Generation of Urine Cell-Derived Non-integrative Human iPSCs and iNSCs: A Step-by-Step Optimized Protocol

Abstract

Objective: Establishing a practical procedure to generate induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) from human urine cells (UCs). In this report, we optimized a non-integrative protocol to generate patient-specific iPSC and iNSC lines with high reprogramming efficiency. Methods: UCs were electroporated with the pEP4-EO2S-ET2K and pEP4-M2L plasmids containing the OCT4, SOX2, KLF4, SV40LT, c-MYC, and LIN28 genes, and then cultured with N2B27 medium plus four small molecule compounds (A83-01, PD0325901, Thiazovivin, and CHIR99021). When iPSC or iNSC clones emerged, the medium was replaced with mTeSR1 or neural growth medium. Morphological changes were seen at day 4-7. After day 10, the clones were picked up when the clone diameter exceeded 1 mm. Results: iPSCs and iNSCs were successfully derived from UCs with up to 80 clones/well. These iPSCs and iNSCs showed typical hESC or NSC morphology and were self-renewable. The iPSCs had pluripotency to differentiate into the three germinal layers and displayed high levels of expression of pluripotency markers SOX2, NANOG, OCT4, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase (AP). They maintained normal karyotype and had no transgene expression or genomic integration. The iNSCs were positive for NSC markers NESTIN, PAX6, SOX2, and OLIG2. Conclusion: The optimized protocol is an easy and fast procedure to yield both iPSC and iNSC lines from a convenient source of human urine in a single experiment.

Keywords: iNSC; iPSC; protocol; reprogram; urine cell.

Figure 1

Culture of the exfoliated urine cells. (A) The urine cell culture from day 0 to day 10. Female urine samples contain squamous cells, and occasionally a few of blood cells. Male samples contain round-shaped sediments. After culture for about 3–4 days, small colonies of epithelium-like cells starts to appear (see day 6). After expansion (day 10), the cells become closely packed. (B) The two types of urine cells. Type I cells have smooth-edged contours and cobblestone-like shape. Type II cells are in slender form and more randomly arranged. (C) The comparison of senescent and healthy urine cells. All scale bars: 200 μm.

Figure 2

Reprogramming of non-integrative iPSCs from UCs. (A) The workflow of UC-derived iPSC (UiPSC) reprogramming. The figure illustrates the reprogramming procedures (upper) and the shape change from UCs to iPSCs from day(D)0 to passage(P)10 (lower). Scale bar: 200 μm. (B) Example of successful establishment of an iPSC line. Scale bar: 200 μm. (C) Non-integration of UC-derived iPSCs. The left plasmid cartoons illustrate the two plasmid backbones and the inserted genes. The right image shows a PCR test for non-integration of these plasmids in established iPSC lines. By using UCs and H₂O as negative controls, and UCs at 3 days post-transfection as a positive control, PCR analysis showed that UiPSC lines had no integration of episomal plasmids.

Figure 3

Characteristics of the UiPSCs. (A) Immunofluorescent labeling for pluripotency markers SOX2, NANOG, OCT4, SSEA-4, TRA-1-60, and TRI-1-81. (B) The morphology of UiPSCs cultured in mTeSR1 medium (upper) and differentiation medium (lower). (C) G-band analysis of an UiPSC shows normal karyotype. (D) Immunofluorescent labeling for three germinal layer markers following UiPSC differentiation. (E) AP staining of UiPSCs, with UCs and fibroblast-derived iPSCs (FiPSCs) as negative and positive controls, respectively. (F) qRT-PCR assay for expression of endogenous human pluripotency genes in two UiPSC lines, with UCs as negative control, and FiPSCs and human ES line H1 as positive controls. (G) qRT-PCR analysis for expression of three germ layer marker genes in UiPSCs after differentiation. Scale bar: A, B, D, 200 μm; E, 2 mm.

Figure 4

Teratoma formation of a UiPSC line. (A) UiPSC injection into immune-deficient mice (NOD-SCID) led to teratoma formation. (B) Hematoxylin-eosin (HE)-staining revealed that the generated teratomas contain the three germinal layer tissues. Scale bar: 200 μm.

Figure 5

Reprogramming of iNSCs from UCs and comparison of a successful iPSC reprogramming and an unsuccessful iPSC reprogramming. (A) The morphology of iNSCs derived from UCs. The upper panel shows the UC-derived iPSCs and iNSCs (UiNSCs) reprogrammed at the same time and the UiNSCs after single-colony replating. Scale bar: 100 μm. The middle panel shows higher-magnification images of iPSCs and iNSCs and the neural rosette of iNSCs. Scale bar: 200 μm. The lower panel shows the morphology comparison between iNSCs and iPSCs. Scale bar: 100 μm. (B) Immunofluorescent staining of iNSC markers SOX2, NESTIN, OLIG2, and PAX6, with nuclear DAPI counter-staining. Scale bar: 100 μm. (C) Compared with a successful iPSC reprogramming in the right panel, the left image shows that the UCs underwent shape transformation but were not fully reprogrammed. Some cells had semi-shape transformation (not round) and the clone cells were loosely attached to each other. Scale bar: 200 μm.