Adipose Tissue and Umbilical Cord Tissue: Potential Sources of Mesenchymal Stem Cells for Liver Fibrosis Treatment

Abstract

Background/aims: Mesenchymal stem cells (MSCs) are potential alternatives for liver fibrosis treatment; however, their optimal sources remain uncertain. This study compares the ex-vivo expansion characteristics of MSCs obtained from adipose tissue (AT) and umbilical cord (UC) and assesses their therapeutic potential for liver fibrosis treatment.

Methods: Since MSCs from early to mid-passage numbers (P2-P6) are preferable for cellular therapy, we investigated the growth kinetics of AT-MSCs and UC-MSCs up to P6 and evaluated their therapeutic effects in a rat model of liver fibrosis induced by diethylnitrosamine.

Results: Results from the expansion studies demonstrated that both cell types exhibited bona fide characteristics of MSCs, including surface antigens, pluripotent gene expression, and differentiation potential. However, AT-MSCs demonstrated a shorter doubling time (58.2 ± 7.3 vs. 82.3 ± 4.3 h; P < 0.01) and a higher population doubling level (10.1 ± 0.7 vs. 8.2 ± 0.3; P < 0.01) compared to UC-MSCs, resulting in more cellular yield (230 ± 9.0 vs. 175 ± 13.2 million) in less time. Animal studies demonstrated that both MSC types significantly reduced liver fibrosis (P < 0.05 vs. the control group) while also improving liver function and downregulating fibrosis-associated gene expression.

Conclusion: AT-MSCs and UC-MSCs effectively reduce liver fibrosis. However, adipose cultures display an advantage by yielding a higher number of MSCs in a shorter duration, rendering them a viable choice for scenarios requiring immediate single-dose administration, often encountered in clinical settings.

Keywords: adipose tissue; cellular therapy; liver fibrosis; mesenchymal stem cells; umbilical cord.

Graphical abstract

Figure 1

Morphology and size of AT-MSCs and UC-MSCs (A) morphology at P0: AT-MSCs exhibited heterogeneous morphology, whereas UC-MSCs exhibited homogeneous morphology. (B) Morphology at P2, P4, and P6: Both MSCs exhibited a uniform spindle-shaped morphology. (C) Cell size at P2 and P6: Cells were stained with crystal violet, and the average cell size was analyzed using the Image J software. No changes in cell size were observed in either type of MSCs. All micrographs were taken at 200x magnification and are representative of three AT-MSCs and UC-MSCs cultures from different donors. AT-MSCs, adipose tissue-mesenchymal stem cells; UC-MSCs, umbilical cord-mesenchymal stem cells.

Figure 2

Growth characteristics of AT-MSCs and UC-MSCs were calculated over six passages: (A) cumulative population doubling level; (B) cell doubling time; (C) AT-MSC yield; and (D) UC-MSC yield. The data show that AT-MSCs have a shorter doubling time, a higher population doubling level, and a higher cellular yield than UC-MSCs. The data is presented as the ± SD of three independent cultures. ∗∗ indicates the statistical significance of P < 0.01. AT-MSCs, adipose tissue-mesenchymal stem cells; UC-MSCs, umbilical cord-mesenchymal stem cells.

Figure 3

Self-renewal capacity of AT-MSCs and UC-MSCs at different passage numbers (A) cell proliferation: Cell numbers were counted on days 1, 3, and 5 to determine the growth rate of early (P2) and late (P6) passage cells. (B) CFU–F: Bar diagram showing the number of CFU-F at P2 and P6 counted macroscopically. (C) β-galactosidase staining: In micrographs, β-galactosidase-stained positive bluish-green cells are senescent (200x magnification). Bar diagram showing the number of senescent cells at P2 and P6. Graphical data is presented as the mean ± SD of three independent cultures. ∗P < 0.05; ∗∗∗P < 0.001. AT-MSCs, adipose tissue-mesenchymal stem cells; UC-MSCs, umbilical cord-mesenchymal stem cells; CFU-F, colony-forming units-fibroblasts.

Figure 4

Characterizations of AT-MSCs and UC-MSCs at the 6th passage (A) MSC-specific surface marker expressions: Immunolabeled fluorescence micrographs showed >90% expressions for CD90 and CD105 and <2% expressions for CD34 and CD45 markers. (B) Pluripotent gene expressions: Bar diagrams show the relative mRNA levels of OCT4, SOX2, and NANOG genes normalized to GAPDH, and agarose gels show PCR product bands. (C) Trilineage differentiation potential: Adipogenic differentiation is shown by oil-red O-stained lipid-rich vesicles; osteogenic differentiation is shown by alizarin red S-stained mineralized matrix deposition; and chondrogenic differentiation is shown by alcian blue-stained glycosaminoglycan matrix deposition. Bar diagrams compare stain uptake levels in AT-MSCs and UC-MSCs. All micrographs were taken at 200x magnification and are representative of three independent cultures from different donors. ∗P < 0.05. AT-MSCs, adipose tissue-mesenchymal stem cells; UC-MSCs, umbilical cord-mesenchymal stem cells.

Figure 5

Rat model of liver fibrosis (A) histopathological changes in the model rats: Gross images show a coating of yellowish fibrous material; H&E stain micrographs show degeneration of liver cells; and Masson and Sirius red stain micrographs show the formation of a fibrous septum in model rat liver tissue. (B) Bar graphs show percentages of fibrotic areas measured using Image J software. All micrographs were taken at 200x magnification. Graphical data is expressed as mean ± SD (n = 4 rats per group). ∗∗∗P < 0.001. H&E, hematoxylin and eosin.

Figure 6

Histopathological examination (A) homing of transplanted MSCs: DAPI (blue) represented cell nuclei and was counted as the total number of cells; PKH-26 (red) represented labeled cells and was counted as PKH-26-positive cells (200x magnifications). Bar graph compares the percentage of PKH-26-labeled cells. (B) Gross images, H&E, Masson, and Sirius red micrographs: Both types of MSCs recover hepatic architecture and reduce collagen-positive areas, as illustrated in H&E and collagen-stained micrographs, respectively (200x magnification). (C) Bar graphs compare Sirius- and Masson-stained collagen areas in experimental groups. All graphical data is expressed as mean ± SD (n = 6 rats per group). ∗P < 0.05; ∗∗P < 0.01. MSCs, mesenchymal stem cells; DAPI, 6-diamidino-2-phenylindole; H&E, hematoxylin and eosin.

Figure 7

Expression of fibrogenic markers in rat liver tissues (A) Immunohistochemical staining: Representative micrographs show the α-SMA expression in liver paraffin-embedded sections (200x magnification). (B) Bar graph compares α-SMA-stained areas quantified by Image J. (C–E) Semi-quantitative PCR: relative mRNA levels of Tgf-β1, Col1α1, and α-Sma genes normalized to β-Actin and expressed as fold change versus the control. Data are expressed as mean ± SD (n = 6 rats per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. α-SMA, alpha smooth muscle actin.

Figure 8

Liver function tests. Blood serum profiles of (A) ALT, (B) AST, (C) ALP, and (D) ALB. Data are expressed as mean ± SD (n = 6 rats per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; ALB, albumin.