Modulating cell differentiation with protein-engineered microenvironments

Yeji Kim, Purdue University

Abstract

Tissue damage caused by diseases and injuries requires regeneration to recover proper tissue function. Traditional treatments using autologous tissue have not yet met the need for tissue regeneration. Tissue engineering has emerged as an alternative strategy for tissue regeneration. It utilizes biomaterials that mimic native cellular microenvironments and provide environmental cues to induce specific cell responses, including differentiation. Biochemical and biophysical cues modulate cell behavior and direct construction of specific types of tissue. Thus, this work describes production and characterization of protein-engineered microenvironments that provide biochemical and biophysical cues to modulate stem cell differentiation. We created modular proteins containing two domains, a resilin structural domain and bioactive domains. The resilin domain (RZ) includes ten repeats of a resilin-like sequence, which are derived from the African malaria mosquito and provide mechanical integrity. Depending on the targeted cell response, the bioactive domains include peptide sequences derived from growth factors or extracellular matrix proteins. Protein-engineered microenvironments were created as adsorbed protein layers or crosslinked matrices. First, we produced RZ-BMP proteins containing a peptide derived from bone morphogenetic protein-2 (BMP-2 peptide) and examined the effect of this biochemical cue on osteogenic differentiation of human mesenchymal stem cells (hMSCs). Cells cultured on the RZ-BMP proteins had increased levels of osteogenic markers, such as alkaline phosphatase activity, calcium deposition, and expression of bone-related genes. Our results show that, within the context of our proteins, the BMP-2 peptide was active and accelerated osteogenic differentiation of hMSCs. We also examined whether there is a synergistic effect of the BMP-2 peptide and the RGD cell-binding sequence on osteogenic differentiation but found that there was no synergy within the context of our proteins. Next, we switched the biochemical cue in our microenvironments to achieve a different type of cell differentiation. Instead of the BMP-2 peptide, a peptide derived from vascular endothelial growth factor (QK peptide) was included in the RZ-QK protein to induce endothelial differentiation. We demonstrated that endothelial differentiation of hMSCs was achieved on the RZ-QK proteins in the absence of exogenous growth factors as the RZ-QK proteins promoted endothelial-specific markers and endothelial functions. In particular, cells on our proteins exhibited statistically equivalent network formation as positive control cells. Finally, we evaluated the effect of biophysical cues (i.e., matrix stiffness) on cell responses such as spreading and endothelial differentiation. We used proteins containing the RGD cell-binding sequence to promote cell adhesion. The proteins were crosslinked with transglutaminase (TGase) to create RZ-TGase matrices with varying stiffness, and the stiffness of our matrices was similar to that of subendothelial environments. The matrix stiffness modulated spreading and endothelial differentiation of hMSCs as the stiffest matrices promoted greater cell spreading and higher endothelial function compared to the softest matrices. Of particular note, our RZ-TGase matrices promoted statistically equivalent endothelial function as hard surfaces, which are routinely used for conventional cell differentiation but are much stiffer than the subendothelial matrix. Thus, our tunable matrix system is a promising tool to provide more native subendothelial environments compared to conventional hard culture substrates.

Degree

Ph.D.

Advisors

Liu, Purdue University.

Subject Area

Biomedical engineering

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