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249 HUMAN URINE-DERIVED STEM CELLS ORIGINATE FROM PARIETAL STEM CELLS
Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013
THE JOURNAL OF UROLOGY姞
Stem Cell Research Podium Session 5 Sunday, May 5, 2013
8:00 AM-10:00 AM
249 HUMAN URINE-DERIVED STEM CELLS ORIGINATE FROM PARIETAL STEM CELLS Rongpei Wu, Guihua Liu, Winston Salem, NC; Yuxin Fan, Houston, TX; Jan Rohozinski, Winston Salem, NC; Xinyan Lu, Gilma Rodriguez, Houston, TX; Alan Farney, Anthony Atala, Yuanyuan Zhang*, Winston Salem, NC INTRODUCTION AND OBJECTIVES: The aim of this study is to identify the origin of urine-derived cells (USCs) to better understand their development and optimize their use. METHODS: Urine specimens were collected from 6 healthy male donors, and from 3 women who received male donor kidneys. After isolation, cultured USCs were assessed for expression of mesenchymal stem cells (MSC)/pericytes (CD73, CD 90, CD 105, CD29, CD44 and CD146) and hematopoietic stem cell surface markers (CD 31 and CD 34), using flow cytometry and immunofluorescent staining. Both ⫻ and Y chromosomes in USCs from gender-mismatched kidney transplant patients were assessed with FISH and PCR. In addition, USCs were examined in renal cell transcripts (PAX8, NR3C2, and L1CAM) with real-time PCR, and assessed in podocyte-specific protein markers (podocin and synaptopodin) using Western blotting and immunofluorescemt staining. Normal human kidney, ureter, and bladder tissues from donors were used as controls. RESULTS: USC clones from both healthy individuals and gender-mismatched kidney transplant patients were small and had a rice grain-shaped appearance. USCs expressed MSC markers (CD73, CD 90, CD 105, CD44, CD29, and CD146), but did not express hematopoietic stem cell surface markers (CD34 and CD31). Nine of 12 USC clones from the gender-mismatched kidney transplant patients showed X/Y chromosome character, indicating that the USCs were from the upper urinary system. Real-time PCR showed that USCs expressed genes for kidney-related markers such as PAX8, NR3C2, and L1CAM. Immunofluorescent staining showed that cultured USCs and parietal cells/podocytes in nephron glomeruli within kidney all expressed CD31⫺/CD146⫹and podocyte-specific protein markers (podocin and synaptopodin), but no cells were expressed in ureter or bladder tissues. Western blotting showed that cultured USC at late passages ( p⬎8) expressed podocin and synaptopodin as well. Taken together, USCs appeared to originate from parietal cells or podocytes in the renal glomerulus. CONCLUSIONS: Stem cells isolated from human urine are CD31⫺/CD146⫹ cells with self-renewal and multipotent differentiation characteristics. USCs could be an alternative source of stem cells for regenerative medicine therapy. USCs are most likely of nephron glomerular podocyte origin, probably from parietal stem cells. Source of Funding: None
250 OPTIMIZATION OF CULTURE CONDITIONS FOR THE EXPANSION OF URINE DERIVED STEM CELL USING COLLAGEN I AND HYPOXIC CONDITION Hyun Tae Kim, Bum Soo Kim, Se Yun Kwon, Jun Nyung Lee, Eun Sang Yoo, Sung Kwang Chung, Bup Wan Kim, Yoon Kyu Park, Jae Young Choi, Seock Hwan Choi*, Tae-Hwan Kim, Tae Gyun Kwon, Daegu, Korea, Republic of INTRODUCTION AND OBJECTIVES: Upper urinary tract derived urine stem cells (uUSCs) have been proposed as a valuable stem cell source for urological tissue reconstruction. However, reported culture condition of uUSCs is hard to achieve clinically applicable
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quality and quantity of cell preparation. These drawbacks led us to reconstitute of culture condition by mimic of stem cell niche. This study established the efficient clinical applicable culture condition of uUSCs to use autologous cell therapy and tissue engineering application. METHODS: Urine samples were collected from the upper urinary tract of 7 male bladder cancer patients using a ureteral catheter. The samples were centrifuged and the pellets were plated for primary culture. According to a time-gradient attachment method about 500 cells were plated. Cells at passage 4 were used for following analysis. Cell proliferation, colony formation, stemness and gene expression analyses were performed in various conditions of extracellular matrix (ECM) and oxygen concentration to optimize the best culture conditions. Immunogenicity and tumorigenicity was also evaluated. RESULTS: Cells grown in the collagen-I coated dishes and hypoxia showed highest cell proliferation and maintenance of stem cell properties, respectively. And cells cultured in the reconstitute condition (uUSCsrecon) composed with both collagen-I and 5% hypoxia revealed better growth rate and stemness than collagen-I coating or hypoxic condition only. The uUSCsrecon cultured for 4 passages had a negative HLA-DR with flow cytometry and there was no teratoma formation in tissues retrieved 8 weeks after renal subcapsular injection of uUSCsrecon. CONCLUSIONS: We could optimize culture condition of uUSCs to get clinically applicable quality and quantity of cell preparation using collagen-1 ECM and hypoxia. Source of Funding: None
251 UP-REGULATION OF SONIC HEDGEHOG (SHH) CONTRIBUTES TO TGF-1-MEDIATED EPITHELIAL TO MESENCHYMAL TRANSITION (EMT) AND STEMNESS CHARACTERISTICS IN HTB-9 CELLS Syed Islam, Reza Mokhtari, Elias Wehbi, Niki Kanaroglou, Herman Yeger, Walid Farhat*, Toronto, Canada INTRODUCTION AND OBJECTIVES: The aggressiveness of urothelial bladder carcinoma HTB-9 cells has been shown to be associated with the acquisition of epithelial-to-mesenchymal transition (EMT). The acquisition of this phenotype, induced by TGF-1 in several cancer cells lines, has been implicated in tumor aggressiveness and resistance to conventional therapeutics; however, the molecular mechanism of EMT and tumor aggressiveness in urothelial bladder carcinoma HTB-9 cells remains unknown. METHODS: Bladder transitional cell carcinoma HTB-9 cells were treated with TGF-1 (5 ng/ml) and assessed for Shh and EMT marker expression by immunoflurescence, qRT-PCR and Western Blotting. Shh-specific siRNA (Cyclopamine) and Hh (GDC-0441) inhibitors were used to block Hh signaling pathway and then assessed for migration, invasion and tumorigenesis. RESULTS: The aggressiveness of HTB-9 cells was attenuated by treatment with pharmaceutical inhibitors of Hh signaling in addition to Shh knockdown by siRNA. The inhibition of Hh signaling by pharmacological inhibitors led to the reversal of EMT phenotype as confirmed by the reduction of mesenchymal markers such as N-cadherin, fibronectin and induction of epithelial marker E-cadherin. In addition, knockdown of Shh by siRNA significantly attenuated EMT induction by TGF-1. Stem cell markers CD133, Oct4, Sox and Nanog expression were upregulated in TGF-1 treated xenograft tumors in vivo. CONCLUSIONS: Our results are the first to confirm the transcriptional up regulation of Shh by TGF-1, which is mechanistically associated with TGF-1-induced EMT phenotype and aggressive behavior in HTB-9 bladder cancer cells. Clinically these Shh inhibitors may potentially be used to reverse the EMT phenotype, thereby inhibiting the metastatic potential and aggressiveness of these tumors and possibly sensitizing them to conventional therapies. Source of Funding: None
THE JOURNAL OF UROLOGY姞
Stem Cell Research Podium Session 5 Sunday, May 5, 2013
8:00 AM-10:00 AM
249 HUMAN URINE-DERIVED STEM CELLS ORIGINATE FROM PARIETAL STEM CELLS Rongpei Wu, Guihua Liu, Winston Salem, NC; Yuxin Fan, Houston, TX; Jan Rohozinski, Winston Salem, NC; Xinyan Lu, Gilma Rodriguez, Houston, TX; Alan Farney, Anthony Atala, Yuanyuan Zhang*, Winston Salem, NC INTRODUCTION AND OBJECTIVES: The aim of this study is to identify the origin of urine-derived cells (USCs) to better understand their development and optimize their use. METHODS: Urine specimens were collected from 6 healthy male donors, and from 3 women who received male donor kidneys. After isolation, cultured USCs were assessed for expression of mesenchymal stem cells (MSC)/pericytes (CD73, CD 90, CD 105, CD29, CD44 and CD146) and hematopoietic stem cell surface markers (CD 31 and CD 34), using flow cytometry and immunofluorescent staining. Both ⫻ and Y chromosomes in USCs from gender-mismatched kidney transplant patients were assessed with FISH and PCR. In addition, USCs were examined in renal cell transcripts (PAX8, NR3C2, and L1CAM) with real-time PCR, and assessed in podocyte-specific protein markers (podocin and synaptopodin) using Western blotting and immunofluorescemt staining. Normal human kidney, ureter, and bladder tissues from donors were used as controls. RESULTS: USC clones from both healthy individuals and gender-mismatched kidney transplant patients were small and had a rice grain-shaped appearance. USCs expressed MSC markers (CD73, CD 90, CD 105, CD44, CD29, and CD146), but did not express hematopoietic stem cell surface markers (CD34 and CD31). Nine of 12 USC clones from the gender-mismatched kidney transplant patients showed X/Y chromosome character, indicating that the USCs were from the upper urinary system. Real-time PCR showed that USCs expressed genes for kidney-related markers such as PAX8, NR3C2, and L1CAM. Immunofluorescent staining showed that cultured USCs and parietal cells/podocytes in nephron glomeruli within kidney all expressed CD31⫺/CD146⫹and podocyte-specific protein markers (podocin and synaptopodin), but no cells were expressed in ureter or bladder tissues. Western blotting showed that cultured USC at late passages ( p⬎8) expressed podocin and synaptopodin as well. Taken together, USCs appeared to originate from parietal cells or podocytes in the renal glomerulus. CONCLUSIONS: Stem cells isolated from human urine are CD31⫺/CD146⫹ cells with self-renewal and multipotent differentiation characteristics. USCs could be an alternative source of stem cells for regenerative medicine therapy. USCs are most likely of nephron glomerular podocyte origin, probably from parietal stem cells. Source of Funding: None
250 OPTIMIZATION OF CULTURE CONDITIONS FOR THE EXPANSION OF URINE DERIVED STEM CELL USING COLLAGEN I AND HYPOXIC CONDITION Hyun Tae Kim, Bum Soo Kim, Se Yun Kwon, Jun Nyung Lee, Eun Sang Yoo, Sung Kwang Chung, Bup Wan Kim, Yoon Kyu Park, Jae Young Choi, Seock Hwan Choi*, Tae-Hwan Kim, Tae Gyun Kwon, Daegu, Korea, Republic of INTRODUCTION AND OBJECTIVES: Upper urinary tract derived urine stem cells (uUSCs) have been proposed as a valuable stem cell source for urological tissue reconstruction. However, reported culture condition of uUSCs is hard to achieve clinically applicable
e103
quality and quantity of cell preparation. These drawbacks led us to reconstitute of culture condition by mimic of stem cell niche. This study established the efficient clinical applicable culture condition of uUSCs to use autologous cell therapy and tissue engineering application. METHODS: Urine samples were collected from the upper urinary tract of 7 male bladder cancer patients using a ureteral catheter. The samples were centrifuged and the pellets were plated for primary culture. According to a time-gradient attachment method about 500 cells were plated. Cells at passage 4 were used for following analysis. Cell proliferation, colony formation, stemness and gene expression analyses were performed in various conditions of extracellular matrix (ECM) and oxygen concentration to optimize the best culture conditions. Immunogenicity and tumorigenicity was also evaluated. RESULTS: Cells grown in the collagen-I coated dishes and hypoxia showed highest cell proliferation and maintenance of stem cell properties, respectively. And cells cultured in the reconstitute condition (uUSCsrecon) composed with both collagen-I and 5% hypoxia revealed better growth rate and stemness than collagen-I coating or hypoxic condition only. The uUSCsrecon cultured for 4 passages had a negative HLA-DR with flow cytometry and there was no teratoma formation in tissues retrieved 8 weeks after renal subcapsular injection of uUSCsrecon. CONCLUSIONS: We could optimize culture condition of uUSCs to get clinically applicable quality and quantity of cell preparation using collagen-1 ECM and hypoxia. Source of Funding: None
251 UP-REGULATION OF SONIC HEDGEHOG (SHH) CONTRIBUTES TO TGF-1-MEDIATED EPITHELIAL TO MESENCHYMAL TRANSITION (EMT) AND STEMNESS CHARACTERISTICS IN HTB-9 CELLS Syed Islam, Reza Mokhtari, Elias Wehbi, Niki Kanaroglou, Herman Yeger, Walid Farhat*, Toronto, Canada INTRODUCTION AND OBJECTIVES: The aggressiveness of urothelial bladder carcinoma HTB-9 cells has been shown to be associated with the acquisition of epithelial-to-mesenchymal transition (EMT). The acquisition of this phenotype, induced by TGF-1 in several cancer cells lines, has been implicated in tumor aggressiveness and resistance to conventional therapeutics; however, the molecular mechanism of EMT and tumor aggressiveness in urothelial bladder carcinoma HTB-9 cells remains unknown. METHODS: Bladder transitional cell carcinoma HTB-9 cells were treated with TGF-1 (5 ng/ml) and assessed for Shh and EMT marker expression by immunoflurescence, qRT-PCR and Western Blotting. Shh-specific siRNA (Cyclopamine) and Hh (GDC-0441) inhibitors were used to block Hh signaling pathway and then assessed for migration, invasion and tumorigenesis. RESULTS: The aggressiveness of HTB-9 cells was attenuated by treatment with pharmaceutical inhibitors of Hh signaling in addition to Shh knockdown by siRNA. The inhibition of Hh signaling by pharmacological inhibitors led to the reversal of EMT phenotype as confirmed by the reduction of mesenchymal markers such as N-cadherin, fibronectin and induction of epithelial marker E-cadherin. In addition, knockdown of Shh by siRNA significantly attenuated EMT induction by TGF-1. Stem cell markers CD133, Oct4, Sox and Nanog expression were upregulated in TGF-1 treated xenograft tumors in vivo. CONCLUSIONS: Our results are the first to confirm the transcriptional up regulation of Shh by TGF-1, which is mechanistically associated with TGF-1-induced EMT phenotype and aggressive behavior in HTB-9 bladder cancer cells. Clinically these Shh inhibitors may potentially be used to reverse the EMT phenotype, thereby inhibiting the metastatic potential and aggressiveness of these tumors and possibly sensitizing them to conventional therapies. Source of Funding: None