VSEL cell controversy – testing pluripotency markers

by Alexey Bersenev on July 13, 2013 · 1 comment

in other adult stem cells

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Very Small Embryonic-Like (VSEL) cell population was described as a “pluripotent stem cells” (PSC) in adult tissues of both – mice and human. In the last few years, some independent laboratories reproduced the original protocol for VSEL cell isolation and characterization, but some did not succeed. The question of reproducibility and, validity of VSEL cell as a real stem cell population is still open.

One of the most controversial issues in VSEL story is validation of their pluripotency. Mariusz Ratajczak‘s group labeled VSEL as “pluripotent stem cells”, based on expression of “classical” embryonic stem cell markers (Oct-4, SSEA-4, Sox-2, Nanog…). They also demonstrated the differentiation potential in vitro of VSEL into variety of cell types of all 3 germ layers. Recently, very interesting study was published by Krause’s group, demonstrating differentiation of VSEL in lung cells in vivo.

However, VSEL cells failed the most rigorous test for pluripotency – teratoma formation. Yet another rigorous in vivo test – blastocyst (embryo) chimerization was never described. We don’t have data from “standard gene profiling tests” – Pluritest and recent TaqMan hPSC Scorecard.

In 3 recent independent studies, the expression of pluripotency markers by VSEL cells was challenged.

Jozef Dulak’s group studied expression of pluripotency marker Oct-4 in murine bone marrow-derived VSEL in details. They concluded:

… using qRT-PCR, we evidenced lack of Oct-4A and Oct-4B mRNA expression either in whole adult murine bone marrow or in the sorted of Lin−Sca-1+CD45−FSClow population, even by single-cell qRT-PCR.

To control the experiment, Dulak’s team used exactly the same PCR primers as Ratajczak‘s group. Interestingly, they were able to detect Oct-4 in whole BM and in sorted VSEL, using those primers. However, they showed that the results are false positive:

We suppose that disparity between current and earlier analyzes of Oct-4 expression in VSELs can result from application of different primer sequences. False-positive findings caused by detection of Oct-4 pseudogenes with non-specific primers have already been evidenced [18], [53], [54]. Here we demonstrated that primers used in the first paper by Kucia and co-workers [6] and then in the other reports [9], [24] may easily produce false positive results. Using them, we obtained an Oct-4 signal of expected length both in whole bone marrow and in sorted Lin−Sca-1+CD45−FSClow VSELs, however, the product was transcribed from a pseudogene template. This false-positive reaction was not effectively prevented by DNase I treatment, what is in agreement with data described by Wang and Dai [55].

They discuss all trickery and art of Oct-3/4 detection in the paper. I’d highly recommend you to read this discussion. Additionally, Dulak’s group was not able to reproduce hematopoietic differentiation of VSEL cells on OP-9 stroma and concluded that this cell population can not be called “stem cells”.

Madrigal’s group was not able to detect SSEA-4 expression, but detected Oct-3/4, Sox-2 and Nanog transcripts in human cord blood-derived VSEL. Also, unlike original Ratajczak’s data, they didn’t observed any CD133+ expression on the surface of VSEL cells. The authors were not able to maintain VSEL cells in culture. They concluded:

These populations have been widely called “embryonic-like stem cell” on the basis of their phenotypical similarity to embryonic stem cells. However, the fact they do not seem to be able to self-renew casts some doubt on their identity, and warns against defining them as “embryonic-like stem cell” at this stag

Finally, Alt’s group was not able to detect any pluripotency markers, including Oct-4, Nanog and Sox-2 in human cord blood-derived VSEL. As Madrigal’s group, they were not able to detect CD133 expression of the surface of VSEL and were not able to maintain them in culture in the media, appropriate for growth or pluripotent stem cells. They concluded:

These data support neither an embryonic nor an adult stem cell like phenotype, suggesting rather that hUCB VSEL cells are an aberrant and inactive population that is not comparable to murine VSEL cells.

This controversy demonstrates, that we can not make valid conclusion about pluripotency of cells, based only on markers. Markers expression is very frequent cell culture and staining artifact. All 3 studies, mentioned above, dig more into VSEL population heterogeneity, single cell analysis and used variety of assays to explain possible disparity between studies. I’d encourage you and highly recommend to read all 3 papers.

{ 1 comment… read it below or add one }

Alexey Bersenev July 17, 2013 at 12:43 am

I’ve received this comment via email from Jozef Dulak. I’m posting it with his permission.

The Oct-4 expression in adult stem cells is a matter of debate. Due to the presence of pseudogenes and different isoforms of Oct-4, it is necessary to exclude false positives. In our work (Szade et al. PLoS One 2013) we used the primers proposed by Mizuno et al. (J Biol Chem 2008, 283: 30997–31004). In our hands, these primers did not amplify Oct-4 pseudogenes on genomic DNA template. In contrast, we showed that primers used by Kucia et al. in Leukemia 2006 (Leukemia 20: 857–869. doi:10.1038/sj.leu.2404171) amplified the sequence also in no reverse-transcription control as well as on genomic DNA template what we discussed in details in our paper.

As in the published version of Dr. Krause’s paper we were not able to find the sequence of primers, we asked her for such an information. Dr Krause has kindly provided us those sequences and informed that her group has used the same primers as Kucia et al (Leukemia 20: 857–869. doi:10.1038/
sj.leu.2404171). Moreover, we noted that the antibody they used (mouse anti-Oct-4 Antibody, clone 10H11.2 | MAB4401, Milipore) is described by manufacturer’s data sheet: “[…] clone reacts strongly with specific nuclear binding to human ES cells and cross-reacts weakly with mouse ES cells” (Dr. Krause group has investigated mouse VSELs). Therefore, we are concerned if Oct-4 detection showed by Krause et al. is specific.
We did not test primers for human Oct-4 applied in the new paper by Madrigal’s group (Alvarez-Gonzalez et al. in PLoS One 2013). Those primers, as the authors indicate, come from the paper by Guasti I. et al (Stem Cells Translational Medicine 1: 384–395.). However, examination of the specificity by Primer Blast showed that these primers may give products of the same length on genomic DNA template. Nevertheless, despite the Oct-4 detection, Alvarez-Gonzales et al. paper did not confirm the stem cell capacity of VSELs.

We believe that detection of expression of genes like Oct-4 or Nanog, which are known to exist in various isoforms and possess numerous pseudogenes should be done very carefully. Moreover, the mere expression of any of those markers could not be considered as the proof for pluripotency.

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