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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.

PH20 is not expressed in murine CNS and oligodendrocyte precursor cells