Human pluripotent stem cell (hPSC)-derived intestinal organoids (hIOs) form 3D structures organized into crypt and villus domains, making them an excellent in vitro model system for studying human intestinal development and disease. However, hPSC-derived hIOs still require in vivo maturation to fully recapitulate adult intestine, with the mechanism of maturation remaining elusive. Here, we show that the co-culture with human T lymphocytes induce the in vitro maturation of hIOs, and identify STAT3-activating interleukin-2 (IL-2) as the major factor inducing maturation. hIOs exposed to IL-2 closely mimic the adult intestinal epithelium and have comparable expression levels of mature intestinal markers, as well as increased intestine-specific functional activities. Even after in vivo engraftment, in vitro-matured hIOs retain their maturation status. The results of our study demonstrate that STAT3 signaling can induce the maturation of hIOs in vitro, thereby circumventing the need for animal models and in vivo maturation.

Tracheal resection has limited applicability. Although various tracheal replacement strategies were performed using artificial prosthesis, synthetic stents and tissue transplantation, the best method in tracheal reconstruction remains to be identified. Recent advances in tissue engineering enabled 3D bioprinting using various biocompatible materials including living cells, thereby making the product clinically applicable. Moreover, clinical interest in mesenchymal stem cell has dramatically increased. Here, rabbit bone marrow-derived mesenchymal stem cells (bMSC) and rabbit respiratory epithelial cells were cultured. The chondrogenic differentiation level of bMSC cultured in regular media (MSC) and that in chondrogenic media (d-MSC) were compared. Dual cell-containing artificial trachea were manufactured using a 3D bioprinting method with epithelial cells and undifferentiated bMSC (MSC group, n = 6) or with epithelial cells and chondrogenic-differentiated bMSC (d-MSC group, n = 6). d-MSC showed a relatively higher level of glycosaminoglycan (GAG) accumulation and chondrogenic marker gene expression than MSC in vitro. Neo-epithelialization and neo-vascularization were observed in all groups in vivo but neo-cartilage formation was only noted in d-MSC. The epithelial cells in the 3D bioprinted artificial trachea were effective in respiratory epithelium regeneration. Chondrogenic-differentiated bMSC had more neo-cartilage formation potential in a short period. Nevertheless, the cartilage formation was observed only in a localized area.

Alternative cell sources, such as three‐dimensional organoids and induced pluripotent stem cell–derived cells, might provide a potentially effective approach for both drug development applications and clinical transplantation. For example, the development of cell sources for liver cell–based therapy has been increasingly needed, and liver transplantation is performed for the treatment for patients with severe end‐stage liver disease. Differentiated liver cells and three‐dimensional organoids are expected to provide new cell sources for tissue models and revolutionary clinical therapies. However, conventional experimental methods confirming the expression levels of liver‐specific lineage markers cannot provide complete information regarding the differentiation status or degree of similarity between liver and differentiated cell sources. Therefore, in this study, to overcome several issues associated with the assessment of differentiated liver cells and organoids, we developed a liver‐specific gene expression panel (LiGEP) algorithm that presents the degree of liver similarity as a “percentage.” We demonstrated that the percentage calculated using the LiGEP algorithm was correlated with the developmental stages of in vivo liver tissues in mice, suggesting that LiGEP can correctly predict developmental stages. Moreover, three‐dimensional cultured HepaRG cells and human pluripotent stem cell–derived hepatocyte‐like cells showed liver similarity scores of 59.14% and 32%, respectively, although general liver‐specific markers were detected. Conclusion: Our study describes a quantitative and predictive model for differentiated samples, particularly liver‐specific cells or organoids; and this model can be further expanded to various tissue‐specific organoids; our LiGEP can provide useful information and insights regarding the differentiation status of in vitro liver models. (Hepatology 2017;66:1662–1674).

We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial–mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.

Directed differentiation of hPSCs and transition from 3D intestinal organoids into functional hIECs.