Live births from urine derived cells

Abstract

Here we report urine-derived cell (UDC) culture and subsequent use for cloning which resulted in the successful development of cloned canine pups, which have remained healthy into adulthood. Bovine UDCs were used in vitro to establish comparative differences between cell sources. UDCs were chosen as a readily available and noninvasive source for obtaining cells. We analyzed the viability of cells stored in urine over time and could consistently culture cells which had remained in urine for 48hrs. Cells were shown to be viable and capable of being transfected with plasmids. Although primarily of epithelial origin, cells were found from multiple lineages, indicating that they enter the urine from more than one source. Held in urine, at 4°C, the majority of cells maintained their membrane integrity for several days. When compared to in vitro fertilization (IVF) derived embryos or those from traditional SCNT, UDC derived embryos did not differ in total cell number or in the number of DNA breaks, measured by TUNEL stain. These results indicate that viable cells can be obtained from multiple species' urine, capable of being used to produce live offspring at a comparable rate to other cell sources, evidenced by a 25% pregnancy rate and 2 live births with no losses in the canine UDC cloning trial. This represents a noninvasive means to recover the breeding capacity of genetically important or infertile animals. Obtaining cells in this way may provide source material for human and animal studies where cells are utilized.

Fig 1

Cells number per volume (ml) of urine following micturition collection in A) Canine and B) Bovine samples C) Average cell number per ml, canine numbers displayed divided by 100 for scale. Error bars SEM *(p = 0.036).

Fig 2

Canine Urine and UDCs A) Urine cells following isolation. B) Cellular morphological variation of large and small cells and integrity staining utilizing Propidium Iodide (red) and Hoechst 33342 (blue). Red stained cells indicate membrane permeability and thus non-viable cells; blue stain illustrates nuclei with intact membranes. C) Urine cell integrity indicate by Propidium iodide negative stain over time stored at 4°C (*p<0.05, *** p<0.001).

Fig 3

UDC immunohistochemical characterization with Hoechst indicating nuclei (Blue) A) Vimentine positive cells (Red) B) E-Cadherin positive staining(Green) C Pancytokeratin stain (Green). D. Pancytokeratin stain overlaid on light microscopy image. E) Presense of Mesynchymal cellular marker Vimentin, F) Epithelial marker P-Glycoprotein. G Further markers for cell characterization indicating a heterogeneous cell population in UDCs.

Fig 4

Embryo quality analysis A) Representative images of blastocyst with total cells (Blue) and TUNEL positive cells (Green), indicating DNA breaks B) Total cell number and TUNEL staining in 8 day embryos derived from IVF, SCNT with standard grown cells, and UDC SCNT. C) average ratio of tunnel positive cells to total cell counts. Error bars are SD.

Fig 5. The UDC cloning flow through.

A) Urine collected, B) Analysis of cellular viability, C) UDCs established D) SCNT performed, E) Ultrasonic pregnancy confirmation at ~30 days after embryo transfer, F) Cloned puppies.

Fig 6

Growth and Development of standard SCNT and UDC clone development A) Growth rate until weaning, B) newborns, C) UDC clones at 4 months of age, D) mature UDC clones. Error bars ae SD.