Patients affected by liver diseases and diabetes mellitus are in need for sources of new cells to enable a better transition into clinic programs of cell therapy and regenerative medicine. We demonstrated the presence of multipotent cells expressing a variety of endodermal stem cell markers in (peri)-biliary glands of bile ducts in fetal or adult human tissues, and in crypts of gallbladder epithelium. Biliary tree displays unique characteristics given the possibility to isolate cell populations with a wide spectrum of endodermal differentiation (human biliary tree stem/progenitor cells – hBTSCs) and, the co-existence of endodermal and mesenchymal-derived cells. Thus, the hBTSCs is a unique and highly available cell source contemporarily candidate for the regenerative medicine of both liver and pancreas.

Transplantation of adult hepatocytes showed transient benefits but requires immune-suppression that is a major pitfall in patients with advanced liver diseases. Mesenchymal stem cells and hematopoietic stem cells transplanted into patients with liver diseases are not able to replace resident hepatocytes but rather they target autoimmune or inflammatory processes into the liver. Stem cells isolated from fetal or adult liver have been recently proposed as alternative cell sources for advanced liver cirrhosis and metabolic liver disease. In the first cirrhotic patients treated in our center with biliary tree stem cell therapy, we registered no adverse event but significant benefits.

While several different sources of stem cells including hepatic stem cells, biliary tree stem cells, MSCs, adipose-derived stem cells, umbilical cord cells, amniotic fluid-derived epithelial cells, embryonic stem cells (ESCs) and iPSC have been investigated for their potential as therapy for chronic liver failure, homing of therapeutic cells to the liver remains a challenge. Although historically the injection into hepatic artery showed the greatest percent of engraftment, injection into the liver parenchyma (~10-20%) and into the portal vein (less than 5%) was still consistent with poor engraftment and significant ectopic cell distribution to the vascular beds of other tissues suggesting a high risk of ectopic liver formation. Given that hyaluronic acid is selectively and specifically cleared by the liver and that it has been implicated in various aspects of stem cell therapy optimization, our team hypothesized that it would not only enhance engraftment but also improve the applicability of human biliary three stem cells to treat liver cirrhosis. Interestingly, hyaluronic acid coating of human biliary three stem cells markedly improved viability, colony formation, and population doubling in primary cultures, and resulted in higher expression of integrins that are key players and mediators of cell attachment to the extracellular matrix. When hyaluronic acid-coated biliary three stem cells were transplanted via the spleen into the liver of immunocompromised mice, the engraftment efficiency increased from 3% of uncoated cells to 11%. Notably, hyaluronic acid-coated human biliary three stem cell transplantation in mice resulted in a 10-fold increase of human albumin gene expression in the liver and in a 2-fold increase of human albumin serum levels with respect to uncoated cells. Moreover, when other organs were sectioned and stained to track the cells in question, only minimal ectopic cell distribution was detected. Furthermore, as one of the major limitations of cellular therapies is the need of their long-term storage, researchers are devising strategies to make this possible. To meet this critical aspect of liver cellular therapies and to optimize their sourcing, our group has developed a cryopreservation protocol consisting in the stepwise use of serum-free Kubota's Medium supplemented with 10% dimethyl sulfoxide, 15% human serum albumin and 0.1% hyaluronans. When freshly isolated biliary three stem cells were cultured in vitro and compared with their cryopreserved counterparts, no differences were noted in terms of self-replication, stemness traits, and multipotency. Just like freshly isolated cells, cryopreserved cells were able to differentiate into functional hepatocytes, cholangiocytes or pancreatic islets and to yield similar capacity to secrete albumin and glucose-inducible insulin. This technology may be expanded to multiple cell types and promises to facilitate the establishment of cell banks with obvious logistic advantages.

Finally, cells isolated from fetal biliary tree showed a high tendency to generate organoids with high colony formation efficiency (> 90%). Fetal biliary tree organoids were composed of single layered cuboidal epithelium and inner cell masses, expressing multipotency stem cell markers (SOX2, NANOG, OCT4), endodermal stem/progenitor cell markers (LGR5, EpCAM, PDX1, SOX17), hepatic, pancreatic and ductal markers (ALB, CYPA3, INS, CFTR) and stem/progenitor surface genes (NCAM, CD133, CD44), recapitulating major processes of self-organization during embryonic development.

In the setting of regenerative medicine of liver and pancreas, fetal and adult biliary tree is becoming the most promising and available source of cells.