Studies on stem cells and on their potential sources have been intensified in recent years, given the promise of their clinical application, especially in regenerative medicine [31
Ethical problems regarding the use of human embryos [5
] and the neoplastic risks after their in vivo
] have led to adult stem cells being considered a more acceptable source. Bone marrow is a good source of adult stem cells but the decrease in number of stem cells available with the donor age and invasive procedure required to obtain the cells are the major problems for their utilization [12
]. Research has consequently turned towards finding alternative sources of MSCs, such as adipose tissue [14
] and foetal-derived tissues. Placenta, amniotic fluid and umbilical cord indeed seem to contain undifferentiated cells, due to their embryonal origin [15
In this study we characterized a novel MSCs population obtained from human UC (UCMSCs) and we have induced their differentiation towards hepatic lineages in vitro seeking the best cell support for this purpose.
Phenotypic analysis showed a profile compatible with MSCs and the simultaneous high expression of CD166, CD105 and CD73 demonstrated that our cells were a novel MSCs population.
We found that these UCMSCs constitutively express mRNA coding for specific hepatic markers such as AFP, albumin and TDO. Then, for the first time, we unexpectedly demonstrated that undifferentiated UCMSCs constitutively expressed mRNA coding for MTP, a transfer protein localized in the endoplasmic reticulum of mature hepatocytes and enterocytes; this protein catalyses the transport of triglycerides, cholesteryl esters and phospholipids [33
We believe that these findings reinforce the conviction that UCMSCs have a strong potential for differentiating into hepatic-lineage cells in vitro.
In fact, when UCMSCs were stimulated to differentiate towards a hepatic lineage, reached a hepatocyte-like phenotype, amazingly, when seeded on a simple untreated plastic support.
Further confirmation comes from flow cytometric analysis on differentiated cells that demonstrated a conspicuous drop in almost all the typical MSCs markers, such as CD73, CD90, and CD105. The only exception was CD166 expression that was spontaneously lost in UCMSCs cultured in proliferative medium while was maintained when the cells were submitted to differentiation protocol. Interestingly, CD29, another typical MSC marker, was expressed higher in differentiated UCMSCs (79,4%) reaching a level comparable with human hepatocytes [7
The morphological features, loss of MSC phenotype, gene expression changes, immunocytochemical staining, albumin secretion, urea production and glycogen storage, all suggested that these cells can grow and differentiate into functional hepatocyte-like cells without any biological support, whereas cells seeded on 3D-supports showed a minor (MatrigelTM
) or negligible (hLAM) differentiation capacity. Indeed, the data that unquestionably confirm our assertions was on TDO mRNA modulation that was found exclusively when the cells were differentiated on petri dishes. Our data would indicate that hLAM is not a suitable support for cell growth and hepatic differentiation considering that the cells lost mRNA coding for MTP and TDO after 7
days of culture and the supplementation with hepatogenic differentiating factors did not influence the basal AFP and albumin mRNA levels during 28
days of stimulation. These findings suggest that, in our experimental conditions, the coating afforded no advantage over an untreated plastic support for the purpose of UCMSCs hepatic differentiation; on the contrary, the simplest support seems to be the most suitable for this aim.
Taken together these results demonstrated that this novel population is an ideal candidate for liver disease treatment by cell therapy.
Mesenchymal stem cell transplantation has been explored as a new clinical approach to repair injured tissue. A growing number of studies have highlighted two important aspects of MSC therapy: MSCs can modulate T-cell mediated immunological responses [9
] and following systemic administration these cells home to sites of ischemia or injury, as was demonstrated in lung [22
], heart [23
], kidney [24
] and liver [35
]. Several different MSC sources have been evaluated for cell therapy in chronic and acute liver diseases, such as bone marrow [36
], amniotic fluid [37
] and human umbilical cord blood [38
]. On the contrary, UCMSCs have been exclusively considered for therapy in chronic liver diseases, such as fibrosis and cirrhosis, and so far liver UCMSCs transplantation in situ
has been the unique administration route [40
It has been documented that multipotent MSCs synthesize a wide variety of growth factors and cytokines, exerting a paracrine effect on local cellular dynamics [42
]. Such trophic effects could be irrespective of direct differentiation of transplanted cells into lineages of the respective tissues as demonstrated in an ischemic acute renal failure model [43
]. The hepatogenic potential and immunomodulatory activity of UCMSCs were further investigated in this study employing systemic transplantation in a murine model of acute liver injury induced by a single administration of carbon tetrachloride, able to induce severe hepatic damage by generation of oxidative stress and activation of immune cells [44
]. In fact, so far the possibility of using human UCMSCs to repair acute liver damage has not been evaluated and they have been transplanted via systemic administration by our group for the first time. MSCs were found to be very resistant to ROS and induced a faster reduction of oxidative stress in recipient mice [35
]. For these reasons, we decided to transplant undifferentiated UCMSCs rather than pre-differentiated cells.
We transplanted UCMSCs after 24 hours from the damage, when 40% of hepatic parenchyma was necrotic. Cells were recruited in the injured tissue and then they were able to engraft the liver (cells did not reach the liver when transplanted in healthy mice) and to regulate the inflammatory process. In fact in transplanted mice as soon as 5
days after the CCl4
injection, the inflammatory process was clearly attenuated, showing a moderated infiltrate, a lower CD68 positivity, a lower pro-inflammatory cytokines expression (primarily TNF-alpha and TGF-beta 1) and a higher level of IL-10 gene expression compared to the CCl4
At day 8 histological analysis of liver of CCl4
treated mice showed the presence of cellular clusters that we identified as mega macrophages and activated Kupffer cells. In transplanted mice it was possible to identify exclusively Kupffer cells only at day 5, whereas after 8
days this activation phase was completely terminated. These findings suggest that transplanted UCMSCs have anti-inflammatory properties or are able to accelerate the kinetic of inflammatory process, leading to liver recovery in a shorter time.
During the pro-inflammatory process induced by CCl4, jointly with Kupffer cells, stellate cells play a pivotal role. Immunofluorescence analysis showed that in CCl4-treated mice there was a conspicuous number of alpha-SMA positive cells. The most of these cells were identified as activated stellate cells since they co-expressed desmin and nestin. Alpha-SMA single positive cells were another subpopulation of liver myofibroblasts. Following cells transplantation there was no activation of stellate cells, therefore, the anti-inflammatory activity of UCMSCs was directed not only towards inflammatory cells, including Kupffer cells but also against stellate cells and myofibroblasts. TGF-beta 1 down-regulation in transplanted mice confirmed our hypothesis.
The liver has several antioxidant enzymatic systems such as superoxide dismutase, catalase and glutathione peroxidase that play a fundamental role during physiological and pathological ROS mediated oxidative stress. We evaluated whether UCMSCs were able to ameliorate hepatic damage also influencing the antioxidant systems, measured by catalase activity within the liver. In CCl4
treated mice livers the activity of catalase was significantly reduced compared with control groups. The enzyme was probably degraded or saturated to block CCl4
-induced massive free radical production. In presence of mesenchymal stem cells catalase activity was higher after 8
days compared to CCl4
treated mice. These finding suggested that UCMSCs reduced catalase consumption, confirming that oxidative damage in transplanted mice was nearly resolved.
Our experiments also showed that catalase activity measured in the liver of UCMSCs transplanted mice without any CCl4 induced damage (MSCs group) was higher compared to PBS group. Therefore we speculate that UCMSCs contribute to scavenging activity against radicals by stimulating the activity of catalase, one of the biological defence system of the liver.
In vivo experiments showed that unquestionably UCMSCs were able to induce total liver recovery acting as an adjuvant or modulating the physiologic defence systems. There is not a direct relationship between the number of cells that are found in the liver and the amazing results in transplanted mice. It was demonstrated that stem cells act through a dual mechanism: cell-to cell contact and modulation mediated by soluble factors produced by cells themselves. Our hypothesis is that cells have immunomodulatory activity both at local level and before homing to damaged tissue. It is possible that cells are able to act also during their permanence in the bloodstream and completing their action upon reaching the liver. Increased catalase activity measured in MSCs group demonstrated that cells were able to influence hepatic antioxidant environment without liver engraftment.