Elsevier

Biomaterials

Volume 35, Issue 2, January 2014, Pages 699-710
Biomaterials

Alveolar epithelial differentiation of human induced pluripotent stem cells in a rotating bioreactor

https://doi.org/10.1016/j.biomaterials.2013.10.018Get rights and content

Abstract

Traditional stem cell differentiation protocols make use of a variety of cytokines including growth factors (GFs) and inhibitors in an effort to provide appropriate signals for tissue specific differentiation. In this study, iPSC-derived type II pneumocytes (iPSC-ATII) as well as native isolated human type II pneumocytes (hATII) were differentiated toward a type I phenotype using a unique air–liquid interface (ALI) system that relies on a rotating apparatus that mimics in vivo respiratory conditions. A relatively homogenous population of alveolar type II-like cells from iPSC was first generated (iPSC-ATII cells), which had phenotypic properties similar to mature human alveolar type II cells. iPSC-ATII cells were then cultured in a specially designed rotating culture apparatus. The effectiveness of the ALI bioreactor was compared with the effectiveness of small molecule-based differentiation of type II pneumocytes toward type 1 pneumocytes. The dynamics of differentiation were examined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), flow cytometry and immunocytochemistry. iPSC-ATII and hATII cells cultured in the ALI bioreactor had higher levels of type I markers, including aquaporin-5(AQ5), caveolin-1, and T1α, at both the RNA and protein levels as compared with the flask-grown iPSC-ATII and hATII that had been treated with small molecules to induce differentiation. In summary, this study demonstrates that a rotating bioreactor culture system that provides an air–liquid interface is a potent inducer of type I epithelial differentiation for both iPS-ATII cells and hATII cells, and provides a method for large-scale production of alveolar epithelium for tissue engineering and drug discovery.

Introduction

The lung has a complex three-dimensional structure that features major differences in the composition of the epithelium along its proximo-distal axis. The most distal region of the lung is organized into a complex system of alveoli that are lined by two primary epithelial cells types: Type I (ATI) and Type II (ATII) pneumocytes. ATI cells line the majority of the alveoli in lung (covering up to 95% of the alveolar surface area) and are primarily responsible for gas-exchange, while ATII pneumocytes secrete alveolar surfactants and are primarily cuboidal in shape [1], [2], [3].

Primary isolations of ATII and ATI pneumocytes have been performed using various protocols [4], [5], but their yield and proliferation rate is low. Typically, freshly isolated pneumocytes are exceedingly difficult to grow and maintain. These primary cells lose many of their characteristics within a few days of standard cell culture [5]. Therefore, a significant emphasis is being placed on identifying a reliable source of functional lung epithelial cells and designing a suitable cell culture system to maintain proper epithelial cell phenotypes [6], [7]. One approach consists of using stem cells that can be manipulated to become alveolar epithelial cells. Induced pluripotent stem cells (iPSC) are the product of adult somatic cell reprogramming to an embryonic-like state by inducing a “forced” expression of specific pluripotent genes [7], [8], [9], [10], [11], [12]. Given that iPS cells can be derived from the patient to be treated, they could provide a cell source that is genetically identical to the patient, allowing tissue that is generated from these cells to avoid immune rejection [9], [13].

Lung epithelia remain among the least studied lineages derived from ESCs and iPSCs in vitro [2], [10], [11], [12], [14], [15], [16]. In our previous study, we showed the feasibility of producing ATII cells with a very high purity from iPSCs [17]. These iPSC-derived ATII cells, referred to as iPSC-ATII, display as a typical cuboidal appearance and express markers associated with ATII cells (Fig. 1) [17]. In this study we specifically focused on the differentiation of the iPSC-ATII cells toward the type I (ATI) alveolar phenotype.

Although ATII cells are differentiated, these cells nonetheless retain a level of plasticity. In peripheral lung injury, ATII undergo proliferation and differentiation toward the ATI phenotype [18], [19], [20]. In fact, ATII are considered to be putative alveolar stem cells and are crucial to the natural regenerative process of the alveoli [3], [18], [19], [21], [22]. Several studies have shown that exposure to air or inhibition of the Wnt/b-catenin signaling pathway can change the marker expression profile of the differentiated ATII-like phenotype to a predominantly ATI-like phenotype [2], [19], [21], [23], [24], [25], [26].

Based upon these facts, following the generation of iPSC-ATII, we examined the impact of air–liquid interface culture, as well as Wnt/β-catenin inhibitors, on the differentiation of iPSC-ATII to the ATI phenotype. iPSC-ATII cells were cultured on a non-cytotoxic mesh, and then transferred to a dynamic rolling system to recapitulate the air–liquid interface. This in vitro biological environment in some ways mimics in vivo respiratory conditions, as the system is continuously rolled to allow cells to spend an equal amount of time in air and liquid. Herein we investigated the impact that the rolling bioreactor culture system (ALI system) has on the differentiation of iPSC-ATII and hATII cells into cells that are ATI-like, either in the absence of or with the addition of soluble factors.

Section snippets

Cultivation of human iPS cells

The human iPSC line IMR90 (which is denoted as “C1” here), and a line derived from neonatal foreskin, denoted as “C2” here, were utilized. Both lines were provided by Prof. James A Thomson, Department of Anatomy, University of Wisconsin–Madison, Madison, WI [9]. Both human iPSC lines were generated by lentiviral infection of isolated human skin fibroblasts with OCT-4, SOX2, Nanog and lin28 genes. These cells have normal karyotypes, express telomerase activity, express cell surface markers and

Results

In our previous studies, we reported the step-wise differentiation method to generate definitive endoderm (DE), anterior foregut endoderm (AFE), and subsequently, a relatively homogeneous population of human alveolar type II cells from human iPSCs [17]. These iPSC-ATII cells not only demonstrate the phenotype of mature human alveolar type II cells, but also express a high percentage of type II cell markers when compared to freshly isolated human primary alveolar type II cells (Fig. 2A–F). Up to

Discussion

The differentiation and maintenance of ATI cells derived from pluripotent stem cells are critically dependent upon providing physiological conditions to maintain proper phenotypic characteristics in vitro [2], [32]. In this paper, we tried to mimic physiological conditions with an air–liquid interface rolling culture system, to provide a differentiation signal to iPSC-derived ATII and to native type II epithelial cells. Current respiratory epithelium differentiation protocols have largely

Conclusions

Our study demonstrates that the differentiation of IPS derived ATII cells towards an ATI phenotype can be achieved with the use of a rotating bioreactor as well as through the use of small molecule inducers. Our data show that use of the rotating bioreactor, in which cells are placed on a membrane, and undergo air/liquid interface is more efficient in differentiating the ATII cells towards the ATI phenotype. A key advantage to using this bioreactor is that the use of small molecules is not

Disclosure statement

L.E.N. has a financial interest in Humacyte, Inc., a regenerative medicine company. Humacyte did not fund these studies, and Humacyte did not affect the design, interpretation, or reporting of any of the experiments herein.

Author contributions

Laura L. Niklason designed the research and wrote the manuscript; Mahboobe Ghaedi differentiated the iPSCs and performed most the analyses and wrote the manuscript. Julio Mendez performed a part of staining and flow cytometry, Peter F. Bove provided isolated human ATII. Amogh Sivarapatna performed cell culture; Micha Sam Brickman Raredon designed and built the bioreactor.

Acknowledgments

We gratefully acknowledge Prof. James Thomson for providing human iPS cells clones. This work was supported by United Therapeutics, Inc. United Therapeutics did not affect the content or conclusions contained in this manuscript. Work also supported by NIH U01 HL111016 and R01 HL098220 (both to LEN). JJM is supported by T32 GM086287.

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