**2. Reconstruction of functional urinary bladders**

Our final goal is to develop treatments for irreversibly damaged urinary bladders resulting from neurogenic bladder associated with brain and spinal cord disease, cystitis, peripheral neuropathy, or radiotherapy-induced injury. To determine the capacity of bone marrowderived cells to fulfill these goals, we have performed preliminary investigations in allogeneic transplantation by using a mouse freeze-injured urinary bladder model [1].

#### **2.1. Freeze-injured urinary bladder model**

Three days prior to implantation, we induce a highly reproducible freeze-injury to the urinary bladders of mice [1]. We apply an iron bar (25 x 3 x 2 mm) chilled by dry ice onto the posterior urinary bladder wall for 30 seconds. Placement of the chilled iron bar causes local freezing of the bladder wall tissues. Within 10 seconds after removal of the bar, the frozen spot thaws due to body and/or room heat and appears to the naked eye similar to the intact normal bladder wall. However, when we monitor blood flow within the capillaries of the frozen area with a charged-couple device (CCD) video microscopy, the local circulation pauses for approximately 20 min after the operation, and then it resumes. It is likely that the freeze-injured urinary bladders experience a period of ischemia followed by reperfusion as described by one of the microcirculation dysfunction models [22]. At 3 days after the freeze-injury operation, the wounded area, which occupies approximately one-third of each urinary bladder, is readily identified by the presence of a hematoma. The freeze-injured urinary bladders have both injured and uninjured regions that are easily observed by histology. The smooth muscle layers of the injured regions are disorganized and readily distinguished from the surrounding uninjured regions (Figure 1A).

#### **2.2. Implantation of cultured bone marrow–derived cells**

factors that accelerate healing in damaged tissues and inhibit apoptosis and the development of fibrosis [19,20]. However, the operation to harvest the bone marrow cells is generally considered to have higher patient risks compared to harvesting adipose cells. The increased risk for harvesting bone marrow cells for autologous transplantation is especially important for elderly patients with lower urinary tract symptoms (LUTS) and who may have other

Equally important as the sources of cells for regenerative medicine are the survival rates for implanted cells, the differentiation into target cell types, and the structural support that enables the reconstruction within the recipient tissues [21]. The survival, differentiation, and reorgan‐ ization of the implanted cells are affected by the microenvironment within the recipient tissues. However, our understanding of these microenvironments is currently insufficient to provide clinically effective and reliable resources for regenerative medicine. Thus, to obtain the optimum microenvironment, we need to investigate the utilization of scaffolds, growth factors,

This chapter has three major topics: (1) implantation of allogenic mouse bone marrow-derived cells into freeze-injured urinary bladders, (2) implantation of autologous bone marrowderived cells into freeze-injured urethral sphincters, and (3) importance of the microenviron‐ ment in reconstructing functional lower urinary tracts. We show that bone marrow-derived cells implanted into freeze-injured bladders or urethras differentiate into smooth muscle and striated muscle cells. These cells become organized into layered structures that are associated with the recovery of tissue function. In injured tissues, we have begun to uncover the important roles that may support the differentiation. Our information leading to future studies will

enable the development of regenerative medicine in urology and other clinical areas.

transplantation by using a mouse freeze-injured urinary bladder model [1].

Our final goal is to develop treatments for irreversibly damaged urinary bladders resulting from neurogenic bladder associated with brain and spinal cord disease, cystitis, peripheral neuropathy, or radiotherapy-induced injury. To determine the capacity of bone marrowderived cells to fulfill these goals, we have performed preliminary investigations in allogeneic

Three days prior to implantation, we induce a highly reproducible freeze-injury to the urinary bladders of mice [1]. We apply an iron bar (25 x 3 x 2 mm) chilled by dry ice onto the posterior urinary bladder wall for 30 seconds. Placement of the chilled iron bar causes local freezing of the bladder wall tissues. Within 10 seconds after removal of the bar, the frozen spot thaws due to body and/or room heat and appears to the naked eye similar to the intact normal bladder wall. However, when we monitor blood flow within the capillaries of the frozen area with a charged-couple device (CCD) video microscopy, the local circulation pauses for approximately 20 min after the operation, and then it resumes. It is likely that the freeze-injured urinary

**2. Reconstruction of functional urinary bladders**

**2.1. Freeze-injured urinary bladder model**

diseases as well.

412 Regenerative Medicine and Tissue Engineering

and combinations of these materials.

We harvest mouse bone marrow cells by flushing them out from both ends of femurs, and the recovered cells are cultured on collagen-coated dishes for 7 days. During the culture period, we completely replace the 15% fetal bovine serum-containing medium every day and remove non-attached cells. Immediately after plating in dishes, the bone marrow cells consist of heterogeneous, spindle-shaped, round, and polygonal cell types along with red blood cells. At 5 days after seeding, the cells achieve approximately 80% confluence, and at that time we transfect them with the green fluorescence protein (GFP) gene for identification in the recipient tissues. After 7 days of culture (i.e., 2 days after transfection), the adhered proliferating cells are relatively homogenous in spindle shaped appearance, and they expressed GFP. We confirm that the cultured cells do not differentiate into smooth muscle cells during the culture period.

The culture conditions readily promote attachment and proliferation of bone marrow cells. The cultured bone marrow-derived cells contain a variety of stem cell types, such as hemato‐ poietic, mesenchymal, and stromal stem cells [17,18]. Marker proteins on cells can be used to sort the ones that will differentiate into specific target cells [23-27]. However, which of the sorted cells are best for clinical use is unknown [28]. The simplicity of our selection procedure, based only on attachment and proliferation of bone marrow cells on collagen, would be a significant advantage for clinical applications.

On Day 7 of culture, we dissociate the cultured bone marrow-derived cells and allotransplant 2.0x106 cells with a 30-guage micro-syringe into the center of the injured region of the 3-dayold freeze-injured urinary bladders. As controls, we inject cell-free solution. The implantation cell number and volume are chosen to avoid further damaging the cells with shear stress or the recipient tissues by bursting the bladder wall. Each operation is performed under a stereomicroscope where we visually confirm the presence of a small swelling, indicating that the implanted cells remain at the site.

#### **2.3. Reconstructed smooth muscle layers**

At 14 days after implantation, we estimate the effect of the bone marrow-derived cells. The regions implanted with these cells have numerous alpha-smooth muscle actin (SMA)-positive smooth muscle cells compared to the control regions injected with the cell-free solution (Figure 1B). The cells are organized into distinct smooth muscle layers. In contrast, the few SMApositive cells present in the regions injected with the cell-free solution are not organized into layers.

At 14 days, SMA mRNA expression in the implanted regions is significantly higher than that in the cell-free injected regions, which supports the immunohistochemical observations [1]. In fact, the expression level of SMA mRNA in the implanted regions is not significantly different from that in the normal urinary bladder. Expression levels of other smooth muscle cell differentiation marker genes in the implanted region are also elevated. In the implanted regions, smooth muscle myosin heavy chain (MHC) and calponin I mRNA expression are significantly higher than in the cell-free injected control regions [1]. There are no significant differences in MHC and calponin I mRNA expression levels when the implanted regions are compared to the normal urinary bladders. Desmin mRNA expression is significantly higher than either control or normal regions. Thus the implanted regions have a larger number of mature smooth muscle cells and developing smooth muscle layers compared to the control regions [29-33]. Collectively, the immunohistochemical and gene expression results show that the implanted regions have formed smooth muscle layers composed of regenerated smooth muscle cells during the 14 days of the study period, while the control regions have only minimal recovery.

#### **2.4. Progress of implanted bone marrow–derived cells**

We detect the implanted bone marrow-derived cells in the recipient tissues by the presence of GFP labeling. Some of the GFP-labeled implanted cells are positive for proliferating cell nuclear antigen (PCNA), a marker of proliferating cells. In addition, within

the newly formed smooth muscle layers, some GFP-labeled cells that do not express smooth muscle cell differentiation markers are organized into cord-like structures. The cells may have differentiated into cell types that provide histoarchitectural elements for the blood vascular system [24] or the nervous system [25]. The implanted bone marrow-derived cells may participate in as yet other unknown changes in the various tissues of the urinary bladder.

We perform double staining with the smooth muscle cell differentiation markers and GFP antibody to identify the regenerated smooth muscle cells that are derived from the implanted cells. Both GFP- and SMA-positive cells show that the implanted bone marrow-derived cells differentiate into smooth muscle cells within the injured urinary bladders (Figure 1C). Other implanted GFP-labeled cells are also positive for MHC, desmin, and calponin I. Some of the GFP-labeled, differentiated smooth muscle cells contact each other, and they also contact non-GFP-labeled smooth muscle cells of the host that surround the implanted regions. Collectively, these cells form smooth muscle layers. The differentiation toward smooth muscle cells occurs after implantation because none of the cells expressed detectable levels of the marker proteins while in culture.

#### **2.5. Recovery of bladder contractions**

Cystometric investigations at 3 days after injury show that the mice do not have defined regular bladder contractions. The bladder contractions at 14 days after cell-free control injection also remain disrupted. However 14 days after cell implantation, there are distinct regular bladder contractions that are similar to those of normal mice without injury [1]. Thus, cystometric Bone Marrow–Derived Cells Regenerate Structural and Functional Lower Urinary Tracts http://dx.doi.org/10.5772/55558 415

At 14 days, SMA mRNA expression in the implanted regions is significantly higher than that in the cell-free injected regions, which supports the immunohistochemical observations [1]. In fact, the expression level of SMA mRNA in the implanted regions is not significantly different from that in the normal urinary bladder. Expression levels of other smooth muscle cell differentiation marker genes in the implanted region are also elevated. In the implanted regions, smooth muscle myosin heavy chain (MHC) and calponin I mRNA expression are significantly higher than in the cell-free injected control regions [1]. There are no significant differences in MHC and calponin I mRNA expression levels when the implanted regions are compared to the normal urinary bladders. Desmin mRNA expression is significantly higher than either control or normal regions. Thus the implanted regions have a larger number of mature smooth muscle cells and developing smooth muscle layers compared to the control regions [29-33]. Collectively, the immunohistochemical and gene expression results show that the implanted regions have formed smooth muscle layers composed of regenerated smooth muscle cells during the 14 days of the study period, while the control regions have only

We detect the implanted bone marrow-derived cells in the recipient tissues by the presence of GFP labeling. Some of the GFP-labeled implanted cells are positive for proliferating cell nuclear

the newly formed smooth muscle layers, some GFP-labeled cells that do not express smooth muscle cell differentiation markers are organized into cord-like structures. The cells may have differentiated into cell types that provide histoarchitectural elements for the blood vascular system [24] or the nervous system [25]. The implanted bone marrow-derived cells may participate in as yet other unknown changes in the various tissues of the urinary bladder.

We perform double staining with the smooth muscle cell differentiation markers and GFP antibody to identify the regenerated smooth muscle cells that are derived from the implanted cells. Both GFP- and SMA-positive cells show that the implanted bone marrow-derived cells differentiate into smooth muscle cells within the injured urinary bladders (Figure 1C). Other implanted GFP-labeled cells are also positive for MHC, desmin, and calponin I. Some of the GFP-labeled, differentiated smooth muscle cells contact each other, and they also contact non-GFP-labeled smooth muscle cells of the host that surround the implanted regions. Collectively, these cells form smooth muscle layers. The differentiation toward smooth muscle cells occurs after implantation because none of the cells expressed detectable levels of the marker proteins

Cystometric investigations at 3 days after injury show that the mice do not have defined regular bladder contractions. The bladder contractions at 14 days after cell-free control injection also remain disrupted. However 14 days after cell implantation, there are distinct regular bladder contractions that are similar to those of normal mice without injury [1]. Thus, cystometric

minimal recovery.

414 Regenerative Medicine and Tissue Engineering

while in culture.

**2.5. Recovery of bladder contractions**

**2.4. Progress of implanted bone marrow–derived cells**

antigen (PCNA), a marker of proliferating cells. In addition, within

**Figure 1.** Implantation of bone marrow-derived cells into freeze-injured urinary bladders. (A) At 3 days after opera‐ tion, the freeze-injured regions (arrowheads and asterisk in inset) lose typical smooth muscle layers. Black bar: 1 cm. (B) At 14 days, the bone marrow-derived cell-implanted regions have numerous SMA-positive smooth muscle cells (red, asterisks) that are organized into layers (green, urothelium; blue, nuclei). (C) The cells detected with GFP antibody (upper left inset, green cells) are simultaneously positive for the smooth muscle marker SMA (bottom left inset, red cells) within the newly formed smooth muscle layers. The merged images (right, yellow cells) show that the implanted GFP-labeled cells have differentiated into cells expressing smooth muscle markers. Blue, nuclei.

investigations indicate that implanted bone marrow-derived cells have the potential to restore some or all normal bladder functions. We believe that the smooth muscle layers reconstructed by the implantation of the cells contribute to the restoration of bladder contractions.
