**3. Stem cell properties of urine-derived cells**

Tissue-specific stem cells are rare in each tissue or organ. These cells play an important role in replacing aged, injured, and diseased cells and promoting tissue regeneration. However, it is a challenge to isolate these cells for therapeutic purposes. Recently, we successfully established a primary culture system to isolate and repopulate stem cells from regular voided human urine [21]. These cells possess stem cell properties, such as robust proliferation potential and multipotential differentiation capabilities, and are termed urine-derived stem cells (USCs) [22] (Fig.1). USCs expressed a whole set of MSC/pericyte markers and some key cell surface markers, such as CD 105, CD 90, CD 73, and CD 146, but not hematopoietic stem cell makers such as CD 31 and CD34 [21, 23](Fig.2). These cells maintain high telomerase activity and a normal karyotype in culture medium, even after several passages. The cells can differentiate into mesodermal cell lineages, such as osteocytes, chondrocytes, adipocytes, and myocytes, including smooth muscle cell differentiation and endodermal lineages (e.g. endothelial and urothelial cells) [23,24]. Additionally, USCs can be isolated and cultured from almost every urine sample. Our recent data demonstrate that 100 ml of urine contains about 3-10 USC clones [23]. On average, 12 USC clones can be isolated from one urine sample (about 200 ml). To induce stem cells to differentiate into skeletal muscle cells more efficiently, cells at the early passage are recommended. Usually, it takes 4 weeks for one cell clone to generate 3.2X107 cells at an early passage (p4) [24]. Therefore, at least 4x108 USCs (12x3.2 x 107 cells) can be generated from one urine sample, which are enough cells for use in the treatment of any grade of SUI.

**Figure 1.** Morphology of USC: Phase contrast microscopic appearances of urine derived stem cell clones showing "rice-grain" like morphology.

**Figure 2.** Stem cell surface marker of USC cell clones.

reinforce weak muscle tissue at the pelvic floor or around the urethra, deficient urethral sphincter function remains. Furthermore, surgery for SUI sometimes causes complications such as infection and postoperative voiding difficulty [11]. Autologous adult stem cell injection therapy for SUI has recently provided a promising alternative for sphincter tissue regeneration for repair of SUI. Stem cells obtained from skeletal muscle [12], bone marrow [13-15], umbilical cord [16], adipose tissue [17], and more recently, induced pluripotent cells [18] are regarded as possible candidates for use in this therapy. However, harvesting these types of cells is invasive and may cause complications. Furthermore, the amount of tissue that can be safely harvested in some patients, such as skeletal muscle-derived progenitor cells or bone marrow stem cells (BMSCs) in older individuals, limits clinical applications [19]. Thus, an autologous stem cell source that can be obtained using non invasive techniques would be desirable.

Mesechymal stem cells (MSCs) are often used as a cell source for cell therapy in two ways. First, stem cells are implanted directly into the tissues where repair is needed. By secreting paracrine factors, MSCs promote angiogenesis, decrease fibrosis, and recruit stem cells from native tissues to complete the repair, replacement, and regeneration processes at the injured sites. In addition, the surrounding normal cell- and tissue-based signals from the host envi‐ ronment guide the undifferentiated stem cells to give rise to the specific target cells required for tissue regeneration [20]. Second, stem cells are induced to differentiate into the target cells or tissue–like cells *in vitro*. The induced cells are then implanted into defective sites where

In clinical settings, most patients with SUI acquire chronic injuries related to urethral dys‐ function spanning years or even decades. In cases with extensive injuries and fibroblast formation, it would be better to induce the stem cells to differentiate into a myogenic lineage before injection, since the unhealthy or diseased environment (e.g. muscular dystrophy) may not be able to provide efficient differentiation cues required for efficient stem cells differen‐ tiation. Therefore, our strategy is to guide USCs to give rise to myogenic differentiation and then lead to repairing the deficiency, and also determine whether USCs can secrete paracrine

Tissue-specific stem cells are rare in each tissue or organ. These cells play an important role in replacing aged, injured, and diseased cells and promoting tissue regeneration. However, it is a challenge to isolate these cells for therapeutic purposes. Recently, we successfully established a primary culture system to isolate and repopulate stem cells from regular voided human urine [21]. These cells possess stem cell properties, such as robust proliferation potential and multipotential differentiation capabilities, and are termed urine-derived stem cells (USCs) [22] (Fig.1). USCs expressed a whole set of MSC/pericyte markers and some key cell surface markers, such as CD 105, CD 90, CD 73, and CD 146, but not hematopoietic stem cell makers such as CD 31 and CD34 [21, 23](Fig.2). These cells maintain high telomerase activity and a normal karyotype in culture medium, even after several passages. The cells can differentiate

factors to recruit the resident cells from the host to participate in tissue repair.

**3. Stem cell properties of urine-derived cells**

normal cells and tissues are not available.

788 Regenerative Medicine and Tissue Engineering
