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Keywords:

  • Mesenchymal stem cells;
  • Apoptosis;
  • Flow cytometry;
  • Cell culture

Rebuttal

  1. Top of page
  2. Rebuttal
  3. Disclosure of Potential Conflicts of Interest
  4. Author contributions
  5. References

Hui and colleagues have raised the concern that the detection of cell surface Annexin V staining on mesenchymal stem cells (MSC) following treatment with the enhancer of Zeste homology 2 (EZH2) inhibitor, DZNep (Sequoia Research, Pangbourne, United States, http://www.seqchem.com/) may have been compromised due to the use of Trypsin/EDTA to detach the adherent cells in our recent publication “EZH2 and KDM6A Act as an Epigenetic Switch to Regulate MSC lineage specification” [1]. Specifically, it is proposed that EDTA could affect the structure of the plasma membrane and chelate Ca2+ preventing the binding of Annexin V protein to the phosphatidylserine [2-4]. However, different manufacturers specify the use of Trypsin/EDTA, and not EDTA-free trypsin for detaching adherent cells prior to staining for Annexin V, where Trypsin/EDTA has been used by various studies for a wide range of adherent cell types [5-12].

While the Letter to the Editor by Hui and colleagues, published in this article, use the example that DZNep has been previously shown to induced apoptosis in human colon cancer stem cells [13], our study [1] found that DZNep had no effect on the apoptosis status of human bone marrow-derived MSC, in accord with other studies reporting that DZNeP is not harmful for nontransformed cells at tumor-inhibiting doses [14]. The discrepancy in DZNep sensitivity between normal stem cells and cancer stem cells may be due to aberrant levels of EZH2 in cancer cells required for survival [15, 16].

In order to determine whether EDTA influences Annexin V binding on MSC, we performed the Annexin V protocol with either Trypsin/EDTA (Sigma Aldrich, NSW, Australia, http://www.sigmaaldrich.com/australia.html) as described in our published study [1], or with EDTA-free Collagenase I (2 mg/ml Worthington Biochemical Corporation, NJ, United States of America, http://www.worthington-biochem.com/default.html). Two different human MSC cultures were treated with 5 µM of the potent apoptotic drug, doxorubicin (Sapphire Biosciences, NSW, Australia, http://www.sapphirebioscience.com/) for 24 hours prior to Annexin V staining. Single-cell suspensions were harvested by either Trypsin/EDTA or Collagenase I treatment then washed with Hanks Balanced Salt Solution (Sigma Aldrich, NSW, Australia, http://www.sigmaaldrich.com/australia.html) supplemented with 5% fetal calf serum (FCS; SAFC Biosciences, Sydney, NSW, Australia, http://www.safcglobal.com/safc-global/en-us/home.html). The cells were further washed twice with cold phosphate buffered saline prior to cell staining for Annexin V as specified in the manufacturer's protocol (Annexin V conjugates Alexa Fluro 488, for apoptosis detection, Life Technologies, VIC, Australia, http://www.lifetechnologies.com/au/). Flow cytometry was used to determine the levels of Annexin V staining under each condition. Cells treated with doxorubicin without Annexin V staining were used for negative gating. Flow cytometric analysis showed that there was no significant difference in the mean fluorescence levels between Trypsin/EDTA and collagenase I-treated MSC (Fig. 1). These results support recent findings by another group that reported no difference in Annexin V staining of apoptotic cells, following Trypsin/EDTA and Trypsin-free EDTA treatment, using two different adherent cell lines [6]. Therefore, we believe the use of Trypsin/EDTA for cell detachment has little influence on the measurement of early cell apoptosis by Annexin V staining on human MSC as we have previously reported [10].

image

Figure 1. Comparable expression of Annexin V on doxorubicin treated MSC detached by Trypsin/EDTA or Collagenase I. Two different human bone marrow-derived MSC cultures were treated with 5 µM doxorubicin for 24 hours. Single-cell suspensions were harvested by either Trypsin/EDTA or Collagenase I treatment then stained for Annexin V. Assessment of Annexin V levels was performed by flow cytometric analysis. Data represent mean fluorescence intensity from three replicate experiments. Abbreviation: MSC, mesenchymal stem cells.

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Disclosure of Potential Conflicts of Interest

  1. Top of page
  2. Rebuttal
  3. Disclosure of Potential Conflicts of Interest
  4. Author contributions
  5. References

The authors indicate no potential conflicts of interest.

Author contributions

  1. Top of page
  2. Rebuttal
  3. Disclosure of Potential Conflicts of Interest
  4. Author contributions
  5. References

S.H.: experimental design, experimental procedures, data analysis, manuscript preparation; D.C.: experimental design, experimental procedures, data analysis, manuscript preparation; S.G.: project development, experimental design, data analysis, manuscript preparation, financial support.

References

  1. Top of page
  2. Rebuttal
  3. Disclosure of Potential Conflicts of Interest
  4. Author contributions
  5. References
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