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Reciprocal Induction of Simple Organogenesis by Mouse Kidney Progenitor Cells in Three-Dimensional Co-Culture

2011; Elsevier BV; Volume: 180; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2011.11.002

ISSN

1525-2191

Autores

Chakradhar Velagapudi, Rune-Par Nilsson, Myung Ja Lee, Hannah Burns, Jill M. Ricono, Mazen Arar, Veronique L. Barnes, Hanna E. Abboud, Jeffrey L. Barnes,

Tópico(s)

Organ Donation and Transplantation

Resumo

Kidney development is regulated by a coordinated reciprocal induction of metanephric mesenchymal (MM) and ureteric bud (UB) cells. Here, established MM and UB progenitor cell lines were recombined in three-dimensional Matrigel implants in SCID mice. Differentiation potential was examined for changes in phenotype, organization, and the presence of specialized proteins using immunofluorescence and bright-field and electron microscopy. Both cell types, when grown alone, did not develop into specialized structures. When combined, the cells organized into simple organoid structures of polarized epithelia with lumens surrounded by capillary-like structures. Tracker experiments indicated the UB cells formed the tubuloid structures, and the MM cells were the source of the capillary-like cells. The epithelial cells stained positive for pancytokeratin, the junctional complex protein ZO-1, collagen type IV, as well as UB and collecting duct markers, rearranged during transfection (RET), Dolichos biflorus lectin, EndoA cytokeratin, and aquaporin 2. The surrounding cells expressed α-smooth muscle actin, vimentin, platelet endothelial cell adhesion molecule 1 (PECAM), and aquaporin 1, a marker of vasculogenesis. The epithelium exhibited apical vacuoles, microvilli, junctional complexes, and linear basement membranes. Capillary-like structures showed endothelial features with occasional pericytes. UB cell epithelialization was augmented in the presence of MM cell–derived conditioned medium, glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), or fibronectin. MM cells grown in the presence of UB-derived conditioned medium failed to undergo differentiation. However, UB cell–derived conditioned medium induced MM cell migration. These studies indicate that tubulogenesis and vasculogenesis can be partially recapitulated by recombining individual MM and UB cell lineages, providing a new model system to study organogenesis ex vivo. Kidney development is regulated by a coordinated reciprocal induction of metanephric mesenchymal (MM) and ureteric bud (UB) cells. Here, established MM and UB progenitor cell lines were recombined in three-dimensional Matrigel implants in SCID mice. Differentiation potential was examined for changes in phenotype, organization, and the presence of specialized proteins using immunofluorescence and bright-field and electron microscopy. Both cell types, when grown alone, did not develop into specialized structures. When combined, the cells organized into simple organoid structures of polarized epithelia with lumens surrounded by capillary-like structures. Tracker experiments indicated the UB cells formed the tubuloid structures, and the MM cells were the source of the capillary-like cells. The epithelial cells stained positive for pancytokeratin, the junctional complex protein ZO-1, collagen type IV, as well as UB and collecting duct markers, rearranged during transfection (RET), Dolichos biflorus lectin, EndoA cytokeratin, and aquaporin 2. The surrounding cells expressed α-smooth muscle actin, vimentin, platelet endothelial cell adhesion molecule 1 (PECAM), and aquaporin 1, a marker of vasculogenesis. The epithelium exhibited apical vacuoles, microvilli, junctional complexes, and linear basement membranes. Capillary-like structures showed endothelial features with occasional pericytes. UB cell epithelialization was augmented in the presence of MM cell–derived conditioned medium, glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), or fibronectin. MM cells grown in the presence of UB-derived conditioned medium failed to undergo differentiation. However, UB cell–derived conditioned medium induced MM cell migration. These studies indicate that tubulogenesis and vasculogenesis can be partially recapitulated by recombining individual MM and UB cell lineages, providing a new model system to study organogenesis ex vivo. Development of the kidney is governed by a well-orchestrated series of reciprocal inductive events between the ureteric bud (UB) epithelium and the metanephric mesenchyme (MM)1Saxen L. Sariola H. Early organogenesis of the kidney.Pediatr Nephrol. 1987; 1 (385–192)Crossref PubMed Scopus (299) Google Scholar, 2Costantini F. Renal branching morphogenesis: concepts, questions, and recent advances.Differentiation. 2006; 74: 402-421Crossref PubMed Scopus (146) Google Scholar, 3Dressler G.R. The cellular basis of kidney development.Ann Rev Cell Dev Biol. 2006; 22: 509-529Crossref PubMed Scopus (476) Google Scholar, 4Kanwar Y.S. Wada J. Lin S. Danesh F.R. Chugh S.S. Yang Q. Banerjee T. Lomasney J.W. Update of extracellular matrix, its receptors, and cell adhesion molecules in mammalian nephrogenesis.Am J Physiol Renal. 2004; 286: F202-F215Crossref PubMed Scopus (58) Google Scholar, 5Monte J.C. Sakurai H. Bush K.T. Nigam S.K. The developmental nephrome: systems biology in the developing kidney.Curr Opin Nephrol Hypertens. 2007; 16: 3-9Crossref PubMed Scopus (30) Google Scholar, 6Nigam S.K. Shah M.M. How does the ureteric bud branch.J Am Soc Nephrol. 2009; 20: 1465-1469Crossref PubMed Scopus (48) Google Scholar, 7Abrahamson D.R. Development of kidney glomerular endothelial cells and their role in basement membrane assembly.Organogenesis. 2009; 5: 275-287Crossref PubMed Scopus (43) Google Scholar, 8Ricono J.M. Xu Y.C. Arar M. Jin D.C. Barnes J.L. Abboud H.E. Morphological insights into the origin of glomerular endothelial and mesangial cells and their precursors.J Histochem Cytochem. 2003; 51: 141-150Crossref PubMed Scopus (46) Google Scholar (Figure 1). The UB, an outgrowth of the Wolffian duct, invades and interacts with the MM.2Costantini F. Renal branching morphogenesis: concepts, questions, and recent advances.Differentiation. 2006; 74: 402-421Crossref PubMed Scopus (146) Google Scholar, 3Dressler G.R. The cellular basis of kidney development.Ann Rev Cell Dev Biol. 2006; 22: 509-529Crossref PubMed Scopus (476) Google Scholar, 4Kanwar Y.S. Wada J. Lin S. Danesh F.R. Chugh S.S. Yang Q. Banerjee T. Lomasney J.W. Update of extracellular matrix, its receptors, and cell adhesion molecules in mammalian nephrogenesis.Am J Physiol Renal. 2004; 286: F202-F215Crossref PubMed Scopus (58) Google Scholar, 5Monte J.C. Sakurai H. Bush K.T. Nigam S.K. The developmental nephrome: systems biology in the developing kidney.Curr Opin Nephrol Hypertens. 2007; 16: 3-9Crossref PubMed Scopus (30) Google Scholar, 6Nigam S.K. Shah M.M. How does the ureteric bud branch.J Am Soc Nephrol. 2009; 20: 1465-1469Crossref PubMed Scopus (48) Google Scholar The MM induces UB branching morphogenesis, eventually giving rise to the collecting duct system, renal pelvis, and ureter.1Saxen L. Sariola H. Early organogenesis of the kidney.Pediatr Nephrol. 1987; 1 (385–192)Crossref PubMed Scopus (299) Google Scholar, 3Dressler G.R. The cellular basis of kidney development.Ann Rev Cell Dev Biol. 2006; 22: 509-529Crossref PubMed Scopus (476) Google Scholar In turn, the mesenchyme is induced to form aggregates around the advancing tips of the UB, eventually forming the renal vesicle, committing the mesenchyme to epithelialize, and give rise to the visceral and parietal epithelial cells of the glomerulus, proximal tubule, loop of Henle, and distal tubule.1Saxen L. Sariola H. Early organogenesis of the kidney.Pediatr Nephrol. 1987; 1 (385–192)Crossref PubMed Scopus (299) Google Scholar, 7Abrahamson D.R. Development of kidney glomerular endothelial cells and their role in basement membrane assembly.Organogenesis. 2009; 5: 275-287Crossref PubMed Scopus (43) Google Scholar MM cells may also differentiate into vascular and stromal structures throughout the developing kidney, including mesangial cells and endothelium of the developing glomerulus.4Kanwar Y.S. Wada J. Lin S. Danesh F.R. Chugh S.S. Yang Q. Banerjee T. Lomasney J.W. Update of extracellular matrix, its receptors, and cell adhesion molecules in mammalian nephrogenesis.Am J Physiol Renal. 2004; 286: F202-F215Crossref PubMed Scopus (58) Google Scholar, 8Ricono J.M. Xu Y.C. Arar M. Jin D.C. Barnes J.L. Abboud H.E. Morphological insights into the origin of glomerular endothelial and mesangial cells and their precursors.J Histochem Cytochem. 2003; 51: 141-150Crossref PubMed Scopus (46) Google Scholar, 9Levinson R. Mendelsohn C. Stromal progenitors are important for patterning epithelial and mesenchymal cell types in the embryonic kidney.Sem Cell Dev Biol. 2003; 14: 225-231Crossref PubMed Scopus (39) Google Scholar Recent state-of-the-art methods such as targeted disruption of genes, in vivo delivery of test substances, and the examination of whole embryonic kidney explants have been especially informative in defining roles for growth factors, signaling pathways, and genes involved in inductive events during nephrogenesis.3Dressler G.R. The cellular basis of kidney development.Ann Rev Cell Dev Biol. 2006; 22: 509-529Crossref PubMed Scopus (476) Google Scholar, 5Monte J.C. Sakurai H. Bush K.T. Nigam S.K. The developmental nephrome: systems biology in the developing kidney.Curr Opin Nephrol Hypertens. 2007; 16: 3-9Crossref PubMed Scopus (30) Google Scholar, 6Nigam S.K. Shah M.M. How does the ureteric bud branch.J Am Soc Nephrol. 2009; 20: 1465-1469Crossref PubMed Scopus (48) Google Scholar, 10Bouchard M. Transcriptional control of kidney development.Differentiation. 2004; 72: 295-306Crossref PubMed Scopus (61) Google Scholar Also, developmental defects may result in death of transgenic animals before the onset of nephrogenesis, precluding the study of important developmental processes in vivo, making development of simple organotypic culture systems desirable. In vitro experiments using intact MM or UB explants or isolated cells in monolayer or three-dimensional gels have been instrumental in examining the direct effect of soluble factors on the induction of differentiation. Factors known to induce MM cell differentiation include extracts of pituitary, nervous and salivary gland tissue, UB cell–conditioned media, as well as specific growth factors such as bone morphogenic protein-7 (BMP-7), epidermal growth factor (EGF), transforming growth factor α (TGF-α), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF).3Dressler G.R. The cellular basis of kidney development.Ann Rev Cell Dev Biol. 2006; 22: 509-529Crossref PubMed Scopus (476) Google Scholar, 11Wallner E.I. Kumar A. Carone F.A. Kanwar Y.S. Growth factors in metanephric development.Ren Fail. 1998; 20: 331-341Crossref PubMed Scopus (2) Google Scholar, 12Karavanova I.D. Dove L.F. Resau J.H. Perantoni A.O. Conditioned medium from a rat ureteric bud cell line in combination with bFGF induces complete differentiation of isolated metanephric mesenchyme.Development. 1996; 122: 4159-4167PubMed Google Scholar, 13Simon M. Maresh J.G. Harris S.E. Hernandez J.D. Arar M. Olson M.S. Abboud H.E. Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney.Am J Physiol Renal. 1999; 276: F382-F389PubMed Google Scholar, 14Vukicevic S. Kopp J.B. Luyten F.P. Sampath T.K. Induction of nephrogenic mesenchyme by osteogenic protein 1 (bone morphogenetic protein 7).Proc Natl Acad Sci U S A. 1996; 93: 9021-9026Crossref PubMed Scopus (157) Google Scholar, 15Woolf A.S. Kolatsi-Joannou M. Hardman P. Andermarcher E. Moorby C. Fine L.G. Jat P.S. Noble M.D. Gherardi E. Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros.J Cell Biol. 1995; 128: 171-184Crossref PubMed Scopus (299) Google Scholar, 16Rogers S.A. Padanilam B.J. Hruska K.A. Giachelli C.M. Hammerman M.R. Metanephric osteopontin regulates nephrogenesis in vitro.Am J Physiol Renal. 1997; 272: F469-F476PubMed Google Scholar Similarly, UB branching can be induced by conditioned medium derived from MM cells and specifically with the growth factors glial-derived neurotrophic factor (GDNF) and HGF and extracellular matrix proteins, including fibronectin, collagen, and laminin,17Sakai T. Larsen M. Yamada K.M. Fibronectin requirement in branching morphogenesis.Nature. 2003; 423: 876-881Crossref PubMed Scopus (408) Google Scholar, 18Ye P. Habib S.L. Ricono J.M. Kim N.H. Choudhury G.G. Barnes J.L. Abboud H.E. Arar M.Y. Fibronectin induces ureteric bud cells branching and cellular cord and tubule formation.Kidney Int. 2004; 66: 1356-1364Crossref PubMed Scopus (33) Google Scholar, 19George E.L. Georges-Labouesse E.N. Patel-King R.S. Rayburn H. Hynes R.O. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin.Development. 1993; 119: 1079-1091Crossref PubMed Google Scholar, 20Zent R. Bush K.T. Pohl M.L. Quaranta V. Koshikawa N. Wang Z. Kreidberg J.A. Sakurai H. Stuart R.O. Nigam S.K. Involvement of laminin binding integrins and laminin-5 in branching morphogenesis of the ureteric bud during kidney development.Dev Biol. 2001; 238: 289-302Crossref PubMed Scopus (77) Google Scholar that are known to be abundant in the mesenchyme of the developing kidney.4Kanwar Y.S. Wada J. Lin S. Danesh F.R. Chugh S.S. Yang Q. Banerjee T. Lomasney J.W. Update of extracellular matrix, its receptors, and cell adhesion molecules in mammalian nephrogenesis.Am J Physiol Renal. 2004; 286: F202-F215Crossref PubMed Scopus (58) Google Scholar, 21Barnes V.L. Musa J. Mitchell R.J. Barnes J.L. Expression of embryonic fibronectin isoform EIIIA parallels alpha-smooth muscle actin in maturing and diseased kidney.J Histochem Cytochem. 1999; 47: 787-798Crossref PubMed Scopus (22) Google Scholar To date, in vitro studies have relied on isolated nephrogenic explants or growth of progenitor cells as single-cell cultures in monolayer or in three-dimensional matrices. The studies described herein were designed to mimic the conditions of nephrogenesis by co-culturing pre-existing mouse MM and UB cell lines in three-dimensional gels implanted in SCID mice. Such a format provides a microenvironment allowing for intermingling and direct cell–cell contact, reciprocal induction, and stimulation of morphogenesis in three-dimensional culture. Three-dimensional co-culture models have been widely used to emulate a more physiologically relevant microenvironment for the study of genes and signaling pathways in the induction of gliogenesis and neurogenesis,22Yen B.L. Chien C.C. Chen Y.C. 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Pulmonary artery adventitial fibroblasts cooperate with vasa vasorum endothelial cells to regulate vasa vasorum neovascularization: a process mediated by hypoxia and endothelin-1.Am J Pathol. 2006; 168: 1793-1807Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar and stromal–epithelial interactions in endometrial26Arnold J.T. Kaufman D.G. Seppala M. Lessey B.A. Endometrial stromal cells regulate epithelial cell growth in vitro: a new co-culture model.Hum Reprod. 2001; 16: 836-845Crossref PubMed Scopus (211) Google Scholar and prostatic epithelial27Lang S.H. Stark M. Collins A. Paul A.B. Stower M.J. Maitland N.J. Experimental prostate epithelial morphogenesis in response to stroma and three-dimensional matrigel culture.Cell Growth Diff. 2001; 12: 631-640PubMed Google Scholar differentiation. Recent studies also indicate that adult kidney stem cells in Matrigel (BD Biosciences, Bedford, MA) differentiate into tubular profiles complete with lumens and junctional complexes,28Bussolati B. Bruno S. Grange C. Buttiglieri S. Deregibus M.C. Cantino D. Camussi G. Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar verifying an important tool in the study of kidney cell induction/differentiation. In this study, we report that co-culture of established MM and UB cell lines in three-dimensional matrices results in the reciprocal induction of the cells to differentiate into simple organoid structures comprised of collecting duct–like epithelia with accompanying cells at their periphery in early stages of vasculogenesis and capillary differentiation. Mouse MM cells and UB cells (Probetex, San Antonio, TX) were grown and maintained at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as originally described by Wagner et al29Wagner B. Ricono J.M. Gorin Y. Block K. Arar M. Riley D. Choudhury G.G. Abboud H.E. Mitogenic signaling via platelet-derived growth factor beta in metanephric mesenchymal cells.J Am Soc Nephrol. 2007; 18: 2903-2911Crossref PubMed Scopus (27) Google Scholar and Ye et al.18Ye P. Habib S.L. Ricono J.M. Kim N.H. Choudhury G.G. Barnes J.L. Abboud H.E. Arar M.Y. Fibronectin induces ureteric bud cells branching and cellular cord and tubule formation.Kidney Int. 2004; 66: 1356-1364Crossref PubMed Scopus (33) Google Scholar The cells were characterized according to cell type as described previously18Ye P. Habib S.L. Ricono J.M. Kim N.H. Choudhury G.G. Barnes J.L. Abboud H.E. Arar M.Y. Fibronectin induces ureteric bud cells branching and cellular cord and tubule formation.Kidney Int. 2004; 66: 1356-1364Crossref PubMed Scopus (33) Google Scholar, 29Wagner B. Ricono J.M. Gorin Y. Block K. Arar M. Riley D. Choudhury G.G. Abboud H.E. Mitogenic signaling via platelet-derived growth factor beta in metanephric mesenchymal cells.J Am Soc Nephrol. 2007; 18: 2903-2911Crossref PubMed Scopus (27) Google Scholar and further examined by Western blot analysis and immunohistochemistry for additional mesenchymal and ureteric bud or collecting duct markers. For co-culture experiments, MM and UB cells were then trypsinized, washed with Hanks' balanced salt solution, mixed in equal numbers, and then reseeded in monolayer and examined for alterations in structure using mesenchymal and ureteric bud markers by immunofluorescence microscopy (see below). Additionally, the cells were grown to confluency, trypsinized, and then washed for subsequent growth in three-dimensional Matrigel implants as described below. Immunoblotting was performed as previously described.29Wagner B. Ricono J.M. Gorin Y. Block K. Arar M. Riley D. Choudhury G.G. Abboud H.E. Mitogenic signaling via platelet-derived growth factor beta in metanephric mesenchymal cells.J Am Soc Nephrol. 2007; 18: 2903-2911Crossref PubMed Scopus (27) Google Scholar, 30Faulkner J.L. Szcykalski L.M. Springer F. Barnes J.L. Origin of interstitial fibroblasts in an accelerated model of angiotensin II-induced renal fibrosis.Am J Pathol. 2005; 167: 1193-1205Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar Cells grown in monolayer to confluency were lysed in 0.5 mL of radioimmunoprecipitation assay (RIPA) buffer [50 mmol/L Tris-HCl (pH 7.5); 1 mmol/L EGTA; 140 mmol/L NaCl; 1.0% NP-40] containing 1 μg/mL leupeptin and aprotinin, 1 mmol/L sodium fluoride, 0.1 mmol/L sodium orthovanadate, and 1.0 mmol/L PMSF. Insoluble proteins were removed by centrifugation at 10,000 × g. Protein concentrations were determined using the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA). Protein lysates were boiled in sample buffer for 10 minutes, then equal amounts of samples were loaded onto 7.5% SDS-PAGE gels and electrophoretically separated. The proteins were transferred to polyvinylidene fluoride membranes using a Bio-Rad Trans-Blot cell followed by blocking with 5% nonfat dry milk in PBS containing 0.1% Tween 20 and incubated overnight in primary antibody diluted into ECL Advance Blocking Agent (Amersham Pharmacia Biotech, Piscataway, NJ). The antigens were detected and identified by enhanced chemiluminescence using standard enhanced chemiluminescence techniques as recommended by the manufacturer (Amersham). Signal was detected using a Syngene ChemiHR16 photo documentation system (Frederick, MD) or by film radiography. GAPDH or actin was used as loading control. Details of antibodies used for the identification of mesenchymal, endothelial, and tubular markers are listed in Table 1.Table 1Differentiation Markers: Antibody Sources, Targets, Species, and ConcentrationsMarkerPrimary antibodyTarget cellSourceSpecies/concentrationGeneral epithelialPancytokeratinEpithelialSanta Cruz BiotechnologyRabbit/10 μg/mLZO-1 (R26.4c)Epithelial tight junctionsDSHBRat (1:5)Collagen IVEpithelial basement membraneMilliporeRabbit/10 μg/mLUBRETUBSanta Cruz BiotechnologyRabbit/10 μg/mLD. biflorus lectinUB, collecting ductVector LaboratoriesLectinEndoA cytokeratinUB, collecting ductDSHBRat (1:50)Aquaporin 2Mature collecting ductSanta Cruz BiotechnologyGoat/10 μg/mLMMα-SMA (1A4)MM, pericytesSigma-AldrichMouse/10 μg/mLVimentin (V13.2)MM, pericytesSigma-AldrichMouse/10 μg/mLPDGFR-βMM, pericytesSanta Cruz BiotechnologyRabbit/10 μg/mLPECAMEndotheliumSanta Cruz BiotechnologyRabbit/10 μg/mLAquaporin 1Proximal tubule, limb of Henle, differentiating endotheliumSanta Cruz BiotechnologyRabbit/10 μg/mLAminopeptidaseProximal tubuleSanta Cruz BiotechnologyRabbit/10 μg/mLDSHB, Developmental Studies Hybridoma Bank; MM, metanephric mesenchyme; UB, ureteric bud. Open table in a new tab DSHB, Developmental Studies Hybridoma Bank; MM, metanephric mesenchyme; UB, ureteric bud. Each cell line was grown to 50% to 70% confluence in multiwell plastic Lab-Tek chamber microscope slides (Nalge Nunc International, Naperville, IL) and examined for expression of the mesenchymal, endothelial, and epithelial markers listed in Table 1, using previously described immunohistochemical techniques.8Ricono J.M. Xu Y.C. Arar M. Jin D.C. Barnes J.L. Abboud H.E. Morphological insights into the origin of glomerular endothelial and mesangial cells and their precursors.J Histochem Cytochem. 2003; 51: 141-150Crossref PubMed Scopus (46) Google Scholar, 13Simon M. Maresh J.G. Harris S.E. Hernandez J.D. Arar M. Olson M.S. Abboud H.E. Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney.Am J Physiol Renal. 1999; 276: F382-F389PubMed Google Scholar, 18Ye P. Habib S.L. Ricono J.M. Kim N.H. Choudhury G.G. Barnes J.L. Abboud H.E. Arar M.Y. Fibronectin induces ureteric bud cells branching and cellular cord and tubule formation.Kidney Int. 2004; 66: 1356-1364Crossref PubMed Scopus (33) Google Scholar, 21Barnes V.L. Musa J. Mitchell R.J. Barnes J.L. Expression of embryonic fibronectin isoform EIIIA parallels alpha-smooth muscle actin in maturing and diseased kidney.J Histochem Cytochem. 1999; 47: 787-798Crossref PubMed Scopus (22) Google Scholar, 30Faulkner J.L. Szcykalski L.M. Springer F. Barnes J.L. Origin of interstitial fibroblasts in an accelerated model of angiotensin II-induced renal fibrosis.Am J Pathol. 2005; 167: 1193-1205Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 31Arar M. Xu Y.C. Elshihabi I. Barnes J.L. Choudhury G.G. Abboud H.E. Platelet-derived growth factor receptor beta regulates migration and DNA synthesis in metanephric mesenchymal cells.J Biol Chem. 2000; 275: 9527-9533Crossref PubMed Scopus (30) Google Scholar The cells were washed in PBS and fixed in cold (−20°C) methanol for 5 minutes, then briefly rinsed with 0.02 mol/L phosphate-buffered saline (pH 7.4). The slides were blocked with PBS containing 0.1% bovine serum albumin, and then the specific protein of interest was detected by indirect immunofluorescence using primary antibodies (Table 1) followed by a Cy3- or FITC-labeled secondary antibody appropriate for the primary antibody (Millipore, Billerica, MA). The sections were viewed and photographed under epifluorescence microscopy using band-pass filters optimal for red or green wavelengths using an Olympus BX51 Research microscope equipped with a DP-71 digital camera (Melville, NY). Paired digital images representing each fluorochrome were color balanced and merged using Image-Pro 4.5 software as previously described.8Ricono J.M. Xu Y.C. Arar M. Jin D.C. Barnes J.L. Abboud H.E. Morphological insights into the origin of glomerular endothelial and mesangial cells and their precursors.J Histochem Cytochem. 2003; 51: 141-150Crossref PubMed Scopus (46) Google Scholar, 13Simon M. Maresh J.G. Harris S.E. Hernandez J.D. Arar M. Olson M.S. Abboud H.E. Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney.Am J Physiol Renal. 1999; 276: F382-F389PubMed Google Scholar, 18Ye P. Habib S.L. Ricono J.M. Kim N.H. Choudhury G.G. Barnes J.L. Abboud H.E. Arar M.Y. Fibronectin induces ureteric bud cells branching and cellular cord and tubule formation.Kidney Int. 2004; 66: 1356-1364Crossref PubMed Scopus (33) Google Scholar, 30Faulkner J.L. Szcykalski L.M. Springer F. Barnes J.L. Origin of interstitial fibroblasts in an accelerated model of angiotensin II-induced renal fibrosis.Am J Pathol. 2005; 167: 1193-1205Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 31Arar M. Xu Y.C. Elshihabi I. Barnes J.L. Choudhury G.G. Abboud H.E. Platelet-derived growth factor receptor beta regulates migration and DNA synthesis in metanephric mesenchymal cells.J Biol Chem. 2000; 275: 9527-9533Crossref PubMed Scopus (30) Google Scholar To test for phenotypic changes of MM and UB cells grown in two-dimensional co-culture, initial experiments were conducted in chamber slides in which the cells were grown together and compared to each cell line grown alone. The cells were allowed to grow for sequential time periods of 1, 2, and 3 days, and then fixed and stained by dual-label immunohistochemistry. MM and UB cells were detected by staining for vimentin and EndoA cytokeratin, respectively, using dual-label immunohistochemistry methods as previously described.8Ricono J.M. Xu Y.C. Arar M. Jin D.C. Barnes J.L. Abboud H.E. Morphological insights into the origin of glomerular endothelial and mesangial cells and their precursors.J Histochem Cytochem. 2003; 51: 141-150Crossref PubMed Scopus (46) Google Scholar, 30Faulkner J.L. Szcykalski L.M. Springer F. Barnes J.L. Origin of interstitial fibroblasts in an accelerated model of angiotensin II-induced renal fibrosis.Am J Pathol. 2005; 167: 1193-1205Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar Differentiation potential of MM and UB progenitor cells in three-dimensional co-culture was conducted in a similar fashion as described for adult kidney stem cells by Bussolati et al.28Bussolati B. Bruno S. Grange C. Buttiglieri S. Deregibus M.C. Cantino D. Camussi G. Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar For homogeneous suspensions, 1 × 106 cells of each line were dispersed in 250 μL of medium, then combined with an equal volume of cold Matrigel, and immediately injected subcutaneously into the nape of the neck of 6-week-old ICR-SCID mice (Taconic Farms, Hudson, NY). Co-culture was performed by mixing an equal number of each of the cell lines, not exceeding a combined total of 1 × 106. Handling of cells, supplies, and Matrigel was conducted on ice to prevent gelling of the matrix before implantation. Once injected, the Matrigel solidifies, with cells dispersed throughout the three-dimensional gel. At the end of the incubation period, the implant was excised and frozen or fixed for subsequent histological analysis as described below. All animal protocols were performed in accordance with National Institutes of Health guidelines and reviewed by the University of Texas Health Science Center Institutional Animal Care and Use Committee. After removal, the implants were fixed in 10% neutral-buffered formalin overnight then processed for paraffin embedment. Three-micron-thick sections were cut and stained with hematoxylin and eosin (H&E), and then viewed and photographed using an Olympus BX51 research microscope and DP71 digital camera. Assessment of the differentiation potential of the cells grown in the three-dimensional matrix showed varying degrees of organization characterized by no organization, development of small round aggregates of cells without lumens (spheroids), tubuloid structures with lumens, or profiles showing one or more spheroid or tubuloid cross sections surrounded by capillary-like cells (organoid). The degree of organization of the cells in 10-day implants was quantified by counting the number of each type of profile in three random fields/slide (×20 objective magnification) of at least three experiments. The MM and UB cells were labeled with PKH26 (red) or PKH67 (green) fluorescent linkers (Sigma Chemical Co., St Louis, MO) according to the manufacturer's instructions. In an additional experiment, the color labeling of the cells was reversed. Briefly, the cells were grown to confluence, detached with trypsin, and washed in serum-free medium using standard culture technique. A total of 2 × 107 cells were suspended in labeling diluent, then added to an equal volume of freshly prepared diluent containing PKH dye to make a final concentration of 2 × 10−6 mol/L at 25°C. The reaction was terminated by addition of buffer containing 1% bovine serum albumin followed by washing the cells

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