**4.2.1 Bone marrow-derived stem cells (BMSCs)**

Bone marrow-derived stem cells (BMSCs) have been most widely studied of all mesenchymal stem cells. BMSCs have been used for the regeneration of cardiac muscle **(Sadek et al, 2009),** bladder detrusor muscle **(Kanematsu et al, 2005),** anal sphincter muscle **(Lorenzi et al, 2008)**, and many other structures **(El Backly and Cancedda, 2010)**. Drost et al. in a pilot study transplanted autologous BMSCs into injured rat urethral sphincter. The cultured BMSCs were injected periurethrally 1 week after the urethral injury. Both histological and immunohistochemistry evaluation showed that transplanted BMSCs survived and differentiated into peripheral nerve cells and striated muscle cells compared to the cell-free group **(Drost et al, 2009).** 

#### **4.2.2 Adipose-derived stem cells**

Adipose derived stem cells (ADSCs), is a pluripotent cells which have the ability to differentiate into cells of the same and of another germ layer, such as adipogenic, chondrogenic, neurogenic, myogenic and osteogenic cells **(Roche et al, 2010).** In the field of urinary incontinence ADSCs are of special interest for mesodermal and neuronal regeneration and to promote revascularization. Bacou et al, reported that transplantation of ADSCs increases mass and functional capacity of damaged skeletal muscle. They can express specific striated muscle markers (eg, desmin, myod1, myogenin, myosin heavy chain), form multinucleated cells characteristic of myotubules **(Bacou et al, 2004)**. Also, ADSCs can express nerve growth factor at the time of neural differentiation. Zhang et al, reported that neural-differentiated ADSCs present glial characteristics and promote nerve regeneration, after 7 days of transplantation in a rat model in vivo **(Zhang et al, 2010).**  Periurethral injection of ADSCs in an immune-competent, incontinent rat model with SUI, exhibited in vivo differentiation into smooth muscle cells and improved urethral resistance **(Lin et al, 2010).** Fu et al. injected predifferentiated ADSCs with 5-azacitidine periurethrally into incontinent rats. A significant difference in bladder capacity and leak point pressure was observed after 3 months of follow-up between the control group and the pretreated group. Also increased number of myoblasts under the mucosa and expression of α-smooth muscle actin was observed 3 months after implantation **(Fu et al, 2010).** 

#### **4.2.3 Muscle derived stem cells (MDCs)**

Muscle-derived stem cells (MDSCs) can naturally differentiate to multinucleated muscle fibers and display stem cell characteristics **(Seale and Rudnicki, 2000).** MDSCs have also the ability to undergo long-term proliferation, self-renewal, and multipotent differentiation, including differentiation toward endothelial and neuronal lineages **(Lee et al, 2000; Qu-Petersen et al, 2002).** Cannon et al, reported that MDSCs are capable of restoring muscular contraction of the urethral sphincter 2 weeks after injection **(Cannon et al, 2003)**. MDSCs

anesthesia, and low number of (MSCs) are obtained **(Pittenger et al. 1999**). Muscle derived stem cells (MDCs) and adipose derived stem cells (ADSCs) are advantageous because they can be easily obtained in large quantities under local anesthesia **(Rodríguez et al,. 2006;** 

Bone marrow-derived stem cells (BMSCs) have been most widely studied of all mesenchymal stem cells. BMSCs have been used for the regeneration of cardiac muscle **(Sadek et al, 2009),** bladder detrusor muscle **(Kanematsu et al, 2005),** anal sphincter muscle **(Lorenzi et al, 2008)**, and many other structures **(El Backly and Cancedda, 2010)**. Drost et al. in a pilot study transplanted autologous BMSCs into injured rat urethral sphincter. The cultured BMSCs were injected periurethrally 1 week after the urethral injury. Both histological and immunohistochemistry evaluation showed that transplanted BMSCs survived and differentiated into peripheral nerve cells and striated muscle cells compared to

Adipose derived stem cells (ADSCs), is a pluripotent cells which have the ability to differentiate into cells of the same and of another germ layer, such as adipogenic, chondrogenic, neurogenic, myogenic and osteogenic cells **(Roche et al, 2010).** In the field of urinary incontinence ADSCs are of special interest for mesodermal and neuronal regeneration and to promote revascularization. Bacou et al, reported that transplantation of ADSCs increases mass and functional capacity of damaged skeletal muscle. They can express specific striated muscle markers (eg, desmin, myod1, myogenin, myosin heavy chain), form multinucleated cells characteristic of myotubules **(Bacou et al, 2004)**. Also, ADSCs can express nerve growth factor at the time of neural differentiation. Zhang et al, reported that neural-differentiated ADSCs present glial characteristics and promote nerve regeneration, after 7 days of transplantation in a rat model in vivo **(Zhang et al, 2010).**  Periurethral injection of ADSCs in an immune-competent, incontinent rat model with SUI, exhibited in vivo differentiation into smooth muscle cells and improved urethral resistance **(Lin et al, 2010).** Fu et al. injected predifferentiated ADSCs with 5-azacitidine periurethrally into incontinent rats. A significant difference in bladder capacity and leak point pressure was observed after 3 months of follow-up between the control group and the pretreated group. Also increased number of myoblasts under the mucosa and expression of α-smooth

Muscle-derived stem cells (MDSCs) can naturally differentiate to multinucleated muscle fibers and display stem cell characteristics **(Seale and Rudnicki, 2000).** MDSCs have also the ability to undergo long-term proliferation, self-renewal, and multipotent differentiation, including differentiation toward endothelial and neuronal lineages **(Lee et al, 2000; Qu-Petersen et al, 2002).** Cannon et al, reported that MDSCs are capable of restoring muscular contraction of the urethral sphincter 2 weeks after injection **(Cannon et al, 2003)**. MDSCs

muscle actin was observed 3 months after implantation **(Fu et al, 2010).** 

**4.2.3 Muscle derived stem cells (MDCs)** 

**Strem et al, 2005).** 

**4.2.1 Bone marrow-derived stem cells (BMSCs)** 

the cell-free group **(Drost et al, 2009).** 

**4.2.2 Adipose-derived stem cells** 

also may improve neurogenic bladder dysfunction in animal models by reconstitution of damaged peripheral nerve cells (eg, Schwann cells, perineum) and vascular cells (eg, vascular smooth muscle cells, pericytes, endothelial cells) **(Nitta et al, 2010)**. For the treatment of SUI myoblasts have been injected to the striated urinary sphincter of a pig model. The animals have shown an increase in urethral pressure profile and muscular myofibrils **(Mitterberger et al, 2008).** 

Advantages of MDSCs injection therapy over conventional treatments for SUI:


#### **4.3 Isolation of muscle-derived stem cells in humans**

Mitterberger M et al. and Strasser H et al. reported biopsy in the right or left upper limb (biceps muscle) to obtain 0.32 to 2 cm3 of muscle tissue (figure 11). At the same time, 250 mL of blood were drawn for autologous serum **(Mitterberger et al, 2008; Strasser et al, 2007b).**

Fig. 11. Diagram showing autologous stem cell injection therapy for SUI. Autologous stem cells are obtained with a biopsy of tissue, the cells are dissociated and expanded in culture, and the expanded cells are implanted into the same host. MDSCs = muscle derived stem cells **(Jankowski et al, 2008)** 

Futuristic Concept in Management

of Female SUI: Permanent Repair Without Permanent Material 311

At the beginning of the cell injection, the TUUS probe (8 Ch, 15– 20 MHz) was carefully inserted into the urethra. The urethral wall and the rhabdosphincter were visualized. A specially designed patent-pending injection device was used for precisely adjusted injection of several small portions. First, 15–18 portions of the myoblast suspension were injected directly into the omega-shaped rhabdosphincter at two different levels. Then, 25–30 depots of the fibroblast/collagen suspension were injected into the submucosa circumferentially at three levels (Figure 13). After implantation of the cells the patients were instructed to perform PFT and FES for 4 wk postoperatively to support integration of the cells and to

Fig. 13. Cross-sectional ultrasonography images of the urethra and the rhabdosphincter (A) The tip of the needle (marked with an arrow) is positioned at the inner aspect of the rhabdosphincter (RS) for injection of myoblasts. (B) The tip of the needle is placed at the outer aspect of the submucosa (SM) for injection of fibroblasts **(Strasser et al, 2007b).**

Few human trials have been conducted using autologous derived stem cells in the treatment of female SUI, which mainly involved the use of MDSCs. Stem cell therapy, shows an early encouraging results and these results suggest the ability of pure cellular therapy to treat

Strasser et al. conducted the first clinical experiments in women with SUI. 42 women suffering from SUI were recruited and subsequently treated with transurethral ultrasonography- guided injections of autologous myoblasts and fibroblasts obtained from skeletal muscle biopsies. After a follow-up of 12 months incontinence was cured in 39 women **(Strasser et al, 2007a)**. In another trial 42 women were randomly assigned to receive transurethral ultrasonography guided injections of autologous myoblasts and fibroblasts, at 12-months' follow-up, 38 of the 42 women injected with autologous cells were completely continent **(Strasser et al, 2007b).** Mitterbarger et al. studied 20 female patients suffering from SUI after TUUS guidance injection of autologous myoblasts and fibroblasts. At 1 year follow-up 18 patients were cured and 2 patients improved. At 2 years after therapy 16 of the

**4.4 Transurethral ultrasound (TUUS) guided injection of stem cell** 

improve formation of new muscle tissue **(Strasser et al, 2007).** 

**4.5 Clinical results of stem cell therapy** 

female SUI (Table 8).

Myoblasts and fibroblasts are separated from connective tissue by centrifugation and enzymatic digestion with type I collagenase. Myoblasts are cultured in Ham's F10 medium supplemented with 20% autologous serum, and fibroblasts in DMEM (Dulbecco's modified Eagle medium) and Ham's F12 medium with 10% autologous serum. Cells are accepted when they reach 80% confluence. After 6-8 weeks in culture, fibroblasts and myoblasts are harvested separately by tripsinization and washing with centrifugation. Cell quality is assessed by immunohistochemistry, immunofluorescence, and fluorescence-activated cell sorting. Anti-desmin, vimentin, CD56, CD34 and ASO2 antibodies are used to differentiate myoblasts from fibroblasts. Fusion capacity of myoblasts is measured in differentiation medium without autologous serum to assess their viability, and cells are counted in each culture using the Neubauer chamber (figure 12). Once the cells are "harvested", they are transferred in adequate numbers to sterile syringes, separating myoblasts from fibroblasts. Myoblasts are suspended in 1.4 mL of DMEM/F12 with 20% autologous serum, and fibroblasts in 1 mL of DMEM/F12 with 20% autologous serum mixed with collagen as transport material to prevent cell migration from the injection site, because fibroblasts are mobile following application. Collagen has been shown to stabilize cells so that they remain in place and produce their own extracellular matrix **(Strasser et al, 2007b).** 

Fig. 12. Characterisation of cells: (A) Immunohistochemical image of human myoblasts stained with antidesmin antibodies. (B) Immunofl uorescence image of human fibroblasts stained with antivimetin antibodies. (C) Fluorescent antibody cell sorter (FACS) analysis of myoblast cell culture showing that 97% of the myoblasts are positive for CD56 antibodies. (D) Phase-contrast microscope image of multinucleated myotubes (marked with arrows) that have formed after fusion of mononucleated myoblasts in diff erentiation medium **(Strasser et al, 2007b).** 

Myoblasts and fibroblasts are separated from connective tissue by centrifugation and enzymatic digestion with type I collagenase. Myoblasts are cultured in Ham's F10 medium supplemented with 20% autologous serum, and fibroblasts in DMEM (Dulbecco's modified Eagle medium) and Ham's F12 medium with 10% autologous serum. Cells are accepted when they reach 80% confluence. After 6-8 weeks in culture, fibroblasts and myoblasts are harvested separately by tripsinization and washing with centrifugation. Cell quality is assessed by immunohistochemistry, immunofluorescence, and fluorescence-activated cell sorting. Anti-desmin, vimentin, CD56, CD34 and ASO2 antibodies are used to differentiate myoblasts from fibroblasts. Fusion capacity of myoblasts is measured in differentiation medium without autologous serum to assess their viability, and cells are counted in each culture using the Neubauer chamber (figure 12). Once the cells are "harvested", they are transferred in adequate numbers to sterile syringes, separating myoblasts from fibroblasts. Myoblasts are suspended in 1.4 mL of DMEM/F12 with 20% autologous serum, and fibroblasts in 1 mL of DMEM/F12 with 20% autologous serum mixed with collagen as transport material to prevent cell migration from the injection site, because fibroblasts are mobile following application. Collagen has been shown to stabilize cells so that they remain

in place and produce their own extracellular matrix **(Strasser et al, 2007b).** 

Fig. 12. Characterisation of cells: (A) Immunohistochemical image of human myoblasts stained with antidesmin antibodies. (B) Immunofl uorescence image of human fibroblasts stained with antivimetin antibodies. (C) Fluorescent antibody cell sorter (FACS) analysis of myoblast cell culture showing that 97% of the myoblasts are positive for CD56 antibodies. (D) Phase-contrast microscope image of multinucleated myotubes (marked with arrows) that have formed after fusion of mononucleated myoblasts in diff erentiation medium

**(Strasser et al, 2007b).** 
