Blog

5 giugno 2018

In vitro generation of human pluripotent stem cell derived lung organoids

Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, Dye BR and colleagues from Department of Cell and Developmental Biology (University of Michigan Medical School) expose the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, scientists show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease.

Several reports have demonstrated that directed differentiation of human pluripotent stem cells (hPSCs), which include embryonic (hESCs) and induced (iPSCs) stem cells, is one of the most efficient approaches to achieving differentiation of a cell or tissue of interest (D’Amour et al., 2005; Kroon et al., 2008; Si-Tayeb et al., 2009; Spence et al., 2011; Wong et al., 2012). Using this approach, differentiation of hPSCs into lung lineages has been achieved using diverse methodology with varying degrees of success (Kadzik and Morrisey, 2012; Longmire et al., 2012; Mou et al., 2012; Wong et al., 2012; Ghaedi et al., 2013; Huang et al., 2013; Firth et al., 2014). Thus far, the majority of efforts to differentiate lung lineages from hPSCs have focused on using 2-dimensional (2D) monolayer cultures. Several recent advances in generating 3-dimensional (3D) organ-like tissues, called ‘organoids’, have been reported (Meyer et al., 2011; Spence et al., 2011; Nakano et al., 2012; Takebe et al., 2013; Lancaster et al., 2013; McCracken et al., 2014).

Here, Dye et al. developed a new three-dimensional model of the human lung by coaxing human stem cells to become specific types of cells that then formed complex tissues in a petri dish. To make these lung organoids, Dye et al. manipulated several of the signaling pathways that control the formation of organs during the development of animal embryos. First, the stem cells were instructed to form a type of tissue called anterior foregut endoderm (by using ActivinA), which is found in early embryos and gives rise to the lung, liver and other several other internal organs.

By inhibiting two other key developmental pathways at the same time (BMP and TGFβ signaling), the endoderm became tissue that resembles the early lung found in embryos instead.

This early lung-like tissue formed three-dimensional spherical structures as it developed. The next challenge was to make these structures develop into lung tissue. Dye et al. worked out a method to do this, which involved the modulation of FGF and HH signalling that are involved in lung development. The resulting human lung organoids (HLOs) survived in laboratory cultures for over 100 days and developed into well-organized structures that contain many of the types of cells found in the lung. They show striking epithelial structures resembling proximal airways, expressing proximal cell type-specific markers, including basal cells, ciliated cells and club cells. Proximal-like and distal-like airway tissues were often surrounded by a smooth muscle actin positive mesenchyme compartment. Accumulating evidence suggests that HLOs are immature and further analysis revealed the gene activity in the lung organoids resembles that of the lung of a developing human fetus, suggesting that lung organoids grown in the dish are not fully mature. Dye et al.’s findings provide a new approach for creating human lung organoids in culture that may open up new avenues for investigating lung development and diseases.

 

Figure 5 from Dye BR et al., 2015. Lung organoids possess multiple types of mesenchymal cells. (A) D65 HLOs have PDGFRα+ (green) VIM+ (white) double-positive myofibroblasts and PDGFRα−/VIM+ fibroblasts. Scale bar represents 50 μm. (B) D65 HLOs also possesses PDGFRα+ (green) SMA+ (white) double-positive myofibroblasts and PDGFRα−/SMA+ smooth muscle and myofibrblasts. Scale bar represents 50 μm. (C) D65 HLOs do not contain any cartilage whereas positive control iPSC derived teratoma had clear Safranin O staining specific to cartilage. Fast green marks the cytoplasm and hematoxylin the nuclei of both tissues. Scale bar represents 100 μm
 

References

 D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. 2005. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nature Biotechnology 23:1534–1541.

Firth AL, Dargitz CT, Qualls SJ, Menon T, Wright R, Singer O, Gage FH, Khanna A, Verma IM. 2014. Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells. Proceedings of the National Academy of Sciences of USA 111:E1723–E1730

Ghaedi M, Calle EA, Mendez JJ, Gard AL, Balestrini J, Booth A, Bove PF, Gui L, White ES, Niklason LE. 2013. Human iPS cell-derived alveolar epithelium repopulates lung extracellular matrix. The Journal of Clinical Investigation 123:4950–4962

Huang SX, Islam MN, O’Neill J, Hu Z, Yang YG, Chen YW, Mumau M, Green MD, Vunjak-Novakovic G, Bhattacharya J, Snoeck HW. 2013. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nature Biotechnology 32:84–91

Kadzik RS, Morrisey EE. 2012. Directing lung endoderm differentiation in pluripotent stem cells. Cell Stem Cell 10: 355–361.

Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S, Young H, Richardson M, Smart NG, Cunningham J, Agulnick AD, D’Amour KA, Carpenter MK, Baetge EE. 2008. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nature Biotechnology 26:443–452.

Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME, Homfray T, Penninger JM, Jackson AP, Knoblich JA. 2013. Cerebral organoids model human brain development and microcephaly. Nature 501: 373–379.

Longmire TA, Ikonomou L, Hawkins F, Christodoulou C, Cao Y, Jean JC, Kwok LW, Mou H, Rajagopal J, Shen SS, Dowton AA, Serra M, Weiss DJ, Green MD, Snoeck HW, Ramirez MI, Kotton DN. 2012. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell 10:398–411

McCracken KW, Howell JC, Wells JM, Spence JR. 2011. Generating human intestinal tissue from pluripotent stem cells in vitro. Nature Protocols 6:1920–1928.

Meyer JS, Howden SE, Wallace KA, Verhoeven AD, Wright LS, Capowski EE, Pinilla I, Martin JM, Tian S, Stewart R, Pattnaik B, Thomson JA, Gamm DM. 2011. Optic vesicle-like structures derived from human pluripotentstem cells facilitate a customized approach to retinal disease treatment. Stem Cells 29:1206–1218.

Nakano T, Ando S, Takata N, Kawada M, Muguruma K, Sekiguchi K, Saito K, Yonemura S, Eiraku M, Sasai Y. 2012. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell

Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, North PE, Dalton S, Duncan SA. 2009. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51:297–305.

Spence JR, Mayhew CN, Rankin SA, Kuhar MF, Vallance JE, Tolle K, Hoskins EE, Kalinichenko VV, Wells SI, Zorn AM, Shroyer NF, Wells JM. 2011. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470:105–109.

Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, Zhang RR, Ueno Y, Zheng YW, Koike N, Aoyama S, Adachi Y, Taniguchi H. 2013. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499:481–484

Wong AP, Bear CE, Chin S, Pasceri P, Thompson TO, Huan LJ, Ratjen F, Ellis J, Rossant J. 2012. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nature Biotechnology 30:876–882.

Blog
About Cell_Dynamics