Liver transplantation is the most common treatment for patients suffering from liver failure that is caused by congenital diseases, infectious agents, and environmental factors. Despite a high rate of patient survival following transplantation, organ availability remains the key limiting factor. As such, research has focused on the transplantation of different cell types that are capable of repopulating and restoring liver function. The best cellular mix capable of engrafting and proliferating over the long-term, as well as the optimal immunosuppression regimens, remain to be clearly well-defined. Hence, alternative strategies in the field of regenerative medicine have been explored. Since the discovery of induced pluripotent stem cells (iPSC) that have the potential of differentiating into a broad spectrum of cell types, many studies have reported the achievement of iPSCs differentiation into liver cells, such as hepatocytes, cholangiocytes, endothelial cells, and Kupffer cells. In parallel, an increasing interest in the study of self-assemble or matrix-guided three-dimensional (3D) organoids have paved the way for functional bioartificial livers. In this review, we will focus on the recent breakthroughs in the development of iPSCs-based liver organoids and the major drawbacks and challenges that need to be overcome for the development of future applications.

We generated patient-specific disease-free induced pluripotent stem cells (iPSCs) from peripheral blood CD34+ cells and differentiated them into functional endothelial cells (ECs) secreting factor VIII (FVIII) for gene and cell therapy approaches to cure hemophilia A (HA), an X-linked bleeding disorder caused by F8 mutations. iPSCs were transduced with a lentiviral vector carrying FVIII transgene driven by an endothelial-specific promoter (VEC) and differentiated into bona fide ECs using an optimized protocol. FVIII-expressing ECs were intraportally transplanted in monocrotaline-conditioned non-obese diabetic (NOD) severe combined immune-deficient (scid)-IL2rγ null HA mice generating a chimeric liver with functional human ECs. Transplanted cells engrafted and proliferated in the liver along sinusoids, in the long term showed stable therapeutic FVIII activity (6%). These results demonstrate that the hemophilic phenotype can be rescued by transplantation of ECs derived from HA FVIII-corrected iPSCs, confirming the feasibility of cell-reprogramming strategy in patient-derived cells as an approach for HA gene and cell therapy.

Hemophilia A (HA) is a rare bleeding disorder caused by deficiency/dysfunction of the FVIII protein. As current therapies based on frequent FVIII infusions are not a definitive cure, long-term expression of FVIII in endothelial cells through lentiviral vector (LV)-mediated gene transfer holds the promise of a one-time treatment. Thus, here we sought to determine whether LV-corrected blood outgrowth endothelial cells (BOECs) implanted through a prevascularized medical device (Cell Pouch) would rescue the bleeding phenotype of HA mice. To this end, BOECs from HA patients and healthy donors were isolated, expanded, and transduced with an LV carrying FVIII driven by an endothelial-specific promoter employing GMP-like procedures. FVIII-corrected HA BOECs were either directly transplanted into the peritoneal cavity or injected into a Cell Pouch implanted subcutaneously in NSG-HA mice. In both cases, FVIII secretion was sufficient to improve the mouse bleeding phenotype. Indeed, FVIII-corrected HA BOECs reached a relatively short-term clinically relevant engraftment being detected up to 16 weeks after transplantation, and their genomic integration profile did not show enrichment for oncogenes, confirming the process safety. Overall, this is the first preclinical study showing the safety and feasibility of transplantation of GMP-like produced LV-corrected BOECs within an implantable device for the long-term treatment of HA.

Endothelial cells (ECs) are crucial for vascular health, regulating blood flow, nutrient exchange, and modulating immune responses and inflammation. The impairment of these processes causes the endothelial dysfunction (ED) characterized by oxidative stress, inflammation, vascular permeability, and extracellular matrix remodeling. While primary ECs have been widely used to study ED in vitro, their limitations—such as short lifespan and donor variability—pose challenges. In this context, induced iECs derived from induced pluripotent stem cells offer an innovative solution, providing an unlimited source of ECs to explore disease-specific features of ED. Recent advancements in 3D models and microfluidic systems have enhanced the physiological relevance of iEC-based models by better mimicking the vascular microenvironment. These innovations bridge the gap between understanding ED mechanisms and drug developing and screening to prevent or treat ED. This review highlights the current state of iEC technology as a model to study ED in vascular and non-vascular disorders, including diabetes, cardiovascular, and neurodegenerative diseases.

A major challenge in the development of a gene therapy for hemophilia A (HA) is the selection of cell type- or tissue-specific promoters to ensure factor VIII (FVIII) expression without eliciting an immune response. As liver sinusoidal endothelial cells (LSECs) are the major FVIII source, understanding the transcriptional F8 regulation in these cells would help optimize the minimal F8 promoter (pF8) to efficiently drive FVIII expression. In silico analyses predicted several binding sites (BS) for the E26 transformation-specific (Ets) transcription factors Ets-1 and Ets-2 in the pF8. Reporter assays demonstrated a significant up-regulation of pF8 activity by Ets-1 or Ets-1/Est-2 combination, while Ets2 alone was ineffective. Moreover, Ets-1/Ets-2-DNA binding domain mutants (DBD) abolished promoter activation only when the Ets-1 DBD was removed, suggesting that pF8 up-regulation may occur through Ets-1/Ets-2 interaction with Ets-1 bound to DNA. pF8 carrying Ets-BS deletions unveiled two Ets-BS essential for pF8 activity and response to Ets overexpression. Lentivirus-mediated delivery of GFP or FVIII cassettes driven by the shortened promoters led to GFP expression mainly in endothelial cells in the liver and to long-term FVIII activity without inhibitor formation in HA mice. These data strongly support the potential application of these promoters in HA gene therapy.

The engraftment of transplanted islets depends on the rapid establishment of a novel vascular network. The present study evaluated the effects of cord blood-derived blood outgrowth endothelial cells (BOECs) on the viability of neonatal porcine islets (NPIs) and the post-transplant outcome of grafted NPIs. Dispersed NPIs and human BOECs were reaggregated on microwell cell culture plates and tested for their anti-apoptotic and pro-angiogenic capacity by qRT-PCR and immunohistochemistry. The in vivo functionality was analyzed after transplantation into diabetic NOD-SCID IL2rγ^−/− (NSG) mice. The spheroids, which contained reaggregated neonatal porcine islet cells (REPIs) and BOECs, exhibited enhanced viability and a significantly elevated gene expression of VEGFA, angiopoetin-1, heme oxygenase-1, and TNFAIP3 (A20) in vitro. The development of normoglycemia was significantly faster in animals transplanted with spheroids in comparison to the only REPI group (median 51.5 days versus 60 days) (p < 0.05). Furthermore, intragraft vascular density was substantially increased (p < 0.01). The co-transplantation of prevascularized REPI-BOEC spheroids resulted in superior angiogenesis and accelerated in vivo function. These findings may provide a novel tool to enhance the efficacy of porcine islet xenotransplantation.