Open AccessPH20 is not expressed in murine CNS and oligodendrocyte precursor cells
Author(s) -
Marella Mathieu,
Ouyang Joe,
Zombeck Jonathan,
Zhao Chunmei,
Huang Lei,
Connor Robert J.,
Phan Kim B.,
Jorge Michael C.,
Printz Marie A.,
Paladini Rudolph D.,
Gelb Arnold B.,
Huang Zhongdong,
Frost Gregory I.,
Sugarman Barry J.,
Steinman Lawrence,
Wei Ge,
Shepard H. Michael,
Maneval Daniel C.,
Lapinskas Paula J.
Publication year - 2017
Publication title -
annals of clinical and translational neurology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.824
H-Index - 42
ISSN - 2328-9503
DOI - 10.1002/acn3.393
Subject(s) - in vitro , recombinant dna , microbiology and biotechnology , in vivo , basic fibroblast growth factor , oligodendrocyte , immunolabeling , biology , biochemistry , chemistry , central nervous system , myelin , immunology , immunohistochemistry , growth factor , gene , receptor , endocrinology
Objective Expression of Spam1 / PH 20 and its modulation of high/low molecular weight hyaluronan substrate have been proposed to play an important role in murine oligodendrocyte precursor cell ( OPC ) maturation in vitro and in normal and demyelinated central nervous system ( CNS ). We reexamined this using highly purified PH 20. Methods Steady‐state expression of mRNA in OPC s was evaluated by quantitative polymerase chain reaction; the role of PH 20 in bovine testicular hyaluronidase ( BTH ) inhibition of OPC differentiation was explored by comparing BTH to a purified recombinant human PH 20 ( rHuPH 20). Contaminants in commercial BTH were identified and their impact on OPC differentiation characterized. Spam1 / PH 20 expression in normal and demyelinated mouse CNS tissue was investigated using deep RNA sequencing and immunohistological methods with two antibodies directed against recombinant murine PH20. Results BTH , but not rHuPH 20, inhibited OPC differentiation in vitro. Basic fibroblast growth factor ( bFGF ) was identified as a significant contaminant in BTH , and bFGF immunodepletion reversed the inhibitory effects of BTH on OPC differentiation. Spam1 mRNA was undetected in OPC s in vitro and in vivo; PH 20 immunolabeling was undetected in normal and demyelinated CNS. Interpretation We were unable to detect Spam1 / PH 20 expression in OPC s or in normal or demyelinated CNS using the most sensitive methods currently available. Further, “ BTH ” effects on OPC differentiation are not due to PH 20, but may be attributable to contaminating bFGF . Our data suggest that caution be exercised when using some commercially available hyaluronidases, and reports of Spam1 / PH 20 morphogenic activity in the CNS may be due to contaminants in reagents.