hPSC-derived lung and intestinal organoids as models of human fetal tissue

Abstract

In vitro human pluripotent stem cell (hPSC) derived tissues are excellent models to study certain aspects of normal human development. Current research in the field of hPSC derived tissues reveals these models to be inherently fetal-like on both a morphological and gene expression level. In this review we briefly discuss current methods for differentiating lung and intestinal tissue from hPSCs into individual 3-dimensional units called organoids. We discuss how these methods mirror what is known about in vivo signaling pathways of the developing embryo. Additionally, we will review how the inherent immaturity of these models lends them to be particularly valuable in the study of immature human tissues in the clinical setting of premature birth. Human lung organoids (HLOs) and human intestinal organoids (HIOs) not only model normal development, but can also be utilized to study several important diseases of prematurity such as respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), and necrotizing enterocolitis (NEC).

Fig. 1

Directed Differentiation of hPSCs to a Lung and Intestinal Organoid FatehPSC with the use of ActivinA can be differentiated into predominantly endoderm (in yellow) with some proportion of cells adopting a mesoderm fate (in red). When exposed to foregut (Noggin, small molecule TGFB inhibitor) or hindgut (WNT3A, FGF4) specific growth factors the endoderm is patterned accordingly. WNT3A and FGF4 appear to be critical for 3-dimensionality and spheroid formation, so that when added to foregut specific growth factors, foregut spheroids are formed. Spheroids collected and placed in Matrigel droplets are allowed to grow in three dimensions into organoid tissue. These organoids are maintained and passaged in tissue specific growth factors (lung: SAG and FGF10) (Intestinal: EGF, Noggin, R-Spondin [ENR]) for up to a year.

Fig. 2

Alveolar like Tissue in Lung Organoids contains Alveoli type 1 and 2 Pneumocytes and Lamellar Bodies. Lung organoids contain cell types associated with alveoli such as alveolar type 1 and 2 pneumocytes (AECI and AECII cells) surrounded by fibroblasts. AECII cells are more rounded in shape and found located between elongated AECI cells. These AECII cells contain lamellar bodies, which are responsible for manufacturing surfactant proteins both in the lung organoids and in vivo human lung tissue.

Fig. 3

HIO Barrier Function Assay using FITC-dextran Dye Microinjection. Cystic HIOs are placed in Matrigel droplets to secure them in place. Glass pulled microinjection pipettes are filled with FITC-dextran dye and carefully inserted into the cystic organoid without causing significant disturbance to the epithelium. Experimental conditions could include adding treatments, insults, or microbes to the media or the intraluminal compartment through microinjection. Intact epithelial barrier will result in dye retention over time, while a damaged epithelium will result in dye leakage and decreased luminescence over time.