Engineered hydrogel biomaterials facilitate lung progenitor cell differentiation from induced pluripotent stem cells

Abstract

Lung progenitor (LP) cells identified by the expression of transcription factor NK2 homeobox 1 (NKX2.1) are essential for development of all lung epithelial cell types and hold tremendous potential for pulmonary research and translational regenerative medicine applications. Here we present engineered hydrogels as a promising alternative to the naturally derived materials that are often used to differentiate human induced pluripotent stem cells (iPSCs) into LP cells. Poly(ethylene glycol) norbornene (PEGNB) hydrogels with defined composition were used to systematically investigate the role of microenvironmental stiffness, cell origin, and splitting during the differentiation process. Results demonstrated each factor impacted LP differentiation efficiency. Soft hydrogels replicating healthy lung stiffness (Elastic modulus (E), E = 4.00 {+/-} 0.25 kPa) produced the highest proportion of LP cells (54% by flow cytometry), stiff hydrogels (E = 18.83 {+/-} 2.24 kPa) resulted in 48% differentiation efficiency, and a thin coating of Matrigel on tissue culture (TC) plastic (E[~]3 GPa) resulted in the lowest proportion of LP cells (32%) at the end of the non-split differentiation protocol. Collectively these results showed that engineered hydrogels enabled control over parameters that impacted differentiation and produced LP cells using well-defined microenvironments that may improve our ability to translate iPSC-derived LP cells into clinical applications. NEW & NOTEWORTHYStandard iPSC differentiation protocols rely on Matrigel, a basement membrane extract from mouse sarcoma cells that is poorly defined and exhibits significant batch-to-batch variation. Due to these limitations Matrigel-derived products have never been approved by the Food and Drug Administration. This study introduces a novel method for differentiating iPSCs into lung progenitor cells using well-defined hydrogel substrates. These biomaterials not only enhance differentiation efficiency, but also streamline the regulatory pathway, facilitating their potential therapeutic application.