Using molecular beacons for isolation of human osteogenic progenitors from adipose SVF

by Alexey Bersenev on October 9, 2014 · 1 comment

in adipose, methods

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Molecular beacons (MB) is a new technique for cell isolation, based on capture of genetic (intracellular or intranuclear) markers. MB have been used in research for isolation of embryonic stem cells and cardiomyocytes since 2011. I’m dreaming about using MB for clinical cell isolation. Recently, a group of researchers from Brown University published a study, which makes one step closer to clinical application of MB.

The authors used MB, which captures mRNA of alkaline phosphatase (ALPL) in osteogenic progenitors, derived from human adipose tissue stromal vascular fraction (SVF). ALPL is a good intracellular marker of osteogenic lineage. MB has a fluorescent probe, which “activates” when beacon binds to target mRNA. Therefore, FACS-based sorting could be used as the next step of cell purification.

Some observations from this study, related to potential clinical application:

  • MB sorting required “cell culture priming” (will be regulated as “a drug” and require GMP facility), electroporation (clinical electroporation is very expansive) and FACS sorting (not on “clinical market” yet).
  • ALPL beacon-based sorting did not deplete multipotent cells – besides enrichment for osteogenic progenitors, cells also differentiated into chondro-/ adipo- lineages under appropriate conditions.
  • Importantly, ALPL beacon did not affect cell differentiation and growth and degraded with time.
  • ALPL sorted cells outcompete all controls (unsorted, non-primed, sorted via surface markers) in “osteogenic assays” in vitro, but in vivo comparison was not performed. So, we still don’t know if MB can provide us superior “therapeutic value”.
  • ALPL gene expression-based sorting allows to get more purified mesenchymal stromal cells than traditional FACS sorting via surface markers.
  • ALPL sorting allows to overcome donor-to-donor variability in differentiation capacity.
  • Osteogenesis of ALPL+ cells, sorted directly from fresh SVF (2 donors) was not different from those SVF, primed in culture. So, maybe we don’t need any culture!
  • ALPL+ cells, sorted from ex vivo propagated adipose MSC (~81%) were still more potent than unsorted cells.
  • Non-specific fluorescence is a still a limitation of MB technique.

I think, some controls were not adequate, for example, ALPL+ sorted cells versus sorted CD34+/CD45-/CD31- SVF cells after osteogenic priming in culture. I didn’t find a fresh total SVF control. In the future work, multiple controls and in vivo studies are important in order to evaluate advantages of MB in comparison with conventional techniques.

{ 1 comment… read it below or add one }

Touret February 10, 2015 at 5:35 am

Hello, first of all congratulations, I’m very interested in this study, as part of my doctoral thesis, where can I find it? (in pdf)
thank you in advance

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