Bioprocessing considerations for generation of iPSCs intended for clinical application: perspectives from the ISCT Emerging Regenerative Medicine Technology working group.

Approval of induced pluripotent stem cells (iPSCs) for the manufacture of cell therapies to support clinical trials is now becoming realized after 20 years of research and development. In 2022 the International Society for Cell and Gene Therapy (ISCT) established a Working Group on Emerging Regenerative Medicine Technologies, an area in which iPSCs-derived technologies are expected to play a key role. In this article, the Working Group surveys the steps that an end user should consider when generating iPSCs that are stable, well-characterised, pluripotent, and suitable for making differentiated cell types for allogeneic or autologous cell therapies. The objective is to provide the reader with a holistic view of how to achieve high-quality iPSCs from selection of the starting material through to cell banking. Key considerations include: (i) intellectual property licenses; (ii) selection of the raw materials and cell sources for creating iPSC intermediates and master cell banks; (iii) regulatory considerations for reprogramming methods; (iv) options for expansion in 2D vs. 3D cultures; and (v) available technologies and equipment for harvesting, washing, concentration, filling, cryopreservation, and storage. Some key process limitations are highlighted to help drive further improvement and innovation, and includes recommendations to close and automate current open and manual processes.

A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors.

Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T cell (CAR-T) therapies, have generated significant clinical and commercial outcomes due to their unparalleled response rates against relapsed and refractory blood cancers. However, the development and scalable manufacture of these novel therapies remains challenging and further process understanding and optimisation is required to improve product quality and yield. In this study, we employ a quality-by-design (QbD) approach to systematically investigate the impact of critical process parameters (CPPs) during the expansion step on the critical quality attributes (CQAs) of CAR-T cells. Utilising the design of experiments (DOE) methodology, we investigated the impact of multiple CPPs, such as number of activations, culture seeding density, seed train time, and IL-2 concentration, on CAR-T CQAs including, cell yield, viability, metabolism, immunophenotype, T cell differentiation, exhaustion and CAR expression. Initial studies undertaken in G-Rex® 24 multi-well plates demonstrated that the combination of a single activation step and a shorter, 3-day, seed train resulted in significant CAR-T yield and quality improvements, specifically a 3-fold increase in cell yield, a 30% reduction in exhaustion marker expression and more efficient metabolism when compared to a process involving 2 activation steps and a 7-day seed train. Similar findings were observed when the CPPs identified in the G-Rex® multi-well plates studies were translated to a larger-scale automated, controlled stirred-tank bioreactor (Ambr® 250 High Throughput) process. The single activation step and reduced seed train time resulted in a similar, significant improvement in CAR-T CQAs including cell yield, quality and metabolism in the Ambr® 250 High Throughput bioreactor, thereby validating the findings of the small-scale studies and resulting in significant process understanding and improvements. This study provides a methodology for the systematic investigation of CAR-T CPPs and the findings demonstrate the scope and impact of enhanced process understanding for improved CAR-T production.

Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors.

Combining the sustainable culture of billions of human cells and the bioprinting of wholly cellular bioinks offers a pathway toward organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, the suspension culture of human induced pluripotent stem cell-derived aggregates (hAs) is optimized using an automated 250 mL stirred tank bioreactor system. Cell yield, aggregate morphology, and pluripotency marker expression are maintained over three serial passages in two distinct cell lines. Furthermore, it is demonstrated that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. This 4-day culture results in a 16.6- to 20.4-fold expansion of cells, generating approximately 4 billion cells per vessel, while maintaining >94% expression of pluripotency markers. The pluripotent aggregates can be subsequently differentiated into derivatives of the three germ layers, including cardiac aggregates, and vascular, cortical and intestinal organoids. Finally, the aggregates are compacted into a wholly cellular bioink for rheological characterization and 3D bioprinting. The printed hAs are subsequently differentiated into neuronal and vascular tissue. This work demonstrates an optimized suspension culture-to-3D bioprinting pipeline that enables a sustainable approach to billion cell-scale organ engineering.

[Human pluripotent stem cells and liver disorders]. / Cellules souches pluripotentes humaines et modélisation de maladies hépatiques.

The liver is associated with many diseases including metabolic and cholestatic diseases, cirrhosis as well as chronic and acute hepatitis. However, knowledge about the mechanisms involved in the pathophysiology of these diseases remains limited due to the restricted access to liver biopsies and the lack of cellular models derived from patients. The liver is the main organ responsible for the elimination of xenobiotics and thus hepatocytes have a key role in toxicology and pharmacokinetics. The induced pluripotent stem cells generated from patients with monogenic metabolic disorders, for which the corresponding gene is identified, are relevant in vitro models for the study of the mechanisms involved in generation of pathologies and also for drug screening. Towards this aim, robust protocols for generating liver cells, such as hepatocytes and cholangiocytes, are essential. Our study focused on familial hypercholesterolemia disease modeling, as well as on establishing a protocol for generation of functional cholangiocytes from pluripotent stem cells.

The potential of induced pluripotent stem cell derived hepatocytes.

Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers.

An atypical human induced pluripotent stem cell line with a complex, stable, and balanced genomic rearrangement including a large de novo 1q uniparental disomy.

Human induced pluripotent stem cells (hiPSCs) hold great promise for cell therapy through their use as vital tools for regenerative and personalized medicine. However, the genomic integrity of hiPSCs still raises some concern and is one of the barriers limiting their use in clinical applications. Numerous articles have reported the occurrence of aneuploidies, copy number variations, or single point mutations in hiPSCs, and nonintegrative reprogramming strategies have been developed to minimize the impact of the reprogramming process on the hiPSC genome. Here, we report the characterization of an hiPSC line generated by daily transfections of modified messenger RNAs, displaying several genomic abnormalities. Karyotype analysis showed a complex genomic rearrangement, which remained stable during long-term culture. Fluorescent in situ hybridization analyses were performed on the hiPSC line showing that this karyotype is balanced. Interestingly, single-nucleotide polymorphism analysis revealed the presence of a large 1q region of uniparental disomy (UPD), demonstrating for the first time that UPD can occur in a noncompensatory context during nonintegrative reprogramming of normal fibroblasts.

Midkine lacking its last 40 amino acids acts on endothelial and neuroblastoma tumor cells and inhibits tumor development.

Midkine (MDK) is a member of a new family of neurotrophic factors considered as rate-limiting growth and angiogenic factors implicated in the onset, invasion, and metastatic process of neuronal tumors, including neuroblastoma. We showed that all neuroblastoma cell lines highly expressed MDK, indicating that it is a critical player in tumor development, which may henceforth represent an attractive therapeutic target. We showed that the knockdown of MDK expression by siRNA led to a marked and significant decrease in neuroblastoma (IGR-N91 and SH-SY5Y) cell proliferation in vitro. Using a new strategy, we then evaluated the antitumor effect of a truncated MDK protein, lacking the C-terminal 81-121 portion of the molecule (MDKΔ81-121), which may act as a dominant-negative effector for its mitogenic, angiogenic, and tumorigenic activities by heterodimerizing with the wild-type protein. In vitro studies showed that MDKΔ81-121 selectively inhibited MDK-dependent tumor cells and was able to strongly reduce endothelial cell proliferation and migration and to induce ER stress-mediated apoptosis. We then investigated the effects of MDKΔ81-121 in vivo using electrotransfer of a plasmid encoding a secretable form of MDKΔ81-121 into tibialis cranialis muscles of nude mice. We showed that MDKΔ81-121 dramatically inhibited (up to 91%) tumor development and growth. This inhibition was correlated with the detection of the MDKΔ81-121 molecule in plasma and the suppression of intratumor neovascularization. Our findings demonstrate that MDK inhibition is a tractable therapeutic target for this lethal pediatric malignancy.

Messenger RNA- versus retrovirus-based induced pluripotent stem cell reprogramming strategies: analysis of genomic integrity.

The use of synthetic messenger RNAs to generate human induced pluripotent stem cells (iPSCs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of DNA-based or virus-based reprogramming strategies, including transgene integration in particular. We compared the genomic integrity of mRNA-derived iPSCs with that of retrovirus-derived iPSCs generated in strictly comparable conditions, by single-nucleotide polymorphism (SNP) and copy number variation (CNV) analyses. We showed that mRNA-derived iPSCs do not differ significantly from the parental fibroblasts in SNP analysis, whereas retrovirus-derived iPSCs do. We found that the number of CNVs seemed independent of the reprogramming method, instead appearing to be clone-dependent. Furthermore, differentiation studies indicated that mRNA-derived iPSCs differentiated efficiently into hepatoblasts and that these cells did not load additional CNVs during differentiation. The integration-free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients' iPSCs and their use in the context of stem cell-derived hepatocyte transplantation. Our findings also highlight the need to conduct careful studies on genome integrity for the selection of iPSC lines before using them for further applications.

Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells.

UNLABELLED: Cholangiocytes are biliary epithelial cells, which, like hepatocytes, originate from hepatoblasts during embryonic development. In this study we investigated the potential of human embryonic stem cells (hESCs) to differentiate into cholangiocytes and we report a new approach, which drives differentiation of hESCs toward the cholangiocytic lineage using feeder-free and defined culture conditions. After differentiation into hepatic progenitors, hESCs were differentiated further into cholangiocytes using growth hormone, epidermal growth factor, interleukin-6, and then sodium taurocholate. These conditions also allowed us to generate cholangiocytes from HepaRG-derived hepatoblasts. hESC- and HepaRG-derived cholangiocyte-like cells expressed markers of cholangiocytes including cytokeratin 7 and osteopontin, and the transcription factors SOX9 and hepatocyte nuclear factor 6. The cells also displayed specific proteins important for cholangiocyte functions including cystic fibrosis transmembrane conductance regulator, secretin receptor, and nuclear receptors. They formed primary cilia and also responded to hormonal stimulation by increase of intracellular Ca(2+) . We demonstrated by integrative genomics that the expression of genes, which signed hESC- or HepaRG-cholangiocytes, separates hepatocytic lineage from cholangiocyte lineage. When grown in a 3D matrix, cholangiocytes developed epithelial/apicobasal polarity and formed functional cysts and biliary ducts. In addition, we showed that cholangiocyte-like cells could also be generated from human induced pluripotent stem cells, demonstrating the efficacy of our approach with stem/progenitor cells of diverse origins. CONCLUSION: We have developed a robust and efficient method for differentiating pluripotent stem cells into cholangiocyte-like cells, which display structural and functional similarities to bile duct cells in normal liver. These cells will be useful for the in vitro study of the molecular mechanisms of bile duct development and have important potential for therapeutic strategies, including bioengineered liver approaches.

Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

BACKGROUND: Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. RESULTS: We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. CONCLUSIONS: We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

Human pluripotent stem cells for modelling human liver diseases and cell therapy.

The liver is affected by many types of diseases, including metabolic disorders and acute liver failure. Orthotopic liver transplantation (OLT) is currently the only effective treatment for life-threatening liver diseases but transplantation of allogeneic hepatocytes has now become an alternative as it is less invasive than OLT and can be performed repeatedly. However, this approach is hampered by the shortage of organ donors, and the problems related to the isolation of high quality adult hepatocytes, their cryopreservation and their absence of proliferation in culture. Liver is also a key organ to assess the pharmacokinetics and toxicology of xenobiotics and for drug discovery, but appropriate cell culture systems are lacking. All these problems have highlighted the need to explore other sources of cells such as stem cells that could be isolated, expanded to yield sufficiently large populations and then induced to differentiate into functional hepatocytes. The presence of a niche of "facultative" progenitor and stem cells in the normal liver has recently been confirmed but they display no telomerase activity. The recent discovery that human induced pluripotent stem cells can be generated from somatic cells has renewed hopes for regenerative medicine and in vitro disease modelling, as these cells are easily accessible. We review here the present progresses, limits and challenges for the generation of functional hepatocytes from human pluripotent stem cells in view of their potential use in regenerative medicine and drug discovery.