In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model.

BACKGROUND AND AIMS: Pluripotent stem cell-derived hepatocytes differentiated in monolayer culture are known to have more fetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies, or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity. Here we used the complex production and most importantly the activity of the coagulation factor IX (FIX) produced by mature hepatocytes to assess the differentiation of hemophilia B (HB) patient's induced pluripotent stem cells (iPSCs) in both monolayer culture and organoids. APPROACH AND RESULTS: Indeed, HB is an X-linked monogenic disease due to an impaired activity of FIX synthesized by hepatocytes in the liver. We have developed an in vitro model of HB hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. We used CRISPR/Cas9 technology to target the genomic insertion of a coagulation factor 9 minigene bearing the Padua mutation to enhance FIX activity. Noncorrected and corrected iPSCs were differentiated into hepatocytes under both two-dimensional and three-dimensional differentiation protocols and deciphered the production of active FIX in vitro. Finally, we assessed the therapeutic efficacy of this approach in vivo using a mouse model of HB. CONCLUSIONS: Functional FIX, whose post-translational modifications only occur in fully mature hepatocytes, was only produced in corrected iPSCs differentiated in organoids. Immunohistochemistry analyses of mouse livers indicated a good cell engraftment, and the FIX activity detected in the plasma of transplanted animals confirmed rescue of the bleeding phenotype.

Advanced Techniques and Awaited Clinical Applications for Human Pluripotent Stem Cell Differentiation into Hepatocytes.

Liver transplantation is currently the only curative treatment for several liver diseases such as acute liver failure, end-stage liver disorders, primary liver cancers, and certain genetic conditions. Unfortunately, despite improvements to transplantation techniques, including live donor transplantation, the number of organs available remains insufficient to meet patient needs. Hepatocyte transplantation has enabled some encouraging results as an alternative to organ transplantation, but primary hepatocytes are little available and cannot be amplified using traditional two-dimensional culture systems. Indeed, although recent studies have tended to show that three-dimensional culture enables long-term hepatocyte culture, it is still agreed that, like most adult primary cell types, hepatocytes remain refractory to in vitro expansion. Because of their exceptional properties, human pluripotent stem cells (hPSCs) can be amplified indefinitely and differentiated into any cell type, including liver cells. While many teams have worked on hepatocyte differentiation, there has been a consensus that cells obtained after hPSC differentiation have more fetal than adult hepatocyte characteristics. New technologies have been used to improve the differentiation process in recent years. This review discusses the technical improvements made to hepatocyte differentiation protocols and the clinical approaches developed to date and anticipated in the near future.

Successful Derivation of Hepatoblasts, Cholangiocytes and Hepatocytes from Simian Induced Pluripotent Stem Cells.

The use of primary cells in human liver therapy is limited by a lack of cells. Induced pluripotent stem cells (iPSCs) represent an alternative to primary cells as they are infinitely expandable and can be differentiated into different liver cell types. The aim of our work was to demonstrate that simian iPSCs (siPSCs) could be used as a new source of liver cells to be used as a large animal model for preclinical studies. We first differentiated siPSCs into a homogenous population of hepatoblasts (siHBs). We then separately differentiated them into hepatocytes (siHeps) and cholangiocytes (siChols) expressing respective specific markers and displaying epithelial polarity. Moreover, we showed that polarized siChols can self-organize into 3D structures. These results should facilitate the deciphering of liver development and open the way to exploring co-culture systems that could be assessed during preclinical studies, including in autologous monkey donors, for regenerative medicine purposes.

Liver Regeneration and Recanalization Time Course following Repeated Reversible Portal Vein Embolization in Swine.

INTRODUCTION: Portal vein embolization (PVE) is an accepted technique to preoperatively increase the volume of the future remnant liver before major hepatectomy. A permanent material is usually preferred since its superiority to induce liver hypertrophy over absorbable material has been demonstrated. Nevertheless, the use of an absorbable material generates a reversible PVE (RPVE) capable of inducing significant liver hypertrophy. In small animal models, the possibility to proceed to a repeated RPVE (RRPVE) has shown to boost liver hypertrophy further. The aim of this preliminary study was to assess the feasibility and the tolerance of RRPVE in a large animal model, in comparison with permanent PVE (PPVE) and single RPVE. METHODS: Six swine (2 per group) were assigned either to single RPVE group (using powdered gelatin sponge), RRPVE group (2 RPVEs separated by 14 days) or PPVE group (using N-butyl-cyanoacrylate). The feasibility and tolerance of the procedures were evaluated using portography, liver function tests and histological analysis. Evolution of liver volumes was assessed with volumetric imaging by computed tomography. RESULTS: Embolization of portal branches corresponding to 75% of total liver volume was performed successfully in all animals. Procedures were well tolerated, inducing moderate changes in portal pressure and transient aminotransferase increase. None of the animals developed portal vein thrombosis. After RPVE, complete recanalization occurred at day 11. RRPVE showed a trend for higher hypertrophy, the non-embolized liver to total liver ratio reaching 5.2 ± 1.0% in the RPVE group, 6.8 ± 0.1% in the RRPVE group and 5.0 ± 0.3% in the PPVE group. DISCUSSION/CONCLUSION: In this preliminary comparative study, RRPVE was as feasible and as well tolerated as the other procedures, and resulted in higher liver hypertrophy.

Improving Hepatocyte Engraftment Following Hepatocyte Transplantation Using Repeated Reversible Portal Vein Embolization in Rats.

Hepatocyte transplantation (HT) has emerged as a promising alternative to orthotopic liver transplantation, yet liver preconditioning is needed to promote hepatocyte engraftment. A method of temporary occlusion of the portal flow called reversible portal vein embolization (RPVE) has been demonstrated to be an efficient method of liver preconditioning. By providing an additional regenerative stimulus, repeated reversible portal vein embolization (RRPVE) could further boost liver engraftment. The aim of this study was to determine the efficiency of liver engraftment of transplanted hepatocytes after RPVE and RRPVE in a rat model. Green fluorescent protein-expressing hepatocytes were isolated from transgenic rats and transplanted into 3 groups of syngeneic recipient rats. HT was associated with RPVE in group 1, with RRPVE in group 2, and with sham embolization in the sham group. Liver engraftment was assessed at day 28 after HT on liver samples after immunostaining. Procedures were well tolerated in all groups. RRPVE resulted in increased engraftment rate in total liver parenchyma compared with RPVE (3.4% ± 0.81% versus 1.4% ± 0.34%; P < 0.001). In conclusion, RRPVE successfully enhanced hepatocyte engraftment after HT and could be helpful in the frame of failure of HT due to low cell engraftment.

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.

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.

In vitro generated Rh(null) red cells recapitulate the in vivo deficiency: a model for rare blood group phenotypes and erythroid membrane disorders.

Lentiviral modification combined with ex vivo erythroid differentiation was used to stably inhibit RhAG expression, a critical component of the Rh(rhesus) membrane complex defective in the Rh(null) syndrome. The cultured red cells generated recapitulate the major alterations of native Rh(null) cells regarding antigen expression, membrane deformability, and gas transport function, providing the proof of principle for their use as model of Rh(null) syndrome and to investigate Rh complex biogenesis in human primary erythroid cells. Using this model, we were able to reveal for the first time that RhAG extinction alone is sufficient to explain ICAM-4 and CD47 loss observed on native Rh(null) RBCs. Together with the effects of RhAG forced expression in Rh(null) progenitors, this strongly strengthens the hypothesis that RhAG is critical to Rh complex formation. The strategy is also promising for diagnosis purpose in order to overcome the supply from rare blood donors and is applicable to other erythroid defects and rare phenotypes, providing models to dissect membrane biogenesis of multicomplex proteins in erythroid cells, with potential clinical applications in transfusion medicine.

Microencapsulated Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells: Optimizing 3D Culture for Tissue Engineering Applications.

Liver cell therapy and in vitro models require functional human hepatocytes, the sources of which are considerably limited. Human induced pluripotent stem cells (hiPSCs) represent a promising and unlimited source of differentiated human hepatocytes. However, when obtained in two-dimensional (2D) cultures these hepatocytes are not fully mature and functional. As three-dimensional culture conditions offer advantageous strategies for differentiation, we describe here a combination of three-dimensional (3D) approaches enabling the successful differentiation of functional hepatocytes from hiPSCs by the encapsulation of hiPSC-derived hepatoblasts in alginate beads of preformed aggregates. The resulting encapsulated and differentiated hepatocytes (E-iHep-Orgs) displayed a high level of albumin synthesis associated with the disappearance of α-fetoprotein (AFP) synthesis, thus demonstrating that the E-iHep-Orgs had reached a high level of maturation, similar to that of adult hepatocytes. Gene expression analysis by RT-PCR and immunofluorescence confirmed this maturation. Further functional assessments demonstrated their enzymatic activities, including lactate and ammonia detoxification, as well as biotransformation activities of Phase I and Phase II enzymes. This study provides proof of concept regarding the benefits of combining three-dimensional techniques (guided aggregation and microencapsulation) with liver differentiation protocols as a robust approach to generate mature and functional hepatocytes that offer a permanent and unlimited source of hepatocytes. Based on these encouraging results, our combined conditions to produce mature hepatocytes from hiPSCs could be extended to liver tissue engineering and bioartificial liver (BAL) applications at the human scale for which large biomasses are mandatory.

Evidence of Adult Features and Functions of Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells and Self-Organized as Organoids.

BACKGROUND: Human-induced pluripotent stem cell-derived hepatocytes (iHeps) have been shown to have considerable potential in liver diseases, toxicity, and pharmacological studies. However, there is a growing need to obtain iHeps that are truly similar to primary adult hepatocytes in terms of morphological features and functions. We generated such human iHeps, self-assembled as organoids (iHep-Orgs). METHODS: iPSC-derived hepatoblasts were self-assembled into spheroids and differentiated into mature hepatocytes modulating final step of differentiation. RESULTS: In about four weeks of culture, the albumin secretion levels and the complete disappearance of α-fetoprotein from iHep-Orgs suggested the acquisition of a greater degree of maturation than those previously reported. The expression of apical transporters and bile acid secretion evidenced the acquisition of complex hepatocyte polarity as well as the development of a functional and well-defined bile canalicular network confirmed by computational analysis. Activities recorded for CYP450, UGT1A1, and alcohol dehydrogenase, response to hormonal stimulation, and glucose metabolism were also remarkable. Finally, iHep-Orgs displayed a considerable ability to detoxify pathological concentrations of lactate and ammonia. CONCLUSIONS: With features similar to those of primary adult hepatocytes, the iHep-Orgs thus produced could be considered as a valuable tool for the development and optimization of preclinical and clinical applications.

Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein.

Expansion of human hematopoietic stem cells (HSCs) is a major challenge in cellular therapy, and currently relies on the use of recombinant cytokines or on gene transfer of transcription factors. Of these, the HOXB4 homeoprotein protein is of particular interests as it promotes the expansion of mouse HSCs without inducing the development of leukemia. To eliminate any deleterious effects that might be associated with stable HOXB4 gene transfer into human cells, we took advantage of the ability of HOX proteins to passively translocate through cell membranes. Here we show that when cultured on stromal cells genetically engineered to secrete HOXB4, human long-term culture-initiating cells (LTC-ICs) and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mouse repopulating cells (SRCs) were expanded by more than 20- and 2.5-fold, respectively, over their input numbers. This expansion was associated with enhanced stem cell repopulating capacity in vivo and maintenance of pluripotentiality. This method provides a basis for developing cell therapy strategies using expanded HSCs that are not genetically modified.

[Hepatic organoids: What are the challenges?] / Les organoïdes hépatiques - Quels sont les enjeux ?

The study and understanding of liver organogenesis have allowed the development of protocols for pluripotent stem cells differentiation to overcome the lack of primary cells, providing an almost unlimited source of liver cells. However, as their differentiation in conventional 2D culture systems has shown serious limits, hepatic organoids derived from human pluripotent stem cells represent a promising alternative. These complex and organized structures, containing one or more cell types, make it possible to recapitulate in vitro some of the organ functions, thus enabling numerous applications such as the study of the liver development, the mass production of functional liver cells for transplantation or the development of bioartificial livers, as well as the in vitro modeling of hepatic pathologies allowing high throughput applications in drug screening or toxicity studies. Economic and ethical issues must also be taken into account before using these organoids in therapeutic applications.

TITLE: Les organoïdes hépatiques - Quels sont les enjeux ? ABSTRACT: L'étude et la compréhension de l'organogenèse du foie ont permis le développement de protocoles de différenciation des cellules souches pluripotentes afin de pallier le manque de cellules primaires, offrant ainsi une source quasi illimitée de cellules hépatiques. La différenciation de ces cellules dans des systèmes de culture conventionnels en deux dimensions (2D) ayant cependant montré ses limites, des organoïdes hépatiques ont été dérivés de cellules souches pluripotentes humaines et représentent désormais une alternative prometteuse. Ces structures 3D, complexes et organisées, intégrant un ou plusieurs types cellulaires, permettent de reproduire in vitro une ou plusieurs fonctions de l'organe, et ouvrent ainsi la voie à de nombreuses applications, comme l'étude du développement du foie, la production en masse de cellules hépatiques fonctionnelles pour la transplantation ou le développement de foies bioartificiels, sans oublier la modélisation de pathologies hépatiques permettant le criblage à haut débit de médicaments ou des études de toxicité. Des enjeux économiques et éthiques doivent également être pris en considération avant une utilisation de ces organoïdes pour des applications thérapeutiques.

A versatile microfluidic tool for the 3D culture of HepaRG cells seeded at various stages of differentiation.

The development of livers-on-a-chip aims to provide pharmaceutical companies with reliable systems to perform drug screening and toxicological studies. To that end, microfluidic systems are engineered to mimic the functions and architecture of this organ. In this context we have designed a device that reproduces series of liver microarchitectures, each permitting the 3D culture of hepatocytes by confining them to a chamber that is separated from the medium conveying channel by very thin slits. We modified the structure to ensure its compatibility with the culture of hepatocytes from different sources. Our device was adapted to the migratory and adhesion properties of the human HepaRG cell line at various stages of differentiation. Using this device, it was possible to keep the cells alive for more than 14 days, during which they achieved a 3D organisation and acquired or maintained their differentiation into hepatocytes. Albumin secretion as well as functional bile canaliculi were confirmed on the liver-on-a-chip. Finally, an acetaminophen toxicological assay was performed. With its multiple micro-chambers for hepatocyte culture, this microfluidic device architecture offers a promising opportunity to provide new tools for drug screening applications.

FOXO1 regulates L-Selectin and a network of human T cell homing molecules downstream of phosphatidylinositol 3-kinase.

In T cells, the PI3K pathway promotes proliferation and survival induced by Ag or growth factors, in part by inactivating the FOXO transcription factor 1. We now report that FOXO1 controls the expression of L-selectin, an essential homing molecule, in human T lymphocytes. This control is already operational in unprimed T cells and involves a transcriptional regulation process that requires the FOXO1 DNA-binding domain. Using transcriptional profiling, we demonstrate that FOXO1 also increases transcripts of EDG1 and EDG6, two sphingosine-1-phosphate receptors that regulate lymphocyte trafficking. Additionally, FOXO1 binds the promoter of the cell quiescence and homing regulator Krüppel-like factor 2 and regulates its expression. Together, these results reveal a new function of FOXO1 in the immune system and suggest that PI3K controls a coordinated network of transcription factors regulating both cell quiescence and homing of human T lymphocytes.

Pluripotent stem cell-derived cholangiocytes and cholangiocyte organoids.

The development of protocols for pluripotent stem cell (PSC) differentiation into cholangiocytes and cholangiocyte organoids in three-dimensional structures represent a huge advance in both research and medical fields because of the limited access to primary human cholangiocytes and the potential bias induced by animal models used to study cholangiopathies in vivo. PSC-derived cholangiocyte organoids consisting of either cysts with luminal space or branching tubular structures are composed of cells with apico-basal polarity that can fulfill cholangiocyte functions like the transport of bile salts. Several protocols of PSC differentiation have already been published but we added to the detailed protocol we describe here some notes or advice to facilitate its handling by new users. We also propose detailed protocols to carry out some of the characterization analyses using immunofluorescence to study the expression of specific markers and a functionality test to visualize bile acid transport using cholyl-lysyl-fluorescein (CLF).

Pluripotent-Stem-Cell-Derived Hepatic Cells: Hepatocytes and Organoids for Liver Therapy and Regeneration.

The liver is a very complex organ that ensures numerous functions; it is thus susceptible to multiple types of damage and dysfunction. Since 1983, orthotopic liver transplantation (OLT) has been considered the only medical solution available to patients when most of their liver function is lost. Unfortunately, the number of patients waiting for OLT is worryingly increasing, and extracorporeal liver support devices are not yet able to counteract the problem. In this review, the current and expected methodologies in liver regeneration are briefly analyzed. In particular, human pluripotent stem cells (hPSCs) as a source of hepatic cells for liver therapy and regeneration are discussed. Principles of hPSC differentiation into hepatocytes are explored, along with the current limitations that have led to the development of 3D culture systems and organoid production. Expected applications of these organoids are discussed with particular attention paid to bio artificial liver (BAL) devices and liver bio-fabrication.

HepaRG Self-Assembled Spheroids in Alginate Beads Meet the Clinical Needs for Bioartificial Liver.

In liver tissue engineering, cell culture in spheroids is now well recognized to promote the maintenance of hepatic functions. However, the process leading to spheroids formation is time consuming, costly, and not easy to scale-up for further use in human bioartificial liver (BAL) applications. In this study, we encapsulated HepaRG cells (precursors of hepatocyte-like cells) in 1.5% alginate beads without preforming spheroids. Starting from a given hepatic biomass, we analyzed cell differentiation and metabolic performance for further use in a fluidized-bed BAL. We observed that cells self-rearranged as aggregates within the beads and adequately differentiated over time, in the absence of any differentiating factors classically used. On day 14 postencapsulation, cells displayed a wide range of hepatic features necessary for the treatment of a patient in acute liver failure. These activities include albumin synthesis, ammonia and lactate detoxification, and the efficacy of the enzymes involved in the xenobiotic metabolism (such as CYP1A1/2). Impact statement It has been recognized that culturing cells in spheroids (SPHs) is advantageous as they better reproduce the three-dimensional physiological microenvironment. This approach can be exploited in bioartificial liver applications, where obtaining a functional hepatic biomass is the major challenge. Our study describes an original method for culturing hepatic cells in alginate beads that makes possible the autonomous formation of SPHs after 3 days of culture. In turn, the cells differentiate adequately and display a wide range of hepatic features. They are also capable of treating a pathological plasma model. Finally, this setup can easily be scaled-up to treat acute liver failure.

Genomic integrity of human induced pluripotent stem cells: Reprogramming, differentiation and applications.

Ten years after the initial generation of induced pluripotent stem cells (hiPSCs) from human tissues, their potential is no longer questioned, with over 15000 publications listed on PubMed, covering various fields of research; including disease modeling, cell therapy strategies, pharmacology/toxicology screening and 3D organoid systems. However, despite evidences that the presence of mutations in hiPSCs should be a concern, publications addressing genomic integrity of these cells represent less than 1% of the literature. After a first overview of the mutation types currently reported in hiPSCs, including karyotype abnormalities, copy number variations, single point mutation as well as uniparental disomy, this review will discuss the impact of reprogramming parameters such as starting cell type and reprogramming method on the maintenance of the cellular genomic integrity. Then, a specific focus will be placed on culture conditions and subsequent differentiation protocols and how their may also trigger genomic aberrations within the cell population of interest. Finally, in a last section, the impact of genomic alterations on the possible usages of hiPSCs and their derivatives will also be exemplified and discussed. We will also discuss which techniques or combination of techniques should be used to screen for genomic abnormalities with a particular focus on the necessary quality controls and the potential alternatives.