Urine-derived stem cells serve as a robust platform for generating native or engineered extracellular vesicles

Abstract

Background Mesenchymal stromal cell (MSC) therapy holds great potential yet efficacy and safety concerns with cell therapy persist. The beneficial effects of MSCs are often attributed to their secretome that includes extracellular vesicles (EVs). EVs carry biologically active molecules, protected by a lipid bilayer. However, several barriers hinder large-scale MSC EV production. A serum-free culturing approach is preferred for producing clinical-grade MSC-derived EVs but this can affect both yield and purity. Consequently, new strategies have been explored, including genetically engineering MSCs to alter EV compositions to enhance potency, increase circulation time or mediate targeting. However, efficient transfection of MSCs is challenging. Typical sources of MSC include adipose tissue and bone marrow, which both require invasive extraction procedures. Here, we investigate the use of urine-derived stem cells (USCs) as a non-invasive and inexhaustible source of MSCs for EV production. Methods We isolated, expanded, and characterized urine-derived stem cells (USCs) harvested from eight healthy donors at three different time points during the day. We evaluated the number of clones per urination, proliferation capacity and conducted flow cytometry to establish expression of surface markers. EVs were produced in chemically defined media and characterized. PEI/DNA transfection was used to genetically engineer USCs using transposon technology. Results There were no differences between time points for clone number, doubling time or viability. USCs showed immunophenotypic characteristics of MSCs, such as expression of CD73, CD90 and CD105, with no difference at the assessed time points, however, male donors had reduced CD73+ cells. Expanded USCs were incubated without growth factors or serum for 72 h without a loss in viability and EVs were isolated. USCs were transfected with high efficiency and after 10 days of selection, pure engineered cell cultures were established. Conclusions Isolation and expansion of MSCs from urine is non-invasive, robust, and without apparent sex-related differences. The sampling time point did not affect any measured markers or USC isolation potential. USCs offer an attractive production platform for EVs, both native and engineered.

1. Introduction

Mesenchymal stromal cells (MSCs) are an attractive cell-based treatment, based on their regenerative and immunomodulatory properties [1–3]. Autologous and allogeneic MSC therapies have been used and are accepted as immunologically privileged or immune evasive respectively [4, 5]. However, the production of donor-specific antibodies has been observed for allogeneic therapies, which could cause MSC clearance by host recipient immune cells and long-term effects remain to be investigated [6]. Other risks, such as uncontrollable growth of transplanted cells and chromosomal instability, have also been reported [7, 8].

The therapeutic effects of MSCs, when treating immune-mediated diseases, are often attributed to the secretome [9]. MSCs secrete molecules such as prostaglandin E2, various cytokines, and extracellular vesicles (EVs), which collectively contribute to the immunomodulatory effects [9, 10]. As such, a secretome-based and therefore cell-free therapy could circumvent some of the risks associated with MSC therapy. EVs have gained extensive attention in this context, as they are complex entities interacting with multiple modulatory pathways. EVs serve as a physiological drug delivery system that can alter the immune system more subtly and sustainably than individual molecules as they contain several bioactive molecules such as lipids, regulatory RNAs and proteins [11, 12].

MSCs can be isolated from various tissues and fluids of either neonatal-associated or adult origin [13]. EVs derived from the most investigated MSCs, bone marrow-, neonatal-associated, and adipose-derived MSCs (AD-MSCs), have been assessed for their modulatory effects in both pre-clinical and clinical settings [14, 15]. EVs from MSCs derived from less investigated sources such as dental pulp, central nervous system, menstrual blood and urine-derived stem cells (USCs) have mostly been investigated in preclinical trials.