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Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells.

Ma W, Tavakoli T, Derby E, Serebryakova Y, Rao MS, Mattson MP - BMC Dev. Biol. (2008)

Bottom Line: We found that the five substrates instructed neural progenitors followed by neuronal differentiation to differing degrees.Glia did not appear until 4 weeks later.Neural progenitor and neuronal generation and neurite outgrowth were significantly greater on laminin and laminin-rich Matrigel substrates than on other 3 substrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stem Cell Center, Developmental Biology, American Type Culture Collection, Manassas, VA, USA. wma@atcc.org

ABSTRACT

Background: Interactions of cells with the extracellular matrix (ECM) are critical for the establishment and maintenance of stem cell self-renewal and differentiation. However, the ECM is a complex mixture of matrix molecules; little is known about the role of ECM components in human embryonic stem cell (hESC) differentiation into neural progenitors and neurons.

Results: A reproducible protocol was used to generate highly homogenous neural progenitors or a mixed population of neural progenitors and neurons from hESCs. This defined adherent culture system allowed us to examine the effect of ECM molecules on neural differentiation of hESCs. hESC-derived differentiating embryoid bodies were plated on Poly-D-Lysine (PDL), PDL/fibronectin, PDL/laminin, type I collagen and Matrigel, and cultured in neural differentiation medium. We found that the five substrates instructed neural progenitors followed by neuronal differentiation to differing degrees. Glia did not appear until 4 weeks later. Neural progenitor and neuronal generation and neurite outgrowth were significantly greater on laminin and laminin-rich Matrigel substrates than on other 3 substrates. Laminin stimulated hESC-derived neural progenitor expansion and neurite outgrowth in a dose-dependent manner. The laminin-induced neural progenitor expansion was partially blocked by the antibody against integrin alpha6 or beta1 subunit.

Conclusion: We defined laminin as a key ECM molecule to enhance neural progenitor generation, expansion and differentiation into neurons from hESCs. The cell-laminin interactions involve alpha6beta1 integrin receptors implicating a possible role of laminin/alpha6beta1 integrin signaling in directed neural differentiation of hESCs. Since laminin acts in concert with other ECM molecules in vivo, evaluating cellular responses to the composition of the ECM is essential to clarify further the role of cell-matrix interactions in neural derivation of hESCs.

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Laminin stimulates hESC-derived neural progenitor expansion and neuronal neurite outgrowth in a dose-dependent manner. Dark EB-derived neural populations at 3 days postplating on PDL/laminin substrates with 5 different concentrations: 1 μg/ml, 10 μg/ml, 30 μg/ml, 60 μg/ml and 100 μg/ml. The cultures were double-immunostained for nestin and TuJ1. (A, B) Bar plots summarizing the responses of number of nestin+ neural progenitors (A) and total neurite length of TuJ1+ neurons (B) to different laminin concentrations. The neural progenitor expansion and total neurite length of neurons per cell increases with concentrations and reach a peak at 60 μg/ml. Values are expressed as mean ± SEM of 3 independent experiments. Statistical differences for cells/mm2 and total neurite length per cell between 60 μg/ml or 100 μg/ml and 1 μg/ml or 10 μg/ml or 30 μg/ml are significant * p < 0.05; ** p < 0.01.
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Figure 9: Laminin stimulates hESC-derived neural progenitor expansion and neuronal neurite outgrowth in a dose-dependent manner. Dark EB-derived neural populations at 3 days postplating on PDL/laminin substrates with 5 different concentrations: 1 μg/ml, 10 μg/ml, 30 μg/ml, 60 μg/ml and 100 μg/ml. The cultures were double-immunostained for nestin and TuJ1. (A, B) Bar plots summarizing the responses of number of nestin+ neural progenitors (A) and total neurite length of TuJ1+ neurons (B) to different laminin concentrations. The neural progenitor expansion and total neurite length of neurons per cell increases with concentrations and reach a peak at 60 μg/ml. Values are expressed as mean ± SEM of 3 independent experiments. Statistical differences for cells/mm2 and total neurite length per cell between 60 μg/ml or 100 μg/ml and 1 μg/ml or 10 μg/ml or 30 μg/ml are significant * p < 0.05; ** p < 0.01.

Mentions: Results from above show that laminin is a particularly effective ECM substrate for stimulating hESC-derived neural progenitor expansion and neuronal neurite outgrowth. To examine a possible dose-dependent relationship between laminin concentrations and hESC-derived neurogenesis, we evaluated the hESC-derived neural progenitor expansion and total neurite length of neurons on culture dishes coated with PDL/laminin substrates. Laminin was used at the following 5 concentrations: 1 μg/ml (0.25 μg/cm2), 10 μg/ml (2.5 μg/cm2), 30 μg/ml (7.5 μg/cm2), 60 μg/ml (15 μg/cm2) and 100 μg/ml (25 μg/cm2). We observed that both neural progenitor expansion and total neurite length increased in response to increased laminin concentrations from1 μg/ml to 60 μg/ml. The cell expansion and neurite outgrowth declined at higher laminin concentration (100 μg/ml) (Fig. 9A, B).


Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells.

Ma W, Tavakoli T, Derby E, Serebryakova Y, Rao MS, Mattson MP - BMC Dev. Biol. (2008)

Laminin stimulates hESC-derived neural progenitor expansion and neuronal neurite outgrowth in a dose-dependent manner. Dark EB-derived neural populations at 3 days postplating on PDL/laminin substrates with 5 different concentrations: 1 μg/ml, 10 μg/ml, 30 μg/ml, 60 μg/ml and 100 μg/ml. The cultures were double-immunostained for nestin and TuJ1. (A, B) Bar plots summarizing the responses of number of nestin+ neural progenitors (A) and total neurite length of TuJ1+ neurons (B) to different laminin concentrations. The neural progenitor expansion and total neurite length of neurons per cell increases with concentrations and reach a peak at 60 μg/ml. Values are expressed as mean ± SEM of 3 independent experiments. Statistical differences for cells/mm2 and total neurite length per cell between 60 μg/ml or 100 μg/ml and 1 μg/ml or 10 μg/ml or 30 μg/ml are significant * p < 0.05; ** p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570688&req=5

Figure 9: Laminin stimulates hESC-derived neural progenitor expansion and neuronal neurite outgrowth in a dose-dependent manner. Dark EB-derived neural populations at 3 days postplating on PDL/laminin substrates with 5 different concentrations: 1 μg/ml, 10 μg/ml, 30 μg/ml, 60 μg/ml and 100 μg/ml. The cultures were double-immunostained for nestin and TuJ1. (A, B) Bar plots summarizing the responses of number of nestin+ neural progenitors (A) and total neurite length of TuJ1+ neurons (B) to different laminin concentrations. The neural progenitor expansion and total neurite length of neurons per cell increases with concentrations and reach a peak at 60 μg/ml. Values are expressed as mean ± SEM of 3 independent experiments. Statistical differences for cells/mm2 and total neurite length per cell between 60 μg/ml or 100 μg/ml and 1 μg/ml or 10 μg/ml or 30 μg/ml are significant * p < 0.05; ** p < 0.01.
Mentions: Results from above show that laminin is a particularly effective ECM substrate for stimulating hESC-derived neural progenitor expansion and neuronal neurite outgrowth. To examine a possible dose-dependent relationship between laminin concentrations and hESC-derived neurogenesis, we evaluated the hESC-derived neural progenitor expansion and total neurite length of neurons on culture dishes coated with PDL/laminin substrates. Laminin was used at the following 5 concentrations: 1 μg/ml (0.25 μg/cm2), 10 μg/ml (2.5 μg/cm2), 30 μg/ml (7.5 μg/cm2), 60 μg/ml (15 μg/cm2) and 100 μg/ml (25 μg/cm2). We observed that both neural progenitor expansion and total neurite length increased in response to increased laminin concentrations from1 μg/ml to 60 μg/ml. The cell expansion and neurite outgrowth declined at higher laminin concentration (100 μg/ml) (Fig. 9A, B).

Bottom Line: We found that the five substrates instructed neural progenitors followed by neuronal differentiation to differing degrees.Glia did not appear until 4 weeks later.Neural progenitor and neuronal generation and neurite outgrowth were significantly greater on laminin and laminin-rich Matrigel substrates than on other 3 substrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stem Cell Center, Developmental Biology, American Type Culture Collection, Manassas, VA, USA. wma@atcc.org

ABSTRACT

Background: Interactions of cells with the extracellular matrix (ECM) are critical for the establishment and maintenance of stem cell self-renewal and differentiation. However, the ECM is a complex mixture of matrix molecules; little is known about the role of ECM components in human embryonic stem cell (hESC) differentiation into neural progenitors and neurons.

Results: A reproducible protocol was used to generate highly homogenous neural progenitors or a mixed population of neural progenitors and neurons from hESCs. This defined adherent culture system allowed us to examine the effect of ECM molecules on neural differentiation of hESCs. hESC-derived differentiating embryoid bodies were plated on Poly-D-Lysine (PDL), PDL/fibronectin, PDL/laminin, type I collagen and Matrigel, and cultured in neural differentiation medium. We found that the five substrates instructed neural progenitors followed by neuronal differentiation to differing degrees. Glia did not appear until 4 weeks later. Neural progenitor and neuronal generation and neurite outgrowth were significantly greater on laminin and laminin-rich Matrigel substrates than on other 3 substrates. Laminin stimulated hESC-derived neural progenitor expansion and neurite outgrowth in a dose-dependent manner. The laminin-induced neural progenitor expansion was partially blocked by the antibody against integrin alpha6 or beta1 subunit.

Conclusion: We defined laminin as a key ECM molecule to enhance neural progenitor generation, expansion and differentiation into neurons from hESCs. The cell-laminin interactions involve alpha6beta1 integrin receptors implicating a possible role of laminin/alpha6beta1 integrin signaling in directed neural differentiation of hESCs. Since laminin acts in concert with other ECM molecules in vivo, evaluating cellular responses to the composition of the ECM is essential to clarify further the role of cell-matrix interactions in neural derivation of hESCs.

Show MeSH
Related in: MedlinePlus