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Simple Substrates Aid Stem Cell Differentiation

Review of “Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins” from PNAS by Stuart P. Atkinson

Protocols used to differentiate human pluripotent stem cells (hPSCs) mainly concentrate on the addition of soluble factors to the growth medium, but the importance of the substrate on which cells are grown is now becoming understood. Recombinant proteins, fully synthetic polymers, and peptide-modified surfaces have all been utilized previously and these promote the adhesion of specific cell types, or activate specific signaling pathways to aid differentiation into specific lineages. This is opposed to many currently used growth substrates which represent complex, undefined mixtures of proteins which interact in a complex manner with differentiation signals and display multiple adhesion molecules. Now, in a study in PNAS, researchers from the laboratory of Laura L. Kiessling (University of Wisconsin–Madison, USA) have assessed defined simple growth substrates in the lineage specific differentiation of hPSCs for their ability to control differentiation and further understand the pathways which mediate this differentiation [1].

The researchers first assessed ectodermal differentiation (via dual Smad inhibition) of various human embryonic stem cell (hESC) lines using Matrigel; a widely used complex substrate that engages multiple signaling and adhesion receptors [2, 3]. Expression analysis of genes related to cell adhesion or the extracellular matrix demonstrated an ectodermal-specific increase in several integrin subunits and integrin-binding ECM proteins, suggesting that these may be a more effective substratum for ectodermal cell binding. From this data set the group assessed a cyclic RGD (arginylglycylaspartic acid)-containing peptide (cRGD) known to interact with high affinity with the αVβ3 and αVβ5 integrins, as well as peptides derived from bone sialoprotein and vitronectin. This strategy involved the display of peptides as biotinylated proteins on tissue culture plastic-immobilized streptavidin. Of these, a surface composed of the integrin-binding peptide and the glycosaminoglycan-binding peptide (GBP), known to promote long-term hPSC self-renewal, mediated good ectodermal cell adhesion after 5 days of cell differentiation on a Matrigel substrate. Furthermore, ectodermal differentiation was highly efficient and cells expanded well on this surface, generating distinct cytoskeletal stress fibers (filamentous actin) indicative of robust cell–matrix interactions. Encouragingly, this surface also allowed for the differentiation of more mature ectodermal cell types (spinal motor neurons), at an efficiency similar to Matrigel-based differentiation.

The directed differentiation of hPSCs to definitive endoderm (via activin A signaling) was significantly enhanced when using the GBP substrate alone as compared to integrin-containing Matrigel. GBP mediated a quicker downregulation of pluripotency gene expression, and an enhanced upregulation definitive, but not extra-embryonic, endoderm genes. Integrin-mediated signaling is known to activate Akt signaling which inhibits mesendodermal differentiation, the common progenitor for definitive endoderm and mesoderm. Therefore, the lack of Integrin signaling is likely to be behind this boost in endodermal differentiation. Mesoderm differentiation (activin A followed by BMP4 and bFGF treatment) was also boosted on a GBP surface as compared with Matrigel, with 50% of cells expressing the mesoderm marker PDGFRα on GBP, as compared to only 3% for Matrigel. The GBP substratum also significantly boosted differentiation to contractile cardiomyocytes, a mature mesodermal cell type, as compared to Matrigel.

The final piece of the research tried to understand how these simple culture substrates affected cell signaling in response to differentiation signals, a feat difficult to undertake using a complex undefined substratum. Vitronectin-coated surfaces, which react with integrin, mediated high Akt-phosphorylation in response to activin A and led to cell proliferation (instead of differentiation). However, growth on GBP surfaces led to less Akt-phosphorylation, a cessation of proliferation, and a slight increase in apoptosis, which may enhance the differentiation efficiency. Inhibition of the integrin-linked kinase (ILK), which transmits signals from integrins to the cell interior, also greatly reduced Akt phosphorylation and induced endoderm induction, again suggesting that integrin-mediated extracellular signals can influence endodermal differentiation.

The strategy applied in this study has led to the discovery of some potentially useful cell growth substrates, and should give an impetus for the search for more. They are cheap, simple, defined and, ultimately, highly useful. The simple biotin/streptavidin method of peptide display should allow for high throughput combinatorial assessments of differing peptide and small molecule combinations which could greatly enhance the efficiency and quality of specific cell types.


  1. Wrighton PJ, Klim JR, Hernandez BA, et al. Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins. PNAS 2014;111:18126-18131.
  2. Ludwig TE, Levenstein ME, Jones JM, et al. Derivation of human embryonic stem cells in defined conditions. Nature biotechnology 2006;24:185-187.
  3. Hughes CS, Postovit LM, and Lajoie GA Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics 2010;10:1886-1890.