Promethera publications

Guillaume Rommelaere, Kris Gellynck, Tuba Baran, Sarah Snykers, Luca Falciola, Philippe Stordeur, Mustapha Najimi, Giuseppe Mazza, Etienne Sokal

Human hepatobiliary progenitor cells as new candidates for liver cell therapy

Background: Orthotopic liver transplantation is the only treatment for patients with end-stage liver diseases. However, this methodology is limited due to a shortage of organ donors and liver cell therapy has been suggested as an alternative treatment. Injection of hepatocytes has been tested but this method suffers from practical limitations (difficulty in expansion and cryopreservation). As a solution to this problem, we isolated human hepatobiliary progenitor cells (H2Stem) from unsuitable livers for transplantation. H2Stem proliferate in culture, show high hepatic functionality in vitro and engraft in vivo in pre-clinical models.

Methods: Human livers segments were dissociated using a two-step perfusion method and the parenchymal cell fraction isolated by low speed centrifugations. When put in adherent culture, a population of epithelial-like proliferating cells develops from the liver cell suspensions and grows for several passages in vitro. The expression profile of hepatobiliary stem cells was determined pre- and post-differentiation by a full transcriptomic analysis and confirmed by immunostaining and flow cytometry. In addition, the hepatic functionality of this cell population was evaluated after in vitro differentiation in 2D as well as in 3D cultures. Lastly, their ability to repopulate and differentiate in damaged livers was assessed by the infusion in FRG mice models.

Results: Gene expression analysis and flow cytometry analysis indicated that the proliferative cell population isolated from human livers expresses both biliary and hepatic markers. After in vitro differentiation (2D or 3D), H2Stem cells present high CYP3A4 activities and the ability to secrete hepatic markers such as albumin or alpha-1-antitrypsin. Notably, H2Stem cells are able to repopulate livers from the immunodeficient FRG mice after intra-splenic injection. Engrafted islets stain negatively for CK19 and positively for albumin, alpha-1-antitrypsin, ornithine transcarbamylase and arginase. Furthermore, human albumin and alpha-1-Antitrypsin are detected in the blood of infused animals thus confirming in vivo differentiation.

Conclusion: In this study hepatobiliary progenitor cells isolated from human livers present an ability to grow in culture and to engraft in livers of FRG mice as well as showing hepatic functionality in vivo. Developments are currently ongoing to further expand H2Stem stem cells in vitro under GMP-like conditions before evaluating safety and efficacy in patients affected by liver diseases.

Kris Gellynck, Guillaume Rommelaere, Mustapha Najimi, John Tchelingerian, Catherine Lombard, Joelle Thonnard, Giuseppe Mazza, Etienne Sokal

Clinical-grade human liver mesenchymal stem cells for the treatment of NASH-Fibrosis through immunomodulation

Background: Nonalcoholic steatohepatitis (NASH), a severe form of nonalcoholic liver diseases (NAFLD), is one of the prominent liver diseases worldwide. There is currently no approved drug for the treatment of NASH and liver transplantation is the only therapeutic approach for advanced NASH. Mesenchymal stem cells (MSCs) are promising candidates to modulate the pro-inflammatory and pro-fibrogenic environment of chronic liver because of their immunomodulatory properties. HepaStem, Human adult liver-derived MSCs isolated from organs unsuitable for transplantation can be GMP-manufactured, cryopreserved and reconstituted at the bedside as an off-the-shelf product. Safety and tolerability have been shown in a phase I/II clinical trial in patients with metabolic disorders. The proposed mechanism of action in NASH is a systemic hit-and-run suppression of inflammation and stellate cell activation through the secretion of several cytokines in response to the liver inflammation.

Method: The secretion of pro-inflammatory and anti-inflammatory cytokines including HGF, IDO, PGE2 was measured using multiplexed immunoassays in cell culture with or without inflammation cocktail. The anti-inflammatory effect of HepaStem was investigated in co-culture systems with T-lymphocytes in a mixed leukocyte reaction as well as with immature and mature dendritic cells. In a preclinical high-fat model, the potency of HepaStem was compared to a vehicle, either with or without the use of immunosuppression (cyclosporine), to factor in the use of human cells in an animal model.

Results: Secretion of anti-inflammatory and anti-fibrotic cytokines was increased with the addition of an inflammatory cytokines in the culture medium HepaStem inhibited both T-lymphocyte response and the dendritic cell generation and function in co-culture experiments. In the NASH model, while the immunosuppression by itself did not affected the disease progression, cell-based treatment (3 IV injections 12.5x106 cells/kg) significantly and dose-dependently decreased collagen deposition in the pericentral region as shown by Sirius red staining. A single HepaStem injection significantly decreased the NAS score, which was mainly attributed to reduction in inflammation and thus supporting the proposed mechanism of action.  

Conclusion: Our results suggest that clinical grade liver progenitor cells have anti-fibrosis and anti-NASH effects, both in vitro and in a pre-clinical NASH model. This observation provides significant evidences to open new phase I/II studies in NASH patients as well as to apply MSCs for the treatment of chronic liver disorders.

Kris Gellynck, Giuseppe Mazza, Joelle Thonnard, Mustapha Najimi, Etienne Sokal

Human liver-derived cell therapy for Immuno-mediated and inflammatory liver diseases

HepaStem is a human liver derived progenitor cell manufactured under  GMP conditions by Promethera Biosciences. A phase I/II clinical trial already showed the cell therapy was safe and well tolerated in 20 paediatric patients. The cell's metabolic properties were used to treat genetic diseases like urea cycle  disorder, but as HepaStem has similarities to mesenchymal stem cells, it also has immunomodulatory properties.

This immunomodulation will be now be applied in a clinical trial with patients suffering from acute-on-chronic-liver-failure (ACLF). They have a chronic liver fibrosis/cirrhosis,  but are admitted to the hospital with an acute liver decompensation on top of the cirrhosis.  ACLF is defined by an imbalance of the immune system, which progresses the diseases towards multiple organ failure. The survival of these ACLF patients drops below  50% in the first 3 months after hospital admission, but once ACLF progression is overturned, patients can return to a normal state, where the liver fibrosis is chronic only.  Rebalancing the immune system, involved a complex mix of immuno-cytokines and cells.

MSCs, and our liver progenitor cells are able to suppress T lymphocyte activation and proliferation, by soluble factors and cell-cell contact is not required for this inhibition to happen. Secreted cytokines like TGF-β, HGF, PGE2, IDO are involved in this mechanism. Apart from the T-lymphocytes, dendritic cells (DCs) play a key role in the induction of immunity and tolerance, depending on the activation and maturation stage.  MSCs have been demonstrated to interfere with DC differentiation, maturation and function, through Il-6 and M-CSF.

The abovementioned molecules are all cytokines that are well secreted by HepaStem, affirming its immunomodulatory properties.  Even more, MSCs and our HepaStem cells lack surface expression of costimulatory molecules, such as CD80 and CD86 ,and it is believed that MSCs can render T cells anergic. When a cocktail of inflammatory cytokines (IFN-ɣ, IL1b, TNF-α) is added to the in vitro culture of liver progenitor cells we can see that surface markers (CD54, CD274, ..) are increasing, thus the cells are adapting to the inflamed environment.  This adaptation is the difference between using a molecular drug substance and a cell therapy; Human MSCs cells like HepaStem can interact with the different environment that will be patient-specific.

HepaStem is liver derived and has shown to migrate towards the liver when infused in the peripheral vein, and a homing to inflamed body areas was observed; both helping the cells to migrate to the liver fibrotic areas that are the subject of this project.

Promethera is planning a clinical trial involving multiple centres spread over Europe, in which ACLF patients will receive 4 injections of HepaStem. Blood and tissue analysis will be evaluated to investigate the effect of the HepaStem modulation on the immune system and the ACLF progression of these patients.

Bayon Y., Vertès A., Ronfard V., Culme-Seymour E., Mason C., Stroemer P. Najimi M., Sokal E., Wilson C., Barone J., Aras R., Chiesi A.

Turning Regenerative Medicine Breakthrough Ideas and Innovations into Commercial Products.

The TERMIS-Europe (EU) Industry committee intended to address the two main critical issues in the clinical/commercial translation of Advanced Therapeutic Medicine Products (ATMP): (1) entrepreneurial exploitation of breakthrough ideas and innovations, and (2) regulatory market approval. Since January 2012, more than 12,000 publications related to regenerative medicine and tissue engineering have been accepted for publications, reflecting the intense academic research activity in this field. The TERMIS-EU 2014 Industry Symposium provided a reflection on the management of innovation and technological breakthroughs in biotechnology first proposed to contextualize the key development milestones and constraints of allocation of financial resources, in the development life-cycle of radical innovation projects. This was illustrated with the biofuels story, sharing similarities with regenerative medicine. The transition was then ensured by an overview of the key identified challenges facing the commercialization of cell therapy products as ATMP examples. Real cases and testimonies were then provided by a palette of medical technologies and regenerative medicine companies from their commercial development of cell and gene therapy products. Although the commercial development of ATMP is still at the proof-of-concept stage due to technology risks, changing policies, changing markets, and management changes, the sector is highly dynamic with a number of explored therapeutic approaches, developed by using a large diversity of business models, both proposed by the experience, pitfalls, and successes of regenerative medicine pioneers, and adapted to the constraint resource allocation and environment in radical innovation projects.

Snykers, S., P. Willemsen, et al.

Ensuring supply chain of cell therapy products during commercialization: easy reconstitution at the bedside

Promethera   HepaStem  is the  company's  therapy  product  to  treat  serions metabolic liver disorders. An European  phase IIII  clinical trial is ongoing  to treat  Crigler-Najjar and  Urea  Cycle Disorders  in a pediatric setting. As  of today, 20 patients have been treated with HepaStem.  Cnrrently,  Promethera is preparing  its next clinical phases in US and Europe.  Th.e challenge is to provide  a  drug   product  easy-to-reconstitute  at  clinical  sites.  In  order   to guarantee  a flexible, highly quali tative, and economically sustainable supply chain  during  commercialization,   Promethera   has developed  a  ready-to-use off-the-shelf  product for direct reconstitution  at the hospitals. The  product preparation  is simply carried out  like conventional  reconstitution  of fi·eeze­ dried sterile  medicinal products (e.g. vaccine). The  challenge was to find an appropriate   final  container   (i)  compliant   to  liquid  nitrogen   storage,   (ii) allowing automated in-line filling and (iii) reconstitt1tion without changing the product  quality (safety/identity/purity/potency). The  Aseptic Technologies closed  vials perfectly  fit within  this concept. This  approach  does not  only simplify  the  preparation  procedure  and  operation  time, it also reduced  the storage  footprint  and  improved  the  product's  quality  in  terms  of viability, yield, content  uniformity and batch-to-batch  consistency. Shelf-life, the key­ bottleneck of most cell therapeutic  products, is substantially less critical. The idea for the upcoming clinical phases is to provide an ali-in-one  kit, including the cryopreserved cell tl1erapeutic product HepaStem  and all material needed for  reconstitution   (syringe/adaptor/uisinfectantlprefilled  vial with  reconsti­ tution   media).  In  conclusion,   Promethera   has  developed  a    ready-to-use product,   allowing  worldwide  availability  and   easy  reconstitution   at   the bedside. This technology guarantees a sustainable supply chain during commercialization  and offers opportunities  in the field of cell-based products with limited shelf-life.

Sokal et al. JIMD Rep. 2013 Oct 20.

Liver Engraftment and Repopulation by In Vitro Expanded Adult Derived Human Liver Stem Cells in a Child with Ornithine Carbamoyltransferase Deficiency.

A 3-year-old girl suffering from ornithine carbamoyltransferase (OTC) deficiency was poorly equilibrated under conventional diet and scavenger treatment. Following unsuccessful cryopreserved hepatocyte transplantation, she received two infusions of Adult Derived Human Liver Stem/Progenitor Cells (ADHLSCs) expanded in vitro under GMP settings, the quantity being equivalent to 0.75% of her calculated liver mass. Using FISH immunostaining for the Y chromosome, the initial biopsy did not detect any male nuclei in the recipient liver. Two liver biopsies taken 100 days after ADHLSC transplantation showed 3% and 5% of male donor cells in the recipient liver, thus suggesting repopulation by donor cells. Although limited follow-up did not allow us to draw conclusions on long-term improvement, these results provide a promising proof of concept that this therapy is feasible in an OTC patient.

Scheers et al. Cell Transplant. 2012 Apr 17. [Epub ahead of print]

Adult derived human liver progenitor cells in long term culture maintain appropriate gatekeeper mechanisms against transformation.

Background and aims: The use of human liver progenitor cells in the development of clinical cell therapy depends on their constant availability and unaltered properties during culture. The present study investigates the effects of long-term in vitro culture on the specific characteristics of these cells and on their genetic stability. Methods: Adult-derived human liver progenitor cells (ADHLPCs) were isolated from 12 donors and cultured until senescence and cell death. Cells were analyzed at different time points for their phenotype stability and differentiation potential. In addition, growth characteristics, chromosomal karyotype, telomere maintenance mechanisms and activity of cell cycle related genes were studied. Finally, their in vivo tumorigenicity was investigated in a xenograft assay.Results: The long-term culture of ADHLPCs revealed a variable proliferation capacity. Cells maintained their original phenotype and acquired hepatocyte-like metabolic functions after differentiation. Eight of the 12 cell populations grew fast (doubling time of 6.3 days), during a limited time period (mean 116.2 days) and mainly presented normal cytogenetic features. The four other cell cultures presented an early decline in growth potential (doubling time of 28.6 days) and premature senescence. Chromosomal alterations were detected in 3 out of 4 cultures at passage 6. Cytogenetic anomalies were not correlated with tumorigenic potential in vitro or in vivo and expression of cell cycle related genes was appropriately upregulated, inducing senescence.Conclusions: Although chromosomal anomalies may occur in long-term cell cultures, neither transformation nor alteration of their characteristics was noted during in vitro expansion. All ADHLPCs reached senescence and growth arrest. Presenescent ADHLPCs might therefore be considered as a suitable source for liver-based cell therapy.

Review papers

Promethera summer 2010

Clinical Perspectives of Stem and Progenitor Cells for Liver Regenerative Medicine, summer 2010

Because the liver is the site of many vital functions, impairment of only one protein within a complex metabolic pathway is usually highly deleterious. Such a condition is called inborn error of metabolism, and concerns many genetic diseases linked to a non-functional enzyme in the liver. Treatments, and longterm management, are currently not efficient enough, and patients would greatly benefit from an innovative therapy that meets this medical need. Orthotopic liver transplantation is the only radical treatment of severe defects, and/or end stage diseases. Cell therapy has been identified as the best alternative tool to overcome scarcity of organ donation. Several cell types are under investigation, and adult liver stem/progenitor cells represent an attractive cell source for liver regenerative medicine.