Isolation, Culture and Comprehensive Characterization of Biological Properties of Human Urine-Derived Stem Cells

Abstract

Mesenchymal stem cells (MSCs) represent an attractive source within the field of tissue engineering. However, their harvesting often requires invasive medical procedures. Urine-derived stem cells (UDSCs) display similar properties to MSCs, and their obtention and further processing is non-invasive for the donors as well as low cost. Here, we offer a comprehensive analysis of their biological properties. The goal of this study was to analyze their morphology, stemness, differentiation potential and cytokine profile. We have successfully isolated UDSCs from 25 urine samples. First colonies emerged up to 9 days after the initial seeding. Cell doubling time was 45 ± 0.24 SD, and when seeded at the density of 100 cells/cm², they formed 42 ± 6.5 SD colonies within 10 days. Morphological analyzes revealed that two different types of the cell populations have been present. The first type had a rice-grain shape and the second one was characterized by a polyhedral shape. In several cell cultures, dome-shaped cells were observed as well. All examined UDSCs expressed typical MSC-like surface markers, CD73, CD90 and CD105. Moreover, conditioned media from UDSCs were harvested, and cytokine profile has been evaluated showing a significantly higher secretory rate of IL-8, IL-6 and chemokines MCP-1 and GM-CSF. We have also successfully induced human UDSCs into chondrogenic, osteogenic and myogenic cell lineages. Our findings indicate that UDSCs might have immense potential in the regeneration of the damaged tissues.

Keywords: biological characteristics; cytokine profile; differentiation capacity; morphology; stemness; urine-derived stem cells.

Figure 1

Isolation and basic biological characterization of the urine-isolated cells. (A) Image depicting freshly processed female urine sample. Multiple epithelial cells, forming non-adherent layer in a culture dish, could be detected. Non-adherent cells were removed after the first change of the culture medium. (B) Primary cell colonies observed after 5–9 days after initial seeding. (C) Representative growth curve of the isolated stem cells. Growth kinetics was studied over the period of 12 days. Exponential growth phase, in which cells proliferated rapidly, lasted for 6 days. (D) Representative figure of colony forming unit experiment. After 10 days, 42 ± 6.5 SD adherent cell colonies were observed.

Figure 2

Morphological analysis of the human UDSCs. (A) Two different cell morphologies could be microscopically observed within one sample: rice-grain shape (thin arrows) and polyhedral-shape (thick arrows). (B) Image of the cell culture depicting dome-like structures, which could be spontaneously formed in confluent cell populations. These vesicles are filled with liquid and are capable of enlarging their volume, which could lead to the bursting of cells. (C) electron micrograph showing proteosynthetically and metabolically active cell with presence of endoplasmic reticulum, mitochondria and lysosomes. Multivesicular bodies (MVBs) were clearly visible as well. (D) Electronogram of the second type of UDSCs. Cytoplasmic organelles were mostly absent, which might have indicated the inactive form of the cell population.

Figure 3

Representative histograms of the specific surface markers by flow cytometry. Isolated human UDSCs expressed typical MSC-like surface markers. Statistically, the highest expression was detected within CD73 (99.65% ± 0.38 SD), followed by CD44 (98.05% ± 2.37 SD), CD146 (97.91% ± 3.01 SD), CD90 (95.42% ± 5.44 SD), CD105 (91.77% ± 4.93 SD) and CD271 (91.72% ± 10.68 SD). The positivity for non-mesenchymal and hematopoietic surface markers (CD14/CD20/CD34/CD45 cocktail) was approximately 1.76% ± 1.57 SD.

Figure 4

Cytokine profile of the human UDSCs. Analysis of the cells’ secretory activity revealed that IL-8 was secreted at the highest concentration (6958.33 pg/mL) followed by MCP-1 (3789.54 pg/mL), IL-6 (3650.03 pg/mL), and GM-CSF (817.89 pg/mL). Moderate levels within conditioned media were measured for TNF-alpha (62.4 pg/mL), IL-4 (31.29 pg/mL), RANTES (27.99 pg/mL) and INF-alpha2 (10.35 pg/mL). Concentration of other cytokines and chemokines was lower compared to the control (unconditioned) medium.

Figure 5

Multilineage differentiation of human UDSCs. (A) Chondrogenic differentiation. Image depicting glycosaminoglycan deposits which were formed after 28 days of induction in specific differentiation media. Results obtained from RT-PCR also confirmed high expression of collagen I and collagen II (* p < 0.05). (B) Alizarin Red was applied to stain calcium nodules and thereby determine the successful osteogenic induction. RT-PCR analysis revealed significantly higher expression levels of collagen I and osteopontin when compared to the control group (* p < 0.05). (C) Oil Red O staining together with RT-PCR were applied in order to assess successful adipogenic differentiation. However, isolated UDSCs did not differentiate into this cell lineage. (D) UDSCs induced into smooth cell lineage. Specific marker myosin (green) was confirmed by immunofluorescence. Nuclei were stained with DAPI. Moreover, significantly higher expression of alpha smooth actin and calponin I was detected as well (* p < 0.05).