MUSE Stem Cells Can Be Isolated from Stromal Compartment of Mouse Bone Marrow, Adipose Tissue, and Ear Connective Tissue: A Comparative Study of Their In Vitro Properties

Abstract

The cells present in the stromal compartment of many tissues are a heterogeneous population containing stem cells, progenitor cells, fibroblasts, and other stromal cells. A SSEA3(+) cell subpopulation isolated from human stromal compartments showed stem cell properties. These cells, known as multilineage-differentiating stress-enduring (MUSE) cells, are capable of resisting stress and possess an excellent ability to repair DNA damage. We isolated MUSE cells from different mouse stromal compartments, such as those present in bone marrow, subcutaneous white adipose tissue, and ear connective tissue. These cells showed overlapping in vitro biological properties. The mouse MUSE cells were positive for stemness markers such as SOX2, OCT3/4, and NANOG. They also expressed TERT, the catalytic telomerase subunit. The mouse MUSE cells showed spontaneous commitment to differentiation in meso/ecto/endodermal derivatives. The demonstration that multilineage stem cells can be isolated from an animal model, such as the mouse, could offer a valid alternative to the use of other stem cells for disease studies and envisage of cellular therapies.

Keywords: adipose tissue; bone marrow; differentiation; fibroblasts; mesenchymal stem cells.

Figure 1

MUSE cells were isolated from bone marrow MSCs (bmMSC-MUSE), subcutaneous adipose tissue MSCs (aMSC-MUSE), and ears fibroblast (eFIB-MUSE). (A) Microscopy pictures of MUSE cell clusters showing different sizes. The picture shows representative samples. Scale bars = 50 µm. The graph displays the size of MUSE cell clusters. Each dot corresponds to one MUSE cell cluster. The red bar represents the mean size value in the different cell populations. (B) Representative microscopic field of SSEA3 staining (red) in MUSE cells. The nuclei were counterstained with DAPI (blue). The histogram shows the percentage of SSEA3-positive cells in each population. The data are expressed with standard deviation (n = 5). (C–E) Expression of CD44, CD90, and CD105 surface markers measured by flow cytometry in MUSE cells derived from mouse stromal tissues. The data are expressed with standard deviation (n = 5). We compared the three MUSE cell populations. The statistical differences among bmMSC-MUSE and the other MUSE cell populations are indicated with * (* p < 0.05).

Figure 2

MUSE cells isolated from different stromal tissues showed similar biological properties. (A) MUSE cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) colorimetric assay. (B) Representative cell cycle analysis of the MUSE cells isolated from different tissues. The percentages of different cell populations (G1, S, and G2/M) are indicated. (C) Representative analysis of MUSE cells apoptosis. The assay identifies early (Annexin V + and 7ADD −) and late (Annexin V + and 7ADD +) apoptosis. Apoptosis is a continuous process, and we calculated the percentage of apoptosis as the sum of early and late apoptotic cells. The histogram shows the mean percentage of Annexin V-positive cells. (D) The pictures show representative microscopic fields of senescence-associated beta-galactosidase-positive cells (blue) in MUSE cells cultures. The histograms show the percentage of senescent cells. All experimental data are represented as mean ± standard deviation (SD) of five independent replicates (n = 5). We compared the three MUSE cells populations. The statistical differences among bmMSC-MUSE and the other MUSE cell populations are indicated with * (* p < 0.05, ** p < 0.01).

Figure 3

(A) Representative cell cycle analysis of the non-MUSE cells isolated from different tissues. The percentages of different cell populations (G1, S, and G2/M) are indicated. (B) Representative analysis of non-MUSE cells’ apoptosis. The assay identifies early (Annexin V + and 7ADD −) and late (Annexin V + and 7ADD +) apoptosis. Apoptosis is a continuous process, and we calculated the percentage of apoptosis as the sum of early and late apoptotic cells. The histogram shows the mean percentage of Annexin V-positive cells. (C) The pictures show representative microscopic fields of senescence-associated beta-galactosidase-positive cells (blue) in non-MUSE cell cultures. The histograms show the percentage of senescent cells. All experimental data are represented as mean ± standard deviation (SD) of five independent replicates (n = 5). We compared the three non-MUSE cells’ populations.

Figure 4

MUSE cells expressed stem cell markers. (A) The pictures are representative images of immunocytochemistry for stemness markers: SOX2, OCT3/4, and NANOG, performed on bmMSC-MUSE, aMSC-MUSE and eFIB-MUSE. We performed ICC only with secondary antibodies, as reaction negative control. The nuclei were counterstained with DAPI (blue). The histograms show the percentage of SOX2, OCT3/4, and NANOG-positive cells. The data are expressed with standard deviation (n = 5). In the last column is reported the immunostaining performed only with secondary antibody (negative control). The bar indicates 50 μm. (B) mRNA expression levels of stem cell genes. The histograms show the quantitative RT-PCR analysis of a group of genes involved in stemness: SOX2, OCT3/4, NANOG and TERT. The mRNA levels were normalized to GAPDH mRNA expression, which was selected as an internal control. Histograms show expression levels in the different MUSE cell and non-MUSE cell populations. Data are expressed as fold changes with standard error (n = 5). For each gene, the expression level of bmMSC-MUSE is set as the baseline (the arbitrary value is 1). All experimental data are represented as mean ± standard deviation (SD) of five independent replicates (n = 5). We compared the three MUSE cell populations. The statistical differences among bmMSC-MUSE (first column) and the other MUSE cell population are indicated with * (* p < 0.05, ** p < 0.01, *** p < 0.001). The statistical differences among aMSC-MUSE (second column) and the other MUSE cell populations are indicated with # (# p < 0.05, ## p < 0.01).

Figure 5

Stem cell markers in non-MUSE cells. The pictures are representative images of immunocytochemistry for stemness markers: SOX2, OCT3/4, and NANOG, performed on bmMSC-non-MUSE, aMSC-non-MUSE and eFIB-non-MUSE. The nuclei were counterstained with DAPI (blue). The histograms show the percentage of SOX2, OCT3/4, and NANOG-positive cells. The data are expressed with standard deviation (n = 5). Scale bar = 50 µm.

Figure 6

MUSE cells showed spontaneous meso/endo/ectodermal differentiation capacity. (A) Representative picture of ICC staining after spontaneous differentiation of MUSE cells. Desmin (red) was used as a mesodermal marker; muscle tissue was used as positive control. CK-7 (red) was used as endodermal marker; liver tissue was used as positive control. NF-L (red) was used as ectodermal marker; brain tissue was used as positive control. We performed ICC only with secondary antibodies, as reaction negative control. The nuclei were counterstained with DAPI (blue). The histograms show the percentage of Desmin, CK-7, and NF-L-positive cells. The data are expressed with standard deviation (n = 5). (B) mRNA expression levels of meso/endo/ectodermal lineage markers. The histograms show the quantitative RT-PCR analysis of desmin (mesodermal marker), GLUT2 (endodermal marker), and MAP2 (ectodermal marker). The mRNA levels were normalized to GAPDH mRNA expression, which was selected as an internal control. Histograms show expression levels in the different MUSE cell, non-MUSE cell population, and tissues. Muscle, liver, and brain were used as reference tissues for mesodermal, endodermal, and ectodermal markers respectively. For each gene, the expression level of bmMSC-MUSE was set as the baseline (the arbitrary value is 1). All experimental data are represented as mean ± standard deviation (SD) of five independent replicates (n = 5). We compared the three MUSE cells populations. The statistical differences among bmMSC-MUSE (first column) and the other MUSE cell populations are indicated with * (* p < 0.05, ** p < 0.01). The statistical differences among aMSC-MUSE (second column) and the other MUSE cell populations are indicated with # (# p < 0.05, ## p < 0.01).

Figure 7

Representative picture of ICC staining after spontaneous differentiation of non-MUSE cells. Desmin (red) was used as a mesodermal marker. CK-7 (red) was used as endodermal marker. NF-L (red) was used as ectodermal marker. The nuclei were counterstained with DAPI (blue). The histograms show the percentage of desmin, CK-7, and NF-L positive cells. The data are expressed with standard deviation (n = 5).