Human Umbilical Cord Blood Mononuclear Cell Transplantation in Rats with Intrinsic Sphincter Deficiency

Animals

All experiments were performed on normal female, 5-6 week-old Sprague-Dawley rats (Orient Bio Inc. Seongnam, Korea), weighing 200 to 250 g. ISD was induced by electrocauterization in all animals. One week after electro-cauterization, the animals were injected with either HUCB mononuclear cells (experimental group, n=28) or normal saline (control group, n=22). The rats were kept in individual cages and had free access to water and food. Eight and 14 rats from the control group and 9 and 19 rats from the experimental group were studied at 2 and 4 weeks after injection, respectively.

Induction of ISD by electro-cauterization

ISD was induced by electro-cauterization as previously described (8, 11). Briefly, rats were anesthetized by intraperitoneal injection of ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (15 mg/kg). Under anesthesia, they were placed in the supine position with the lower legs abducted. The bladder and urethra were exposed through a lower midline abdominal incision. Tissues, 1 cm lateral to the mid-urethra were cauterized on both sides to produce sphincter injury. A fine tip, high-temperature cautery (Aaron Medical, St. Petersburg, FL, USA) was used. Each side was cauterized for 30 sec.

Human umbilical cord blood mononuclear cell separation

After thawing at 37℃, cord blood cells were separated into a low-density mononuclear fraction (<1,077 g/mL) by Ficoll-Paque Plus (GE Healthcare AB, Uppsala, Sweden, http://www.amersham.com). Briefly, after adding and mixing the same amount of normal saline into the cord blood, poured the diluted solution on Ficoll in 15 mL tubes (Ficoll: diluted blood=3:4), and centrifuged at 1,800 rpm for 30 min. After carefully extracting the mononuclear cells from the mononuclear cell layer on the Ficoll, gathered the cells in 50 mL tubes and added a 3×volume of normal saline and collected the target cells by centrifugation at 1,800 rpm for 10 min. After removing the upper layer solution, 50 mL of normal saline was added and mixed into the remaining cell pellet and two more centrifugation processes at 1,000 rpm for 10 min were performed. Finally, the cell pellet with 50 mL of normal saline was elutriated and cell counting in 10 µL of the suspension was performed.

Labeling of human umbilical cord blood mononuclear cells

To track the migration and engraftment of HUCB mononuclear cells at the site of injection, cells were double labeled with iron oxides and the fluorescent cell tracker 1,10-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) dye according to the method developed by Arbab et al. (12). Briefly, HUCB mononuclear cells were grown in 75 cm² culture flasks to 80-90% confluence before labeling with iron oxides (Feridex®, Berlex Laboratories Inc., Wayne, NJ, USA) and fluorescent dye DiI (Molecular Probes, Eugene, OR, USA). On the day of labeling, the old media was discarded, replaced by 50% of fresh complete BMSC media and 50% serum free media containing iron oxides-protamine sulfate complexes (50 and 2 µg/mL, respectively), and incubated overnight. Cells were harvested and resuspended in serum free media at a concentration of 1-2 million/mL. 1×10⁶ iron-labeled cells were further labeled with fluorescent tracker DiI (Cell tracker CM-DiI, C-7000; Molecular Probes) dye for 30 min at 37℃ in 1 mL of serum-free medium containing 10 mL of DiI stock solution. After labeling with DiI, cells were washed twice with PBS before injection. Cell viability was established by the Trypan blue exclusion method before and after fluorescent labeling.

Human umbilical cord blood mononuclear cell injection

One week after electro-cauterization, the experimental rats underwent ketamine/xylazine anesthesia and a low midline incision was made to expose the bladder and urethra. A 30 µL Hamilton syringe was used to inject a total of 20 µL of HUCB mononuclear cell suspension in saline solution (2×10⁶ cells per 20 µL). Two injections per rat (10 µL each) were performed on each lateral wall of the mid-urethra and then the abdomen was sutured. Control rats were injected with normal saline only.

Leak point pressure measurement

At 2 and 4weeks after HUCB mononuclear cells injection the leak point pressure (LPP) were measured using the vertical tilt/intravesical pressure clamp model of stress urinary incontinence (11). The animals were anesthetized with urethane (1.2 g/kg) injected subcutaneously. And all rats underwent spinal cord transection at the T9-T10 level following laminectomy in order to eliminate spontaneous bladder activity in response to increasing intravesical pressures. This manipulation does not interfere with the spinal continence reflexes of the bladder neck and urethra. The overlying skin was then closed with a suture. Next, through a midline abdominal incision the distal colon was cleared of feces by gentle massage, and a loose suture was secured around the proximal end of the distal colon to prevent further fecal migration to the pelvic floor during the course of the experiment. A transvesical catheter with a fire-flared tip (PE-90, Clay Adams, Parsippany, NJ, USA) was inserted into the dome of the bladder and secured with a ligature for bladder filling and pressure recording. The abdominal wall and overlying skin were closed with a suture. The rat was then mounted on a tilt table with the axis of rotation positioned for constant bladder height in relation to the pressure transducer, either in the supine or vertical position. To measure the intravesical pressure, normal saline was infused through the polyethylene catheter that was inserted into the animal's bladder. The catheter is connected to the microsyringe pump (KD Scientific, Holliston, MA, USA) and the pressure measurement system via a 3-way stopcocks. Data generated by the pressure sensor (ADInstruments, Colorado-springs, CO, USA) and bridge amplifier (ADInstruments, Powerlab® 2/25, Coloradosprings, CO, USA) were analyzed.

All bladder pressure was referenced to air pressure at the level of bladder. The rats were placed at the level of zero pressure while bladders were filled with room temperature saline at 5 mL/hr through the bladder catheter. The rats were then mounted on a tilt table and placed in the vertical position. The pressure at leak point was taken as the LLP. The average of three consecutive LPP was taken as a data point for each animal.

Tissue preparation and histology

Immediately after the LPP measurement, the rats were euthanized, and the proximal urethra was removed. The tissue samples for fluorescent microscopy were prepared by snap freezing of the urethra, embedding in OCT compound (Tissue Tek; Sakura Finekek USA, Torrance, CA, USA). Sections were cut (5 µm) and used for fluorescent dye staining. Specimens for light microscopy were fixed in 10% formaldehyde, embedded in paraffin, and cut to obtain 5 µm thick sections. These sections were deparaffinated, hydrated, and stained with hematoxylin-eosin and Masson's trichrome. All staining were performed using standard methods, and all reagents were from Sigma-Aldrich (St.Louis, MO, USA).

Fluorescent microscopy and prussian blue staining

Tissues were cut into 10 mm pieces (axial) and snap frozen for sectioning at 5 µm thickness. Tissue sections were cover slipped using aqueous mounting media containing 40, 6-diamidino-2-phenylindole (DAPI; Vector Shield, Vector Laboratories Inc., Burlingame, CA, USA) to outline the nucleus. Fluorescent-labeled cells were detected and photographed using a fluorescent microscope. Following fluorescent photomicrography, slides were dismounted and tissue sections were stained for iron using Prussian blue staining technique. Bright field photomicrographs were obtained from the same sites with same magnification to determine whether fluorescent positive cells were also iron positive.

All immunofluorescent labeling and dyes were examined using a Carl Zeiss LSM 510 Meta confocal laser scanning microscope equipped with 488 nm argon ion, 543 nm green helium-neon, and 633 nm red helium-neon laser lines. Three-dimensional images were reconstructed by several consecutive optical sections of various thicknesses (0.25-0.38 µm).

Immunohistochemistry for detecting desmin

Formalin-fixed, paraffin-embedded tissues were sectioned at 5 µm thickness. These sections were deparaffinated, rehydrated, and pretreated with 3% H₂O₂ for 10 min at room temperature. Then antigen retrieval was performed in boiling 10mM citrate acid buffer (pH6.0) for 10 min followed by cooling at room temperature for 20 min. Then the sections were incubated in 10% normal goat serum in PBS for 1 hr at room temperature followed by incubation with primary antibody (mouse anti-desmin monoclonal antibody, abcam ab17156, Abcam Inc, Cambridge, MA) at 4℃ overnight. Sections were incubated with horseradish peroxidase-conjugated goat-anti mouse IgG (Vector Laboratory Inc. Burlingame, CA, USA) for 15 min at room temperature. DAB chromogen was used for color development under the light microscope and the sections were counterstained with hematoxylin.

Statistical analysis

LPP data are presented as means±SD. Overall comparisons between groups were performed using a one-way analysis of variance for within time-point analyses and two-way analysis of variance for across time-point analyses. Individual comparisons were made using Student's t-test. A P value of less than 0.05 was accepted as significant.

The LPP in HUCB mononuclear cell-injected rats

Fig. 1 shows the LPP values measured using vertical tilt/intravesical pressure clamp technique at 2 and 4 weeks after the injection. The LPP values were slightly higher in the experimental group, but not statistically different at 2 weeks. The mean value for the control group (n=8) was 46.13±12.14 mmHg, and the value for the experimental group (n=9) was 55.72±9.99 mmHg at 2 weeks. But, the LPP values were significantly increased in the experimental group at 4 weeks while the control group did not show significant change in LPP value (mean LPP for control group (n=14): 65.02±22.09 mmHg, for the experimental group (n=19): 91.75±18.99 mmHg, P=0.001, t=-3.728, within 95% validity interval).

Fig. 1
Comparative effect of HUCB mononuclear cell injection on leak point pressure between groups at 2 and 4 weeks. ^*is significantly different from each other (P<0.05) at each time. Compared with control group, the leak point pressure in the experimental group at 4 week after HUCB mononuclear cell injection was significantly increased.

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HUCB mononuclear cells in the urethral wall in the experimental group at 2 weeks and 4 weeks

Iron-stained, fluorescent-labeled HUCB mononuclear cells were found in the lamina propria and in the muscular urethral sphincter in the experimental group at 2 weeks (Figs. 2, 3). The human cells were mainly found in the lamina propria just beneath the urethral mucosa. A few cells were also noted in the sphincter skeletal muscle layer. Some of the cells were small, round cells with scant cytoplasm and some were bigger with abundant cytoplasm. A few of the cells just beneath the mucosa were spindle shaped. We could not find any iron-stained or fluorescent-labeled cells in the rat urethral tissue at 4 weeks. The control group rats did not show any iron-stained or fluorescent-labeled human cells at 2 weeks and 4 weeks.

Fig. 2
Photographs show Feridex®-labeled HUCB mononuclear cells in urethral tissue at 2 weeks post-injection. A number of cells considered human cells within the tissue sections were detected by blue-colored signals. (A, C) Control group; (B,D) Experimental group (Magnification ×200, 400).

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Fig. 3
Photographs show Di-I labeled HUCB mononuclear cells in urethral tissue at 2 weeks post-injection. A number of cells considered human cells within the tissue sections were detected by red-colored Di-I signals (A,B, Di-I stained cells; C, Control group; D-F, Experimental group. A,D are DIC images. B and E are fluorescence images).

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The morphological integrity of the internal urethral sphincter

No significant morphological difference in the urethral sphincter muscle was observed at 2 weeks between the experimental group and control group. At 4 weeks after the electro-cauterization, however, obvious disruption of the urethral sphincter muscle was apparent in the control group, while the sphincter muscle in the experimental group injected with HUCB mononuclear cells was largely restored to the morphological integrity of the intact urethral sphincter (Figs. 2-4). The immunohistochemical staining to desmin showed the disrupted arrangement of skeletal muscle layer of the urethral sphincter in the control group while in the experimental group, the sphincter remained well-organized (Fig. 5). In the control group, collagen was deposited at the disrupted area, which was confirmed by the blue color on trichrome staining. On the other hand, collagen deposit was not observed in the experimental group injected with HUCB mononuclear cells (Fig. 5).

Fig. 4
Photographs show sphincter muscle disruption and collagen deposit in the control group (A) at 4 weeks after injection. Sphincter muscles in the experimental group (B) were well preserved and intact, and with no collagen deposit (H&E, ×400).

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Fig. 5
The immunohistochemical stainings for desmin and trichrome stain show apparent sphincter muscle disruption and collagen deposit in the control group (A&C, control group, B&D, experimental group, ×400).

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