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Expandable Lung Epithelium Differentiated from Human Embryonic Stem Cells

  • Original Article
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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Background

The progenitors to lung airway epithelium that are capable of long-term propagation may represent an attractive source of cells for cell-based therapies, disease modeling, toxicity testing, and others. Principally, there are two main options for obtaining lung epithelial progenitors: (i) direct isolation of endogenous progenitors from human lungs and (ii) in vitro differentiation from some other cell type. The prime candidates for the second approach are pluripotent stem cells, which may provide autologous and/or allogeneic cell resource in clinically relevant quality and quantity.

Methods

By exploiting the differentiation potential of human embryonic stem cells (hESC), here we derived expandable lung epithelium (ELEP) and established culture conditions for their long-term propagation (more than 6 months) in a monolayer culture without a need of 3D culture conditions and/or cell sorting steps, which minimizes potential variability of the outcome.

Results

These hESC-derived ELEP express NK2 Homeobox 1 (NKX2.1), a marker of early lung epithelial lineage, display properties of cells in early stages of surfactant production and are able to differentiate to cells exhibitting molecular and morphological characteristics of both respiratory epithelium of airway and alveolar regions.

Conclusion

Expandable lung epithelium thus offer a stable, convenient, easily scalable and high-yielding cell source for applications in biomedicine.

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Acknowledgements

This work was supported by the Czech Science Foundation (grant no. 18-00145S), by the Ministry of Health of the Czech Republic (grant no. 16-31501A (AH), NV18-08-00299 (PV), 18-08-00245 (KS)), by Masaryk University (MUNI/A/1390/2020, MUNI/A/1689/2020), by the European Regional Development Fund—Project INBIO (No. CZ.02.1.01/0.0/0.0/16_026/0008451). LM is supported by funds from the Faculty of Medicine MU to junior researcher (Lukáš Moráň, ROZV/28/LF/2020), supported by MH CZ-DRO (Masaryk Memorial Cancer Institute, 00209805) and Brno PhD Talent scholarship holder, funded by the Brno City Municipality. The authors thank Katarína Marečková and Dobromila Klemová for assistance with laboratory techniques and Nina Tokanová and Ráchel Víchová for maintenance of the animal facility and Eva Slabáková for TEER.

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Authors and Affiliations

  1. Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic

    Hana Kotasová, Michaela Capandová, Vendula Pelková, Jana Dumková, Zuzana Koledová, Zuzana Garlíková, Veronika Sedláková, Anas Rabata, Petr Vaňhara, Lukáš Moráň, Volodymyr Porokh, Martin Kučírek & Aleš Hampl

  2. International Clinical Research Center, St. Anne’s University Hospital, Brno, Czech Republic

    Hana Kotasová, Ján Remšík, Karel Souček, Petr Vaňhara, Lukáš Pečinka, Josef Havel & Aleš Hampl

  3. Institute of Biophysics, The Czech Academy of Sciences, Brno, Czech Republic

    Ján Remšík & Karel Souček

  4. Research Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Brno, Czech Republic

    Lukáš Moráň

  5. Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic

    Lukáš Pečinka & Josef Havel

  6. Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

    Ján Remšík & Karel Souček

  7. Current Address: Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York City, NY, USA

    Ján Remšík

  8. Department of Plastic and Cosmetic Surgery, Faculty of Medicine, Masaryk University, Brno, Czech Republic

    Libor Streit

  9. Department of Plastic and Cosmetic Surgery, St. Anne’s Faculty Hospital, Brno, Czech Republic

    Libor Streit

Authors
  1. Hana Kotasová
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  2. Michaela Capandová
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Contributions

HK: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript. MC: Collection and/or assembly of data, data analysis and interpretation, manuscript writing. VP: Collection and/or assembly of data, data analysis and interpretation, manuscript writing. JD: Collection and/or assembly of data, data analysis and interpretation, manuscript writing. ZK: Collection and/or assembly of data, data analysis and interpretation, manuscript writing, other (animal work). JR: Collection and/or assembly of data, other (animal work). KS: Conception and design, manuscript writing. ZG: Collection and/or assembly of data. VS: Collection and/or assembly of data, data analysis and interpretation, manuscript writing. AR: Collection and/or assembly of data other (animal work). PV: Collection and/or assembly of data, data analysis and interpretation, manuscript writing. LM: Collection and/or assembly of data, data analysis and interpretation. LP: Collection and/or assembly of data, data analysis and interpretation. VP: Collection and/or assembly of data. MK: Telomerase assay, collection and/or assembly of data. LS: Surgical samples of human skin, collection and/or assembly of data. JH: Data analysis and interpretation, manuscript writing. AH: Conception and design, financial support, administrative support, manuscript writing, final approval of manuscript.

Corresponding author

Correspondence to Aleš Hampl.

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Conflict of interest

The authors have no financial conflicts of interest.

Ethical statement

Human lung tissue samples were obtained from therapeutical lung surgery based on the written informed consent by the patient and approval of Ethics Committee of the University Hospital Brno (28–170621/EK). Human skin samples were obtained from healthy donors undergoing surgical procedures based on the written informed consent by the patient and approval of Ethics Committee St. Anne’s University Hospital Brno (8 V/2020). For animal experiments, all European Union Animal Welfare lines (EU Directive 2010/63/EU for animal experiments) were respected. Animal experiments were approved by the Academy of Sciences of the Czech Republic (AVCR 13/2015), supervised by the local ethical committee and performed by certified individuals (JR, AR, ZK).

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13770_2022_458_MOESM1_ESM.tif

S1. Western blot analysis of phosphorylated (pSMAD2, pSMAD1/5/9) or total (SMAD1, SMAD2) SMADs in DE cells cultivated for 1 hour in the absence ( − ) or presence (+) of ITS, FBS, 50 ng/ml of Activin A and 50 ng/ml of BMP4, respectively.This analysis has shown the activation of BMP signaling after treatment with FBS and/or BMP4 and activation of Activin A/Nodal signaling after treatment with FBS and/or Activin A as opposed to inactive signaling after using serum free media. (TIF 1812 KB)

13770_2022_458_MOESM2_ESM.tif

S2. Analysis of forebrain neuronal marker FOXG1 expression during differentiation of hESC into ELEP. Relative gene expression of FOXG1 in nondifferentiated hESC, definitive endoderm cells (DE), foregut endoderm cells (FE), ELEP low and ELEP high, human lung and human brain tissue, normalized to GAPDH and related to hESC. Data are presented as the mean + SEM, log transformed data were used for statistical analysis, graphs show non-transformed data. *P < 0.05, **P < 0.01, ***P < 0.001. (TIF 844 KB)

13770_2022_458_MOESM3_ESM.tif

S3. Transepithelial electrical resistance (TEER) and dextran permeability. TEER (A) was measured on day 1, 5, and 10 in ELEP, differentiating ELEP, and H441 cells cultured at A-Li. TEER was determined as resistance (Ωcm2) = (Rsample – Rblank) related to effective membrane area (cm2). Permeability to FITC-conjugated dextran (B) was measured on day 6 and 10 in ELEP, differentiating ELEP and H441 cells cultured at A-Li. (TIF 2021 KB)

13770_2022_458_MOESM4_ESM.tif

S4. Histological and immunohistochemistry analysis of outgrowths formed from ELEP. Histological analysis (A) shows kidney parenchyma (ki) and outgrowth tissue (ou). Immunohistochemistry analysis shows cytokeratin 5 and 8 (B and C, respectively) confirming epithelial nature of ELEP, pro-SPC, marker of pneumocytes II (D) and human nucleolar antigen (E). (TIF 11016 KB)

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Kotasová, H., Capandová, M., Pelková, V. et al. Expandable Lung Epithelium Differentiated from Human Embryonic Stem Cells. Tissue Eng Regen Med 19, 1033–1050 (2022). https://doi.org/10.1007/s13770-022-00458-0

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