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INJECTED MYOBLASTS FORM MYOFIBERS AND INDUCE ANGIOGENESIS AS WELL AS FORMATION OF MOTORIC ENDPLATES
74
THE JOURNAL OF UROLOGY®
VXSSOHPHQWVXQWLOSDVVDJH7KH06&VREWDLQHGZHUHLGHQWL¿HGE\ LPPXQRÀXRUHVFHQFH XVLQJ PDUNHUV VXFK DV 6FD &' &' &'&'DQG&'7KH\ZHUHIXUWKHUFKDUDFWHUL]HGE\SHUIRUPLQJ FRORQ\IRUPLQJXQLW¿EUREODVW &)8) DVVD\E\DQDO\]LQJGLIIHUHQWLDWLRQ potential into bone or fat and by measuring expression of the stemnessrelated transcription factor Oct-4. Furthermore, MSCs were evaluated IRU 60& PDUNHUV VXFK DV ĮVPRRWK PXVFOH DFWLQ Į60$ P\RVLQ heavy chain (MHC) and calponin. Their potential role as progenitors was investigated by inducing their differentiation into SMCs with growth IDFWRUV 3'*)%%DQG7*)ȕ 'LIIHUHQWLDWLRQRI06&VLQWR60&VZDV DVVHVVHG E\ GHWHFWLQJ WKH H[SUHVVLRQ RI WKH 60& SURWHLQV Į60$ calponin, and MHC as functional markers of fully differentiated SMCs. RESULTS: Mouse MSCs expressed Oct-4, gave rise to CFU-Fs in vitro (1:250000) and could be differentiated into bone or fat. FACS analysis showed that 70% of MSCs were Sca-1 positive. 0RUHRYHU LPPXQRÀXRUHVFHQFH VKRZHG WKDW WKH PDMRULW\ RI 6FD cells co-expressed CD44, CD29 and were negative for CD34, CD31 DQG&'WKXVFRQ¿UPLQJWKHLUPHVHQFK\PDORULJLQ06&VZHUHDOVR SRVLWLYH IRU Į60$ EXW QHJDWLYH IRU PDWXUH 60& PDUNHUV 0+& DQG calponin. Induced differentiation of MSCs into SMCs gave rise to cells VKRZLQJD60&SKHQRW\SHZLWKDQHI¿FLHQF\RIHI¿FLHQF\LQSULPDU\ FXOWXUHVDVFRQ¿UPHGE\0+&DQGFDOSRQLQH[SUHVVLRQ CONCLUSIONS: MSCs obtained from mouse bone marrow GH¿QHDPXOWLSRWHQWVWHPFHOOSRSXODWLRQFDSDEOHRIGLIIHUHQWLDWLQJLQWR ERQHIDWDQGVPRRWKPXVFOHFHOOV7KHVROHH[SUHVVLRQRIĮ60$LV QRWVXI¿FLHQWWRFRQ¿UPWKHVPRRWKPXVFOHFHOOSKHQRW\SHVLQFHLWZDV also expressed in MHC and calponin-negative MSCs. Further studies LQZKLFKWKHVHFHOOVDUHSXUL¿HGDQGH[SDQGHGPD\EHQHFHVVDU\WR study their potential for tissue regeneration. Source of Funding: None
208 INJECTED MYOBLASTS FORM MYOFIBERS AND INDUCE ANGIOGENESIS AS WELL AS FORMATION OF MOTORIC ENDPLATES Hannes Strasser*, Michael Mitterberger, Orietta Dalpiaz, Andrea Kerschbaumer, Germar M Pinggera, Georg Bartsch, Eva Margreiter, Rainer Marksteiner. Innsbruck, Austria. INTRODUCTION AND OBJECTIVE: The aim of the present study was to investigate in vivo survival, growth, tissue integration and takeover of muscle function of myoblasts that have previously been expanded in vitro. Special emphasis was laid upon the investigation if newly formed muscle tissue was both vascularised and innervated. METHODS: In 12 Fisher rats myoblasts were mixed with collagen (Contigen®, Bard, USA) as carrier material and injected into a polyethersulfon (PES) tube that was permeable for trophic factors but impermeable for cells. The tube was implanted into the region of the right groin. A motoric branch of the femoral nerve as well as the accompanying vessels were then implanted into the tube, and the tube was then closed. Therefopre, an isolated compartment, i.e. a PES tube containing only implanted cells, was created. 4 or 8 weeks after implantation the whole tube was removed. The whole tube was then analysed histologically XVLQJ KDHPDWR[\OLQHRVLQ FKROLQHVWHUDVH QHXRU¿ODPHQWVSHFL¿F DQG bungarotoxin stainings. RESULTS: It could be demonstrated that the young myoblasts that had been outgrown in vitro on a three-dimensional carrier structure IRUPHGQHZPXVFOH¿EHUV,QDGGLWLRQLWFRXOGEHVKRZQWKDWWKHQHZ PXVFOH ¿EHUV DFKLHYHG V\QDSWLF FRQQHFWLRQ ZKHQ LPSODQWHG FORVH WR DGMDFHQW QHUYHV 1HXUR¿ODPHQWVSHFL¿F VWDLQLQJ UHYHDOHG YLWDOO\ VSURXWLQJ QHUYH ¿EUHV %\ PHDQV RI FKROLQHVWHUDVHVWDLQLQJ LW ZDV SRVVLEOHWRYLVXDOL]HPRWRULFHQGSODWHV0RQRFORQDODQWLERGLHVDJDLQVW EXQJDURWR[LQKDYLQJDKLJKDI¿QLW\WRDFHW\OFKROLQUHFHSWRUVVKRZHG VLPLODUSRVLWLYHUHVXOWVLQÀXRUHVFHQFHPLFURVFRS\,QDGGLWLRQDGHQVH network of newly formed vessels could be observed inside the PES tube after implantation of the cells. CONCLUSIONS: On the basis of the present animal model the demonstration of a mutual, positive interaction of re-implanted myoblasts on the one hand and nerves as well as blood vessels on the other hand was made possible in vivo. This interaction and formation of new nerves and motoric endplates does not depend on predetermined,
Vol. 179, No. 4, Supplement, Sunday, May 18, 2008
given structures. This is also true for the formation of new vessels that provide the blood supply for newly formed muscle tissue. Source of Funding: Innovacell Biotechnologie GmbH.
209 MITOGENIC ACTIVITY OF FIBROBLAST GROWTH FACTOR-10 ON HUMAN UROTHELIAL CELLS IS DEPENDENT ON ITS NUCLEAR LOCALIZATION SIGNAL James A Bassuk*. Seattle, WA. INTRODUCTION AND OBJECTIVE: Fibroblast growth factor-10 (FGF-10), a mitogen for the epithelial cells lining the lower XULQDU\ WUDFW KDV EHHQ LGHQWL¿HG LQVLGH XURWKHOLDO FHOOV GHVSLWH LWV acknowledged role as an extracellular signaling ligand. The goal of this study was to identify the mechanism of this alternative, intracellular signaling pathway. METHODS: Recombinant (r)FGF-10 was determined by ÀXRUHVFHQFHPLFURVFRS\RSWLFDOVHFWLRQLQJWRORFDOL]HVWURQJO\WRQXFOHL inside cultured urothelial cells. To clarify the possible role of a nuclear ORFDOL]DWLRQVLJQDO 1/6 LQWKLVWUDQVORFDWLRQDYDULDQWRIU)*)ZDV constructed which lacked this sequence. RESULTS: rFGF-10(no NLS) was found in cytoplasm to a far greater degree than rFGF-10, identifying this motif as a possible NLS. Furthermore, this variant displayed poor or nonexistent bioactivity compared to the wild-type protein in triggering mitogenesis in quiescent urothelial cells. The presence of rFGF-10(no NLS) in the nucleus suggested that additional interactions were also responsible for the nuclear accumulation of rFGF-10. The FGF-10 receptor was observed in cell nuclei regardless of the presence or concentration of exogenous U)*)OLJDQG&RORFDOL]DWLRQVWXGLHVEHWZHHQU)*)DQGWKH)*) receptor revealed a strong intracellular relationship between the two. This FRORFDOL]DWLRQZDVVHHQLQQXFOHLIRUERWKU)*)DQGIRUU)*) QR NLS), although the correlation was weaker for rFGF-10(no NLS).
CONCLUSIONS: These data show that an NLS-like motif of rFGF-10 is a partial determinant of its intracellular distribution and is necessary for its mitogenic activity. These advancements in the understanding of the activity of FGF-10 present an opportunity to engineer the growth factor as a therapeutic agent for the healing of damaged urothelial tissue. Source of Funding: NIDDK R01-DK62251.
210 THE EFFECTS OF HEPATOCYTE GROWTH FACTOR AND INSULIN-LIKE GROWTH FACTOR-1 ON THE MYOGENIC DIFFERENTIATION OF SATELLITE CELLS IN HUMAN URETHRAL RHABDOSPHINCTER Yasuhiro Sumino*, Yuji Hirata, Mari Hanada, Fuminori Sato, Hiromitsu Mimata. Oita, Japan. INTRODUCTION AND OBJECTIVE: Satellite cells have been considered a population of muscle-derived stem cells responsible for WKHGHYHORSPHQWDQGUHQHZDORIVWULDWHGPXVFOH¿EHUV:HSUHYLRXVO\ reported that the proliferation of human urethral rhabdosphincter (RS) satellite cells was primarily enhanced by both the endogenous and exogenous actions of hepatocyte growth factor (HGF) and insulinlike growth factor-1 (IGF-1) (Neurourol Uroldyn. 2007). In the present study, we also examined the effects of HGF and IGF-1 on the myogenic differentiation of these cells. METHODS: Human RS was obtained from the patients undergoing radical prostatectomy for prostate cancer. Primary cells were VHOHFWLYHO\FXOWXUHGE\PDJQHWLFDI¿QLW\FHOOVRUWLQJXVLQJDQDQWLQHXUDO cell adhesion molecule antibody. Selectively cultured cells, transfected with temperature sensitive simian virus-40 T antigen (ts-SV40Tag) to
THE JOURNAL OF UROLOGY®
VXSSOHPHQWVXQWLOSDVVDJH7KH06&VREWDLQHGZHUHLGHQWL¿HGE\ LPPXQRÀXRUHVFHQFH XVLQJ PDUNHUV VXFK DV 6FD &' &' &'&'DQG&'7KH\ZHUHIXUWKHUFKDUDFWHUL]HGE\SHUIRUPLQJ FRORQ\IRUPLQJXQLW¿EUREODVW &)8) DVVD\E\DQDO\]LQJGLIIHUHQWLDWLRQ potential into bone or fat and by measuring expression of the stemnessrelated transcription factor Oct-4. Furthermore, MSCs were evaluated IRU 60& PDUNHUV VXFK DV ĮVPRRWK PXVFOH DFWLQ Į60$ P\RVLQ heavy chain (MHC) and calponin. Their potential role as progenitors was investigated by inducing their differentiation into SMCs with growth IDFWRUV 3'*)%%DQG7*)ȕ 'LIIHUHQWLDWLRQRI06&VLQWR60&VZDV DVVHVVHG E\ GHWHFWLQJ WKH H[SUHVVLRQ RI WKH 60& SURWHLQV Į60$ calponin, and MHC as functional markers of fully differentiated SMCs. RESULTS: Mouse MSCs expressed Oct-4, gave rise to CFU-Fs in vitro (1:250000) and could be differentiated into bone or fat. FACS analysis showed that 70% of MSCs were Sca-1 positive. 0RUHRYHU LPPXQRÀXRUHVFHQFH VKRZHG WKDW WKH PDMRULW\ RI 6FD cells co-expressed CD44, CD29 and were negative for CD34, CD31 DQG&'WKXVFRQ¿UPLQJWKHLUPHVHQFK\PDORULJLQ06&VZHUHDOVR SRVLWLYH IRU Į60$ EXW QHJDWLYH IRU PDWXUH 60& PDUNHUV 0+& DQG calponin. Induced differentiation of MSCs into SMCs gave rise to cells VKRZLQJD60&SKHQRW\SHZLWKDQHI¿FLHQF\RIHI¿FLHQF\LQSULPDU\ FXOWXUHVDVFRQ¿UPHGE\0+&DQGFDOSRQLQH[SUHVVLRQ CONCLUSIONS: MSCs obtained from mouse bone marrow GH¿QHDPXOWLSRWHQWVWHPFHOOSRSXODWLRQFDSDEOHRIGLIIHUHQWLDWLQJLQWR ERQHIDWDQGVPRRWKPXVFOHFHOOV7KHVROHH[SUHVVLRQRIĮ60$LV QRWVXI¿FLHQWWRFRQ¿UPWKHVPRRWKPXVFOHFHOOSKHQRW\SHVLQFHLWZDV also expressed in MHC and calponin-negative MSCs. Further studies LQZKLFKWKHVHFHOOVDUHSXUL¿HGDQGH[SDQGHGPD\EHQHFHVVDU\WR study their potential for tissue regeneration. Source of Funding: None
208 INJECTED MYOBLASTS FORM MYOFIBERS AND INDUCE ANGIOGENESIS AS WELL AS FORMATION OF MOTORIC ENDPLATES Hannes Strasser*, Michael Mitterberger, Orietta Dalpiaz, Andrea Kerschbaumer, Germar M Pinggera, Georg Bartsch, Eva Margreiter, Rainer Marksteiner. Innsbruck, Austria. INTRODUCTION AND OBJECTIVE: The aim of the present study was to investigate in vivo survival, growth, tissue integration and takeover of muscle function of myoblasts that have previously been expanded in vitro. Special emphasis was laid upon the investigation if newly formed muscle tissue was both vascularised and innervated. METHODS: In 12 Fisher rats myoblasts were mixed with collagen (Contigen®, Bard, USA) as carrier material and injected into a polyethersulfon (PES) tube that was permeable for trophic factors but impermeable for cells. The tube was implanted into the region of the right groin. A motoric branch of the femoral nerve as well as the accompanying vessels were then implanted into the tube, and the tube was then closed. Therefopre, an isolated compartment, i.e. a PES tube containing only implanted cells, was created. 4 or 8 weeks after implantation the whole tube was removed. The whole tube was then analysed histologically XVLQJ KDHPDWR[\OLQHRVLQ FKROLQHVWHUDVH QHXRU¿ODPHQWVSHFL¿F DQG bungarotoxin stainings. RESULTS: It could be demonstrated that the young myoblasts that had been outgrown in vitro on a three-dimensional carrier structure IRUPHGQHZPXVFOH¿EHUV,QDGGLWLRQLWFRXOGEHVKRZQWKDWWKHQHZ PXVFOH ¿EHUV DFKLHYHG V\QDSWLF FRQQHFWLRQ ZKHQ LPSODQWHG FORVH WR DGMDFHQW QHUYHV 1HXUR¿ODPHQWVSHFL¿F VWDLQLQJ UHYHDOHG YLWDOO\ VSURXWLQJ QHUYH ¿EUHV %\ PHDQV RI FKROLQHVWHUDVHVWDLQLQJ LW ZDV SRVVLEOHWRYLVXDOL]HPRWRULFHQGSODWHV0RQRFORQDODQWLERGLHVDJDLQVW EXQJDURWR[LQKDYLQJDKLJKDI¿QLW\WRDFHW\OFKROLQUHFHSWRUVVKRZHG VLPLODUSRVLWLYHUHVXOWVLQÀXRUHVFHQFHPLFURVFRS\,QDGGLWLRQDGHQVH network of newly formed vessels could be observed inside the PES tube after implantation of the cells. CONCLUSIONS: On the basis of the present animal model the demonstration of a mutual, positive interaction of re-implanted myoblasts on the one hand and nerves as well as blood vessels on the other hand was made possible in vivo. This interaction and formation of new nerves and motoric endplates does not depend on predetermined,
Vol. 179, No. 4, Supplement, Sunday, May 18, 2008
given structures. This is also true for the formation of new vessels that provide the blood supply for newly formed muscle tissue. Source of Funding: Innovacell Biotechnologie GmbH.
209 MITOGENIC ACTIVITY OF FIBROBLAST GROWTH FACTOR-10 ON HUMAN UROTHELIAL CELLS IS DEPENDENT ON ITS NUCLEAR LOCALIZATION SIGNAL James A Bassuk*. Seattle, WA. INTRODUCTION AND OBJECTIVE: Fibroblast growth factor-10 (FGF-10), a mitogen for the epithelial cells lining the lower XULQDU\ WUDFW KDV EHHQ LGHQWL¿HG LQVLGH XURWKHOLDO FHOOV GHVSLWH LWV acknowledged role as an extracellular signaling ligand. The goal of this study was to identify the mechanism of this alternative, intracellular signaling pathway. METHODS: Recombinant (r)FGF-10 was determined by ÀXRUHVFHQFHPLFURVFRS\RSWLFDOVHFWLRQLQJWRORFDOL]HVWURQJO\WRQXFOHL inside cultured urothelial cells. To clarify the possible role of a nuclear ORFDOL]DWLRQVLJQDO 1/6 LQWKLVWUDQVORFDWLRQDYDULDQWRIU)*)ZDV constructed which lacked this sequence. RESULTS: rFGF-10(no NLS) was found in cytoplasm to a far greater degree than rFGF-10, identifying this motif as a possible NLS. Furthermore, this variant displayed poor or nonexistent bioactivity compared to the wild-type protein in triggering mitogenesis in quiescent urothelial cells. The presence of rFGF-10(no NLS) in the nucleus suggested that additional interactions were also responsible for the nuclear accumulation of rFGF-10. The FGF-10 receptor was observed in cell nuclei regardless of the presence or concentration of exogenous U)*)OLJDQG&RORFDOL]DWLRQVWXGLHVEHWZHHQU)*)DQGWKH)*) receptor revealed a strong intracellular relationship between the two. This FRORFDOL]DWLRQZDVVHHQLQQXFOHLIRUERWKU)*)DQGIRUU)*) QR NLS), although the correlation was weaker for rFGF-10(no NLS).
CONCLUSIONS: These data show that an NLS-like motif of rFGF-10 is a partial determinant of its intracellular distribution and is necessary for its mitogenic activity. These advancements in the understanding of the activity of FGF-10 present an opportunity to engineer the growth factor as a therapeutic agent for the healing of damaged urothelial tissue. Source of Funding: NIDDK R01-DK62251.
210 THE EFFECTS OF HEPATOCYTE GROWTH FACTOR AND INSULIN-LIKE GROWTH FACTOR-1 ON THE MYOGENIC DIFFERENTIATION OF SATELLITE CELLS IN HUMAN URETHRAL RHABDOSPHINCTER Yasuhiro Sumino*, Yuji Hirata, Mari Hanada, Fuminori Sato, Hiromitsu Mimata. Oita, Japan. INTRODUCTION AND OBJECTIVE: Satellite cells have been considered a population of muscle-derived stem cells responsible for WKHGHYHORSPHQWDQGUHQHZDORIVWULDWHGPXVFOH¿EHUV:HSUHYLRXVO\ reported that the proliferation of human urethral rhabdosphincter (RS) satellite cells was primarily enhanced by both the endogenous and exogenous actions of hepatocyte growth factor (HGF) and insulinlike growth factor-1 (IGF-1) (Neurourol Uroldyn. 2007). In the present study, we also examined the effects of HGF and IGF-1 on the myogenic differentiation of these cells. METHODS: Human RS was obtained from the patients undergoing radical prostatectomy for prostate cancer. Primary cells were VHOHFWLYHO\FXOWXUHGE\PDJQHWLFDI¿QLW\FHOOVRUWLQJXVLQJDQDQWLQHXUDO cell adhesion molecule antibody. Selectively cultured cells, transfected with temperature sensitive simian virus-40 T antigen (ts-SV40Tag) to