Stem Cell Research

Podium Session 5

Sunday, May 5, 2013 8:00 AM-10:00 AM

249HUMAN URINE-DERIVED STEM CELLS ORIGINATE FROMPARIETAL 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 isto identify the origin of urine-derived cells (USCs) to better understandtheir development and optimize their use.

METHODS: Urine specimens were collected from 6 healthymale donors, and from 3 women who received male donor kidneys.After isolation, cultured USCs were assessed for expression of mes-enchymal stem cells (MSC)/pericytes (CD73, CD 90, CD 105, CD29,CD44 and CD146) and hematopoietic stem cell surface markers (CD31 and CD 34), using flow cytometry and immunofluorescent staining.Both � and Y chromosomes in USCs from gender-mismatched kidneytransplant patients were assessed with FISH and PCR. In addition,USCs were examined in renal cell transcripts (PAX8, NR3C2, andL1CAM) with real-time PCR, and assessed in podocyte-specific proteinmarkers (podocin and synaptopodin) using Western blotting and im-munofluorescemt staining. Normal human kidney, ureter, and bladdertissues from donors were used as controls.

RESULTS: USC clones from both healthy individuals and gen-der-mismatched kidney transplant patients were small and had a ricegrain-shaped appearance. USCs expressed MSC markers (CD73, CD90, CD 105, CD44, CD29, and CD146), but did not express hemato-poietic stem cell surface markers (CD34 and CD31). Nine of 12 USCclones from the gender-mismatched kidney transplant patients showedX/Y chromosome character, indicating that the USCs were from theupper urinary system. Real-time PCR showed that USCs expressedgenes for kidney-related markers such as PAX8, NR3C2, and L1CAM.Immunofluorescent staining showed that cultured USCs and parietalcells/podocytes in nephron glomeruli within kidney all expressedCD31�/CD146�and podocyte-specific protein markers (podocin andsynaptopodin), 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, USCsappeared to originate from parietal cells or podocytes in the renalglomerulus.

CONCLUSIONS: Stem cells isolated from human urine areCD31�/CD146� cells with self-renewal and multipotent differentiationcharacteristics. USCs could be an alternative source of stem cells forregenerative medicine therapy. USCs are most likely of nephron glo-merular podocyte origin, probably from parietal stem cells.

Source of Funding: None

250OPTIMIZATION OF CULTURE CONDITIONS FOR THEEXPANSION OF URINE DERIVED STEM CELL USINGCOLLAGEN 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 de-rived urine stem cells (uUSCs) have been proposed as a valuable stemcell source for urological tissue reconstruction. However, reportedculture condition of uUSCs is hard to achieve clinically applicable

quality and quantity of cell preparation. These drawbacks led us toreconstitute of culture condition by mimic of stem cell niche. This studyestablished the efficient clinical applicable culture condition of uUSCsto use autologous cell therapy and tissue engineering application.

METHODS: Urine samples were collected from the upper uri-nary tract of 7 male bladder cancer patients using a ureteral catheter.The samples were centrifuged and the pellets were plated for primaryculture. According to a time-gradient attachment method about 500cells were plated. Cells at passage 4 were used for following analysis.Cell proliferation, colony formation, stemness and gene expressionanalyses were performed in various conditions of extracellular matrix(ECM) and oxygen concentration to optimize the best culture condi-tions. Immunogenicity and tumorigenicity was also evaluated.

RESULTS: Cells grown in the collagen-I coated dishes andhypoxia showed highest cell proliferation and maintenance of stem cellproperties, respectively. And cells cultured in the reconstitute condition(uUSCsrecon) composed with both collagen-I and 5% hypoxia revealedbetter growth rate and stemness than collagen-I coating or hypoxiccondition only. The uUSCsrecon cultured for 4 passages had a negativeHLA-DR with flow cytometry and there was no teratoma formation intissues retrieved 8 weeks after renal subcapsular injection of uUSCsrecon.

CONCLUSIONS: We could optimize culture condition ofuUSCs to get clinically applicable quality and quantity of cell prepara-tion using collagen-1 ECM and hypoxia.

Source of Funding: None

251UP-REGULATION OF SONIC HEDGEHOG (SHH) CONTRIBUTESTO TGF-�1-MEDIATED EPITHELIAL TO MESENCHYMALTRANSITION (EMT) AND STEMNESS CHARACTERISTICS INHTB-9 CELLS

Syed Islam, Reza Mokhtari, Elias Wehbi, Niki Kanaroglou,Herman Yeger, Walid Farhat*, Toronto, Canada

INTRODUCTION AND OBJECTIVES: The aggressiveness ofurothelial bladder carcinoma HTB-9 cells has been shown to be asso-ciated with the acquisition of epithelial-to-mesenchymal transition(EMT). The acquisition of this phenotype, induced by TGF-�1 in severalcancer cells lines, has been implicated in tumor aggressiveness andresistance to conventional therapeutics; however, the molecular mech-anism of EMT and tumor aggressiveness in urothelial bladder carci-noma HTB-9 cells remains unknown.

METHODS: Bladder transitional cell carcinoma HTB-9 cellswere treated with TGF-�1 (5 ng/ml) and assessed for Shh and EMTmarker expression by immunoflurescence, qRT-PCR and WesternBlotting. Shh-specific siRNA (Cyclopamine) and Hh (GDC-0441) inhib-itors were used to block Hh signaling pathway and then assessed formigration, invasion and tumorigenesis.

RESULTS: The aggressiveness of HTB-9 cells was attenuatedby treatment with pharmaceutical inhibitors of Hh signaling in additionto Shh knockdown by siRNA. The inhibition of Hh signaling by phar-macological inhibitors led to the reversal of EMT phenotype as con-firmed 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 byTGF-�1. Stem cell markers CD133, Oct4, Sox and Nanog expressionwere upregulated in TGF-�1 treated xenograft tumors in vivo.

CONCLUSIONS: Our results are the first to confirm the tran-scriptional up regulation of Shh by TGF-�1, which is mechanisticallyassociated with TGF-�1-induced EMT phenotype and aggressive be-havior in HTB-9 bladder cancer cells. Clinically these Shh inhibitorsmay potentially be used to reverse the EMT phenotype, thereby inhib-iting the metastatic potential and aggressiveness of these tumors andpossibly sensitizing them to conventional therapies.

Source of Funding: None

Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013 THE JOURNAL OF UROLOGY� e103