The Generation of Two Induced Pluripotent Cell Lines from Patients with an Atypical Familial Form of Lung Fibrosis

Abstract

Background: Pulmonary fibrosis is a major disease that leads to the progressive loss of lung function. The disease manifests early, resulting in type 2 respiratory failure. This is likely due to the bronchocentric fibrosis around the major airways, which causes airflow limitation. It affects approximately three million patients worldwide and has a poor prognosis. Skin fibroblasts isolated from patients offer valuable insights into understanding the disease mechanisms, identifying the genetic causes, and developing personalized therapies. However, the use of skin fibroblasts to study a disease that exclusively impacts the lungs is often questioned, particularly since lung fibrosis primarily affects the alveolar epithelium. Method: We report the reprogramming of skin fibroblasts from patients with an atypical early-onset form of lung fibrosis into induced pluripotent stem cells (iPSCs) and subsequently into alveolar epithelial cells. This was achieved using a Sendai virus approach. Results: We show that the reprogrammed cells carry mutations in the calcium-binding protein genes S100A3 and S100A13, leading to diminished protein expression, thus mimicking the patients' cells. Additionally, we demonstrate that the generated patient iPSCs exhibit aberrant calcium and mitochondrial functions. Conclusions: Due to the lack of a suitable animal model that accurately resembles the human disease, generating patient lung cells from these iPSCs can provide a valuable "disease in a dish" model for studying the atypical form of inherited lung fibrosis. This condition is associated with mutations in the calcium-binding protein genes S100A3 (NM_002960) and S100A13 (NM_001024210), aiding in the understanding of its pathogenesis.

Keywords: S100A3/S100A13; atypical lung fibrosis; iPSCs; intracellular calcium homeostasis; mitochondrial mutation; reprogramming.

Figure 1

Characterization of IPF.PT1-iPSC and IPF.PT2-iPSC. (A) The iPSCs show normal hESC-like morphology with the expression of SOX2 and SSEA4 pluripotency markers. The nuclei were counterstained with DAPI. (B) The expression of endodermal alpha-fetoprotein (AFP), ectodermal Nestin, and mesodermal α-SMA, counterstained with DAPI. (C) Agarose gel of sample results after mycoplasma testing. (D) Embryoid body formation and alkaline phosphatase test at passage 15. Scale bar 10 μm.

Figure 2

The effect of S100A3 and S100A13 mutations on mitochondria. FITC: fluorescein isothiocyanate. (A) Flow cytometry of the induced pluripotent stem cell- (top) and induced pluripotent stem cell-generated alveolar progenitors (bottom) from patient and control cells stained with MitoTracker Green FM. The inset shows the mean ± SEM of the fluorescence intensity in the patient and control cells. The experiments were performed in triplicate and are representative of at least three independent experiments using one million cells per sample. (B) Confocal fluorescence micrographs of induced pluripotent stem cell- and induced pluripotent stem cell-generated alveolar progenitors labeled with MitoTracker Red CMXRos (1 µM); the corresponding three-dimensional intensity maps are color coded so that warm colors indicate high intensity and cold colors indicate low intensity. Scale bar: 20 µm. FAM-iPSCs is a control cell line generated from control male donor skin fibroblasts. **** p < 0.0001.

Figure 3

The effect of S100A3 and S100A13 mutations on intracellular calcium changes following the stimulation of cultured control FAM.iPSCs (A), IPF-PT1.iPSCs (B), IPF-PT1.iPSCs (C). The cells were stimulated with bradykinin (50 µM) followed by ionomycin (2 µM) and EGTA (1 mM). The histograms show the maximum response to bradykinin (D). The experiments were performed on live single cells using confocal laser scanning microscopy. The data are expressed as mean ± SEM (n = 6 and 5 for the control and patient cells, respectively). The data are expressed as the normalized fluorescence intensity ratio (F/F0) relative to the averaged three images obtained prior to the addition of the stimulus and are representative of three independent experiments. p-values are indicated (*** p < 0.001). FAM-iPSCs is a control cell line generated from control male donor skin fibroblast.

Figure 4

Inflammatory cytokine mediator secretion of patient-derived and normal donor iPSCs. (A–C) Constitutive release of MCP-1, IL-8, and IL-6 in the patient-derived iPSCs compared to the control-derived iPSCs (FAM). The experiments were performed on both patients (n = 3 each). *** p < 0.0001; ***** p < 0.000001; ****** p < 0.0000001.

Figure 4

Inflammatory cytokine mediator secretion of patient-derived and normal donor iPSCs. (A–C) Constitutive release of MCP-1, IL-8, and IL-6 in the patient-derived iPSCs compared to the control-derived iPSCs (FAM). The experiments were performed on both patients (n = 3 each). *** p < 0.0001; ***** p < 0.000001; ****** p < 0.0000001.

Figure 5

Putative lung epithelial cells: (A) Cells expressing definitive endoderm marker SOX17, followed by anteriorization (day 8) cells expressing SOX2, and finally by day 13 Nkx2.1 progenitor (Nkx 2.1 ⁺⁺) cell expression. (B) Day 30, after the maturation of the progenitor cells into epithelial cells expressing markers such as podaplanin and RAGE, with the disappearance of Nkx2.1 ⁺⁺ cells. (C) By day 50, the cells showed maturation toward lung epithelial cells by expressing EP-CAM membranous epithelial cell adhesion molecule and CC10, a non-ciliated airway lung epithelial cell protein. Scale bar: 10 µm.

Figure 5

Putative lung epithelial cells: (A) Cells expressing definitive endoderm marker SOX17, followed by anteriorization (day 8) cells expressing SOX2, and finally by day 13 Nkx2.1 progenitor (Nkx 2.1 ⁺⁺) cell expression. (B) Day 30, after the maturation of the progenitor cells into epithelial cells expressing markers such as podaplanin and RAGE, with the disappearance of Nkx2.1 ⁺⁺ cells. (C) By day 50, the cells showed maturation toward lung epithelial cells by expressing EP-CAM membranous epithelial cell adhesion molecule and CC10, a non-ciliated airway lung epithelial cell protein. Scale bar: 10 µm.

Figure 6

The effect of S100A3 and S100A13 mutations on intracellular calcium changes following stimulation of the cultured control FAM.iPSC lung cells (A), the IPF-PT1.iPSC lung cells (B), or the IPF-PT1.iPSC lung cells (C). The cells were stimulated with bradykinin (50 µM) followed by ionomycin (2 µM) and EGTA (1 mM). The histograms show the maximum response to bradykinin (D). The experiments were performed on live single cells using confocal laser scanning microscopy. The data are expressed as mean ± SEM (n = 4). The data are expressed as the normalized fluorescence intensity ratio (F/F0) relative to the averaged three images obtained prior to the addition of the stimulus and are representative of three independent experiments. ** p < 0.01.