XB-ART-58033Stem Cell Res 2021 May 01;53:102341. doi: 10.1016/j.scr.2021.102341.
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Isolation and characterization of bone marrow-derived mesenchymal stem cells in Xenopus laevis.
Mesenchymal stem cells (MSCs) are multipotent cells that exist in mesenchymal tissues such as bone marrow and are able to differentiate into osteocytes, chondrocytes, and adipocytes. MSCs are generally collected as adherent cells on a plastic dish, and are positive for markers such as CD44, CD73, CD90, CD105 and CD166, and negative for CD11b, CD14, CD19, CD31, CD34, CD45, CD79a and HLA-DR. MSCs have been established from many kinds of mammals, but MSCs from amphibians have not yet been reported. We cultured adherent cells from the bone marrow of Xenopus laevis by modifying the protocol for culturing mammalian MSCs. The morphology of these cells was similar to that of mammalian MSCs. The amphibian MSCs were positive for cd44, cd73, cd90 and cd166, and negative for cd11b, cd14, cd19, cd31, cd34, cd45, cd79a and hla-dra. Moreover, they could be induced to differentiate into osteocyte-, chondrocyte-, and adipocyte-lineage cells by cytokine induction systems that were similar to those used for mammalian MSC differentiation. Thus, they are considered to be similar to mammalian MSCs. Unlike mammals, amphibians have high regenerative capacity. The findings from the present study will allow for future research to reveal how Xenopus MSCs are involved in the amphibian regenerative capacity and to elucidate the differences in the regenerative capacity between mammals and amphibians.
PubMed ID: 33892293
Article link: Stem Cell Res
Species referenced: Xenopus laevis
Genes referenced: alcam bglap2 cd19 cd34 cd44 cd79a eng hla-dra itgam msc nt5e odc1 pecam1 pparg ptprc slc26a3.1 thy1
GO keywords: chondrocyte differentiation
Article Images: [+] show captions
|Fig. 1. Isolation of bone marrow stromal cells from Xenopus laevis. (A) Froglets with a snout-vent length of ~ 5 cm were used. Scale bar = 2 cm. (B) Five froglets were used for each 10-cm plastic dish. (C) Two femurs and two tibiofibulas were collected from each froglet. #1 and #2 indicate femurs, and #3 and #4 indicate tibiofibulas. Black arrowhead indicates the removed femur #1. Scale bar = 2 cm. (D) Ten femurs and Ten tibiofibulas were collected from 5 froglets. (E) The collected bones were crushed in the mortar. (F) Crushed bones were treated with collagenase and dispase. The mixture was filtered through the cell strainer, then centrifuged. Finally, the bone marrow stromal cell pellet was collected (black arrowhead).|
|Fig. 2. Characteristics of Xl-BM-MSCs. (A) Comparison of the cell morphology between Xl-BM-MSCs and human BM-MSCs (scale bar = 500 μm). The black box is a magnified view (scale bar = 100 μm). (B) Comparison of the cell morphology among passaged Xl BM-MSCs. Black arrowheads indicated cells with flat shape and spread cytoplasm (scale bar = 500 μm). The black box is a magnified view (scale bar = 100 μm). (C) Growth curve of Xl-BM-MSCs. Cells growth was observed until P13. Data show the mean of triplicate independent samples and the error bars represent the SD. (D) Growth curve and PDT of each passage. Cells were reseeded in 6-well culture plates at a density of 1 × 105 cells/well to observe cell growth and PDT at each passage. PDT in the log phase (cells increased exponentially) was calculated by the formula: PDT = T × ln(2)/ln(Xe/Xb). T: log phase time (from day 1 to day 3). Xb: cell number at the beginning of log phase. Xe: cell number at the end of log phase. Data show the mean of triplicate independent samples and error bars represent the SD. (E) CFU assay of Xl-BM-MSCs. 2 × 105 fresh Xl-BM-derived mononuclear cells seeded in a 10-cm plastic dish. After 12 days, the cells were fixed and stained with Giemsa stain solution.|
|Fig. 3. Expression of cd markers. odc was used as an internal control for Xenopus laevis. Data show the mean of triplicate independent samples and error bars represent the SD. Statistical significance was calculated by a 2-tailed Student t-test, and defined as *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with each positive control, or †, p < 0.05; ††, p < 0.01; †††, p < 0.001 compared with each negative control. N.D. means not detected.|
|Fig. 4. Differentiation capacity of Xl-BM-MSCs. (A) Each sample was stained with Alizarin Red S (red color) (a-d), Alcian Blue (blue color) combined with hematoxylin (bluish purple color) (e-h), and Oil Red O (red color) (i-l). (a, e, i) Naïve Xl-BM-MSCs before induction. Scale bar = 200 μm. The black box is a magnified view (scale bar = 100 μm). (b, f, j) Samples after differentiation (scale bar = 100 μm). The black box is a magnified view (scale bar = 50 μm). Black arrowhead indicates chondrocyte. (c, g, k) Positive controls for each staining. Scale bar = 100 μm. The black box is a magnified view (scale bar = 25 μm). Black arrowhead indicates chondrocytes. (d, h, l) Negative controls for each staining. Scale bar = 100 μm. The black box is a magnified view (scale bar = 25 μm). (B) RT-PCR of differentiation marker expression. osteocalcin, collagen type Ⅱ, and pparg were used as osteocyte, chondrocyte, and adipocyte markers, respectively. odc was used as an internal control.|