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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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Neurite outgrowth of hESC-derived neurons are greater on PDL/laminin and matrigel substrates than other substrates. Light EBs were cultured on 5 different substrates: PDL, PDL/laminin, PDL/fibronectin, collagen and Matrigel for 3 days in the NDM and immunostained for TuJ1. (A) A panel of immunofluorescent images showing representative fields of TuJ1+ cells expanded on PDL, PDL/fibronectin (PDL/FN), PDL/laminin (PDL/LN), collagen and Matrigel for 3 days. (B, C) Bar plots summarizing the effect of different substrates on number of neuritis (B) and total neurite length (C) per cell. The laminin (LN)- and matrigel (MG)-induced number of neuritis and total neurite length per cell are significantly higher than on PDL, fibronectin (FN), and collagen (CG). Values are expressed as mean ± SEM of 4 independent experiments. Statistical differences for number of neuritis and for total neurite length per cell between LM or MG and PDL or FN or CG are significant ** p < 0.01. Scale bar in (A) = 30 μm.
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Figure 8: Neurite outgrowth of hESC-derived neurons are greater on PDL/laminin and matrigel substrates than other substrates. Light EBs were cultured on 5 different substrates: PDL, PDL/laminin, PDL/fibronectin, collagen and Matrigel for 3 days in the NDM and immunostained for TuJ1. (A) A panel of immunofluorescent images showing representative fields of TuJ1+ cells expanded on PDL, PDL/fibronectin (PDL/FN), PDL/laminin (PDL/LN), collagen and Matrigel for 3 days. (B, C) Bar plots summarizing the effect of different substrates on number of neuritis (B) and total neurite length (C) per cell. The laminin (LN)- and matrigel (MG)-induced number of neuritis and total neurite length per cell are significantly higher than on PDL, fibronectin (FN), and collagen (CG). Values are expressed as mean ± SEM of 4 independent experiments. Statistical differences for number of neuritis and for total neurite length per cell between LM or MG and PDL or FN or CG are significant ** p < 0.01. Scale bar in (A) = 30 μm.

Mentions: To evaluate the effect of substrates on the neurite outgrowth of hESC-derived neurons, quantification of the number of primary neurites and total neurite length per cell was carried out on neurons derived from the light EBs. The hESC-derived neurons were identified using immunocytochemistry with the antibody against TuJ1 (Fig. 8A). hESC-derived neurons on laminin and Matrigel had significantly greater numbers of neurites and total neurite length per cell than neurons on other substrates (Fig. 8B, C). The total neurite length per cell on laminin slightly greater than on matrigel, but this difference was not statistically significant (total neurite length per cell ± SEM: laminin 85 ± 5.8, Matrigel 81 ± 3.8; P ≥ 0.1 for both comparisons). Therefore, laminin-supported neurite outgrowth was comparable to Matrigel.


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)

Neurite outgrowth of hESC-derived neurons are greater on PDL/laminin and matrigel substrates than other substrates. Light EBs were cultured on 5 different substrates: PDL, PDL/laminin, PDL/fibronectin, collagen and Matrigel for 3 days in the NDM and immunostained for TuJ1. (A) A panel of immunofluorescent images showing representative fields of TuJ1+ cells expanded on PDL, PDL/fibronectin (PDL/FN), PDL/laminin (PDL/LN), collagen and Matrigel for 3 days. (B, C) Bar plots summarizing the effect of different substrates on number of neuritis (B) and total neurite length (C) per cell. The laminin (LN)- and matrigel (MG)-induced number of neuritis and total neurite length per cell are significantly higher than on PDL, fibronectin (FN), and collagen (CG). Values are expressed as mean ± SEM of 4 independent experiments. Statistical differences for number of neuritis and for total neurite length per cell between LM or MG and PDL or FN or CG are significant ** p < 0.01. Scale bar in (A) = 30 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Neurite outgrowth of hESC-derived neurons are greater on PDL/laminin and matrigel substrates than other substrates. Light EBs were cultured on 5 different substrates: PDL, PDL/laminin, PDL/fibronectin, collagen and Matrigel for 3 days in the NDM and immunostained for TuJ1. (A) A panel of immunofluorescent images showing representative fields of TuJ1+ cells expanded on PDL, PDL/fibronectin (PDL/FN), PDL/laminin (PDL/LN), collagen and Matrigel for 3 days. (B, C) Bar plots summarizing the effect of different substrates on number of neuritis (B) and total neurite length (C) per cell. The laminin (LN)- and matrigel (MG)-induced number of neuritis and total neurite length per cell are significantly higher than on PDL, fibronectin (FN), and collagen (CG). Values are expressed as mean ± SEM of 4 independent experiments. Statistical differences for number of neuritis and for total neurite length per cell between LM or MG and PDL or FN or CG are significant ** p < 0.01. Scale bar in (A) = 30 μm.
Mentions: To evaluate the effect of substrates on the neurite outgrowth of hESC-derived neurons, quantification of the number of primary neurites and total neurite length per cell was carried out on neurons derived from the light EBs. The hESC-derived neurons were identified using immunocytochemistry with the antibody against TuJ1 (Fig. 8A). hESC-derived neurons on laminin and Matrigel had significantly greater numbers of neurites and total neurite length per cell than neurons on other substrates (Fig. 8B, C). The total neurite length per cell on laminin slightly greater than on matrigel, but this difference was not statistically significant (total neurite length per cell ± SEM: laminin 85 ± 5.8, Matrigel 81 ± 3.8; P ≥ 0.1 for both comparisons). Therefore, laminin-supported neurite outgrowth was comparable to Matrigel.

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