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What Kind of Signaling Maintains Pluripotency and Viability in Human-Induced Pluripotent Stem Cells Cultured on Laminin-511 with Serum-Free Medium?

Nakashima Y, Omasa T - Biores Open Access (2016)

Bottom Line: In hiPSCs, the interaction of laminin-511/α6β1 integrin with the cell-cell adhesion molecule E-cadherin confers protection against apoptosis through the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathways for cell death) and the proto-oncogene tyrosine-protein kinase Fyn (Fyn)-RhoA-ROCK signaling pathway.A combination of growth factors, medium constituents, cell membrane-located E-cadherin, and α6β1 integrin-induced signaling is required for pluripotent cell proliferation and for optimal cell survival on a laminin-511 scaffold.In this review, we discuss and explore the influence of growth factors on the cadherin and integrin signaling pathways in serum-free and xeno-free cultures of hiPSCs during the preparation of products for regenerative medicinal therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University , Osaka, Japan .

ABSTRACT
Xeno-free medium contains no animal-derived components, but is composed of minimal growth factors and is serum free; the medium may be supplemented with insulin, transferrin, and selenium (ITS medium). Serum-free and xeno-free culture of human-induced pluripotent stem cells (hiPSCs) uses a variety of components based on ITS medium and Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12) that contain high levels of iron salt and glucose. Culture of hiPSCs also requires scaffolding materials, such as extracellular matrix, collagen, fibronectin, laminin, proteoglycan, and vitronectin. The scaffolding component laminin-511, which is composed of α5, β1, and γ1 chains, binds to α3β1, α6β1, and α6β4 integrins on the cell membrane to induce activation of the PI3K/AKT- and Ras/MAPK-dependent signaling pathways. In hiPSCs, the interaction of laminin-511/α6β1 integrin with the cell-cell adhesion molecule E-cadherin confers protection against apoptosis through the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathways for cell death) and the proto-oncogene tyrosine-protein kinase Fyn (Fyn)-RhoA-ROCK signaling pathway. The expression levels of α6β1 integrin and E-cadherin on cell membranes are controlled through the activation of insulin receptor/insulin, FGF receptor/FGF2, or activin-like kinase 5 (ALK5)-dependent TGF-β signaling. A combination of growth factors, medium constituents, cell membrane-located E-cadherin, and α6β1 integrin-induced signaling is required for pluripotent cell proliferation and for optimal cell survival on a laminin-511 scaffold. In this review, we discuss and explore the influence of growth factors on the cadherin and integrin signaling pathways in serum-free and xeno-free cultures of hiPSCs during the preparation of products for regenerative medicinal therapies. In addition, we suggest the optimum serum-free medium components for use with laminin-511, a new scaffold for hiPSC culture.

No MeSH data available.


Related in: MedlinePlus

Signaling pathways in serum-free and feeder cell-free culture systems (on laminin-511). Cadherin is activated by a cell junction. When N-cadherin is activated, transcriptional enhancement of FGFR occurs. As a result, the FGF2 signal pathway is activated. This signaling pathway has an important role in the expression of FGFR attachment to the cell membrane. In cultures using a laminin-511 scaffold, the E-cadherin-α6β1 integrin signal pathway is required to control cell death in hiPSCs. PI3K signaling promotes expression of Rac1. Rac1 binds to cadherins, and cadherins prevent endocytosis. When α6β1 integrin is combined with laminin-511, Fyn-RhoA-ROCK signaling is induced. As a result, hiPSC death caused by ROCK is repressed. Laminin-511 can directly bind to α3β1 integrin and, thereby, activate the PI3K/AKT signaling pathway through Ras. In addition, linkage of α6β4 activates the MAP kinase pathways directly through Ras.
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f3: Signaling pathways in serum-free and feeder cell-free culture systems (on laminin-511). Cadherin is activated by a cell junction. When N-cadherin is activated, transcriptional enhancement of FGFR occurs. As a result, the FGF2 signal pathway is activated. This signaling pathway has an important role in the expression of FGFR attachment to the cell membrane. In cultures using a laminin-511 scaffold, the E-cadherin-α6β1 integrin signal pathway is required to control cell death in hiPSCs. PI3K signaling promotes expression of Rac1. Rac1 binds to cadherins, and cadherins prevent endocytosis. When α6β1 integrin is combined with laminin-511, Fyn-RhoA-ROCK signaling is induced. As a result, hiPSC death caused by ROCK is repressed. Laminin-511 can directly bind to α3β1 integrin and, thereby, activate the PI3K/AKT signaling pathway through Ras. In addition, linkage of α6β4 activates the MAP kinase pathways directly through Ras.

Mentions: Human albumin has many biological and physical roles in hiPSC culture and is necessary for StemFit medium (Ajinomoto Co., Inc.) to enable the efficient formation of colonies by hiPSCs on recombinant laminin-511.45 However, albumin in the culture medium promotes the secretion of ECM molecules such as collagen I, collagen IV, and HSPG. Consequently, protein lytic enzymes such as the trypsin are necessary to break the strong cell-to-plate adhesion (Fig. 3).


What Kind of Signaling Maintains Pluripotency and Viability in Human-Induced Pluripotent Stem Cells Cultured on Laminin-511 with Serum-Free Medium?

Nakashima Y, Omasa T - Biores Open Access (2016)

Signaling pathways in serum-free and feeder cell-free culture systems (on laminin-511). Cadherin is activated by a cell junction. When N-cadherin is activated, transcriptional enhancement of FGFR occurs. As a result, the FGF2 signal pathway is activated. This signaling pathway has an important role in the expression of FGFR attachment to the cell membrane. In cultures using a laminin-511 scaffold, the E-cadherin-α6β1 integrin signal pathway is required to control cell death in hiPSCs. PI3K signaling promotes expression of Rac1. Rac1 binds to cadherins, and cadherins prevent endocytosis. When α6β1 integrin is combined with laminin-511, Fyn-RhoA-ROCK signaling is induced. As a result, hiPSC death caused by ROCK is repressed. Laminin-511 can directly bind to α3β1 integrin and, thereby, activate the PI3K/AKT signaling pathway through Ras. In addition, linkage of α6β4 activates the MAP kinase pathways directly through Ras.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834485&req=5

f3: Signaling pathways in serum-free and feeder cell-free culture systems (on laminin-511). Cadherin is activated by a cell junction. When N-cadherin is activated, transcriptional enhancement of FGFR occurs. As a result, the FGF2 signal pathway is activated. This signaling pathway has an important role in the expression of FGFR attachment to the cell membrane. In cultures using a laminin-511 scaffold, the E-cadherin-α6β1 integrin signal pathway is required to control cell death in hiPSCs. PI3K signaling promotes expression of Rac1. Rac1 binds to cadherins, and cadherins prevent endocytosis. When α6β1 integrin is combined with laminin-511, Fyn-RhoA-ROCK signaling is induced. As a result, hiPSC death caused by ROCK is repressed. Laminin-511 can directly bind to α3β1 integrin and, thereby, activate the PI3K/AKT signaling pathway through Ras. In addition, linkage of α6β4 activates the MAP kinase pathways directly through Ras.
Mentions: Human albumin has many biological and physical roles in hiPSC culture and is necessary for StemFit medium (Ajinomoto Co., Inc.) to enable the efficient formation of colonies by hiPSCs on recombinant laminin-511.45 However, albumin in the culture medium promotes the secretion of ECM molecules such as collagen I, collagen IV, and HSPG. Consequently, protein lytic enzymes such as the trypsin are necessary to break the strong cell-to-plate adhesion (Fig. 3).

Bottom Line: In hiPSCs, the interaction of laminin-511/α6β1 integrin with the cell-cell adhesion molecule E-cadherin confers protection against apoptosis through the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathways for cell death) and the proto-oncogene tyrosine-protein kinase Fyn (Fyn)-RhoA-ROCK signaling pathway.A combination of growth factors, medium constituents, cell membrane-located E-cadherin, and α6β1 integrin-induced signaling is required for pluripotent cell proliferation and for optimal cell survival on a laminin-511 scaffold.In this review, we discuss and explore the influence of growth factors on the cadherin and integrin signaling pathways in serum-free and xeno-free cultures of hiPSCs during the preparation of products for regenerative medicinal therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University , Osaka, Japan .

ABSTRACT
Xeno-free medium contains no animal-derived components, but is composed of minimal growth factors and is serum free; the medium may be supplemented with insulin, transferrin, and selenium (ITS medium). Serum-free and xeno-free culture of human-induced pluripotent stem cells (hiPSCs) uses a variety of components based on ITS medium and Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12) that contain high levels of iron salt and glucose. Culture of hiPSCs also requires scaffolding materials, such as extracellular matrix, collagen, fibronectin, laminin, proteoglycan, and vitronectin. The scaffolding component laminin-511, which is composed of α5, β1, and γ1 chains, binds to α3β1, α6β1, and α6β4 integrins on the cell membrane to induce activation of the PI3K/AKT- and Ras/MAPK-dependent signaling pathways. In hiPSCs, the interaction of laminin-511/α6β1 integrin with the cell-cell adhesion molecule E-cadherin confers protection against apoptosis through the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathways for cell death) and the proto-oncogene tyrosine-protein kinase Fyn (Fyn)-RhoA-ROCK signaling pathway. The expression levels of α6β1 integrin and E-cadherin on cell membranes are controlled through the activation of insulin receptor/insulin, FGF receptor/FGF2, or activin-like kinase 5 (ALK5)-dependent TGF-β signaling. A combination of growth factors, medium constituents, cell membrane-located E-cadherin, and α6β1 integrin-induced signaling is required for pluripotent cell proliferation and for optimal cell survival on a laminin-511 scaffold. In this review, we discuss and explore the influence of growth factors on the cadherin and integrin signaling pathways in serum-free and xeno-free cultures of hiPSCs during the preparation of products for regenerative medicinal therapies. In addition, we suggest the optimum serum-free medium components for use with laminin-511, a new scaffold for hiPSC culture.

No MeSH data available.


Related in: MedlinePlus