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. 2018 Oct-Dec;41(4):870-877.
doi: 10.1590/1678-4685-GMB-2018-0012. Epub 2018 Nov 29.

Isolation and characterization of mesenchymal stem/stromal cells from Ctenomys minutus

Mayra Ramos de Jesus Pereira  1 , Valéria Rodrigues Pinhatti  1 , Maiele Dornelles da Silveira  1 , Cristina Araujo Matzenbacher  2 , Thales Renato Ochotorena de Freitas  2 , Juliana da Silva  1 , Melissa Camassola  1 , Nance Beyer Nardi  1
Affiliations

Isolation and characterization of mesenchymal stem/stromal cells from Ctenomys minutus

Mayra Ramos de Jesus Pereira et al. Genet Mol Biol. 2018 Oct-Dec.

Abstract

Mesenchymal stem/stromal cells (MSCs) are multipotent cells distributed in all tissues and characterized by adherence, morphology, immunophenotype and trilineage differentiation potential. The present study aimed to isolate and characterize adherent MSC-like populations from different tissues of Ctenomys minutus, a threatened wildlife rodent popularly known as tuco-tuco. Adherent cells were isolated from bone marrow, brain, liver, pancreas and adipose tissue of three adult animals collect in southern Brazil. Cultures showed typical morphology and proliferation potential. Adipose-derived MSCs showed trilineage potential. Cultures derived from adipose tissue, bone marrow and brain were immunophenotyped with negative results for CD31, CD44, CD45, CD106, and MHC class II, as well as strong positive results for CD29. Low fluorescence levels were seen for CD49d, CD90.2 and CD117. Cultures were negative for CD49e, except for brain-derived cultures that were weakly positive. CD11b was negative in adipose-derived MSCs, but positive in brain and bone marrow-derived cultures. The scratch assay showed high migration potential for pancreas and adipose tissue-derived cells. This study represents the first report of isolation and characterization of cultures having characteristics of MSCs from Ctenomys minutus. The collection of biological information for biobanks represents an important contribution to the creation of strategies for prevention of loss of genetic diversity.

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Figures

Figure 1
Figure 1. Morphology of cultures. Cultures established from all organs and tissues presented the typical fibroblastoid morphology of mesenchymal stem/stromal cells: (A) brain; (B) adipose tissue; (C) bone marrow; (D) liver; (E) pancreas. Scale bar = 50 μm.
Figure 2
Figure 2. Mean values of population doublings. Viable cells were counted at each passage (P3 to P8) of cultures derived from adipose tissue (n = 3), pancreas (n = 2), bone marrow (BM, n = 2) and brain (n = 2), for determination of population doubling times.
Figure 3
Figure 3. Differentiation potential of adipose-derived MSCs. Representative results show trilineage differentiation potential of cultures. Scale bar = 50 μm.
Figure 4
Figure 4. Immunophenotype of adipose-derived (n = 3) and brain- and bone marrow (BM)-derived (n = 2) MSCs. Representative results show that all cultures were negative for CD31, CD44, CD45, CD106, and MHC class II. Cultures were strongly positive for CD29 and weakly positive for CD49d, CD90.2, and CD117. Adipose- and BM-derived MSCs were negative for CD49e, and brain (Br)-derived cells were weakly positive. Two of the anti-CD11b antibodies tested showed positive marking for Br- and BM-derived cultures.
Figure 5
Figure 5. In vitro scratch assay. Migration of cells into a scratch produced in adipose tissue and pancreas-derived cultures from two donors (#1 and #2) was evaluated by images recorded at 0, 24, 48, and 72 h. The percent of closed scratch (or “healed wound”) was determined with ImageJ software. Values are mean ± standard deviation of three independent experiments.
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