Phenylketonuria (PKU), a common autosomal recessive metabolic disorder, arises from diverse pathogenic variants in the phenylalanine hydroxylase (PAH) gene, causing phenylalanine accumulation and neurological impairment. The vast spectrum of over 2,200 PAH variants and frequent compound heterozygosity complicate genotype-phenotype prediction, highlighting the need for deeper mechanistic insight. We generated human induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells of four PKU patients with distinct PAH genotypes using non-integrating Sendai viruses. The iPSC lines were validated for pluripotency, vector clearance, and genomic integrity. These patient-specific iPSCs provide a valuable platform for elucidating PKU pathophysiology and advancing personalized therapeutic development.

We describe here a human pluripotent stem cell line (hPSC) designed to serve as an acceptor (a "chassis" cell line) for the recombination-mediated cassette exchange (RMCE)-mediated precise insertion of DNA fragments into a safe harbor locus. To enable the use of Flp recombinase for directional insertion, we targeted one of the AAVS1 safe harbor alleles with an excisable negative selection cassette flanked with FRT and FRT3 sites. The generated hPSC line displayed typical colony morphology, pluripotency signatures and normal karyotype. We expect it to provide a useful AAVS1 safe harbor "landing site" for FRT/FRT3-flanked transgenes.

Background aims: With the continuous development and advancement of human pluripotent stem cell (PSC)derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. To mitigate these limitations, in vitro analytical methods can be utilized as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared the sensitivity of three in vitro assays: qPCR, ddPCR and RT-LAMP. Methods: The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A. Results: The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts. Discussion: In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy. (c) 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

An ability to monitor cell cycle progression in real time has numerous applications in pluripotent stem cell-based models and therapeutics development. Fluorescence Ubiquitination-based Cell Cycle Indicator (FUCCI) systems enable live monitoring of the cell cycle in stem cells and their differentiated progenies in a non-invasive manner. We describe the generation and characterisation of a cell line with a doxycycline-inducible reporter system, iFUCCI, in the human embryonic stem cell (hESC) line MEL-1. MEL-1 iFUCCI hESCs exhibited a normal karyotype, expressed markers of the undifferentiated state, and demonstrated expected differentiation potential by giving rise to cell populations from all three germ layers.

Phenylketonuria is a rare autosomal recessive metabolic disorder mainly due to a significant reduction in the enzyme phenylalanine hydroxylase, resulting in elevation of phenylalanine in the blood. Here, we have established two fibroblast-derived induced pluripotent stem cell lines using Sendai virus-based reprogramming. The established induced pluripotent stem cell lines exhibited a normal karyotype and expressed markers of pluripotency assessed through quantitative PCR, flow cytometry and immunocytochemistry. These cell lines also demonstrated the ability to differentiate into the three primary germ layers of the human body, including ectoderm, endoderm, and mesoderm.

Background & AimCardiovascular disease (CVD) remains a major cause of death and disease burden worldwide. Myocardial infarction (MI) and ischaemic heart disease are prominent contributors to this. With improved survival following MI, many patients are now living with reduced heart function and are at serious risk of heart failure due to a significant loss of heart muscle following MI. Recent studies have shown that cardiomyocytes derived from induced pluripotent stem cells (iPSCs) have the potential to engraft and electromechanically couple with recipient heart tissue, providing a strong candidate for regenerative therapies. However, given the large number of cells lost following MI (∼1x109) cell replacement therapy will require the manufacture of very large numbers of cardiomyocytes, best addressed by a scalable 3D bioreactor based process.Methods, Results & ConclusionHere we present a scalable process for the expansion and subsequent differentiation of iPSCs to produce cardiomyocytes in vertical wheel bioreactors (VWB). Process development took place in PBS 100mL VWBs investigating seeding density, agitation rates, aggregate size, and timing of small molecule additions. iPSCs were seeded into bioreactors containing mTeSR 3D media (Stem Cell Technologies) on Day 0 and received supplement additions on Day 1 and Day 2 of expansion. On the final day of pluripotent expansion (Day 4) reactors received a full exchange with E8 media (Thermo Fisher). After pluripotent expansion, differentiation towards the cardiac mesoderm lineage was induced using the GSK3β inhibitor CHIR99021 followed by treatment with the WNT pathway inhibitor IWP-2. After 15 days in cardiomyocyte differentiation media, cells were harvested and analysed for total cell number and purity using the pan-cardiomyocyte marker cardiac troponin T (CTNT). The reactors averaged 1.37 x 106 cells per mL of reactor volume, of which an average of 95.75% were positive for CTNT as measured by flow cytometry. Cardiomyocyte subset characterisation was performed via scRNAseq along with flow cytometry and immunocytochemistry staining for the ventricular marker MLC2v. Cells produced in 500mL PBS VWBs were succesfully engrafted into a porcine model of MI. In conclusion, these experiments have shown that induced pluripotent stem cells can be expanded and differentiated to produce relatively pure populations of cardiomyocytes in VWBs.

Background Exciting pre-clinical data have confirmed that human pluripotent stem cell derived cardiomyocytes (PSC-CMs) can remuscularise the injured or diseased heart, with several clinical trials now in planning or recruitment stages worldwide. However, ventricular arrhythmias are a predictable complication following engraftment of intramyocardially injected PSC-CMs. Therefore, there is an urgent unmet need to gain mechanistic insights and treatment strategies to control or prevent these engraftment arrhythmias (EAs). Methods We used a porcine model of myocardial infarction and PSC-CM transplantation to investigate efficacy of pharmacologic and catheter based anti-arrhythmic strategies in mitigating EAs. Furthermore, cell doses were robustly phenotyped using single cell ribonucleic acid sequencing and high parameter flow cytometry to identify cellular characteristics predictive of arrhythmogenesis. Results Combination therapy with amiodarone and ivabradine significantly reduced EA rate and burden following PSC-CM transplantation. Catheter ablation was also a feasible and effective treatment strategy which could be considered in the case of pharmacologically refractory arrhythmias. In addition, we show that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically, we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. We further describe two unique surface marker signatures, SIRPA + /CD90 - /CD200 + and SIRPA + /CD90 - /CD200 - , which identify arrhythmogenic and non-arrhythmogenic cardiomyocytes respectively. Conclusion Our data deepens mechanistic understanding of EAs and suggests that modifications to current PSC-CM production and/or selection protocols could ameliorate this problem. We further show that current clinical pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias, an important safety consideration given several impending clinical trials.

To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be capable of quantifying tissue-level functions such as contractile force and be amenable to standard multiwell-plate manipulations. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues - termed Cardiac MicroRings (CaMiRi) - in custom 3D-print-molded multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a circumferential ramp. The wells are seeded with cell-laden collagen, which, in response to the gradual slope of the circumferential ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells, and we used it to determine an optimal tissue formulation. We validated the contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable the measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.

Human pluripotent stem cells (hPSCs) are viewed as promising candidates for applications in regenerative medicine and therapy due to their proliferative and pluripotent properties. However, obtaining clinically significant numbers of hPSCs remains a limiting factor and impedes their use in therapeutic applications. Conventionally, hPSCs are cultured on two-dimensional surfaces coated with a suitable substrate, such as Matrigel™. This method, however, requires a large surface area to generate sufficient cell numbers to meet clinical needs and is therefore impractical as a manufacturing platform for cell expansion. In addition, the use of enzymes for cell detachment and small molecule inhibitors to increase plating efficiency may impact future cell behavior when used for routine subculturing. In this study, we describe a protocol to generate and maintain hPSC aggregates in a three-dimensional suspension culture by utilizing thermoresponsive nanobridges. The property of the polymer used in the nanobridges enables passaging and expansion through a temperature change in combination with mechanically applied shear to dissociate aggregates; thus, we eliminate the need of enzymes or small molecules for cell dissociation and viability, respectively. Utilizing this platform, maintenance of human embryonic stem cells for three continuous passages demonstrated high expression levels in key pluripotent markers.

Human pluripotent stem cells (hPSCs) are a promising cell source for many biomedical applications including cell replacement therapy and drug screening. As such, robust and scalable hPSC manufacturing processes are needed to produce appropriate quantities of high quality hPSCs. Various processes have been proposed, but none are fully characterised and optimised. CCRM has undertaken the task of developing and characterising an efficient and robust aggregate-based hPSC manufacturing process in stirred-tank reactors (STRs). Here we present an hPSC aggregate culture process that has been translated from an orbital shaker system to the ambr®15 platform (Sartorius Stedim), a microscale and controlled STR system. With this process, aggregates can be consistently formed from a single cell suspension in the ambr®15 within 24 hours. These aggregates are 41% smaller than those formed on an orbital shaker, but can achieve 1.5 times greater cell expansion over a 4-day period. Over 75% of the hPSCs produced co-express the pluripotent markers Oct4, Nanog, Sox2 and SSEA4; however, on average, 6% more expression is observed in the hPSC product from the ambr®15. Strategies for routine passaging of the cells are being explored. The ambr®15 is capable of parallel processing and multi-parametric control of 24 bioreactors; thereby enabling the exploration of individual and interacting effects of multiple operating parameters on hPSC culture performance. We have begun to explore the effects of 5 operating parameters on aggregate size and size variability using one-factor-at-a-time and split-plot factorial design experiments. As aggregate size was observed to influence hPSC differentiation trajectory, an understanding of how to control and manipulate aggregation dynamics will allow us to tailor our process for specific end applications. Efficient small-scale studies enabled by the ambr®15 will ultimately be translated to larger scale STR systems for robust and routine production of hPSC.

Despite advances in coronary artery disease treatment and prevention, myocardial damage due to acute myocardial infarction (MI) remains a major cause of morbidity and mortality in the population. Cell-based clinical trials to treat MI have focused on cells derived from the bone marrow or those potentially possessing functional similarities such as skeletal myoblasts or cardiac progenitors isolated from heart biopsies. Any benefits provided by these cells in improving heart function, left ventricular ejection fraction, or extending life expectancy after MI have been credited mostly to paracrine effects. Functional restoration of damaged myocardium will require a functional cell type with similar phenotype and characteristics of the damaged tissue that can also integrate, survive, and electrically couple to the host. Human pluripotent stem cells (hPSCs) have the ability to differentiate into multiple cell types of the adult body. hPSC-derived cardiomyocytes represent a promising target population for cell-based therapies for MI because they are scalable and the product can be defined with a specific set of release criteria. The purpose of this article is to review the rationale for cell therapy in heart disease, discuss the properties of hPSC cardiomyocytes that define their usefulness for regenerative therapy, consider manufacturing issues and preclinical investigation, and finally examine the steps required to establish effective clinical implementation. Pluripotent stem cell-derived cardiomyocyte-based therapies have enormous potential to revolutionize the management of heart disease; expedient but careful development is needed to ensure that this potential is fully realized.

The development of robust suspension cultures of human embryonic stem cells (hESCs) without the use of cell membrane disrupting enzymes or inhibitors is critical for future clinical applications in regenerative medicine. We have achieved this by using long, flexible, and thermoresponsive polymer worms decorated with a recombinant vitronectin subdomain that bridge hESCs, aiding in hESC's natural ability to form embryoid bodies (EBs) and satisfying their inherent requirement for cell-cell and cell-extracellular matrix contact. When the EBs reached an optimal upper size where cytokine and nutrient penetration becomes limiting, these long and flexible polymer worms facilitated EB breakdown via a temperature shift from 37 to 25 °C. The thermoresponsive nature of the worms enabled a cyclical dissociation and propagation of the cells. Repeating the process for three cycles (over eighteen days) provided a >30-fold expansion in cell number while maintaining pluripotency, thereby providing a simple, nondestructive process for the 3D expansion of hESC.

Human embryonic stem cell (hESC) derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction) and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP) are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.

Use of stem cells, whether adult or embryonic for clinical applications to treat diseases such as Parkinson's, macular degeneration or Type I diabetes will require a homogenous population of mature, terminally differentiated cells. A current area of intense interest is the development of defined surfaces for stem cell derivation, maintenance, proliferation and subsequent differentiation, which are capable of replicating the complex cellular environment existing in vivo. During development many cellular cues result from integrin signalling induced by the local extracellular matrix. There are 24 known integrin heterodimers comprised of one of 18 α subunits and one of 8 β subunits and these have a diverse range of functions mediating cell-cell adhesion, growth factor receptor responses and intracellular signalling cascades for cell migration, differentiation, survival and proliferation. We discuss here a brief summary of defined conditions for human embryonic stem cell culture together with a description of integrin function and signalling pathways. The importance of integrin expression during development is highlighted as critical for lineage specific cell function and how consideration of the integrin expression profile should be made while differentiating stem cells for use in therapy. In addition this review summarises the known integrin expression profiles for human embryonic stem cells and 3 common adult stem cell types: mesenchymal, haematopoietic and neural. We then outline some of the possible technologies available for investigating cell-extracellular matrix interactions and subsequent integrin mediated cell responses.

Title of abstract: Abstract: In order to enable the use of human embryonic stem cells (hESC) in clinical applications cost-effective scalable culture platforms will be required that both yield xeno-free genetically stable undifferentiated human pluripotent cells and allow the efficient differentiation of such cells into clinically relevant cells or progenitors. We have begun to address these challenges through an integrated research project combining cell biology, surface modification and reporter gene technology. Control over hESC behavior requires understanding of and control over cell-ECM interactions, hESC maintenance and differentiation pathways and the molecular pathways that control genetic and epigenetic stability of these cells. We have identified vitronectin as a replacement for complex undefined surfaces such as feeder layers or Matrigel™ and Geltrex™ . A small, recombinant, N-terminal fragment of Vitronectin (amino acids 1-54) corresponding to the Somatomedin B "domain" and an integrin binding Arg-Gly-Asp (RGD) sequence was used to functionalise surfaces via either physisorption or binding via a poly-histidine tag. Our results show that these surfaces are able to fully support hESC attachment and maintenance in an equivalent manner to Geltrex™. Using transcriptome comparison of supportive and non-supportive feeder layers we have further identified novel hES maintenance molecules and show that two of these (GDF8 and GDF11) are able to maintain undifferentiated growth of hESC under feeder free conditions 1. Recent work from our laboratory has further shown that the widely used medium supplement ascorbate causes genome wide yet specific demethylation of 1847 CpG islands in hESC 2 and that these epigenetic changes affect the expression of important stem cell genes, including CD30, a biomarker for genetically abnormal HESC 3 . Factorial analysis of Activin A and bFGF in defined serum free medium under feeder free conditions has identified the minimal concentrations of Activin A and bFGF that are sufficient to maintain pluripotency and growth of CD30 negative hESC over long-term culture in the absence of ascorbate. The combination of a defined, ascorbate free media and recombinant Vitronectin based surfaces provide an excellent platform to explore large-scale production of genetically and epigenetically stable hESC for therapeutic applications.

Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible, defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex or Matrigel. In addition we have validated an ascorbate free media capable of supporting CD30(low) populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines, MEL1, MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up.

Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter, technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple, effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent (N-hydroxysulfosuccinimide/N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple, but important step results in remarkable protein deposition efficiencies often exceeding 50%, whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays, and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions, as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies.

Use of flow cytometry to detect pluripotency markers on or in human embryonic stem cells (hESCs) is a powerful analytical tool. However, current staining methodologies for high-content analysis of large numbers of samples utilize large quantities of primary and secondary antibodies, are time consuming, and may suffer from sample-to-sample variability. To circumvent these issues, we have developed a reproducible, quick, and cost-effective method of staining 12 populations of hESCs grown under different conditions by labeling each with a unique optical signature (UOS). The UOS for each population is achieved by combining different combinations and concentrations of 3 esterase activated, live cell, fluorescent indicators. The individually stained populations are then combined and an aliquot of the hESC samples stained for pluripotency or other markers of interest in the far-red region of the spectrum. Based on the unique fluorescent intensity and emission wavelengths of each population, the characteristics of each population are decoded in software after flow cytometric analysis. We have validated both our staining procedure and decoding methods by mixing populations of differentiated and undifferentiated hESCs and successfully quantifying differences in the pluripotency markers SSEA-4, Tra-1-60, GCTM2, and CD9 between the 12 different populations. Our multiplexing approach allows for the addition of internal controls and reduces sample-to-sample variation, while offering a significant reduction in time and reagent consumption. We anticipate that this method will be of great benefit to laboratories conducting high-content flow cytometric analysis of hESCs.

Enumeration of human embryonic stem cell (hESC) numbers through single cell digestion can be time consuming especially in high-throughput or multi-factorial analysis containing 50+ samples. We have developed a reproducible, cost-effective method of counting hESCs in clumps circumventing the need to manually dissociate each sample to single cells. The method is based on the DNA binding capacity of propidium iodide (PI) and subsequent fluorescent signal detection. Standard curves generated for cell numbers versus PI fluorescence as single cells or clumps showed an almost identical relationship in the lines of best fit. The reproducibility of the assay was first demonstrated by seeding hESC clumps at specific cell densities ranging 0.05–2 × 105 cells/well and then secondly by using the assay to count cell numbers after different growth conditions. Validation tests showed that consistent seeding densities are important in maintaining undifferentiated hESC culture and that the assay can be used to estimate relative cell numbers and growth curves with high accuracy.