Int J Stem Cells.  2020 Nov;13(3):394-403. 10.15283/ijsc20072.

Bone Marrow-Derived Mesenchymal Stem Cells Isolated from Patients with Cirrhosis and Healthy Volunteers Show Comparable Characteristics

• Lim YL
• Eom YW
• Park SJ
• Hong T
• Kang SH
• Baik SK
• Park KS
• Kim MY

• KMID: 2508913
• DOI: http://doi.org/10.15283/ijsc20072

Abstract

Background and Objectives
Autologous or allogeneic bone marrow-derived mesenchymal stem cells (BMSCs) have been applied in clinical trials to treat liver disease. However, only a few studies are comparing the characteristics of autologous MSCs from patients and allogeneic MSCs from normal subjects.
Methods and Results
We compared the characteristics of BMSCs (BCs and BPs, respectively) isolated from six healthy volunteers and six patients with cirrhosis. In passage 3 (P3), senescent population and expression of p53 and p21 were slightly higher in BPs, but the average population doubling time for P3–P5 in BPs was approximately 65.3±11.1 h, which is 18.4 h shorter than that in BCs (83.7±9.2 h). No difference was observed in the expression of CD73, CD90, or CD105 between BCs and BPs. Adipogenic differentiation slightly increased in BCs, but the expression levels of leptin, peroxisome proliferator-activated receptor γ, and CCAAT-enhancer-binding protein α did not vary between differentiated BCs and BPs. While ATP and reactive oxygen species levels were slightly lower in BPs, mitochondrial membrane potential, oxygen consumption rate, and expression of mitochondria-related genes such as cytochrome c oxidase 1 were not significantly different between BCs and BPs.
Conclusions
Taken together, there are marginal differences in the proliferation, differentiation, and mitochondrial activities of BCs and BPs, but both BMSCs from patients with cirrhosis and healthy volunteers show comparable characteristics.

Keyword

Mesenchymal stem cells; Cirrhosis; Proliferation; Differentiation; Mitochondria; Senescence

Figure

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  Fig. 1 Positive cell surface markers of BCs and BPs. Positive cell surface markers (CD73, CD90, and CD105) in passage 3 of BCs and BPs were analyzed by flow cytometry, and mean fluorescence intensity (MFI) was compared in BCs and BPs. (A) Positive expression of BMSC markers. (B) MFI of positive cell surface mar-kers. All data are shown as the mean± SE, n=6.

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  Fig. 2 Proliferation potentials of BCs and BPs. (A) Representative PDT changes in BC2 and BP1 during serial passages. (B) Mean PDT of P3, P4, and P5. *p<0.05 (n=6). (C) Cumu-lative cell number, (D∼F) Expression of transcription factors (Nanog, OCT4, Sox2) regulating proliferation. Expre-ssion level was evaluated by qPCR. n=6. (G) Colony forming unit–fibroblast (CFU-F) in normal and cirrhotic SD rat. The CFU-F units counted using a microscope. *p<0.05 (n=5). We defined a CFU-F unit as a colony consisting of more than 100 cells. All data are shown as the mean±SE.

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  Fig. 3 Cellular senescence of BCs and BPs. BCs and BPs of passage 3 were used to detect the cellular senescence and expression of its markers, p21 and p53. (A) SA-β-gal activity in P3 of BCs and BPs. SA-β-gal-positive cells were photographed (100× magnification) with a phase - contrast microscope and enumerated. At least 200 cells were counted from six different fields, and the percentage of positive cells is shown. *p< 0.05 (n=6). (B) Expression of senescence regulators, p21 and p53. All data are shown as the mean±SE (n=6).

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  Fig. 4 Adipogenic differentiation potentials of BCs and BPs. BCs and BPs at passage 3 were differentiated into adipocytes and stained with Oil Red O stain. *p<0.05. Adipogenesis was also evaluated by qPCR to detect adipogenic markers, Leptin, PPARγ, and C/EBPα. (A) Oil Red O staining. (B) qPCR for Leptin, PPARγ, and C/EBPα. All data are shown as the mean±SE (n=6). Adipo.: adipogene-sis.

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  Fig. 5 Parameters for mitochondrial activities of BCs and BPs. To analyze parameters for mitochondrial activities, BCs and BPs at passage 3 were used. (A) Mitochondrial DNA (mtD NA) copy number. (B) Mitochondrial membrane potential (MMP), **p< 0.01 and ***p<0.001. (C) ATP ge-neration. (D∼F) Mitochondrial biogenesis related-gene expression. (G) OCR of BCs. (H) OCR of BPs. (I) Relative OCR in BCs and BPs. (J∼L) ROS-regulating gene expression. (M) ROS generation. **p<0.01. All data are shown as the mean±SE (n=6).

Reference

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