Multipotent stromal cells stimulate liver regeneration by influencing the macrophage polarization in rat

Abstract

AIMTo investigate the influence of the umbilical cord-derived multipotent stromal cells (MSCs) on recovery of the liver after the subtotal resection, that is, removal of 80% of the organ mass, a renowned model of the small-for-size liver remnant syndrome.METHODSThe MSCs were obtained from the intervascular tissue of umbilical cords, dissected from rat fetuses, by the explant culture technique. The vital labeling of MSCs with РКН26 was carried out on the 3rd passage. The subtotal resection was performed on male Sprague-Dawley rats. The experimental group animals received a transplant 106 MSCs infused into the spleen. Hepatocyte proliferation was assessed by counting of either mitotic figures or Ki67-positive cells in microscopic images. MSC differentiation was assessed with antibodies to hepatocyte-specific marker cytokeratin 18 (CK18), cholangiocyte-specific protein CK19, smooth muscle cell-specific protein α-SMA, the endothelial cell marker CD31, or the active fibroblast marker FAPα. Total macrophages of the liver were selectively stained in cryosections incubated with anti-CD68 antibodies (1:100, Abcam), while the M2a and M2c macrophage populations were selectively stained with anti-CD206 antibodies. Expression of interleukin and growth factor genes was evaluated with PCR-RT.RESULTSIntrasplenic allogeneic transplantation of the umbilical cord-derived multipotent stromal cells stimulates reparative processes within the residual liver tissue after subtotal resection (removal of 80% of the organ mass), as indicated by increased rates of hepatocyte proliferation and accelerated organ mass recovery. These effects may result from paracrine influence of the transplanted cells on the resident macrophage population of the liver. The transplantation favors polarization of macrophages to M2 phenotype (the M2-polarized macrophages specifically express CD206; they are known to suppress inflammation and support tissue repair). No differentiation of the transplanted cells into any of the liver cell types have been observed in the study.CONCLUSIONWe found no direct evidence for the paracrine effect of MSCs on liver regeneration after the subtotal liver resection in rats. However, the paracrine mechanism of the therapeutic activity of transplanted MSC is indirectly indicated by a decrease in the total number of CD68 + macrophages and an increase in the proportion of M2 pro-repair macrophages in the regenerating liver as compared to animals in which the transplantation was only mimicked.

Platelet lysate outperforms FCS and human serum for co-culture of primary human macrophages and hMSCs

“…Cells that tightly interact with macrophages in vivo are mesenchymal stromal cells (MSCs) 8–10 . These adult multipotent stem cells can differentiate into various cell types, like osteoblasts 11 , chondrocytes 12 and adipocytes 13 .…”

Molecular mechanisms of splenectomy-induced hepatocyte proliferation

“…Hepatocyte proliferation was assessed by counting mitotic figures or Ki67-positive cells in microscopic images according to a detailed protocol described in our previous work [10]. Hepatocytes were immunostained for Ki67 and CK18 in cryosections.…”

“…Selective immunostaining of liver macrophages in cryosections was implemented with anti-CD68, anti-CD163 or anti-CD206 antibodies (1:100, Abcam) topped with FITC-conjugated secondary antibodies (1:200, Abcam). The nuclei were visualized by DAPI-counterstaining as described previously [10]. The corresponding indexes of macrophage content were determined as relative counts of CD68+, CD163+ and CD206+ cells to the total cell counts in microscopic images.…”

“…The assay was carried out by a previously described protocol [10]. Total RNA was extracted from the tissues by using RNeasy Plus Mini Kit (QIAGEN) according to the manufacturer's protocol and treated with RNase-free DNase I (Thermo Scientific; 1 U per μg of RNA) to remove traces of genomic DNA.…”

Experimental approaches to creating a tissue-specific matrix for a bioartificial liver

“…Присущие мезенхимальным стромальным клеткам (МСК) иммуномодулирующие свойства, способность к стимуляции регенерации и возможность дифференцироваться в гепатоцитоподобные клетки указывают на возможность их применения для создания клеточно-инженерных конструкций (КИК) печени для лечения различных заболеваний печени [10]. Из-за сложности экспансии и низкой жизнеспособности первичных гепатоцитов при культивировании in vitro для исследования взаимодействия специализированных клеток с фрагментами децеллюляризованной печени целесообразно использовать клеточные линии, способные выполнять характерные для печеночных клеток функции [11].…”