An Extracorporeal Bioartificial Liver Embedded With 3D-layered Human Progenitor-like Cells Relieves Acute Liver Failure in Pigs
Li et al. Sci Transl Med. 2020.
Regenerative medicine aims to address the worldwide shortage of donor organs. An excellent example of this is the concept of bioartificial livers (BAL) designed to support those in liver failure by removing waste products and providing synthetic function.1 In the study from Li et al, the authors report on the in vivo safety and efficacy of a novel BAL designed using hepatocyte-derived liver progenitor-like cells (HepLPCs) and an air-liquid interface (Ali) bioreactor.2
HepLPCs were immortalized by the addition of HPV E6/7 genes to increase the number of proliferation cycles (and therefore produce adequate numbers to populate the bioreactor). To increase the expression of mature hepatocytes, transcription factors known to have a role in hepatic fate determination were screened. The overexpression of FOXA3 significantly increased hepatic function. To support the large-scale production of iHepLPCs-FOXA3 cells, the authors designed an Ali-BAL capable of supporting 3D expansion, using polyethylene terephthalate-based microporous carriers. Cells grown on these carriers had improved synthetic functions and elevated urea production, ammonia elimination, and glutamine secretion in comparison to those grown on monolayer culture.
The clinical benefit of the Ali-BAL was tested in a porcine model of acute liver failure (ALF) induced by D-gal injection. Those in the treatment group were attached to the extracorporeal circulation bioreactor system for 3 hours and demonstrated a significant survival benefit (83% versus 16.7%) at the endpoint of 7 days. They had reduced ammonia concentrations at 48 hours posttreatment, improvements in liver enzymes (ALT/AST/bilirubin), and clotting factors (PT/INR/APTT) in comparison with control and no-cell groups. Furthermore, histological examination demonstrated increased numbers of hepatic parenchymal cells and Ki-67+ hepatocytes (indicating proliferation) in the treatment group, suggesting Ali-BAL may even promote regeneration.
The authors explored the mechanism of Ali-BAL on ALF by measuring toxins in the plasma before and after perfusion with the bioreactor. They found urea accumulated in the bioreactor, suggesting it was removed via ureagenesis. In support, there was a reduction in lactate concentrations after perfusion. Although representing a simplified animal model of ALF, the study demonstrates the feasibility, safety, and functionality of the Ali-BAL. Further clinical studies in humans will be required to assess the efficacy of this device, potentially adding to supportive strategies for those patients awaiting liver transplantation.
The Lymph Node Stromal Laminin α5 Shapes Alloimmunity
Li et al. J Clin Invest. 2020.
Lymph nodes are complex and dynamic structures, compartmentalized to facilitate adaptive immune responses through antigen presentation and costimulation and lymphocyte activation and regulation. The function and structure of each area are promoted by stromal cells and structural proteins. Laminins are extracellular matrix proteins produced by stromal cells that contribute as both barriers and coordinators of cellular migration, activation, and differentiation. The ratio of 2 specific subtypes laminin α4 and laminin α5 has divergent roles in immune activation and tolerance.1,2
In the current study by Li et al, the authors dissect the contribution of stromal cells and laminins α4/α5 to the organization of lymph nodes and the coordination of antigen-specific T-cell responses.3 The authors were able to accomplish this by developing laminin α5 knockout mice, in which the Lama5 gene was removed from Pdgfrb-expressing stromal cells such as reticular fibroblasts and blood/lymphatic endothelial cells. Laminin α4 expression was unaffected by this reduction of stromal laminin α5, resulting in a higher α4:α5 ratio.
Steady-state lymph nodes saw an increase in the proportion of regulatory T cells (Treg) and greater density of high endothelial venules, both of which colocalized in paracortex. In parallel, the knockout promoted mobilization of myeloid and plasmacytoid dendritic cells to the cortical ridge. These cellular changes were associated with spatially distinct chemokine and adhesion molecule expressions, indicating that the observed cellular changes may be related to migration.
Laminin α4 and α5 demonstrated opposing effects on mouse and human T-cell motility, attachment, and transendothelial migration. Laminin α5 severely limited T-cell migration, but this could be rescued by laminin α4 or by blocking α5 receptors. No difference in lymphocyte egress was noted between wild-type and knockout mice, so it was concluded that the absence of laminin α5 promoted T-cell recruitment.
Challenging mice with immunizing versus tolerogenic protocols resulted in an increase in allospecific T-cell numbers within peripheral lymph nodes in laminin α5 knockout mice in both conditions. This was associated with a modest decrease in activated effector/memory T-cell phenotype and an increase in the Treg:IL-17+ ratio. In a fully mismatched cardiac transplant model, the knockout of laminin α5 alone was not sufficient to modify rejection kinetics. The reduction in stromal laminin α5 in the knockout mice did, however, work synergistically with laminin α5 receptor blockade, tacrolimus, or anti-CD40L to prolong allograft survival.
The importance of lymph nodes in the presentation of alloantigens and the initiation of allospecific responses is well known. This dissection and manipulation of lymph node stromal cell function demonstrate their capacity to influence transplantation outcome and importantly, present the potential of laminin-targeting therapies as complements to existing immunosuppressive strategies. Despite higher numbers of antigen-specific cells found in the lymph nodes of laminin α5 knockout mice, their quality and function were modulated by laminins and their activation limited. Further research will hopefully help us to understand how such a microenvironment favors reduced activation and a greater proportion of Treg, in the absence of changes in T-cell subset migration. It would also be interesting to assess how laminin-targeting approaches impact intragraft infiltration and the development of tertiary lymphoid structures observed during rejection.
1. Nicolas CT, Hickey RD, Chen HS, et al. Concise review: liver regenerative medicine: from hepatocyte transplantation to bioartificial livers and bioengineered grafts. Stem Cells. 2017; 35:42–50doi: 10.1002/stem.2500
2. Li WJ, Zhu XJ, Yuan TJ, et al. An extracorporeal bioartificial liver embedded with 3D-layered human liver progenitor-like cells relieves acute liver failure in pigs. Sci Transl Med. 2020; 12:eaba5146doi: 10.1126/scitranslmed.aba5146
1. Simon T, Li L, Wagner C, et al. Differential regulation of T-cell immunity and tolerance by stromal laminin expressed in the lymph node. Transplantation. 2019; 103:2075–2089doi: 10.1097/TP.0000000000002774.
2. Warren KJ, Iwami D, Harris DG, et al. Laminins affect T cell trafficking and allograft fate. J Clin Invest. 2014; 124:2204–2218doi: 10.1172/JCI73683.
3. Li L, Gruner K, Tourtellotte WG. Retrograde nerve growth factor signaling abnormalities in familial dysautonomia. J Clin Invest. 2020; 130:2478–2487doi: 10.1172/JCI135099.