Urine-derived cells from the aged donor for the 2D/3D modeling of neural cells via iPSCs

Abstract

Human neural cell models derived from induced pluripotent stem cells (iPSCs) have been widely accepted to model various neurodegenerative diseases such as Alzheimer's disease (AD) in vitro. Although the most common sources of iPSCs are fibroblasts and peripheral blood mononuclear cells, the collection of these cells is invasive. To reduce the donor's burden, we propose the use of urine-derived cells (UDCs), which can be obtained non-invasively from a urine sample. However, the collection of UDCs from elderly donors suffering from age-related diseases such as AD has not been reported, and it is unknown whether these UDCs from the donor aged over 80 years old can be converted into iPSCs and differentiated into neural cells. In this study, we reported a case of using the UDCs from the urine sample of an 89-year-old AD patient, and the UDCs were successfully reprogrammed into iPSCs and differentiated into neural cells in four different ways: (i) the dual SMAD inhibition with small-molecules via the neural progenitor precursor stage, (ii) the rapid induction method using transient expression of Ngn2 and microRNAs without going through the neural progenitor stage, (iii) the cortical brain organoids for 3D culture, and (iv) the human astrocytes. The accumulation of phosphorylated Tau proteins, which is a pathological hallmark of AD, was examined in the neuronal models generated from the UDCs of the aged donor. The application of this cell source will broaden the target population for disease modeling using iPS technology.

Keywords: Astrocytes; Cortical brain organoids; Disease modeling; Induced pluripotent stem cell (iPSC); Neurons; Urine.

Graphical abstract

Fig. 1

Derivation of the UDCs from the very old donor’s urine sample and generation of the iPSCs. (A) The UDCs. Scale bar, 500 μm.; (B) The generated iPSCs. Scale bar, 500 μm.; (C) ALP staining of the iPSCs. Scale bar, 50 μm.; (D) qPCR results of the pluripotency markers (Nanog, Sox2, and Oct4) from the TMGH-1 UDCs (n = 3), the TMGH-1 iPSCs (n = 3), and the control 1210B2 iPSCs (n = 3). Bars, mean ± SEM; (E) Immunocytochemistry of the pluripotency markers (SSEA4, TRA-1–81, SOX2, NANOG, and OCT4) on TMGH-1 iPSCs. Scale bar, 50 μm; (F) Karyotype result of TMGH-1 iPSCs (Female: 46, XX); (G) APOE genotyping results of the generated iPSCs: (H) Trilineage differentiation assay of the iPSCs into the three germ layers (endoderm, mesoderm, and ectoderm). Scale bar, 25 μm.

Fig. 2

2D neuronal models induced from the iPSCs derived from the UDCs using the small-molecule compounds according to the dual SMAD inhibition. (A) Induction method; (B) Induced neurons at PID 45. Scale bar, 50 and 25 μm; (C) Neurons from the dual SMAD inhibition at PID 45 expressed neuronal markers (MAP2, βIII-TUBULIN, TAU, and NEUN). Scale bar, 50 μm.

Fig. 3

2D neuronal models induced from the iPSCs derived from the UDCs using the transient expression of Ngn2 and microRNAs. (A) Establishment of the iPSCs containing Ngn2 and microRNAs; (B) Neuronal induction method; (C) Induced neurons at PID 14. Scale bar, 50 and 25 μm; (D) Neurons from the transient expression of Ngn2 and microRNAs at PID 14 expressed neuronal markers (MAP2, βIII-TUBULIN, TAU, and NEUN). Scale bar, 50 μm.

Fig. 4

Cortical brain organoids induced from the iPSCs derived from the UDCs. (A) Induction method of brain organoids from the iPSCs; (B) Cell aggregates in the 96-well format at PID 3 and 14; (C) Quantification of the diameter of the cell aggregates at PID 3 (n = 9) and 14 (n = 9). Bars, mean ± SEM. ****p < 0.0001 (Mann-Whitney test); (D) Relative GFAP intensity of TMGH-1 cortical brain organoids at 10 weeks and 20 weeks. Bars, mean ± SEM. ****p < 0.0001 (Unpaired t-test); (E) Immunohistochemistry of the neuronal markers (MAP2, βIII-Tubulin, and Tau), astrocyte marker (GFAP and S100β), and oligodendrocyte marker (Olig2) in TMGH-1 cortical brain organoids at 10 weeks and 20 weeks after the differentiation. Scale bar, 50 μm.

Fig. 5

Alzheimer’s Disease (AD)-like phenotypic analysis of the neuronal models generated from TMGH-1 iPSCs. (A) Staining of phosphorylated Tau (pTau) (PHF-1 and CP13) and neuronal markers (MAP2 and Dako Tau) in the 2D neuronal models (Dual SMAD inhibition), 2D neuronal models (Transient expression of Ngn2 and microRNAs), and 3D cortical brain organoids generated from iPSCs of TMGH-1 line and control line. Scale bar, 50 μm; (B) Quantification of pTau expression levels of each neuronal models (n = 20 each) generated from iPSCs of TMGH-1 line and control line. Bars, mean ± SEM. PHF-1 levels of 2D neuronal model (dual SMAD Inhibition): *p = 0.0350; PHF-1 levels of 3D neuronal model (Cortical brain organoid): *p = 0.0460; ****p < 0.0001 (Mann-Whitney test).