Placental stem cell therapy reduces hepatic fibrosis progression

Substances produced by the membrane of the interior part of the placenta could stimulate liver regeneration. The next step is to isolate molecules and discover how they act.

By Karina Toledo

Agência FAPESP – A therapy based on human placental stem cells has reduced the development of hepatic fibrosis by 50% in an experiment conducted with rats.

 

The researchers believe that the benefits are due to substances produced by amniotic membrane cells – the interior part of the placenta – that are capable of stimulating liver regeneration. The next step is identifying and isolating these molecules, which will pave the way for developing new medicines.

 

Hepatic fibrosis is a disease resulting from successive liver aggression, such as that caused by the excessive consumption of alcohol or viral hepatitis, explained Luciana Barros Sant’Anna, researcher at Universidade do Vale do Paraíba (Univap), where the investigation is being conducted with FAPESP funding. 

 

“Although liver cells have an enormous capacity to proliferate and regenerate the organ, they end up dying after recurring inflammation and are substituted by collagen,” explained the researcher. 

 

Cirrhosis is the terminal stage of the disease, and the only treatment available in this case is a liver transplant. However, this option is not viable for many patients, and for this reason, researchers from around the world are seeking a method to impede the worsening of the problem. 

 

The methodology developed by the Univap group, in partnership with Centro di Ricerca E.Menni (CREM) in Italy, consists of wrapping the livers of rats with fresh human amniotic membrane utilized within 48 hours of being collected at hospitals.

 

“This membrane is part of the placenta and is responsible for the production of amniotic liquid during gestation. Normally, all this tissue is discarded after birth,” explains Sant’Anna.

 

After the pregnant donor signs a term of consent, the membrane – which is approximately 20 to 30 centimeters in length and 2 to 3 millimeters in width – is collected, separated from the placenta and taken to the laboratory, where it is washed with an antibiotic and antifungal solution.

 

Next, the tissue is fragmented into 6-9 centimeter pieces, which are large enough to completely wrap the rat liver.

 

To induce fibrosis in the animals, scientists tied the biliary duct, the channel linking the liver to the duodenum that serves to transport bile, in two places. 

 

“In many cases, fibrosis is caused by narrowing of the biliary duct, which can be the result of a congenital problem or a stone. As bile cannot pass, the pressure on the liver increases, and the organ becomes inflamed. The animal model used in the experiment simulates this situation,” explains Sant’Anna.

 

Fifteen days after connecting the duct, the animals began to develop fibrosis. After 28 days, they were already in the advanced stages of the disease.

 

The experiment was conducted in a group of 40 rats. Half received a membrane soon after their biliary duct was sutured. In the other half, the scientists only simulated placement of the tissues, so that all the animal models were submitted to the same surgical stress.

 

“The membrane has very good flexibility and quickly adheres to the liver. The fixation was aided by a special glue,” said the researcher.

 

After four weeks, half of the animals were sacrificed, and their livers were extracted for analysis. In the sixth week after placement of the membrane, the other half had their organs removed.

 

Damage reduction

 

The animals that received the membrane presented 50% less fibrosis than the members of the other group. Comparing the animals in different periods, between four and six weeks, the scientists verified that therapy did not impede the emergence of the disease but reduced the severity and inhibited progression to the cirrhosis stage. 

 

The analyses were conducted with the assistance of Nilson Sant’Anna, head of the Laboratory at the National Institute Space Research (INPE). Fibrosis was quantified through a digital imaging system that allowed for the precise identification, isolation and measurement of the areas of the liver occupied by excess collagen. 

 

“This system of quantitative image analysis operates automatically and affords the rapid and simultaneous analysis of fibrosis in 1,800 histological images,” explains Nilson.

 

The study’s results received awards at the 3rd Tissue Engineering and Regenerative Medicine World Congress, held in Austria in 2012. The results were published in an article in Cell Transplantation magazine.

 

“Some amniotic research groups isolated the stem cells and are working with them separately. We opted to use the amniotic membrane and lower costs, in addition to preserving cells in their habitat, that is, the extracellular matrix,” said Sant’Anna.

 

The objective of the group now, according to researcher, is to discover exactly which substances are produced by the amniotic membrane cells and how they act.

 

“We will have to isolate stem cells and culture them to determine using molecular biology which molecules are being produced. In the future, these molecules could be synthetized in a laboratory and become a medicine,” she said. 

 

First, however, the researchers are conducting a new experiment with rats in which the membrane is applied to the liver two weeks after the biliary duct is tied off, when the process of fibrosis has already begun.

 

“The idea is to simulate a situation that is more similar to what happens with humans. Generally, when the disease is diagnosed, the majority of the organ is compromised,” she explains.

 

The group has not ruled out the possibility of conducting clinical trials in humans. In this case, however, fresh membranes cannot be used due to the risk of infections.

 

“We only collect the placentas of women whose serological exams are negative for diseases, such as syphilis, HIV, toxoplasmosis and hepatitis. However, a pregnant woman could be in an immunological window during the birth,” she affirmed.

 

To avoid these risks, the membrane has to be frozen at a least 70 degrees Celsius after disinfection, and the donor has to be submitted to new exams six months after the birth. Freezing, however, reduces the viability of stem cells by 40%.

 

The major advantage of the method, affirms the researcher, is that the placenta is a secure and easy source of stem cells. As they produce immunomodulating substances that originally function to prevent a baby from being rejected from the maternal organism, there is no risk that they will cause rejection in the recipient, humans or other species.

 

“Furthermore, there are no legal, ethical or religious complications for the collection and use of these cells in research because they are not embryonic cells, and extraction does not require invasive procedures,” adds Sant’Anna.

 

The article Amniotic Membrane Application Reduces Liver Fibrosis in a Bile Duct Ligation Rat Model (doi: 10.3727/096368910X522252), by Luciana Sant'Anna et al, can be read at: www.ingentaconnect.com/content/cog/ct/2011/00000020/00000003/art00008.