Defects in energy metabolism are associated with functional exhaustion of bone marrow mesenchymal stem cells in cirrhosis

Abstract

Objectives: Cellular and functional exhaustion of bone marrow mesenchymal stem cells (BM-MSC) is significantly associated with the loss of HSCs and hepatic osteodystrophy in cirrhosis. The molecular mechanisms underlying the dysfunction of BM-MSCs are not well understood. We investigated the underlying mechanisms of cellular and functional exhaustion of BM-MSCs in cirrhosis.

Methods: The MSCs were isolated retrospectively from bone marrow of decompensated alcoholic cirrhosis patients {(Trial registration: ClinicalTrials.gov NCT01902511) (n=10; MELD=16.2±2.3; CTP=8.7±2.3)} and age and gender-matched healthy controls (n=8). Global gene expression profile of healthy bone marrow MSCs (hBM-MSCs) and cirrhosis patients BM-MSCs (cBM-MSCs) were done by mRNA sequencing. XFe24-bioanalyzer analyzed the bioenergetic potential of cells. Level of different cytokines and growth factors in BM-plasma and MSCs secretome were analyzed by Luminex-based bead array.

Results: Analysis of differentially expressed genes showed significant (P<0.01) up-regulation of genes associated with ubiquitination and catabolism of proteins; TNF signaling, insulin resistance, and down-regulation of genes associated with DNA repair, protein processing, cell cycle, and mitochondrial respiration in cBM-MSCs in comparison to hBM-MSCs. Compared to hBM-MSCs, cBM-MSCs showed a significant defect in glycolysis due to insulin resistance and poor glucose uptake (P=0.002). This led to compromised self-renewal capacity and cellular loss of MSCs in cirrhosis. cBM-MSCs also showed a significant impairment in Oxidative phosphorylation (OXPHOS) due to mitochondrial dysfunction leading to defects in the osteogenic differentiation with early aging and senescence.

Conclusion: Compromised energy metabolism due to inflammatory and metabolic stress-induced insulin resistance underlies the cellular and functional exhaustion of BM-MSCs in cirrhosis.

Keywords: Oxidative phosphorylation; bone marrow mesenchymal stem cells; glycolysis; liver cirrhosis.

Conflict of interest statement

None.

AJSC Copyright © 2022.

Figures

Figure 1

GO-enrichment and KEGG-pathway analysis of differentially expressed genes between healthy and cirrhotic human BM-MSCs. A. Graph showing GO-enrichment and KEGG-pathway analysis of genes significantly upregulated in cBM-MSCs in comparison to hBM-MSCs. B. Graph showing GO-enrichment and KEGG-pathway analysis of genes significantly downregulated in cBM-MSCs in comparison to hBM-MSCs. Note: The bar chart represents % of genes count, and the red dot represents the -log10 (P-value).

Figure 2

Measurement of glycolytic potential and CFU-F of healthy and cirrhotic BM-MSCs: (A) (left) Graph showing the real-time change in ECAR in response to glucose, oligomycin, and 2-deoxyglucose (2DG) to determine glycolysis parameters in healthy and cirrhotic BM-MSCs (right) graph showing glycolysis and glycolytic reserve in healthy and cirrhotic BM-MSCs. (B) Bar-graph showing glucose uptake (2-DG6P pmol/µl) in healthy and cirrhotic BM-MSCs. (C) Individual colonies captured at 4× magnification, for hBM-MSC and cBM-MSCs (left) and Bar graph (right) showing an average number of CFU-F colonies (Mean ± SD) generated per 1000 cells at passage three by hBM-MSC and dBM-MSCs. (D) Graph showing an average number of CFU-F colonies (Mean ± SD) of healthy BM-MSCs with and without 2DG (glycolysis inhibitor). **

Figure 3

Measurement of cytokines and glucose in cirrhotic and control Bone marrow plasma. A. Dot-plot showing level (pg/ml) of pro-inflammatory cytokines TNF-alpha, IL-6, IL-1beta in bone marrow plasma of cirrhotic and control. B. Graph showing level (mg/dl) of glucose in bone marrow plasma of cirrhotic and control. **

Figure 4

Measurement of mitochondrial function and content in healthy and cirrhotic BM-MSCs. A. Graph showing a real-time change in OCR in response to oligomycin, FCCP, and Rotenone/Antimycin to determine mitochondrial respiration parameters in healthy and cirrhotic BM-MSCs. B. Bar-graph showing mitochondrial respiration as a measure of OCR linked to baseline respiration, Maximal reparation, ATP linked respiration in healthy and cirrhotic BM-MSCs. C. Bar-graph showing Proton leak linked respiration in healthy and cirrhotic BM-MSCs. D. Bar-Graph showing mitochondrial and total ROS levels in healthy and cirrhotic BM-MSCs. E. A Representative image showing total mitochondrial mass in healthy and cirrhotic BM-MSCs. F. Graph showing functional mitochondrial mass in healthy and cirrhotic BM-MSCs. ****P

Figure 5

Assessment osteogenic and immunomodulatory function and senescence associated change in in healthy and cirrhotic BM-MSCs. A. Graph showing relative mRNA level of indicated osteogenic marker gene expression in in-vitro osteo-differentiated cBM-MSCs in comparison to hBM-MSCs. B. Bar graph showing the percentage of CD4 and CD8-T cells in CD3/CD28 induced T cell activated PBMCs culture alone or in the presence of hBM-MSCs or cBM-MSCs. C. Bar graph showing relative level of indicated cytokines and growth factors secreted in the culture medium of cBM-MSC with respect to hBM-MSC. D. Bar graph showing population doubling time (in hours) at P3, P4 and P5 of hBM-MSC and cBM-MSCs (*P<0.01). E. Representative picture showing SA-Beta Gal positive cells at passage three culture of hBM-MSCs and cBM-MSCs (Left) and Bar graph (right) showing number of SA-Beta Gal positive cells/1000 of cells (mean ± SD) at P3 and P5 culture of healthy and cirrhotic BM-MSCs (Magnification 40×).

Figure 6

Schematic showing a proposed mechanism of cellular and functional exhaustion of M-MSCs in cirrhosis. Chronic liver injury leads to the accumulation of inflammatory cytokines (TNFα; IL6; IL1β) and hyperglycemia in bone marrow milieu. This induces insulin resistance in cirrhotic BM-MSCs. Poor glucose uptake due to insulin resistance compromises the glycolysis leading to poor self-renewal and loss of MSCs in cirrhotic BM. There is also an accumulation of defective mitochondria leading to a gross defect in mitochondrial respiration that may underline the defect in osteogenic, paracrine, and immunomodulatory function. Mitochondrial damage may also lead to premature aging and senescence in these MSCs. Altogether, broad defect in energy metabolism underlies the cellular and functional exhaustion of BM-MSCs in cirrhotic.