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

Expansion of hESC-derived nestin+ neural progenitors and TuJ1+ neurons is greater on PDL/laminin and Matrigel substrates than on PDL, PDL/fibronectin and collagen substrates. (A) A linear plot summarizing the comparison of neural progenitor expansion on different substrates over time. Values are expressed in the number of nestin+ cells per surface area as mean ± SEM of three independent experiments. The total number of cells per surface area is determined by counting DAPI labeled nuclei which overlapped phase-dark cells. Statistical differences for number of nestin+ cells/mm2 between laminin or Matrigel and PDL or fibrinectin or collagen at 48 h are significant ** p < 0.01. (B) A linear plot summarizing the comparison of neuronal expansion on different substrates over time. Values are expressed in the number of TuJ1+ cells per surface area as mean ± SEM of three independent experiments. The total number of cells per surface area is determined by counting DAPI labeled nuclei which overlapped phase-dark cells. Statistical differences for number TuJ1+ cells/mm2 between laminin or Matrigel and PDL or fibronectin or collagen at 48 h are significant ** p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Expansion of hESC-derived nestin+ neural progenitors and TuJ1+ neurons is greater on PDL/laminin and Matrigel substrates than on PDL, PDL/fibronectin and collagen substrates. (A) A linear plot summarizing the comparison of neural progenitor expansion on different substrates over time. Values are expressed in the number of nestin+ cells per surface area as mean ± SEM of three independent experiments. The total number of cells per surface area is determined by counting DAPI labeled nuclei which overlapped phase-dark cells. Statistical differences for number of nestin+ cells/mm2 between laminin or Matrigel and PDL or fibrinectin or collagen at 48 h are significant ** p < 0.01. (B) A linear plot summarizing the comparison of neuronal expansion on different substrates over time. Values are expressed in the number of TuJ1+ cells per surface area as mean ± SEM of three independent experiments. The total number of cells per surface area is determined by counting DAPI labeled nuclei which overlapped phase-dark cells. Statistical differences for number TuJ1+ cells/mm2 between laminin or Matrigel and PDL or fibronectin or collagen at 48 h are significant ** p < 0.01.

Mentions: To examine the effect of substrates on the overall growth (expansion) of neural progenitors derived from dark EBs, similar sized (diameter) EBs with equal number of neural progenitors were chosen at 3 h postplating. The number of nestin+ cells grown on different substrates was quantified over time at 3 h, 6 h, 12 h, 18 h, 24 h, 36 h and 48 h postplating from randomly selected fields. A comparison of cell counts at these 7 time points showed significantly greater cell expansion on PDL/laminin or Matrigel at 12–48 h postplating than on other substrates (Fig. 7A). At 48 h postplating, the nestin+ cell number increased on PDL/laminin by approximately 4-fold and on Matrigel by about 3-fold. A comparison of TuJ1+ cells grown on these 5 substrates also indicated that laminin and Matrigel stimulated significantly higher numbers of hESC-derived neurons compared to the other substrates (Fig. 7B).


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)

Expansion of hESC-derived nestin+ neural progenitors and TuJ1+ neurons is greater on PDL/laminin and Matrigel substrates than on PDL, PDL/fibronectin and collagen substrates. (A) A linear plot summarizing the comparison of neural progenitor expansion on different substrates over time. Values are expressed in the number of nestin+ cells per surface area as mean ± SEM of three independent experiments. The total number of cells per surface area is determined by counting DAPI labeled nuclei which overlapped phase-dark cells. Statistical differences for number of nestin+ cells/mm2 between laminin or Matrigel and PDL or fibrinectin or collagen at 48 h are significant ** p < 0.01. (B) A linear plot summarizing the comparison of neuronal expansion on different substrates over time. Values are expressed in the number of TuJ1+ cells per surface area as mean ± SEM of three independent experiments. The total number of cells per surface area is determined by counting DAPI labeled nuclei which overlapped phase-dark cells. Statistical differences for number TuJ1+ cells/mm2 between laminin or Matrigel and PDL or fibronectin or collagen at 48 h are significant ** p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Expansion of hESC-derived nestin+ neural progenitors and TuJ1+ neurons is greater on PDL/laminin and Matrigel substrates than on PDL, PDL/fibronectin and collagen substrates. (A) A linear plot summarizing the comparison of neural progenitor expansion on different substrates over time. Values are expressed in the number of nestin+ cells per surface area as mean ± SEM of three independent experiments. The total number of cells per surface area is determined by counting DAPI labeled nuclei which overlapped phase-dark cells. Statistical differences for number of nestin+ cells/mm2 between laminin or Matrigel and PDL or fibrinectin or collagen at 48 h are significant ** p < 0.01. (B) A linear plot summarizing the comparison of neuronal expansion on different substrates over time. Values are expressed in the number of TuJ1+ cells per surface area as mean ± SEM of three independent experiments. The total number of cells per surface area is determined by counting DAPI labeled nuclei which overlapped phase-dark cells. Statistical differences for number TuJ1+ cells/mm2 between laminin or Matrigel and PDL or fibronectin or collagen at 48 h are significant ** p < 0.01.
Mentions: To examine the effect of substrates on the overall growth (expansion) of neural progenitors derived from dark EBs, similar sized (diameter) EBs with equal number of neural progenitors were chosen at 3 h postplating. The number of nestin+ cells grown on different substrates was quantified over time at 3 h, 6 h, 12 h, 18 h, 24 h, 36 h and 48 h postplating from randomly selected fields. A comparison of cell counts at these 7 time points showed significantly greater cell expansion on PDL/laminin or Matrigel at 12–48 h postplating than on other substrates (Fig. 7A). At 48 h postplating, the nestin+ cell number increased on PDL/laminin by approximately 4-fold and on Matrigel by about 3-fold. A comparison of TuJ1+ cells grown on these 5 substrates also indicated that laminin and Matrigel stimulated significantly higher numbers of hESC-derived neurons compared to the other substrates (Fig. 7B).

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