A synthetic nanofibrillar matrix promotes in vitro hepatic differentiation of embryonic stem cells and induced pluripotent stem cells

Abstract

SummaryEmbryonic stem (ES) cells recapitulate normal developmental processes and serve as an attractive source for routine access to a large number of cells for research and therapies. We previously reported that ES cells cultured on M15 cells, or a synthesized basement membrane (sBM) substratum, efficiently differentiated into an endodermal fate and subsequently adopted fates of various digestive organs, such as the pancreas and liver. Here, we established a novel hepatic differentiation procedure using the synthetic nanofiber (sNF) as a cell culture scaffold. We first compared endoderm induction and hepatic differentiation between murine ES cells grown on sNF and several other substrata. The functional assays for hepatocytes reveal that the ES cells grown on sNF were directed into hepatic differentiation. To clarify the mechanisms for the promotion of ES cell differentiation in the sNF system, we focused on the function of Rac1, which is a Rho family member protein known to regulate the actin cytoskeleton. We observed the activation of Rac1 in undifferentiated and differentiated ES cells cultured on sNF plates, but not in those cultured on normal plastic plates. We also show that inhibition of Rac1 blocked the potentiating effects of sNF on endoderm and hepatic differentiation throughout the whole differentiation stages. Taken together, our results suggest that morphological changes result in cellular differentiation controlled by Rac1 activation, and that motility is not only the consequence, but is also able to trigger differentiation. In conclusion, we believe that sNF is a promising material that might contribute to tissue engineering and drug delivery.

Physical cues of cell culture materials lead the direction of differentiation lineages of pluripotent stem cells

“…2; growth on the nanofibers was compared to several other substrata (TCPS coated with collagen type 1, gelatin, Matrigel, or CellStart). 105 The Type E protocol that is shown in Fig. 2, which is similar to protocols reported by Farzaneh et al 83 and Ramasamy et al, 100 was used for the differentiation of hPSCs.…”

“…The morphological changes of ESCs cultured on nanofibers resulted in cellular differentiation that was controlled by Rac1 activation. 105 The nanofibers seemed to recapitulate the structural features of the stem cell niche, which guided the differentiation of hPSCs into specific cell lineages and could contribute to tissue engineering and drug delivery.…”

Extracellular matrix microarrays to study inductive signaling for endoderm specification

“…Together with growth factor signaling, epigenetic changes have additionally been demonstrated to be critical for directing the fate of endoderm cells, for example, increased methylation of distinct promoter regions has been identified to be necessary for hepatic (liver)-lineage commitment [15]. Notably, limited studies have examined the expression of ECM components during liver [16, 17] and pancreas [18] development, or during the in vitro differentiation of stem and progenitor cells [19–21]. Thus, the role of ECM in the differentiation of hepatic and pancreatic lineages remains primarily unclear.…”

Substrate‐mediated commitment of human embryonic stem cells for hepatic differentiation

“…Due to the high degree of control over the fiber structure in the electrospinning process, electrospun meshes have been widely used as micro‐ and nanoscale ECM‐mimicking substrates for stem cell culture . Recently, electrospun nanofibers have been used for differentiation of hESCs into DE‐derived cells such as hepatic cells . It was previously shown that electrospun nanofibers of chitosan/polycaprolactone with controlled nanoscale topography direct the differentiation of hESCs into hepatic endoderm …”

“…20 Recently, electrospun nanofibers have been used for differentiation of hESCs into DE-derived cells such as hepatic cells. [21][22][23] It was previously shown that electrospun nanofibers of chitosan/polycaprolactone with controlled nanoscale topography direct the differentiation of hESCs into hepatic endoderm. 19 This study aimed to evaluate the potential of an Actconjugated nanofibrous substrate for differentiation of hESCs into DE.…”