Tanneberger Alicia E, Blomberg Rachel, Bilousova Ganna, Ryan Amy L, Magin Chelsea M
Department of Bioengineering, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado, United States.
Department of Dermatology, Gates Institute, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States.
Am J Physiol Lung Cell Mol Physiol. 2025 Mar 1;328(3):L379-L388. doi: 10.1152/ajplung.00419.2024. Epub 2025 Jan 30.
Lung progenitor (LP) cells identified by the expression of transcription factor NK2 homeobox 1 (NKX2.1) are essential for the 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 that each factor impacted LP differentiation efficiency and that the soft hydrogels replicating healthy lung stiffness [elastic modulus () = 4.00 ± 0.25 kPa] produced the highest proportion of LP cells based on flow cytometric analysis results (54%) relative to the stiff hydrogels (48%) and Matrigel controls (32%) at the end of the nonsplit differentiation protocol. Collectively, these results showed that engineered hydrogels provide a well-defined microenvironment for iPSC-to-LP differentiation and perform as effectively as the current gold standard Matrigel-coated tissue culture plastic. Adopting engineered biomaterials in cell culture protocols may enable greater control over differentiation parameters and has the potential to enhance the clinical translation of iPSC-derived LP cells. Standard 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.
通过转录因子NK2同源盒1（NKX2.1）的表达鉴定出的肺祖细胞（LP）对于所有肺上皮细胞类型的发育至关重要，在肺部研究和转化再生医学应用中具有巨大潜力。在此，我们展示了工程水凝胶是一种很有前景的替代天然衍生材料的选择，天然衍生材料常用于将人诱导多能干细胞（iPSC）分化为LP细胞。使用具有确定组成的聚（乙二醇）降冰片烯（PEGNB）水凝胶系统地研究了微环境硬度、细胞来源和分化过程中的传代对分化的作用。结果表明，每个因素都影响LP分化效率，并且根据流式细胞术分析结果，复制健康肺硬度[弹性模量（）= 4.00±0.25 kPa]的软水凝胶在非传代分化方案结束时产生的LP细胞比例最高（54%），相对于硬水凝胶（48%）和基质胶对照（32%）。总体而言，这些结果表明，工程水凝胶为iPSC向LP分化提供了一个定义明确的微环境，并且与当前的金标准基质胶包被的组织培养塑料一样有效。在细胞培养方案中采用工程生物材料可能能够更好地控制分化参数，并有可能增强iPSC衍生的LP细胞的临床转化。标准的iPSC分化方案依赖于基质胶，这是一种从小鼠肉瘤细胞中提取的基底膜提取物，其定义不明确且批次间差异显著。由于这些局限性，基质胶衍生产品从未获得美国食品药品监督管理局的批准。本研究介绍了一种使用定义明确的水凝胶底物将iPSC分化为肺祖细胞的新方法。这些生物材料不仅提高了分化效率，还简化了监管途径，促进了它们的潜在治疗应用。
