title: Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.
abstract: The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel activity comparable to bladder SMCs which may be important for urological regenerative medicine applications.
authors.affiliation: Clinical Research Group KFO 273, Department of Urology, University of Tübingen, Tübingen, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Brun
authors.affiliation: Clinical Research Group KFO 273, Department of Urology, University of Tübingen, Tübingen, Germany.
authors.forename: Katrin A
authors.initials: KA
authors.lastname: Lutz
authors.affiliation: Clinical Research Group KFO 273, Department of Urology, University of Tübingen, Tübingen, Germany.
authors.forename: Katharina M H
authors.initials: KM
authors.lastname: Neumayer
authors.affiliation: Center for Medical Research, University Medical Clinic, Department II, University of Tübingen, Tübingen, Germany.
authors.forename: Gerd
authors.initials: G
authors.lastname: Klein
authors.affiliation: Center for Medical Research, University Medical Clinic, Department II, University of Tübingen, Tübingen, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Seeger
authors.affiliation: Siegfried Weller Institute for Trauma Research, Laboratory for Molecular Biomechanics, University of Tübingen, Tübingen, Germany.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Uynuk-Ool
authors.affiliation: Siegfried Weller Institute for Trauma Research, Laboratory for Molecular Biomechanics, University of Tübingen, Tübingen, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Wörgötter
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tübingen, Department of Electrophysiology, Reutlingen, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Schmid
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tübingen, Department of Electrophysiology, Reutlingen, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Kraushaar
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tübingen, Department of Electrophysiology, Reutlingen, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Guenther
authors.affiliation: Siegfried Weller Institute for Trauma Research, Laboratory for Molecular Biomechanics, University of Tübingen, Tübingen, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Rolauffs
authors.affiliation: Clinical Research Group KFO 273, Department of Urology, University of Tübingen, Tübingen, Germany.
authors.forename: Wilhelm K
authors.initials: WK
authors.lastname: Aicher
authors.affiliation: Clinical Research Group KFO 273, Department of Urology, University of Tübingen, Tübingen, Germany.|Siegfried Weller Institute for Trauma Research, Laboratory for Molecular Biomechanics, University of Tübingen, Tübingen, Germany.
authors.forename: Melanie L
authors.initials: ML
authors.lastname: Hart
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000200:Action Potentials; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D006801:Humans; D007473:Ion Channels; D017136:Ion Transport; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D001743:Urinary Bladder / Q000166:cytology; D000096985:Calponins
chemical_list: D000199:Actins; D002118:Calcium; D002135:Calcium-Binding Proteins; D007473:Ion Channels; D008840:Microfilament Proteins; D009124:Muscle Proteins; D018995:Myosin Heavy Chains; D011188:Potassium; D012964:Sodium; D000096985:Calponins; C541116:ACTA2 protein, human; C509903:MYH11 protein, human; C051615:transgelin