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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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hESC-derived neural progenitors are highly proliferative and multipotent. (A-C) Double-immunostaining of differentiated cells at 2 days postplating for nestin (red) and proliferative marker BrdU (greed), indicating approximately 68% of nestin+ cells are BrdU incorporated. (D-F) Double-immunostaining for nestin (red) and A2B5 (green). (G-I) Double-immunostaining for nestin (red) and PS-NCAM (green). The overlapping of nestin and A2B5 or PS-NCAM indicates that hESC-derived nestin+ neural progenitors also express other neural differentiating markers. (J-M) Immunostaining of progeny of hESC-derived neural progenitors for mature neuronal marker MAP2 (J), GABAergic neuronal marker GAD65/67 (K), astrocytic marker GFAP (L) and O4, developing oligodentrocyte marker (M). Scale bars: (A) = 200 μm; (D) = 100 μm; (G) = 100 μm; (J-M) = 30 μm.
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Figure 6: hESC-derived neural progenitors are highly proliferative and multipotent. (A-C) Double-immunostaining of differentiated cells at 2 days postplating for nestin (red) and proliferative marker BrdU (greed), indicating approximately 68% of nestin+ cells are BrdU incorporated. (D-F) Double-immunostaining for nestin (red) and A2B5 (green). (G-I) Double-immunostaining for nestin (red) and PS-NCAM (green). The overlapping of nestin and A2B5 or PS-NCAM indicates that hESC-derived nestin+ neural progenitors also express other neural differentiating markers. (J-M) Immunostaining of progeny of hESC-derived neural progenitors for mature neuronal marker MAP2 (J), GABAergic neuronal marker GAD65/67 (K), astrocytic marker GFAP (L) and O4, developing oligodentrocyte marker (M). Scale bars: (A) = 200 μm; (D) = 100 μm; (G) = 100 μm; (J-M) = 30 μm.

Mentions: Our reproducible protocol generated robust hESC-derived neuroectodermal cells giving rise to highly homogenous nestin+ neural progenitors that undergo extensive cell division before differentiating into neuronal and glial cells. To assay the ability of self-renewal of hESC-derived neural progenitors, cells at 18 days of differentiation (or 3 days postplating on laminin substrate) were exposed to BrdU for 4 hours before double immunostaining for nestin and BrdU. The percentage of nuclei positive for BrdU among the nestin-positive cell population was about 68% (Fig. 6A–C), indicating that hESC-derived neural progenitors were actively synthesizing DNA. To characterize the extent to which rosette cells can differentiate into all three neural cell lineages, we used immunocytochemical analysis of cells cultured on PDL/laminin substrates with a panel of antibodies and demonstrated that hESC-derived neural derivatives sequentially expressed Sox1, nestin, musashi (not shown), A2B5 and PS-NCAM, followed by TuJ1 expression in many cells in first 1–5 days postplating on PDL/laminin substrates. MAP2+ and GABAergic (GAD65,67+) neurons appeared at about 10–15 days on PDL/laminin substrates. GFAP+ astrocytes and O4+ developing oligodendrocytes did not appear until after 30 days of culture on PDL/laminin substrates (Fig. 6).


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)

hESC-derived neural progenitors are highly proliferative and multipotent. (A-C) Double-immunostaining of differentiated cells at 2 days postplating for nestin (red) and proliferative marker BrdU (greed), indicating approximately 68% of nestin+ cells are BrdU incorporated. (D-F) Double-immunostaining for nestin (red) and A2B5 (green). (G-I) Double-immunostaining for nestin (red) and PS-NCAM (green). The overlapping of nestin and A2B5 or PS-NCAM indicates that hESC-derived nestin+ neural progenitors also express other neural differentiating markers. (J-M) Immunostaining of progeny of hESC-derived neural progenitors for mature neuronal marker MAP2 (J), GABAergic neuronal marker GAD65/67 (K), astrocytic marker GFAP (L) and O4, developing oligodentrocyte marker (M). Scale bars: (A) = 200 μm; (D) = 100 μm; (G) = 100 μm; (J-M) = 30 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2570688&req=5

Figure 6: hESC-derived neural progenitors are highly proliferative and multipotent. (A-C) Double-immunostaining of differentiated cells at 2 days postplating for nestin (red) and proliferative marker BrdU (greed), indicating approximately 68% of nestin+ cells are BrdU incorporated. (D-F) Double-immunostaining for nestin (red) and A2B5 (green). (G-I) Double-immunostaining for nestin (red) and PS-NCAM (green). The overlapping of nestin and A2B5 or PS-NCAM indicates that hESC-derived nestin+ neural progenitors also express other neural differentiating markers. (J-M) Immunostaining of progeny of hESC-derived neural progenitors for mature neuronal marker MAP2 (J), GABAergic neuronal marker GAD65/67 (K), astrocytic marker GFAP (L) and O4, developing oligodentrocyte marker (M). Scale bars: (A) = 200 μm; (D) = 100 μm; (G) = 100 μm; (J-M) = 30 μm.
Mentions: Our reproducible protocol generated robust hESC-derived neuroectodermal cells giving rise to highly homogenous nestin+ neural progenitors that undergo extensive cell division before differentiating into neuronal and glial cells. To assay the ability of self-renewal of hESC-derived neural progenitors, cells at 18 days of differentiation (or 3 days postplating on laminin substrate) were exposed to BrdU for 4 hours before double immunostaining for nestin and BrdU. The percentage of nuclei positive for BrdU among the nestin-positive cell population was about 68% (Fig. 6A–C), indicating that hESC-derived neural progenitors were actively synthesizing DNA. To characterize the extent to which rosette cells can differentiate into all three neural cell lineages, we used immunocytochemical analysis of cells cultured on PDL/laminin substrates with a panel of antibodies and demonstrated that hESC-derived neural derivatives sequentially expressed Sox1, nestin, musashi (not shown), A2B5 and PS-NCAM, followed by TuJ1 expression in many cells in first 1–5 days postplating on PDL/laminin substrates. MAP2+ and GABAergic (GAD65,67+) neurons appeared at about 10–15 days on PDL/laminin substrates. GFAP+ astrocytes and O4+ developing oligodendrocytes did not appear until after 30 days of culture on PDL/laminin substrates (Fig. 6).

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