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A non-invasive method to generate induced pluripotent stem cells from primate urine - PubMed

  • ️Fri Jan 01 2021

A non-invasive method to generate induced pluripotent stem cells from primate urine

Johanna Geuder et al. Sci Rep. 2021.

Abstract

Comparing the molecular and cellular properties among primates is crucial to better understand human evolution and biology. However, it is difficult or ethically impossible to collect matched tissues from many primates, especially during development. An alternative is to model different cell types and their development using induced pluripotent stem cells (iPSCs). These can be generated from many tissue sources, but non-invasive sampling would decisively broaden the spectrum of non-human primates that can be investigated. Here, we report the generation of primate iPSCs from urine samples. We first validate and optimize the procedure using human urine samples and show that suspension- Sendai Virus transduction of reprogramming factors into urinary cells efficiently generates integration-free iPSCs, which maintain their pluripotency under feeder-free culture conditions. We demonstrate that this method is also applicable to gorilla and orangutan urinary cells isolated from a non-sterile zoo floor. We characterize the urinary cells, iPSCs and derived neural progenitor cells using karyotyping, immunohistochemistry, differentiation assays and RNA-sequencing. We show that the urine-derived human iPSCs are indistinguishable from well characterized PBMC-derived human iPSCs and that the gorilla and orangutan iPSCs are well comparable to the human iPSCs. In summary, this study introduces a novel and efficient approach to non-invasively generate iPSCs from primate urine. This will extend the zoo of species available for a comparative approach to molecular and cellular phenotypes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

Workflow overview for establishing iPSCs from primate urine. We established the protocol for iPSC generation from human urine based on a previously described protocol. We tested volume, storage and culture conditions for primary cells and compared reprogramming by overexpression of OCT3/4, SOX2, KLF4 and MYC (OSKM) via lipofection of episomal vectors and via transduction of a Sendai virus derived vector (SeV). We used the protocol established in humans and adapted it for unsterile floor-collected samples from non-human primates by adding Normocure to the first passages of primary cell culture and reprogrammed visually healthy and uncontaminated cultures using SeV. Pluripotency of established cultures was verified by marker expression, differentiation capacity and cell type classification using RNA sequencing.

Figure 2
Figure 2

Establishing urinary cell isolation and reprogramming to iPSCs in human samples. (a) Human urine mainly consists of squamous cells and other differentiated cells that are not able to attach and proliferate (upper row). After ~ 5 days, the first colonies become visible and two types of colonies can be distinguished as described in Zhou (2012). Scale bars represent 500 μm. (b) Isolation efficiency of urine varies between samples. The efficiency between 5 ml, 10 ml and 20 ml of starting material is not different (Fisher’s exact test p > 0.5). (c) SeV mediated reprogramming showed significantly higher efficiency than Episomal plasmids (Wilcoxon rank sum test: p = 1.1e−05). (d) Established human colonies transduced with SeV expressed Nanog, Oct4 and Sox2; Scale bars represent 50 μm and (e) differentiated to cell types of the three germ layers; scale bar represents 500 µm in the phase contrast pictures and 100 µm in the fluorescence pictures. See also Supplementary Figure S1.

Figure 3
Figure 3

Isolation and characterization of primate urinary cells. (a) Workflow of cell isolation from primate urine samples. NC Normocure, REMC renal epithelial mesenchymal cell medium. (b) Primary cells obtained from human, gorilla and orangutan samples are morphologically indistinguishable and display similar EmGFP transduction levels. Scale bars represent 400 μm. (c) The package SingleR was used to correlate the expression profiles from six samples of primate urinary cells (passage 1–3) to a reference set of 38 human cell types. Normalized scores of the eight cell types with the highest correlations are shown (MSC mesenchymal stem cells, SM smooth muscle, Epi epithelial, Endo endothelial). Color bar indicates normalized correlation score. (d) Principal component analysis of primary cells from single colony lysates using the 500 most variable genes. (e) Heatmap of normalized SingleR scores show that cluster C is classified as epithelial cell originating from the bladder. The scores for MSCs in Cluster A and B are similarly high, although cluster B also shows higher scores for epithelial cells than cluster A. See also Supplementary Figure S5.

Figure 4
Figure 4

Generation and characterization of primate iPSCs. (a) Workflow for reprogramming of primate urinary cells. Urine collection and cell seeding is carried out in primary medium, then after 5 days changed to REMC medium, and only passaged for the first time after 10–14 days. When the cells reach confluency reprogramming is induced and after 5 days the medium is changed to mTeSR1. Once the reprogrammed cells are ready to be picked, the cells are seeded in StemFit medium. REMC renal epithelial mesenchymal cell medium. (b) Cell morphology of the three species is comparable before (p0), during (p1–3) and after reprogramming (~ p5). Scale bar represents 400 µm. (c) Immunofluorescence analysis of pluripotency associated proteins at passage 10–15: TRA-1-60, SSEA4, OCT4 and SOX2. Nuclei were counterstained with DAPI. Scale bars represent 200 µm. (d) Differentiation potency into the three germ layers. iPSC colony before differentiation, after 8 days of floating culture and after 8 days of attached culture. Scale bar represents 400 µm. (e) Immunofluorescence analyses of ectoderm (β-III Tubulin), mesoderm (α-SMA) and endoderm markers (α-Feto) after EB outgrowth. Nuclei were counterstained with DAPI. Scale bars represent 400 μm. See also Supplementary Figure S7a.

Figure 5
Figure 5

Characterization of primate iPSCs by expression profiling. (a) The package SingleR was used to correlate the expression profiles from seventeen samples of primate iPSCs (passage 1–3) to a reference set of 38 human cell types. The twelve cell types with the highest correlations are shown (MSC mesenchymal stem cells). All lines are similarly correlated to embryonic stem cells and iPS cells. Color bar indicates correlation coefficients. (b) Principal component analysis of primary cells and derived iPSC lines using the 500 most variable genes. PC1 separates the cell types and PC2 separates the species from each other. (c) Correlation coefficient of iPSCs compared to a single cell dataset covering distinct human embryonic stem cell derived progenitor states (Chu et al. 2016). (d) Expression distances of all detected genes are averaged from pairwise distances for six different groups of comparisons. Note that the distance between individuals and between species is calculated within iPSCs and distances between individuals within species. Pairwise t-tests are all below 0.01 (**) for comparisons to the cell-type and species distance and all above 0.05 (n.s.) for comparisons within the species. See also Supplementary Figure S5.

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References

    1. Pecon-Slattery J. Recent advances in primate phylogenomics. Annu. Rev. Anim. Biosci. 2014;2:41–63. doi: 10.1146/annurev-animal-022513-114217. - DOI - PubMed
    1. Enard W. Functional primate genomics-leveraging the medical potential. J. Mol. Med. 2012;90:471–480. doi: 10.1007/s00109-012-0901-4. - DOI - PubMed
    1. Enard W. The molecular basis of human brain evolution. Curr. Biol. 2016;26:R1109–R1117. doi: 10.1016/j.cub.2016.09.030. - DOI - PubMed
    1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292:154–156. doi: 10.1038/292154a0. - DOI - PubMed
    1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676. doi: 10.1016/j.cell.2006.07.024. - DOI - PubMed

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