A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures.

We have generated a monoclonal antibody (R-10G) specific to human induced pluripotent stem (hiPS)/embryonic stem (hES) cells by using hiPS cells (Tic) as an antigen, followed by differential screening of mouse hybridomas with hiPS and human embryonal carcinoma (hEC) cells. Upon western blotting with R-10G, hiPS/ES cell lysates gave a single but an unusually diffuse band at a position corresponding to >250 kDa. The antigen protein was isolated from the induced pluripotent stem (iPS) cell lysates with an affinity column of R-10G. The R-10G positive band was resistant to digestion with peptide N-glycanase F (PNGase F), neuraminidase, fucosidase, chondrotinase ABC and heparinase mix, but it disappeared almost completely on digestion with keratanase, keratanase II and endo-ß-galactosidase, indicating that the R-10G epitope is a keratan sulfate. The carrier protein of the R-10G epitope was identified as podocalyxin by liquid chromatography/mass spectrometry (LC/MS/MS) analysis of the R-10G positive-protein band material obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R-10G epitope is a type of keratan sulfate with some unique properties. (1) The epitope is expressed only on hiPS/ES cells, i.e. not on hEC cells, unlike those recognized by the conventional hiPS/ES marker antibodies. (2) The epitope is a type of keratan sulfate lacking oversulfated structures and is not immunologically cross-reactive with high-sulfated keratan sulfate. (3) The R-10G epitope is distributed heterogeneously on hiPS cells, suggesting that a single colony of undifferentiated hiPS cells consists of different cell subtypes. Thus, R-10G is a novel antibody recognizing hiPS/ES cells, and should be a new molecular probe for disclosing the roles of glycans on these cells.

Effect of chondroitinase ABC on matrix metalloproteinases and inflammatory mediators produced by intervertebral disc of rabbit in vitro.

STUDY DESIGN: Lumbar intervertebral discs in rabbit were cultured in the presence of chondroitinase ABC. The matrix metalloproteinases (MMPs) and inflammatory mediators produced in culture media were then analyzed. OBJECTIVES: To investigate the effect of chondroitinase ABC on MMPs and inflammatory mediators produced by intervertebral disc of rabbit in vitro. SUMMARY OF BACKGROUND DATA: The chemonucleolytic effect of chondroitinase ABC is caused by the decrease in the chondroitin sulfate, hyaluronan, and protein content of the nucleus pulposus in rabbit. The reason for the decreases in protein content remains unclear. METHODS: Anulus fibrosus and nucleus pulposus were cultured for 72 hours with or without chondroitinase ABC stimulated or not stimulated by interleukin-1 after preculture for 4 days. Subsequently, the MMPs (gelatinases MMP-2, MMP-9, and collagenase) and inflammatory mediators (prostaglandin E2 and nitric oxide) produced in the culture media were analyzed. RESULTS: In the anulus fibrosus chondroitinase ABC and interleukin-1 synergistically increased the collagenase activity, which was at a significantly higher level than the increment solely due to interleukin-1. In contrast, chondroitinase ABC counteracted the increase in nitric oxide production by interleukin-1. In the nucleus pulposus the collagenase and nitric oxide productions were not particularly affected by chondroitinase ABC and/or interleukin-1. In zymographic analysis MMP-2 was detected, but MMP-9 was only slightly detected in both tissues. There were no significant differences in both tissues for MMP-2 and prostaglandin E2 following incubation with or without chondroitinase ABC, whether stimulated by interleukin-1 or not. CONCLUSIONS: The collagenase activity in the anulus fibrosus was increased by chondroitinase ABC with interleukin-1. This finding may support the hypothesis that some proteolytic activities are involved in the chemonucleolytic process by chondroitinase ABC treatment.