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

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.

Prime-seq, efficient and powerful bulk RNA-sequencing

“…We have shown above that the power, accuracy and library complexity is similar between prime-seq and TruSeq. The performance and robustness of the prime-seq protocol has been demonstrated by the two examples above as well as its many applications using this or previous versions of the protocol [9,[23][24][25][26][27][28][29][30][31][32][33][34][35]42,43,56,57]. In summary, one could argue that prime-seq performs as well as TruSeq for quantifying gene expression levels.…”

“…To further exemplify the performance of prime-seq, we investigated its ability to detect known differences in a well established differentiation system [54]. We differentiated five human induced pluripotent stem cell (iPSCs) lines [36] to neural progenitor cells (NPCs) and generated expression profiles using prime-seq (Figure 5C). In a hierarchical clustering of well known marker genes [55], the iPSCs and NPCs formed two distinct groups and the expression patterns were in agreement with their cellular identity.…”

“…The human iPSC samples, which were differentiated into the NPCs, were ethically approved by the responsible committee on human experimentation (20-122, Ethikkommission LMU München) as previously published [57].…”

“…Replacing qPCR has been advocated as one example by the authors of BRB-seq [22]. But also other applications, like characterizing cell type composition [36], quality control of libraries, or optimizing experimental procedures can profit considerably from low library costs.…”

Urine-derived induced pluripotent/neural stem cells for modeling neurological diseases

“…Firstly, microorganism contamination in urine remains an issue of USCs sampling compared to other somatic cells. Addition of normocure, a broad-spectrum antibacterial agent that can eliminate microorganism in unsterile floor-collected urine samples without affecting the growth of USCs in culture [ 117 ]. Secondly, it is a double-edged sword that UiPSCs from individuals carrying specific genetic background may impact the differentiation propensities into a specific lineage under the same culture condition that makes comparison difficult, but the distinct genetic differences give us clues about the specific determinants of disease severity and the response to drug treatment on an individual basis that provides an important ground for future personalized medicine.…”