed by increased capillary network formation of endothelial cells stimulated by conditioned media from COL-HA-encapsulated MSCs. Our findings suggest that encapsulation of therapeutic cells in a protective COL-HA hydrogel layer provides the necessary bio-instructive cues to maintain and direct their therapeutic potential. Our customizable hydrogel combines bioactivity and clinically applicable properties such as injectability, on-demand polymerization and tissue-specific elasticity, all features that will support and improve the ability to successfully deliver functional MSCs into patients.

| S-EPMC10024818 | biostudies-literature

Comparative analysis of mesenchymal stem cells cultivated in serum free media

Project description:Differential expression analysis of mRNAs and proteins showed that the expression of genes related with apoptosis, immune response, and inflammatory response were significantly up-regulated in ADSCs cultivated in FBS containing media.

2022-04-05 | GSE200086 | GEO

Both BMP4 and serum have significant roles in differentiation of embryonic stem cells to primitive and definitive endoderm.

Project description:Differentiation of embryonic stem (ES) cells is a heterogeneous process which is influenced by different parameters, including growth and differentiation factors. The aim of the present study was to investigate the effect of bone morphogenetic protein-4 (BMP4) signaling on differentiation of mouse ES cells to endodermal lineages. For this purpose, differentiation of the ES cells was induced by embryoid body (EB) formation through hanging drop method. During the suspension stage, EBs were treated with BMP4 in a medium containing either fetal bovine serum (FBS) or knockout serum replacement (KoSR). After plating, EBs showed differentiation to a heterogeneous population of specialized cell types. Two weeks after plating, all the experimental groups expressed three germ layer markers and some primitive and definitive endoderm-specific genes. Quantitative real-time PCR analysis showed higher expression levels of Sox17, Pdx1, Cdx2 and Villin mRNAs in the KoSR plus BMP4 condition and higher Gata4 and Afp expression levels in the FBS plus BMP4 condition. Formation of visceral endoderm and derivatives of definitive endoderm was detected in the BMP4 treated EBs. In conclusion, we demonstrated that both BMP4 signaling and serum composition have significant roles in differentiation of mouse ES cells towards endodermal lineages.

| S-EPMC4960179 | biostudies-literature

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