To our knowledge, the robustness of USCs as an EV production platform and their ability to maintain high viability over 72 hours at low nutrient conditions has not been previously investigated. Whilst xeno-free EV-production alternatives have been made (24), the absence of serum or growth factors significantly reduces barriers to up and downstream EV production and presents USCs as an ideal low-cost MSC platform easily adaptable to large-scale GMP production (25). We find the particle size and concentrations comparable to MSC EVs reported by others(26). Dot blot showed enrichment of EV marker CD63 in the EV-enriched SEC fractions, validating EVs' presence.
The therapeutic potential of stem cells has been subject to immense attention, yet the development of cell-free advanced therapies, such as EVs, has arisen as an attractive next-generation alternative. The use of EVs as a therapeutic entity offers possibilities of producing engineered drug vehicles with enhanced capabilities and reducing health risks as they are non-replicative when injected. We therefore investigated the feasibility of genetically engineering them. A truncated PTGFRN scaffold has previously been reported to be efficient in either displaying or internalizing recombinant protein in HEK293T cells (27), we, therefore, utilized this scaffold in an attempt to display mCherry on the secreted EVs from engineered USCs. Whilst we saw a high amount of mCherry secreted by HEK293T cells, fluorescence on the secreted EVs from the engineered USCs was limited (Table S1). While the USCs were readily engineerable, and the recombinant protein was abundant in the regions where PTGFRN is most enriched (Fig. S5), such as the Golgi apparatus, ER membrane and endosomes, it was not secreted at high levels which have been previously reported for stem cells (28). We did not validate the expression of PTGFRN in USCs, and this pathway might not be predominant during the biogenesis of EVs in USCs as compared to HEK293 cells; other scaffolds may therefore be better suited to MSCs. Genetic engineering of stem cells is not without challenges as the required culturing time and passages are increased compared to native MSCs, which could lead to chromosomal alterations, phenotypic changes and senescence (29). However, we demonstrated a robust and efficient transfection method and observed a rapid expansion under selection conditions without comparison to other stem cells we have engineered in the lab (personal observation). The engineered USCs expanded to quantities required for therapeutic EV production at a similar rate as non-engineered USCs. As such, USCs could serve as a unique platform for engineered MSCs for both cell-based and secretome-based therapy.
As USC are relevant to several potential clinical applications, we aimed to evaluate some of the factors critical for the suitability and translational aspects of USC in this context. This includes sex-specific differences and an optimal collection time, which could affect parameters such as morphology, proliferation rate, and immunophenotype of the resulting colonies. Although our sample size is not large enough to determine this, we find indications that the aseptic sampling stringency between sexes seems to have an impact on potential contamination. Sex differences in urinary tracts are well described with females have a more complex microbiota, and cellular differences are also apparent (30). As such, it is presumed that the risk of contamination is higher when sampling women. The increased microbiological complexity and the shorter urethra could explain the increased turnover of squamous cells (Fig. S1) released into the urine of women. If autologous cells are a necessity, optimization could therefore be needed to ensure consistency of the quality between donors of different sexes.
Single clones have often been selected in prior works with USCs (31,32). While this strategy could potentially yield very homogeneous phenotypes and secretomes, it could lead to reduced genetic stability as more doublings are needed before reaching EV production cell counts (33). Nonetheless, it has been reported that there were no chromosomal variations at passage 7 for USCs (34). We pooled all clones to keep the potential positive effects of heterogeneity and reduce the number of cell doublings before EV production. Our observations of three distinct phenotypes (Fig. 1) merit further investigation and whether potential modulatory effects on recipient cells differ, but that is beyond the scope of this study. The success rate for urination to large expansions and clones per ml of urine was in line with what has been described, albeit our rates and incidents were somewhat higher than previously reported (35,36). Our data does not suggest that there would be any time during the day that is better to deliver a urine sample when harvesting USCs, nor do they show that there could be an increased risk of contamination in morning urine.
Within the investigated cohort, a relatively homogenous immunophenotype of USCs was demonstrated. Expression of CD73, CD90, and CD105 constitutes a common and shared phenotypic trait of MSCs from different tissue sources and is recommended for minimal characterization of MSC from adipose tissue (37). In this study, CD73 was found to be slightly but significantly upregulated on USC from female donors, compared to the male donors (Fig S2). Donor age and sex have been proposed to affect the therapeutic potential of MSC-based therapies, however, this subject has not been systematically addressed yet(38,39). Whether these features impart a heterogeneity in USC remains to be established in a larger cohort, to allow for adjustment for both sex and age. Nevertheless, the investigated phenotypic attributes are interesting within the scope of clinical translation, as the expression profile of CD73, CD90, and CD105 is accepted by regulatory authorities as a quality parameter for AD-MSCs used in clinical application when combined with the absence of several negative markers (CD14, CD19, CD31, CD45, HLA-DR) (40). In particular, the absence of HLA-DR on the USCs in this study may point to an expected low immunogenicity of these cells, which could support usage in an allogeneic therapeutic strategy. The presence of CD146 on USCs suggests even more shared characteristics with other MSC types, as this marker is also expressed by other types of MSC. CD146-positive MSCs have been shown to have high proliferating capabilities and maintain multipotency regarding differentiation potential into the three classical lineages (41) and even into neurological cells (42). CD146-positive MSCs have also been linked to having higher modulatory and migratory capabilities and the secreted EVs are more potent regarding immunomodulation and regeneration (43,44).
Autologous MSCs have been reported to be a superior therapeutic option for certain clinical indications compared to allogeneic cells. Cell transplants with autologous MSCs have shown higher success rates for engraftment and are not expected lead to any alloimmunity (45,46). MSCs derived from diseased and older individuals could potentially be less potent, but Zhang et al. showed that USCs derived from various end-stage liver disease patients had the same potency in reducing acute and chronic liver injury in a murine model (47). On the contrary to cell transplantation, treatment with non-self EVs does not seem to be as immunogenic (48), yet prolonged treatment with allogenic EVs could lead to immune clearance, reduced circulation time, and lowered therapeutic effects, as previously reported (49). As such, the ease of isolating, growing autologous and the engineering of USCs as we have shown could potentially outweigh the burden and lead to a potent and indefinite treatment form with minimal discomfort for the donors.
In summary, the presented culture strategy is independent of sampling time or sex and yields USCs with a stable proliferation capacity and viability, whilst also producing cells that display a shared stem cell immunophenotype with MSCs derived from other tissue types. These results warrant further investigation to discover if these traits extrapolate into comparable regenerative and immunomodulatory potential. As USC isolation is non-invasive, we see USCs as a unique source of primary MSCs, readily available for all researchers. Lastly, we believe that USCs could serve as a bridge from research to clinical application as they are easily manipulated and are even more robust than cell lines during EV production.