Long-term differentiating primary human airway epithelial cell cultures: how far are we?

Abstract

Background: Human airway epithelial (HAE) cellular models are widely used in applicative studies of the airway physiology and disease. In vitro expanded and differentiated primary HAE cells collected from patients seem to be an accurate model of human airway, offering a quicker and cheaper alternative to the induced pluripotent stem cell (iPSCs) models. However, the biggest drawback of primary HAE models is their limited proliferative lifespan in culture. Much work has been devoted to understand the factors, which govern the HAE cell proliferation and differentiation, both in vivo and in vitro. Here, I have summarized recent achievements in primary HAE culture, with the special emphasis on the models of conditionally reprogrammed cells (CRC), which allow longer in vitro proliferation and differentiation of HAE cells. The review compares the CRC HAE technique variants (feeder culture or HAE mono-culture), based on recently published studies exploiting this model. The advantages and limitations of each CRC HAE model variant are summarized, along with the description of other factors affecting the CRC HAE culture success (tissue type, sampling method, sample quality).

Conclusions: CRC HAE cultures are a useful technique in respiratory research, which in many cases exceeds the iPSCs and organoid culture methods. Until the current limitations of the iPSCs and organoid culture methods will be alleviated, the primary CRC HAE cultures might be a useful model in respiratory research. Airway epithelium (AE) is a type of tissue, which lines the whole length of human airways, from the nose to the bronchi. Improper functioning of AE causes several human airway disorders, such as asthma, chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF). Much work has been devoted to finding the best scientific model of human AE, in order to learn about its functioning in health and disease. Among the popular AE models are the primary in vitro cultured AE cells collected from human donors. Unfortunately, such human AE (HAE) cells do not easily divide (expand) in vitro; this poses a large logistic and ethical problem for the researchers. Here, I summarize recent achievements in the methods for in vitro culture of human AE cells, with special emphasis on the conditionally reprogrammed cell (CRC) models, which allow longer and more effective expansion of primary human AE cells in vitro. The review describes how the specific chemicals used in the CRC models work to allow the increased HAE divisions and compares the effects of the different so-far developed variants of the CRC HAE culture. The review also pinpoints the areas which need to be refined, in order to maximize the usefulness of the CRC AE cultures from human donors in research on human airway disorders. Video abstract.

Keywords: Air–liquid interface culture; Conditional reprogramming; Primary airway cell culture; ROCK inhibitor; SMAD inhibitor; TGF-β1 inhibitor.

Fig. 1

Airway epithelial differentiation pathways. a Differentiation of airway epithelial cells during embryonic development. b Differentiation of airway epithelial cells after birth (from adult stem cells). Names below the different cell types are the markers of the specific cell types (based on recent human single-cell RNAseq studies [21, 24]). TP63, KRT5, KRT6C, KRT14 and KRT15 are basal cell markers, while supra-basal cells are characterized by expression of KRT4, KRT13, KRT16 and KRT23. Within the mucociliary lineage, club cells are distinguished by expression of Scgb1a1, KRT7 and KRT19, mature goblet cells have expression of SPDEF, FOXQ1 and MUC5AC. The deuterosomal cells express PLK4, CCNO, CEP78 and DEUP1, while mature ciliated cells are distinguished by the expression of DNAH5, SPEF2, PIFO and LRRC6. Suprabasal cells can give rise to tuft cells, which express POUF2F3 and TRPM5. These in turn can differentiate either to ionocytes, expressing FOXI1 or CFTR, or to neuroendocrine cells, expressing PSMD5 or NGF

Fig. 2

Existing culturing platforms for HAE expansion and differentiation. ALI – air–liquid interface culture; BC- basal cell

Fig. 3

Mechanisms of conditional HAE reprogramming allowing proliferation of basal cells. ROCKi – ROCK inhibitor, PAK1i – PAK1 inhibitor, Myosin IIi- inhibitor of Myosin II, SMADi – inhibitor of SMAD, mTORi – inhibitor of mTOR pathway

Fig. 4

Choice of the CRC HAE culturing method depending on the experimental application. CBF – ciliary beat frequency