Early cirrhosis and a preserved bone marrow niche favour regenerative response to growth factors in decompensated cirrhosis.

BACKGROUND: Exogenous growth factor-mobilized bone marrow (BM) stem cells have shown a differential response in the management of decompensated cirrhosis (DC). This study was designed to evaluate potential clinical benefit of adding Erythropoietin (EPO) in granulocyte-colony stimulating factor (G-CSF)-mobilized stem cell therapy, possible mechanisms of regeneration and predictive factors of regenerative response. METHODS: Sixty consecutive DC patients received either G-CSF with EPO (Group A; n = 30) or G-CSF and placebo (Group B; n = 30) for 2 months and were carefully followed up for 1 year. Baseline and post-treatment liver biopsy, BM biopsy and BM aspirate were analysed for fibro-inflammatory and regenerative response and BM hematopoietic reservoir. RESULTS: Addition of EPO to G-CSF showed a significant improvement in Child-Pugh score (P = 0.03) and MELD score (P = 0.003) as compared to G-CSF alone, with reduction in mortality (16.6% vs 36.7%, P = 0.09). The combination arm also demonstrated a decreased incidence of acute kidney injury (P < 0.001), encephalopathy (P = 0.005) and refilling of ascites (P = 0.03). Compared to monotherapy, it increased CD163+ macrophages (P = 0.013), Ki67+ index (P < 0.001) with decrease in α-SMA levels (P < 0.001) in liver tissue. The response was better with grade 1 and 2 than with grade 3 ascites; Child B cirrhosis and MELD < 16. Non-responders had lower hematopoietic stem cells (HSCs) at baseline. On multivariate analysis, the liver disease severity (MELD < 16) and a relatively preserved BM (BM-HSCs > 0.4) predicted therapeutic response (AUROC = 0.82). CONCLUSIONS: Early DC (MELD < 16) patients with mild-moderate ascites and those with a healthy cellular baseline BM respond better to growth factor therapy. Addition of EPO to G-CSF provides better regenerative response than G-CSF monotherapy.

Bone marrow stem cells and their niche components are adversely affected in advanced cirrhosis of the liver.

UNLABELLED: Bone marrow (BM) is a reservoir for immune and hematopoietic cells and critical for tissue repair and regeneration. All of these functions are severely altered in cirrhosis. We investigated the cellular and functional state of BM in cirrhosis patients. We studied the histological, cellular, and molecular changes in BM of cirrhosis patients (n = 168) and controls (n = 44). Hematopoietic stem cells (HSCs) and associated niche cells, mesenchymal stem cells, Schwann cells, neural fibers, and endothelial cells were evaluated by immunohistochemistry. Cytokines and growth factors were analyzed in peripheral blood and BM plasma. Cirrhotic BM showed an inverse correlation between cluster of differentiation 34+HSCs and Model of End-Stage Liver Disease (ρ = -0.582, P < 0.001) and Child's scores (P < 0.038). BMs of cirrhosis patients with higher Model of End-Stage Liver Disease (>15) showed significantly decreased HSCs, mesenchymal stem cells, Schwann cells, and neural fibers; increased interleukin-1ß (P = 0.004), tumor necrosis factor-α (P = 0.040), and interferon-γ (P = 0.03); and decreased oncostatin M (P = 0.04), stem cell factor (P = 0.05), and stromal cell-derived factor 1 (P = 0.03) compared to those with lower Model of End-Stage Liver Disease scores (≤15). The cluster of differentiation 34+ cell population was a predictor for the development of sepsis (P < 0.001), and per unit loss increased the probability of sepsis by 16%. Cirrhosis patients with fewer HSCs had lower hemoglobin (P = 0.05) and platelet counts (P = 0.05) and showed early graft dysfunction. CONCLUSIONS: Increasing severity of cirrhosis causes derangement of the hematopoietic niche and loss of HSCs, contributing to the hematological and immunological dysfunctions and reduced potential for regeneration; restoring BM functions could provide new therapeutic options in cirrhosis. (Hepatology 2016;64:1273-1288).

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

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.

Cellular and functional loss of liver endothelial cells correlates with poor hepatocyte regeneration in acute-on-chronic liver failure.

BACKGROUND AND AIM: Acute hepatic insult triggers regeneration. If acute-on-chronic liver failure (ACLF) patients have a poorer regenerative response than acute liver failure (ALF) patients, and if so, the mechanisms underlying this, are not well understood. METHODS: We investigated the status of hepatocyte proliferation, hepatic progenitor cell (HPC) mediated regeneration, non-parenchymal cells (through immunohistochemistery), cytokines and growth factors (cytokine bead array) in liver and peripheral blood of ACLF (n = 29) and ALF (n = 17) patients. Liver endothelial cells, mesenchymal cells and Kupffer cells were isolated from explant livers and analysis of regenerative factors was done by qRT-PCR. RESULTS: Unlike ALF, the ACLF livers showed decreased hepatocyte proliferation (p < 0.001) and profound ductular-reaction with increased CK19 + hepatocytes (p < 0.0001). However, only decrease in Ki67+ hepatocytes was associated with 28 day mortality in ACLF (p < 0.001; HR = 0.78; 95% CI 0.69-0.88). In both groups, increase in plasma hepatocyte growth factor (HGF) (OR = 21.87 p = 0.002;), macrophage colony stimulating factor (MCSF) (OR = 21.73; p = 0.002) and stromal derived factor (SDF1)(OR = 10.2; p = 0.001) were associated with hepatocyte proliferation and decreased (> fivefolds) levels were associated with poor hepatocyte regeneration in ACLF patients. ACLF livers showed decrease in endothelial cells (p < 0.01) and expression of regenerative angiocrine factors C-X-C chemokine receptor type 7 (CXCR7), Inhibitor of DNA Binding 1(IDI) and HGF compared to ALF. In co-culture, while ALF liver mesenchymal stromal cells (LMSCs) induced the expression of CXCR7, IDI and HGF in human umbilical cord endothelial cells (HUVECs), the ACLF LMSCs were defective and showed decreased production of SDF-1, HGF and MCSF compared to ALF. CONCLUSIONS: Decrease in hepatic endothelial cells and their regenerative angiocrine functions indicated by defective CXCR7-ID1 dependent HGF expression underlie the poor hepatocyte proliferation in ACLF compared to ALF patients. A robust hepatocyte self-replication is lacking in the livers of ACLF patients and is associated with poor survival.