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150 MULTIPOTENT STEM CELLS FROM URINE FOR TISSUE ENGINEERED BLADDER
Vol. 183, No. 4, Supplement, Sunday, May 30, 2010
THE JOURNAL OF UROLOGY姞
7-fold more cells than adherent cultures over 5 days. The rapid expansion of cell numbers makes this method attractive for tissue engineering and other biologic studies. Cell type Supernatant cells
CD44v6 67 bright
Parental adherent
10 dim
Mean % positive by flow cytometry ␣6 CD71 4 50 24 16 95
56
92
W6/32 6 96
Source of Funding: K18-DK075843 and P50-DK065313 from NIDDK and R01-DE13417 from NIDCR, NIH
149 TRACKING OF INJECTED ADIPOSE TISSUE-DERIVED STEM CELLS AFTER CAVERNOUS NERVE INJURY IN RATS: INJURYINDUCED HOMING TO THE MAJOR PELVIC GANGLION Thomas M Fandel*, Palo Alto, CA; Maarten Albersen, Guiting Lin, Hongxiu Ning, Ching-Shwun Lin, Tom F Lue, San Francisco, CA INTRODUCTION AND OBJECTIVES: Adipose tissue-derived stem cells (ADSCs) have been utilized in the preservation of erectile function in rats after cavernous nerve injury. We studied the mechanism with which ADSCs facilitate nerve regeneration after cavernous nerve injury in rats. METHODS: ADSCs were procured from 56 male rats for autologous injection and labeled with 5-ethynyl-2-deoxyuridine (EdU). Animals were randomly divided into an injury group undergoing cavernous nerve crush injury (n⫽28) and a sham group (n⫽28). In all rats, ADSCs were injected into both corpora cavernosa of the sham group of rats or immediately after injury in the nerve injury group. Seven animals from each group were euthanized at 1, 2, 3, and 7 days post-injection, and penile tissue and both major pelvic ganglia (MPG) were harvested for histology. The number of EdU-positive cells in the penis and the MPG was quantified by image analysis. Staining for stromal-derivedfactor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in the MPG was performed. Two way analysis of variance with post-testing was used to assess statistical significance. Data are given as mean ⫾ SEM. RESULTS: In penile tissue, there was a significant decrease in EdU-positive cells in the penis of both groups over time (p⬍0.05), however there was no difference between the two groups (p⫽0.4). In the MPG of the sham group, the number of EdU-positive cells remained low at 4.0 ⫾ 0.3, 4.0 ⫾ 0.2, 3.2 ⫾ 0.3, and 4.6 ⫾ 0.2 at 1, 2, 3, and 7 days, respectively, while in the injured group it increased to 25.0 ⫾ 1.3, 42.9 ⫾ 1.5, 32.8 ⫾ 1.3, and 44.3 ⫾ 4.2. ADSC levels in the MPG were significantly higher in the injury group at all time points as compared to sham animals (p⬍0.0001), however these levels did not change significantly over time (p⫽0.2). Both SDF-1 and CXCR4 were upregulated in the MPGs after crush injury as compared to sham controls (p⬍0.01 for both) but this expression did not change significantly over time (p⬎0.05). CONCLUSIONS: ADSCs migrate out of the injection site in a time-dependent manner, which is similar in both groups. Sham animals show low levels of ADSCs at the MPG, while there is significant homing of ADSCs to the MPG in crush injured animals at all time-points tested. Elevated SDF-1 and CXCR4 levels at the MPG after crush injury appear to be responsible for this observation. Increased numbers of ADSCs at the MPG after crush injury may support nerve regeneration by releasing trophic factors to the neurons. Source of Funding: NIDDK/NIH R37 DK045370
150 MULTIPOTENT STEM CELLS FROM URINE FOR TISSUE ENGINEERED BLADDER Shantaram Bharadwaj, Shaofeng Wu, Jan Rohozinski, Mark Furth, Anthony Atala, Winston-Salem, NC; Yuanyuan Zhang*, Winston-Samlem, NC INTRODUCTION AND OBJECTIVES: Urine derived cells can be obtained non-invasively and may represent a potentially significant
e61
source of autologous cells for tissue engineering. One goal of this study was to test the hypothesis that the cell population obtained from urine contains cells that meet the defining criteria of stem cells (self-renewal and multipotency). In particular, the ability of these cells to give rise to smooth muscle cells (SMC) and urothelial cells (UC) for potential use in urinary bladder tissue engineering was tested. METHODS: Human urine derived cells from 9 individual donors (ages 5 to 40 years) were plated on multi-well plates. Single cell growth was monitored using time-lapse microcinematography. The cells were analyzed for expression of pericyte and mesenchymal stem cell (MSC) markers. Expression of telomerase in the isolated cells was assessed by ELISA. Next, urine derived cells were cultured in various induction media for 21 days and assessed for evidence of differentiation into various cell types, including adipocytes, osteocytes, chondrocytes, SMC, and UC. Smooth muscle and urothelium generated in this manner were seeded on scaffold material and implanted into athymic mice for one month. Before and after in vivo implantation, the induced cells were assessed for expression of smooth muscle markers (alphasmooth muscle actin, desmin, myosin, smoothelin and calponin) and urothelial markers (Uroplakin Ia, AE1/AE3, CK7, CK13, CK19 and CK20) using immunofluorescence, RT-PCR and Western Blot. RESULTS: Some urine derived cells grew rapidly from a single cell clone for over 25 population doublings. Six of seven independent clones of urine derived cells expressed detectable levels of telomerase. Urine derived cells were positive for perictye/MSC markers such as CD146, NG2 and PDGF-receptor beta. When placed in appropriate induction media, these cells differentiated towards adipogenic, osteogenic and chrondrogenic lineages. High levels of myogenic growth factors (PDGF-BB and TGF-beta1) and epithelial growth factors can efficiently induce smooth muscle and urothelium differentiation, respectively, of urine derived cells in vitro. When SMC and UC derived from urine derived stem cells were implanted in vivo on a scaffold, tissue resembling native bladder tissue was generated. CONCLUSIONS: Urine-derived cells expressed the phenotypic features of pericytes/ MSC, including self-renewal and multipotency. One urine-derived cell clone can differentiate to multiple cell lineages including bladder SMC and UC, and thus, these cells may be a potential cell source for maintenance and repair of the urinary tract. Source of Funding: None
151 MATURATION OF THE BLADDER WALL DURING IN SITU REGENERATION David Burmeister, Tamer AbouShwareb, Josh Tan, Kerry Link, Karl-Erik Andersson, George Christ*, Winston Salem, NC INTRODUCTION AND OBJECTIVES: We previously reported a model of organ regeneration in adult mammals, using the rodent bladder. Specifically, following subtotal cystectomy (STC; removal of ⬃70% of the bladder), the bladder regenerates to its original volume, while maintaining overall function. In this study, we aim to elucidate some of the mechanisms underlying the growth of the bladder wall and its components including the urothelium, smooth muscle and nerves. METHODS: Female F344 rats underwent subtotal cystectomy (STC) and the regenerative process that followed was characterized with in vivo Magnetic Resonance Imaging (MRI) scans and urodynamic studies. After euthanasia bladders were examined via immunohistochemistry 1,2,4, and 8 weeks post-STC. RESULTS: MRI scans revealed a thinner bladder wall 1 week post-STC (281.96M) compared to controls (493.53 M), which improved greatly by 8 weeks (427.76 M). This wall thickening was associated with hyperplasia, as demonstrated by positive staining for Proliferating Cell Nuclear Antigen (PCNA) in bladder sections at all time points. In vivo urodynamics revealed bladder hyperactivity after afferent nerve activation via intravesical administration of 30 M capsaicin 8 weeks post-STC. In addition to functional sensory nerves, reinnervation of motor nerves was confirmed via positive immunostaining to Vesicular Acetylcholine Transporter (VachT). Mature umbrella cells in the
THE JOURNAL OF UROLOGY姞
7-fold more cells than adherent cultures over 5 days. The rapid expansion of cell numbers makes this method attractive for tissue engineering and other biologic studies. Cell type Supernatant cells
CD44v6 67 bright
Parental adherent
10 dim
Mean % positive by flow cytometry ␣6 CD71 4 50 24 16 95
56
92
W6/32 6 96
Source of Funding: K18-DK075843 and P50-DK065313 from NIDDK and R01-DE13417 from NIDCR, NIH
149 TRACKING OF INJECTED ADIPOSE TISSUE-DERIVED STEM CELLS AFTER CAVERNOUS NERVE INJURY IN RATS: INJURYINDUCED HOMING TO THE MAJOR PELVIC GANGLION Thomas M Fandel*, Palo Alto, CA; Maarten Albersen, Guiting Lin, Hongxiu Ning, Ching-Shwun Lin, Tom F Lue, San Francisco, CA INTRODUCTION AND OBJECTIVES: Adipose tissue-derived stem cells (ADSCs) have been utilized in the preservation of erectile function in rats after cavernous nerve injury. We studied the mechanism with which ADSCs facilitate nerve regeneration after cavernous nerve injury in rats. METHODS: ADSCs were procured from 56 male rats for autologous injection and labeled with 5-ethynyl-2-deoxyuridine (EdU). Animals were randomly divided into an injury group undergoing cavernous nerve crush injury (n⫽28) and a sham group (n⫽28). In all rats, ADSCs were injected into both corpora cavernosa of the sham group of rats or immediately after injury in the nerve injury group. Seven animals from each group were euthanized at 1, 2, 3, and 7 days post-injection, and penile tissue and both major pelvic ganglia (MPG) were harvested for histology. The number of EdU-positive cells in the penis and the MPG was quantified by image analysis. Staining for stromal-derivedfactor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in the MPG was performed. Two way analysis of variance with post-testing was used to assess statistical significance. Data are given as mean ⫾ SEM. RESULTS: In penile tissue, there was a significant decrease in EdU-positive cells in the penis of both groups over time (p⬍0.05), however there was no difference between the two groups (p⫽0.4). In the MPG of the sham group, the number of EdU-positive cells remained low at 4.0 ⫾ 0.3, 4.0 ⫾ 0.2, 3.2 ⫾ 0.3, and 4.6 ⫾ 0.2 at 1, 2, 3, and 7 days, respectively, while in the injured group it increased to 25.0 ⫾ 1.3, 42.9 ⫾ 1.5, 32.8 ⫾ 1.3, and 44.3 ⫾ 4.2. ADSC levels in the MPG were significantly higher in the injury group at all time points as compared to sham animals (p⬍0.0001), however these levels did not change significantly over time (p⫽0.2). Both SDF-1 and CXCR4 were upregulated in the MPGs after crush injury as compared to sham controls (p⬍0.01 for both) but this expression did not change significantly over time (p⬎0.05). CONCLUSIONS: ADSCs migrate out of the injection site in a time-dependent manner, which is similar in both groups. Sham animals show low levels of ADSCs at the MPG, while there is significant homing of ADSCs to the MPG in crush injured animals at all time-points tested. Elevated SDF-1 and CXCR4 levels at the MPG after crush injury appear to be responsible for this observation. Increased numbers of ADSCs at the MPG after crush injury may support nerve regeneration by releasing trophic factors to the neurons. Source of Funding: NIDDK/NIH R37 DK045370
150 MULTIPOTENT STEM CELLS FROM URINE FOR TISSUE ENGINEERED BLADDER Shantaram Bharadwaj, Shaofeng Wu, Jan Rohozinski, Mark Furth, Anthony Atala, Winston-Salem, NC; Yuanyuan Zhang*, Winston-Samlem, NC INTRODUCTION AND OBJECTIVES: Urine derived cells can be obtained non-invasively and may represent a potentially significant
e61
source of autologous cells for tissue engineering. One goal of this study was to test the hypothesis that the cell population obtained from urine contains cells that meet the defining criteria of stem cells (self-renewal and multipotency). In particular, the ability of these cells to give rise to smooth muscle cells (SMC) and urothelial cells (UC) for potential use in urinary bladder tissue engineering was tested. METHODS: Human urine derived cells from 9 individual donors (ages 5 to 40 years) were plated on multi-well plates. Single cell growth was monitored using time-lapse microcinematography. The cells were analyzed for expression of pericyte and mesenchymal stem cell (MSC) markers. Expression of telomerase in the isolated cells was assessed by ELISA. Next, urine derived cells were cultured in various induction media for 21 days and assessed for evidence of differentiation into various cell types, including adipocytes, osteocytes, chondrocytes, SMC, and UC. Smooth muscle and urothelium generated in this manner were seeded on scaffold material and implanted into athymic mice for one month. Before and after in vivo implantation, the induced cells were assessed for expression of smooth muscle markers (alphasmooth muscle actin, desmin, myosin, smoothelin and calponin) and urothelial markers (Uroplakin Ia, AE1/AE3, CK7, CK13, CK19 and CK20) using immunofluorescence, RT-PCR and Western Blot. RESULTS: Some urine derived cells grew rapidly from a single cell clone for over 25 population doublings. Six of seven independent clones of urine derived cells expressed detectable levels of telomerase. Urine derived cells were positive for perictye/MSC markers such as CD146, NG2 and PDGF-receptor beta. When placed in appropriate induction media, these cells differentiated towards adipogenic, osteogenic and chrondrogenic lineages. High levels of myogenic growth factors (PDGF-BB and TGF-beta1) and epithelial growth factors can efficiently induce smooth muscle and urothelium differentiation, respectively, of urine derived cells in vitro. When SMC and UC derived from urine derived stem cells were implanted in vivo on a scaffold, tissue resembling native bladder tissue was generated. CONCLUSIONS: Urine-derived cells expressed the phenotypic features of pericytes/ MSC, including self-renewal and multipotency. One urine-derived cell clone can differentiate to multiple cell lineages including bladder SMC and UC, and thus, these cells may be a potential cell source for maintenance and repair of the urinary tract. Source of Funding: None
151 MATURATION OF THE BLADDER WALL DURING IN SITU REGENERATION David Burmeister, Tamer AbouShwareb, Josh Tan, Kerry Link, Karl-Erik Andersson, George Christ*, Winston Salem, NC INTRODUCTION AND OBJECTIVES: We previously reported a model of organ regeneration in adult mammals, using the rodent bladder. Specifically, following subtotal cystectomy (STC; removal of ⬃70% of the bladder), the bladder regenerates to its original volume, while maintaining overall function. In this study, we aim to elucidate some of the mechanisms underlying the growth of the bladder wall and its components including the urothelium, smooth muscle and nerves. METHODS: Female F344 rats underwent subtotal cystectomy (STC) and the regenerative process that followed was characterized with in vivo Magnetic Resonance Imaging (MRI) scans and urodynamic studies. After euthanasia bladders were examined via immunohistochemistry 1,2,4, and 8 weeks post-STC. RESULTS: MRI scans revealed a thinner bladder wall 1 week post-STC (281.96M) compared to controls (493.53 M), which improved greatly by 8 weeks (427.76 M). This wall thickening was associated with hyperplasia, as demonstrated by positive staining for Proliferating Cell Nuclear Antigen (PCNA) in bladder sections at all time points. In vivo urodynamics revealed bladder hyperactivity after afferent nerve activation via intravesical administration of 30 M capsaicin 8 weeks post-STC. In addition to functional sensory nerves, reinnervation of motor nerves was confirmed via positive immunostaining to Vesicular Acetylcholine Transporter (VachT). Mature umbrella cells in the