Human MSC isolation by MACS – direct comparison of bone marrow versus adipose tissue

by Alexey Bersenev on June 26, 2015 · 0 comments

in adipose, mesenchymal

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Isolation of mesenchymal stromal cells (MSC) from tissues by single step (FACS or MACS) is a good alternative to commonly used adherence-based method. It allows to purify more homogeneous rapidly proliferating population of MSCs. However, it involves use of antibodies and special equipment (sorter). Approaches to sorting MSCs for clinical use are commercialized by some companies (ex: Mesoblast and Orbsen Therapeutics). Despite results of multiple studies, it remains unclear what the best marker or combination of markers for sorting of MSC from particular human tissue. Good marker for bone marrow MSC could not work well for adipose tissue or umbilical cord blood. The recent study, which appears online on Stem Cells and Development journal web-site, investigates these differences between bone marrow (BM) and adipose tissue (AT).

Researchers checked the following markers: MSCA-1, SUSD2, CD271, CD34 and CD44. All populations were isolated by MACS without attempts to increase purity (double sort, depletion of CD45+). Both positive and negative fractions were tested for CFU-F clonogenicity post-MACS. I really like this approach, because it shows how these population are pure for particular marker.

The authors demonstrated that BM MSC can be MACS-sorted by MSCA-1, SUSD2 and CD271 markers with very similar recoveries (0.6-07%). Overall, enrichment by CFU-F colony assay for these 3 markers was ~30 fold, compare to unfractioned marrow. All 3 fractions were highly proliferative in culture with MSCA-1 as a best performer. MSCA-1 demonstrated the highest enrichment ~50-fold on average with max 70-fold and expansion rate of 20×109 after 2 passages (expansion of unselected cells was 100 times less). Purities of MSC, isolated by MACS were lower in relation to described purities by FACS. For example, for CD271 only ~40-50%.

Contrary to BM, the authors were not able to enrich MSC from AT by MSCA-1 and SUSD2. Interestingly, CD271 was good marker for MSC, isolated from lipoaspirates, but no colonies were observed from CD271-selected MSC from resected abdominal fat tissue. Another interesting finding is that only CD34+ was highly selective marker for AT-derived MSC, because was no colonies detected in residual CD34- fraction. Selection by any other marker resulted in detection of CFU-F colonies in both – positive and negative fractions. CD90 and CD146 were significantly enriched in CD34+ and CD271+ fraction of AT, but not in the same fractions of BM.

CD34+ MSC with great clonogenicity were also purified from BM. Therefore, CD34 was the only universal MSC marker for both BM and AT. The study confirms previously described phenomenon – rapid disappearance of CD34+ expanded MSC culture. Enrichment of CD34+ cells was detected in CD271+ fraction for both – BM and AT. Unlike BM, only proliferation of CD34+ MSC was greater than plated unfractioned cells.

One thing is not clear to me – why didn’t they deplete CD45+ before CD34 selection? How can they exclude contamination by hematopoietic cells? CD45+ coexpression was very high on all fractions, isolated from BM.

Overall, it is very interesting study. It highlights significant differences in MSC markers in different tissues in situ. The paper also discusses technical aspects of human MSC isolation by MACS. This study is highly recommended to all “MSC folks”!

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