Limits...
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.

Show MeSH

Related in: MedlinePlus

Neural rosettes generated on collagen (A-D) and matrigel (E-H) substrates. (A-D) A TE03-derived light EB at 3 days postplating on type I collagen substrates was triple-immunostained for nestin (B), TuJ1 (C) and Sox1 (blue in D). Note that TuJ1 stains radial configurations of structures in rosettes (pointed by two solid arrows) and a cluster of neurons (pointed by a dished arrow) in (C). Scale bar: 100 μm. (E-H) Neural rosettes, neural progenitors and neurons are developed in a TE03-derived light EB cultured on matrigel for 2 days. A solid arrow in (E) points to a TuJ1-labeld radial arrangement (G) which overlaps a rosette in phase contrast image (E). The dashed arrow in (G) indicates TuJ1+ neurons with long processes. The majority of cells in the EB are nestin+ (F). Scale bars in (A) and (E) = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2570688&req=5

Figure 5: Neural rosettes generated on collagen (A-D) and matrigel (E-H) substrates. (A-D) A TE03-derived light EB at 3 days postplating on type I collagen substrates was triple-immunostained for nestin (B), TuJ1 (C) and Sox1 (blue in D). Note that TuJ1 stains radial configurations of structures in rosettes (pointed by two solid arrows) and a cluster of neurons (pointed by a dished arrow) in (C). Scale bar: 100 μm. (E-H) Neural rosettes, neural progenitors and neurons are developed in a TE03-derived light EB cultured on matrigel for 2 days. A solid arrow in (E) points to a TuJ1-labeld radial arrangement (G) which overlaps a rosette in phase contrast image (E). The dashed arrow in (G) indicates TuJ1+ neurons with long processes. The majority of cells in the EB are nestin+ (F). Scale bars in (A) and (E) = 100 μm.

Mentions: Neuroectodermal precursors in neural rosettes were induced by replating EBs into the NDM. While the markers of undifferentiated hESC such as Oct3/4 and SSEA4 progressively disappeared (not shown), a number of rosettes gradually emerged throughout the EB. Although neural rosettes were seen in floating EBs, more rosettes were generated after the EBs were plated on PDL/laminin (Fig. 4), Matrigel (Fig. 5A–D) and collagen (Fig. 5E–H) substrates, but not on PDL. These radial neuroectodermal cells were morphologically similar to polarized neuroepithelial structures of the developing CNS. Immunostaining of neuroectodermal cells in rosettes for early neural markers Sox1 and nestin, and the neuronal marker TuJ1, showed that the radially organized columnar neuroepithelial cells co-expressed Sox1 and nestin (Fig. 4A, B). Therefore, neural rosettes are considered as in vitro organized neuroectoderm [13-15]. It was interesting to notice that the antibody against neuronal marker TuJ1 not only stained rosette cell-derived neurons, but also radially distributed fiber-like structures in rosettes (Fig. 5C, D, G and 5H). About 5–10 days later, the rosettes were often transformed into neural tube like structures (Fig. 4C, D).


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)

Neural rosettes generated on collagen (A-D) and matrigel (E-H) substrates. (A-D) A TE03-derived light EB at 3 days postplating on type I collagen substrates was triple-immunostained for nestin (B), TuJ1 (C) and Sox1 (blue in D). Note that TuJ1 stains radial configurations of structures in rosettes (pointed by two solid arrows) and a cluster of neurons (pointed by a dished arrow) in (C). Scale bar: 100 μm. (E-H) Neural rosettes, neural progenitors and neurons are developed in a TE03-derived light EB cultured on matrigel for 2 days. A solid arrow in (E) points to a TuJ1-labeld radial arrangement (G) which overlaps a rosette in phase contrast image (E). The dashed arrow in (G) indicates TuJ1+ neurons with long processes. The majority of cells in the EB are nestin+ (F). Scale bars in (A) and (E) = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Neural rosettes generated on collagen (A-D) and matrigel (E-H) substrates. (A-D) A TE03-derived light EB at 3 days postplating on type I collagen substrates was triple-immunostained for nestin (B), TuJ1 (C) and Sox1 (blue in D). Note that TuJ1 stains radial configurations of structures in rosettes (pointed by two solid arrows) and a cluster of neurons (pointed by a dished arrow) in (C). Scale bar: 100 μm. (E-H) Neural rosettes, neural progenitors and neurons are developed in a TE03-derived light EB cultured on matrigel for 2 days. A solid arrow in (E) points to a TuJ1-labeld radial arrangement (G) which overlaps a rosette in phase contrast image (E). The dashed arrow in (G) indicates TuJ1+ neurons with long processes. The majority of cells in the EB are nestin+ (F). Scale bars in (A) and (E) = 100 μm.
Mentions: Neuroectodermal precursors in neural rosettes were induced by replating EBs into the NDM. While the markers of undifferentiated hESC such as Oct3/4 and SSEA4 progressively disappeared (not shown), a number of rosettes gradually emerged throughout the EB. Although neural rosettes were seen in floating EBs, more rosettes were generated after the EBs were plated on PDL/laminin (Fig. 4), Matrigel (Fig. 5A–D) and collagen (Fig. 5E–H) substrates, but not on PDL. These radial neuroectodermal cells were morphologically similar to polarized neuroepithelial structures of the developing CNS. Immunostaining of neuroectodermal cells in rosettes for early neural markers Sox1 and nestin, and the neuronal marker TuJ1, showed that the radially organized columnar neuroepithelial cells co-expressed Sox1 and nestin (Fig. 4A, B). Therefore, neural rosettes are considered as in vitro organized neuroectoderm [13-15]. It was interesting to notice that the antibody against neuronal marker TuJ1 not only stained rosette cell-derived neurons, but also radially distributed fiber-like structures in rosettes (Fig. 5C, D, G and 5H). About 5–10 days later, the rosettes were often transformed into neural tube like structures (Fig. 4C, D).

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