Medical Hypotheses xxx (2015) xxx–xxx
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Urine – A waste or the future of regenerative medicine? T. Kloskowski a,⇑, M. Nowacki a, M. Pokrywczyn´ska a, T. Drewa a,b a b
Chair of Regenerative Medicine, Department of Tissue Engineering, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland Department of General and Oncological Urology, Nicolaus Copernicus Hospital, Torun, Poland
a r t i c l e
i n f o
Article history: Received 5 February 2014 Accepted 15 January 2015 Available online xxxx
a b s t r a c t In recent years, urine has emerged as a source of urine cells. Two different types of cells can be isolated from urine: urine derived stem cells (USCs) and renal tubular cells called urine cells (UCs). USCs have great differentiation properties and can be potentially used in genitourinary tract regeneration. Within this paper, we attempt to demonstrate that such as easily accessible source of cells, collected during completely non-invasive procedures, can be better utilized. Cells derived from urine can be isolated, stored, and used for the creation of urine stem cell banks. In the future, urine holds great potential to become a main source of cells for tissue engineering and regenerative medicine. Ó 2015 Elsevier Ltd. All rights reserved.
Introduction A great number of recent papers indicate that stem cells are important factors in regenerative medicine development. This new and intriguing medical discipline provides the opportunity to resolve many clinical problems in current surgery [1–3]. Frequently, stem cell-based therapies are perceived as more effective and immunologically better tolerated than standard treatment procedures that are based on full synthetic implantable solutions. Some authors suggest that stem cell-based therapies may contribute to a faster healing process and promote a better long-term outcome [4–7]. An increasing interest in stem cells correlates with a large number of promising in vitro and in vivo studies and, most importantly, with increasing numbers of clinical trials [8–12]. A large number of stem cells derived from different sources have been described up to now. Mesenchymal stem cells derived from bone marrow (BM-MSC) or fat tissue (ADSC – adipose derived stem cells) are the most studied and utilized. Since they can be obtained during minimally invasive procedures (bone marrow aspiration or liposuction), these cells have become a topic of great interest [13–15]. Other stem cells, such as amniotic-fluid derived stem cells (AFS), hair follicle stem cells (HFSC), and cord blood stem cells (CBSCs), are less accessible and their isolation methods are still low-effective [16–19]. An interesting source of stem cells with highly proliferative potential is urine which can be collected during a completely non-invasive procedure, like micturition [20]. Urine is ⇑ Corresponding author at: Chair of Regenerative Medicine, Department of Tissue Engineering, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun´, Karlowicza Str. 24, 85-092 Bydgoszcz, Poland. Tel.: +48 052 585 38 23; fax: +48 052 585 39 56. E-mail address: [email protected] (T. Kloskowski).
also a cell source of adult renal cells that can be transformed into induced pluripotent stem cells (iPSCs) [21]. In the current era of regenerative medicine based on stem cell therapy, there is still a lack of publications fully related to the effective collection and delivery of allogenic or autologous stem cells used in fast-track application procedures. More problematic, however, is the clinical use of allogenic stem cells versus autologous cell implantation [22,23]. Hypothesis Cells present in urine may be isolated, cultured, stored, and used for the creation of urine cell bank. These collected cells may be used in experimental or clinical applications. Evaluation of hypothesis Cell types isolated from urine The standard human urine represents an interesting mix of biological and chemical constituents with varying physical properties [24]. For this reason, during the historical development of medicine, urine has served as an important element in the comprehensive diagnosis of many diseases due to its known presence of proteins, glucose, high level of creatinine, and specific types of markers [25,26]. Besides water and many metabolic waste byproducts, urine contains cells with various potential and physiological meaning. These cells are mainly found in the urine’s sediment. The clinical and scientific viewpoints mostly describe two types of cells: cells of hematological origin and cells of epithelial origin. Erythrocytes (RBCs) are cells of hematological origin that
http://dx.doi.org/10.1016/j.mehy.2015.01.019 0306-9877/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kloskowski T et al. Urine – A waste or the future of regenerative medicine?. Med Hypotheses (2015), http://dx.doi.org/ 10.1016/j.mehy.2015.01.019
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can be present in non-pathological situations ranging from none to fiver per high-power field (hpf). Frequently, leukocytes (WBCs), other cells of hematological origin, may be found in amounts not exceeding five per hpf. Common cells of epithelial origin in nonpathological situations include: renal tubular cells – RTE (zero to one per hpf), transitional epithelial cells (zero to one per hpf) and squamous epithelial cells (fewer than one per hpf) [27–29]. Two cell types derived from urine have been implemented in tissue engineering and regenerative medicine. In 2008, a team from Wake Forest University discovered a population of progenitor cells derived from spontaneous voided urine. These cells can be collected by non-invasive procedures, and according to the authors, cell culture is low-cost and can be easily performed [20]. USCs are stable in cell culture and can be differentiated into cells expressing urothelial, smooth muscle, endothelial, and interstitial markers [20]. The multipotent character of these cells was confirmed by its differentiation into osteocytes, chondrocytes, adipocytes, neurons, and myocytes. USCs have mesenchymal characteristics (positive for CD73, CD90, CD105 and CD133, negative for CD45, CD31 and CD CD34), for this reason, no teratoma formation was observed after implantation on an animal model [30,31]. After 4 passages of one sample of voiding urine (about 200 ml), 3.2 108 viable cells can be obtained [32]. These cells can be stored in 4 °C for up to 24 h by addition of medium containing serum to the urine. Doing so can increase the number of USCs from one donor and ensure that there are enough for clinical use [33]. A single USC has the ability to expand to a large population with 60–70 population doublings [31]. The origin of these cells (probably basal layer of urothelium) makes them a perfect cell source for urinary tract regeneration [34]. Another team isolated a different group of cells from urine, which was derived from the renal tubular system. These are adult cells without stem cell activity. The authors used this easily accessible cell source to create induced pluripotent stem cells (iPSCs) which could be next used in clinical practice [21,35]. In a recent study, this team got one step further; they created iPSCs from urine cells using non-viral vectors without oncogene c-MYC and the feeder-free method. Urine samples were obtained from healthy donors as well as donors with different genetic background (e.g. hemophilia A, B-thalassemia, Parkinson disease). The authors suggested that urine cells are an excellent source for the creation of iPSCs that can then be utilized in disease modeling and regenerative medicine [36]. The next step was to produce footprint- and xeno-free iPSCs using an in vitro culture under special conditions. Such obtained cells have necessary properties for application in clinical practice [37]. A disadvantage of iPSCs includes low-effective cell transformation and its implantation correlates with the possibility of a teratoma formation [37]. Another approach includes directly reprogramming the epithelial cells derived from urine into neural progenitor cells (NPCs) and consequently omitting iPSCs generation. Such cells were successfully obtained using integration-free and feeder-free methods by episomal transfection, which is still low effective ranging 0.2% [38]. As a stem cell source, urine is interesting; however, it is controversial when taking into account its toxic influence on bone marrow mesenchymal stem cells and urothelial cell in in vitro studies [39,40].
detailed autologous stem cell donor criteria does not exist. This situation correlates with the newly discovered urine-derived cells to which we refer to in this publication. Most of the authors describing urine cells refers in their publications mainly to the aspects of their biology and character or its usefulness in cell-based therapies and tissue engineering applications. The relevant data regarding to important aspects from whom, how and when the urine should be collect to get the cells for its future clinical application should be discussed [30,43].
General criteria In our opinion, when urine is collected to obtain urine cells, the procedure should be a combination of standard urine specimen collection procedures and general donor criteria used in transplantology. Also, it is important to determine from whom urine will be collected: from healthy volunteers to obtain allogenic stem cells or directly from patients for a future autologous therapeutic use. In a second situation, the possible negative effects of primary diseases and different medications taken by the patient should be considered [37,44]. Some studies suggest that not only healthy people, but people with different genetic backgrounds can be donors of urine cells; this is especially important for self-individualized stem cell procurement for further regenerative therapies. Those previously diagnosed with urinary tract disorders or suffering from severe bacterial/viral infection, such as HIV or HBV/HCV, should be excluded from potential donor lists [45]. For this reason, during such procedures, the standard tests aimed at avoiding the transmission of infectious and neoplastic diseases. Urine culture and bronchial secretion culture, HIV markers, hepatitis B and C markers, EBV markers, CMV markers, toxoplasmosis, syphilis markers and cancer markers: CEA, AFP, b HCG, CA 19-9, CA 125, should always be performed. There is no clinical evidence that shows the potential gold standards of histocompatibility evaluation when different types of stem cells are derived for future regeneration based on tissue engineering procedures. In our opinion, the standard HLA test should be always performed [46,47]. The best way to organize a complete controlled urine donation for future regenerative medicine is to collect specific cell source during standard blood/bone-marrow donation procedures. However, for this procurement process, a detailed urine donation criteria should be created. The creation process should not be complicated if the standard transplant and blood/bone-marrow donor requirements are extended and the diagnostic procedure are enriched. The potential criteria and excluding factors are present in Table 1. What is interesting is the potential exclusion criteria
Table 1 Hypothetical urine derived stem cells (USCs) donor exclusion criteria created with self-modification based on detailed analysis, selection, and adjustment of current published exclusion criteria used in clinical transplantology. Main potential USCs allogeneic donor exclusion criteria No.
Potential exclusion factor
References
1.
Human immunodeficiency virus infection (HIV) Recent or active malignancy Individuals (carriers) chronically infected with the hepatitis C virus (HCV), hepatitis B virus (HBV), or persons positively diagnosed with other viruses, such as EBV and CMV Serious and complicated urinary tract disorders (with the exception of previously uncomplicated traumas which can regenerate) Severe bacterial infections
Gutiérrez et al. [58]
2. 3.
Potential donors, selection criteria, and urine collection methods Current solid organ transplantation procedures are based on precise guidelines and recommendations regarding donor criteria. The same situation applies to hemato-oncology. The significance of such solutions is inestimable due to their clinical relevance [41,42]. As mentioned before, there is a lack of data referring to donor criteria of potential non-hematopoietic stem cell donor selection, and
4.
5.
Kher et al. [59] Maccarini Jde et al. [60]
Romagnoli et al. [61]
Eastlund [62]
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concerning self-donated urine for future USCs procurement. Perhaps, in the future, such criteria omitting severe bacterial infection, severe intoxication, or active malignancy of the urinary tract will exist. It is possible that a large number of patients, who are unable to currently undergo a solid organ or cell transplantation due to a human immunodeficiency virus, will in fact, be treated with their own cells without any barriers. Standard collection should be always based on hygienic and sterile procedures because it is important for future unproblematic cell cultivation processes and can reduce contamination. Also, the urethral meatus should be previously disinfected and the urine should be collected into a sterile container suitable for subsequent storage. In the case of catheterized patients, a new set should always be used. When dealing with trauma patients, one should use the intraoperatively Cystofix system to obtain stem cells for future potential regeneration procedures. There are publications in which authors inform that USCs can be safely persevered latterly in urine for 24 h without losing their original stem cell properties [33,48,49]. In our work, we can suggest only several practical solutions that will hopefully open new doors for a larger discussion. Future of urine cell donation The average adult generates between 1.5 and 2.0 liters of urine per day. During a single lifetime, this amount of urine can accumulate to fill a small swimming pool (about 60 m3) [50,51]. From a
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single sample of 200 ml of urine, enough stem cells or adult renal cells can be collected and used for SUI treatment, urethra regeneration, or for iPSCs creation. Since each year many of these cells are wasted, we believe that this cell source can be better utilized. To accomplish this, the first step is to create places where volunteers can donate urine following standard physical examinations. In places like blood donor centers or research facilities, urine cells will then be isolated and used for research applications or stored for future clinical purpose (Fig. 1). Similar to blood donation, donation of spontaneous voiding urine is a completely non-invasive procedure which should encourage and increase volunteer numbers. This is of great importance to current transplantology developments because the number of donors is extremely lower than the number of recipients [52,53]. As the simplest and most painless stem cell procurement method, urine donation may potentially eliminate a still larger group of ‘‘marginal donors’’ because there will be no need to use an expanded donor criteria [54,55]. In the future, if in vivo animal model study procedures are proven to correlate with the regeneration of solid organs and exceed all pre-clinical phases to be included in the standard treatment schemes, the regenerative potential of this cheap and easily acquired source of stem cells may revolutionize modern medicine [56,57]. Additionally, this increasing interest in urine-derived stem cells will hopefully expand our knowledge about their biological properties, isolation, expansion and different forms of application. The next step will be to construct devices that will automatically
Fig. 1. Potential applications urine-derived cells. SUI – stress urinary incontinence; VUR - vesicoureteral reflux; ARF – acute renal failure; CRF – chronic renal failure.
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Fig. 2. Isolation and utilization of urine-derived stem cells.
collect and isolate cells form urine. Such devices can be placed in donor centers, hospitals, and even public restrooms. Such devices should be constructed because urine cells are easy to isolate. Urine samples have to be centrifuged, and following suspension in culture medium cells can be seeded in culture bottles [20]. By adding an appropriate amount of DMSO (dimethyl sulfoxide), this sample can be stored in 80 °C until needed (Fig. 1). Potential use of urine derived cells Due to their multi-differentiation properties, urine derived stem cells (USCs) may potentially be used in regenerative medicine applications (Fig. 2). These cells may be used in stress urinary incontinence (SUI) therapy and vesicoureteral reflux (VUR) treatment. In contrast to the BM-MSC, ADSC, muscle-derived progenitor cells or ear chondrocytes, that are used in SUI therapy, USCs do not induce donor side morbidity and anesthesia is not necessary during tissue collection. VEGF expressing USCs, created using viral transformation, were able to differentiate into muscle cells after subcutaneous implantation in collagen type I hydrogel or alginate microspheres. This significantly strengthened neovascularisation and innervations which increased cell survival rate in animal models [43,63,64]. Whether used alone or transfected with FGF2, USCs were used for improving erectile dysfunction in type 2 diabetic rat models. Cells, injected into the cavernosum tissue, enhanced endothelial cell markers, smooth muscle content, and improved erectile dysfunction [65]. These cells were able to differentiate into smooth muscle and urothelial cells in vitro, and after seeding on bacterial cellulose scaffold, were used to create model of urinary conduit [34]. One year later, this same method, using 3-D porous SIS scaffold, was used for experimental urethra construction [32]. Due to its ability to differentiate into urothelial lineages, USCs are an ideal cell source for the regeneration of other urinary tract components like the bladder, or the ureters [20,66]. Recently, the use of USCs in neurology was reported on animal models. These cells were transplanted onto a hydrogel scaffold and into a rat’s brain which resulted in survival and differentiation into neuron-like cells [67]. An in vitro study showed that stem cells from urine can differentiate into cells expressing osteogenic markers. Authors concluded that, together with silver nanoparticles (which additionally promote osteogenic differentiation of USCs), these cells have great potential for bone defect repairs [68]. Stem cells from urine were
also able to differentiate into skeletal muscle lineage cells in vitro and retained this phenotype after implantation making them a promising stem cell source for skeletal muscle regeneration [69]. In theory, iPSCs, derived from urine, have greater application potential when compared to USCs, because they can differentiate into every cell type in the body. In a study conducted by Huang et al., iPSCs successfully differentiated into neural progenitor cells (NPCs) in culture. These cells should not induce immunological response and have the potential to be used in neurogenic disorders treatment. However, additional experiments on animal models are needed to introduce them into clinical practice [70]. Generation of iPSCs can be omitted by the direct generation of neural progenitor cells (NPCs) from urine epithelial cells. Such cells were able to differentiate into neural lineages during in vitro culture, and no teratoma formation was witnessed following implantation into a rats’ brains [38]. The protocol created for the generation of these cells can be used in clinical practice for treatment of neural disorders. iPSCs derived from other cells, such as skin fibroblasts, differentiated into other cell types and were successfully used on animal models with Parkinson’s disease and sickle-cell anemia [71,72]. A second application of epithelial cells, derived from urine, may be for disease modeling. Urine cells, collected from patients suffering from severe hemophilia A, were successfully transformed into iPSCs, and next differentiated into hepatocyte-like cells displaying Factor VIII deficiency. These cells can be used for drug testing, and in the future, may contribute to personalized treatment development [73]. As evidenced by the first registered clinical trial for age-related macular degeneration (AMD) performed by the Institute for Biomedical Research and Innovation in conjunction with the RIKEN Center for Developmental Biology, in Kobe, Japan, iPSCs have great potential in regenerative medicine. Conclusions In this review, we demonstrated the promising capabilities of urine to one day become a main cell source for regenerative medicine. Urine-derived stem cells possess astounding potential in clinical application. Following expansion, these stem cells can be directly used for treatment, as confirmed in experimental studies. Urine cells derived from the renal tubular system have to be transformed into iPSCs or directly into the progenitor lineage. Although this transformation is still low and there is a risk of teratoma
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development, the first registered clinical trial using iPSCs offers hopes to the development and expansion of this technique. Urine is an easily accessible cell source and can be obtained during completely non-invasive procedures. For this reason, urine is a perfect cell source for tissue engineering and regenerative medicine. Construction of devices used to automatically isolate urine cells will significantly increase our knowledge about this cell type and may contribute to the faster development of stem-cell based therapies. It should be noted that all experiments mentioned in the paper were performed in vitro or on animal models by subcutaneous implantation. Additional experiments are needed to develop further clinical applications.
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Urine – A waste or the future of regenerative medicine? T. Kloskowski a,⇑, M. Nowacki a, M. Pokrywczyn´ska a, T. Drewa a,b a b
Chair of Regenerative Medicine, Department of Tissue Engineering, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland Department of General and Oncological Urology, Nicolaus Copernicus Hospital, Torun, Poland
a r t i c l e
i n f o
Article history: Received 5 February 2014 Accepted 15 January 2015 Available online xxxx
a b s t r a c t In recent years, urine has emerged as a source of urine cells. Two different types of cells can be isolated from urine: urine derived stem cells (USCs) and renal tubular cells called urine cells (UCs). USCs have great differentiation properties and can be potentially used in genitourinary tract regeneration. Within this paper, we attempt to demonstrate that such as easily accessible source of cells, collected during completely non-invasive procedures, can be better utilized. Cells derived from urine can be isolated, stored, and used for the creation of urine stem cell banks. In the future, urine holds great potential to become a main source of cells for tissue engineering and regenerative medicine. Ó 2015 Elsevier Ltd. All rights reserved.
Introduction A great number of recent papers indicate that stem cells are important factors in regenerative medicine development. This new and intriguing medical discipline provides the opportunity to resolve many clinical problems in current surgery [1–3]. Frequently, stem cell-based therapies are perceived as more effective and immunologically better tolerated than standard treatment procedures that are based on full synthetic implantable solutions. Some authors suggest that stem cell-based therapies may contribute to a faster healing process and promote a better long-term outcome [4–7]. An increasing interest in stem cells correlates with a large number of promising in vitro and in vivo studies and, most importantly, with increasing numbers of clinical trials [8–12]. A large number of stem cells derived from different sources have been described up to now. Mesenchymal stem cells derived from bone marrow (BM-MSC) or fat tissue (ADSC – adipose derived stem cells) are the most studied and utilized. Since they can be obtained during minimally invasive procedures (bone marrow aspiration or liposuction), these cells have become a topic of great interest [13–15]. Other stem cells, such as amniotic-fluid derived stem cells (AFS), hair follicle stem cells (HFSC), and cord blood stem cells (CBSCs), are less accessible and their isolation methods are still low-effective [16–19]. An interesting source of stem cells with highly proliferative potential is urine which can be collected during a completely non-invasive procedure, like micturition [20]. Urine is ⇑ Corresponding author at: Chair of Regenerative Medicine, Department of Tissue Engineering, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun´, Karlowicza Str. 24, 85-092 Bydgoszcz, Poland. Tel.: +48 052 585 38 23; fax: +48 052 585 39 56. E-mail address: [email protected] (T. Kloskowski).
also a cell source of adult renal cells that can be transformed into induced pluripotent stem cells (iPSCs) [21]. In the current era of regenerative medicine based on stem cell therapy, there is still a lack of publications fully related to the effective collection and delivery of allogenic or autologous stem cells used in fast-track application procedures. More problematic, however, is the clinical use of allogenic stem cells versus autologous cell implantation [22,23]. Hypothesis Cells present in urine may be isolated, cultured, stored, and used for the creation of urine cell bank. These collected cells may be used in experimental or clinical applications. Evaluation of hypothesis Cell types isolated from urine The standard human urine represents an interesting mix of biological and chemical constituents with varying physical properties [24]. For this reason, during the historical development of medicine, urine has served as an important element in the comprehensive diagnosis of many diseases due to its known presence of proteins, glucose, high level of creatinine, and specific types of markers [25,26]. Besides water and many metabolic waste byproducts, urine contains cells with various potential and physiological meaning. These cells are mainly found in the urine’s sediment. The clinical and scientific viewpoints mostly describe two types of cells: cells of hematological origin and cells of epithelial origin. Erythrocytes (RBCs) are cells of hematological origin that
http://dx.doi.org/10.1016/j.mehy.2015.01.019 0306-9877/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kloskowski T et al. Urine – A waste or the future of regenerative medicine?. Med Hypotheses (2015), http://dx.doi.org/ 10.1016/j.mehy.2015.01.019
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can be present in non-pathological situations ranging from none to fiver per high-power field (hpf). Frequently, leukocytes (WBCs), other cells of hematological origin, may be found in amounts not exceeding five per hpf. Common cells of epithelial origin in nonpathological situations include: renal tubular cells – RTE (zero to one per hpf), transitional epithelial cells (zero to one per hpf) and squamous epithelial cells (fewer than one per hpf) [27–29]. Two cell types derived from urine have been implemented in tissue engineering and regenerative medicine. In 2008, a team from Wake Forest University discovered a population of progenitor cells derived from spontaneous voided urine. These cells can be collected by non-invasive procedures, and according to the authors, cell culture is low-cost and can be easily performed [20]. USCs are stable in cell culture and can be differentiated into cells expressing urothelial, smooth muscle, endothelial, and interstitial markers [20]. The multipotent character of these cells was confirmed by its differentiation into osteocytes, chondrocytes, adipocytes, neurons, and myocytes. USCs have mesenchymal characteristics (positive for CD73, CD90, CD105 and CD133, negative for CD45, CD31 and CD CD34), for this reason, no teratoma formation was observed after implantation on an animal model [30,31]. After 4 passages of one sample of voiding urine (about 200 ml), 3.2 108 viable cells can be obtained [32]. These cells can be stored in 4 °C for up to 24 h by addition of medium containing serum to the urine. Doing so can increase the number of USCs from one donor and ensure that there are enough for clinical use [33]. A single USC has the ability to expand to a large population with 60–70 population doublings [31]. The origin of these cells (probably basal layer of urothelium) makes them a perfect cell source for urinary tract regeneration [34]. Another team isolated a different group of cells from urine, which was derived from the renal tubular system. These are adult cells without stem cell activity. The authors used this easily accessible cell source to create induced pluripotent stem cells (iPSCs) which could be next used in clinical practice [21,35]. In a recent study, this team got one step further; they created iPSCs from urine cells using non-viral vectors without oncogene c-MYC and the feeder-free method. Urine samples were obtained from healthy donors as well as donors with different genetic background (e.g. hemophilia A, B-thalassemia, Parkinson disease). The authors suggested that urine cells are an excellent source for the creation of iPSCs that can then be utilized in disease modeling and regenerative medicine [36]. The next step was to produce footprint- and xeno-free iPSCs using an in vitro culture under special conditions. Such obtained cells have necessary properties for application in clinical practice [37]. A disadvantage of iPSCs includes low-effective cell transformation and its implantation correlates with the possibility of a teratoma formation [37]. Another approach includes directly reprogramming the epithelial cells derived from urine into neural progenitor cells (NPCs) and consequently omitting iPSCs generation. Such cells were successfully obtained using integration-free and feeder-free methods by episomal transfection, which is still low effective ranging 0.2% [38]. As a stem cell source, urine is interesting; however, it is controversial when taking into account its toxic influence on bone marrow mesenchymal stem cells and urothelial cell in in vitro studies [39,40].
detailed autologous stem cell donor criteria does not exist. This situation correlates with the newly discovered urine-derived cells to which we refer to in this publication. Most of the authors describing urine cells refers in their publications mainly to the aspects of their biology and character or its usefulness in cell-based therapies and tissue engineering applications. The relevant data regarding to important aspects from whom, how and when the urine should be collect to get the cells for its future clinical application should be discussed [30,43].
General criteria In our opinion, when urine is collected to obtain urine cells, the procedure should be a combination of standard urine specimen collection procedures and general donor criteria used in transplantology. Also, it is important to determine from whom urine will be collected: from healthy volunteers to obtain allogenic stem cells or directly from patients for a future autologous therapeutic use. In a second situation, the possible negative effects of primary diseases and different medications taken by the patient should be considered [37,44]. Some studies suggest that not only healthy people, but people with different genetic backgrounds can be donors of urine cells; this is especially important for self-individualized stem cell procurement for further regenerative therapies. Those previously diagnosed with urinary tract disorders or suffering from severe bacterial/viral infection, such as HIV or HBV/HCV, should be excluded from potential donor lists [45]. For this reason, during such procedures, the standard tests aimed at avoiding the transmission of infectious and neoplastic diseases. Urine culture and bronchial secretion culture, HIV markers, hepatitis B and C markers, EBV markers, CMV markers, toxoplasmosis, syphilis markers and cancer markers: CEA, AFP, b HCG, CA 19-9, CA 125, should always be performed. There is no clinical evidence that shows the potential gold standards of histocompatibility evaluation when different types of stem cells are derived for future regeneration based on tissue engineering procedures. In our opinion, the standard HLA test should be always performed [46,47]. The best way to organize a complete controlled urine donation for future regenerative medicine is to collect specific cell source during standard blood/bone-marrow donation procedures. However, for this procurement process, a detailed urine donation criteria should be created. The creation process should not be complicated if the standard transplant and blood/bone-marrow donor requirements are extended and the diagnostic procedure are enriched. The potential criteria and excluding factors are present in Table 1. What is interesting is the potential exclusion criteria
Table 1 Hypothetical urine derived stem cells (USCs) donor exclusion criteria created with self-modification based on detailed analysis, selection, and adjustment of current published exclusion criteria used in clinical transplantology. Main potential USCs allogeneic donor exclusion criteria No.
Potential exclusion factor
References
1.
Human immunodeficiency virus infection (HIV) Recent or active malignancy Individuals (carriers) chronically infected with the hepatitis C virus (HCV), hepatitis B virus (HBV), or persons positively diagnosed with other viruses, such as EBV and CMV Serious and complicated urinary tract disorders (with the exception of previously uncomplicated traumas which can regenerate) Severe bacterial infections
Gutiérrez et al. [58]
2. 3.
Potential donors, selection criteria, and urine collection methods Current solid organ transplantation procedures are based on precise guidelines and recommendations regarding donor criteria. The same situation applies to hemato-oncology. The significance of such solutions is inestimable due to their clinical relevance [41,42]. As mentioned before, there is a lack of data referring to donor criteria of potential non-hematopoietic stem cell donor selection, and
4.
5.
Kher et al. [59] Maccarini Jde et al. [60]
Romagnoli et al. [61]
Eastlund [62]
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concerning self-donated urine for future USCs procurement. Perhaps, in the future, such criteria omitting severe bacterial infection, severe intoxication, or active malignancy of the urinary tract will exist. It is possible that a large number of patients, who are unable to currently undergo a solid organ or cell transplantation due to a human immunodeficiency virus, will in fact, be treated with their own cells without any barriers. Standard collection should be always based on hygienic and sterile procedures because it is important for future unproblematic cell cultivation processes and can reduce contamination. Also, the urethral meatus should be previously disinfected and the urine should be collected into a sterile container suitable for subsequent storage. In the case of catheterized patients, a new set should always be used. When dealing with trauma patients, one should use the intraoperatively Cystofix system to obtain stem cells for future potential regeneration procedures. There are publications in which authors inform that USCs can be safely persevered latterly in urine for 24 h without losing their original stem cell properties [33,48,49]. In our work, we can suggest only several practical solutions that will hopefully open new doors for a larger discussion. Future of urine cell donation The average adult generates between 1.5 and 2.0 liters of urine per day. During a single lifetime, this amount of urine can accumulate to fill a small swimming pool (about 60 m3) [50,51]. From a
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single sample of 200 ml of urine, enough stem cells or adult renal cells can be collected and used for SUI treatment, urethra regeneration, or for iPSCs creation. Since each year many of these cells are wasted, we believe that this cell source can be better utilized. To accomplish this, the first step is to create places where volunteers can donate urine following standard physical examinations. In places like blood donor centers or research facilities, urine cells will then be isolated and used for research applications or stored for future clinical purpose (Fig. 1). Similar to blood donation, donation of spontaneous voiding urine is a completely non-invasive procedure which should encourage and increase volunteer numbers. This is of great importance to current transplantology developments because the number of donors is extremely lower than the number of recipients [52,53]. As the simplest and most painless stem cell procurement method, urine donation may potentially eliminate a still larger group of ‘‘marginal donors’’ because there will be no need to use an expanded donor criteria [54,55]. In the future, if in vivo animal model study procedures are proven to correlate with the regeneration of solid organs and exceed all pre-clinical phases to be included in the standard treatment schemes, the regenerative potential of this cheap and easily acquired source of stem cells may revolutionize modern medicine [56,57]. Additionally, this increasing interest in urine-derived stem cells will hopefully expand our knowledge about their biological properties, isolation, expansion and different forms of application. The next step will be to construct devices that will automatically
Fig. 1. Potential applications urine-derived cells. SUI – stress urinary incontinence; VUR - vesicoureteral reflux; ARF – acute renal failure; CRF – chronic renal failure.
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Fig. 2. Isolation and utilization of urine-derived stem cells.
collect and isolate cells form urine. Such devices can be placed in donor centers, hospitals, and even public restrooms. Such devices should be constructed because urine cells are easy to isolate. Urine samples have to be centrifuged, and following suspension in culture medium cells can be seeded in culture bottles [20]. By adding an appropriate amount of DMSO (dimethyl sulfoxide), this sample can be stored in 80 °C until needed (Fig. 1). Potential use of urine derived cells Due to their multi-differentiation properties, urine derived stem cells (USCs) may potentially be used in regenerative medicine applications (Fig. 2). These cells may be used in stress urinary incontinence (SUI) therapy and vesicoureteral reflux (VUR) treatment. In contrast to the BM-MSC, ADSC, muscle-derived progenitor cells or ear chondrocytes, that are used in SUI therapy, USCs do not induce donor side morbidity and anesthesia is not necessary during tissue collection. VEGF expressing USCs, created using viral transformation, were able to differentiate into muscle cells after subcutaneous implantation in collagen type I hydrogel or alginate microspheres. This significantly strengthened neovascularisation and innervations which increased cell survival rate in animal models [43,63,64]. Whether used alone or transfected with FGF2, USCs were used for improving erectile dysfunction in type 2 diabetic rat models. Cells, injected into the cavernosum tissue, enhanced endothelial cell markers, smooth muscle content, and improved erectile dysfunction [65]. These cells were able to differentiate into smooth muscle and urothelial cells in vitro, and after seeding on bacterial cellulose scaffold, were used to create model of urinary conduit [34]. One year later, this same method, using 3-D porous SIS scaffold, was used for experimental urethra construction [32]. Due to its ability to differentiate into urothelial lineages, USCs are an ideal cell source for the regeneration of other urinary tract components like the bladder, or the ureters [20,66]. Recently, the use of USCs in neurology was reported on animal models. These cells were transplanted onto a hydrogel scaffold and into a rat’s brain which resulted in survival and differentiation into neuron-like cells [67]. An in vitro study showed that stem cells from urine can differentiate into cells expressing osteogenic markers. Authors concluded that, together with silver nanoparticles (which additionally promote osteogenic differentiation of USCs), these cells have great potential for bone defect repairs [68]. Stem cells from urine were
also able to differentiate into skeletal muscle lineage cells in vitro and retained this phenotype after implantation making them a promising stem cell source for skeletal muscle regeneration [69]. In theory, iPSCs, derived from urine, have greater application potential when compared to USCs, because they can differentiate into every cell type in the body. In a study conducted by Huang et al., iPSCs successfully differentiated into neural progenitor cells (NPCs) in culture. These cells should not induce immunological response and have the potential to be used in neurogenic disorders treatment. However, additional experiments on animal models are needed to introduce them into clinical practice [70]. Generation of iPSCs can be omitted by the direct generation of neural progenitor cells (NPCs) from urine epithelial cells. Such cells were able to differentiate into neural lineages during in vitro culture, and no teratoma formation was witnessed following implantation into a rats’ brains [38]. The protocol created for the generation of these cells can be used in clinical practice for treatment of neural disorders. iPSCs derived from other cells, such as skin fibroblasts, differentiated into other cell types and were successfully used on animal models with Parkinson’s disease and sickle-cell anemia [71,72]. A second application of epithelial cells, derived from urine, may be for disease modeling. Urine cells, collected from patients suffering from severe hemophilia A, were successfully transformed into iPSCs, and next differentiated into hepatocyte-like cells displaying Factor VIII deficiency. These cells can be used for drug testing, and in the future, may contribute to personalized treatment development [73]. As evidenced by the first registered clinical trial for age-related macular degeneration (AMD) performed by the Institute for Biomedical Research and Innovation in conjunction with the RIKEN Center for Developmental Biology, in Kobe, Japan, iPSCs have great potential in regenerative medicine. Conclusions In this review, we demonstrated the promising capabilities of urine to one day become a main cell source for regenerative medicine. Urine-derived stem cells possess astounding potential in clinical application. Following expansion, these stem cells can be directly used for treatment, as confirmed in experimental studies. Urine cells derived from the renal tubular system have to be transformed into iPSCs or directly into the progenitor lineage. Although this transformation is still low and there is a risk of teratoma
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development, the first registered clinical trial using iPSCs offers hopes to the development and expansion of this technique. Urine is an easily accessible cell source and can be obtained during completely non-invasive procedures. For this reason, urine is a perfect cell source for tissue engineering and regenerative medicine. Construction of devices used to automatically isolate urine cells will significantly increase our knowledge about this cell type and may contribute to the faster development of stem-cell based therapies. It should be noted that all experiments mentioned in the paper were performed in vitro or on animal models by subcutaneous implantation. Additional experiments are needed to develop further clinical applications.
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