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Schematic diagram demonstrating an integrated scalable one-unit bioprocess for iPSC expansion, cardiac differentiation, purification and recovery in a continuous stirring bioreactor
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Background:
The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reac...
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Purpose of Review
Human induced pluripotent stem cells (hiPSC) have become one of the most promising cell biology tools over the past decade. With the potential to be differentiated into diverse human cell types, their use ranges from disease modeling and drug development, to future personalized cell therapies and regenerative medicine. One of the...
Citations
... Despite these achievements, challenges persist, particularly in improving patient accessibility and product affordability [4]. Here, allogeneic cell therapies based on human induced pluripotent stem cells (hiPSCs) may prove pivotal, as they allow for both an economy-of-scale approach and the production of a wide range of specialized cell types [5][6][7][8]. Current estimates place the number of cells required for such treatments at up to 10 12 per patient depending on the clinical indication [4]. ...
Given the demands human induced pluripotent stem cell (hiPSC)-based therapeutics place on manufacturing, process intensification strategies which rapidly ensure the desired cell quality and quantity should be considered. Within the context of antibody and vaccine manufacturing, one-step inoculation has emerged as an effective strategy for intensifying the upstream process. This study therefore evaluated whether this approach could be applied to the expansion of hiPSCs in flasks under static and in microcarrier-operated stirred bioreactors under dynamic conditions. Our findings demonstrated that high density working cell banks containing hiPSCs at concentrations of up to 100 × 106 cells mL−1 in CryoStor® CS10 did not impair cell growth and quality upon thawing. Furthermore, while cell distribution, growth, and viability were comparable to routinely passaged hiPSCs, those subjected to one-step inoculation and expansion on microcarriers under stirred conditions were characterized by improved attachment efficiency (≈50%) following the first day of cultivation. Accordingly, the process development outlined in this study establishes the foundation for the implementation of this intensified approach at L-scale.
... The activation of the WNT signaling pathway via small molecule of CHIR99021 leads to the differentiation of PSCs into the mesoderm lineage. [10,11] CHIR99021 increases the activity of the WNT pathway by inhibiting the GSK3β as one of the protein kinases of the WNT signaling pathway, thereby driving PSCs into the mesoderm lineage. [12,13]The main effective factor in WNT signaling is β-catenin. ...
Glycogen Synthase Kinase 3β (GSK3β) is a multifunctional serine/threonine-protein kinase that serves as a pivotal regulator of various human pluripotent stem cell (hPSCs) functions, including self-renewal, adhesion, survival, and differentiation in addition to have an effect on motility of sperm. Despite advancement in understanding the critical roles of GSK3β inhibition in various stem cell functions, the exact molecular basis of its inactivation using various small-molecule inhibitors remains poorly understood. Investigating the mechanistic details of the actions of inhibitors targeting GSK3 proteins, such as CHIR99021, Azakenpaullone, and Tricantin, could be extremely beneficial for improving novel defined stem cell culture systems and cancer research. The present study aimed to predict the binding mode of the aforementioned ligands with GSK3β, by molecular docking and metadynamic simulation, and compare the three-dimensional structure of the inactive conformation of GSK3β in the presence of three inhibitors. Also, the pharmacokinetic or ADMET properties of ligands, such as Lipinski's rule of five violations for drug-likeness, QPlog S, QPlog K, and bioactivity scoring, were predicted. The analysis of protein stability revealed that in the absence of inhibitors, the GSK3β has higher flexibility, while in the presence of CHIR and AZA, the rate of flexibility of most protein regions, especially the envelope area, decreased. It was found that though all small molecules are capable of facilitating the inhibition of GSK3β protein, but the flexibility of protein is a bit higher for CHIR than those for other two ligands.
... At this point, it is worth mentioning that while chemically defined media for cell differentiation are available (Gultian et al. 2022), their application remains limited. On the contrary, the more popular approach is to supplement the media either with fetal bovine serum or a substitute, such as KnockOut™ Serum Replacement (Ackermann et al. 2018), human plasma (Sivalingam et al. 2021), or platelet lysate (Mizukami et al. 2018) alongside other recombinant and synthetic components (Olmer et al. 2018;Haack-Sørensen et al. 2018;Yabe et al. 2019;Laco et al. 2020;Jacobson et al. 2021). Correspondingly, these media compositions have facilitated the differentiation of hiPSCs into various cell types, such as hMSCs (Goetzke et al. 2019), cardiomyocytes (Laco et al. 2020), neurons (Silva et al. 2021), definitive endoderm (Jacobson et al. 2021), and hematopoietic cells (Sivalingam et al. 2021). ...
... On the contrary, the more popular approach is to supplement the media either with fetal bovine serum or a substitute, such as KnockOut™ Serum Replacement (Ackermann et al. 2018), human plasma (Sivalingam et al. 2021), or platelet lysate (Mizukami et al. 2018) alongside other recombinant and synthetic components (Olmer et al. 2018;Haack-Sørensen et al. 2018;Yabe et al. 2019;Laco et al. 2020;Jacobson et al. 2021). Correspondingly, these media compositions have facilitated the differentiation of hiPSCs into various cell types, such as hMSCs (Goetzke et al. 2019), cardiomyocytes (Laco et al. 2020), neurons (Silva et al. 2021), definitive endoderm (Jacobson et al. 2021), and hematopoietic cells (Sivalingam et al. 2021). Moreover, by adjusting composition and leveraging changes in intrinsic metabolic requirements during differentiation, selective pressure could be applied, improving target cell purity (Kehoe et al. 2010;Tohyama et al. 2017;Hsu et al. 2021) prior to downstream processing. ...
Stem cell–based cell therapeutics and especially those based on human mesenchymal stem cells (hMSCs) and induced pluripotent stem cells (hiPSCs) are said to have enormous developmental potential in the coming years. Their applications range from the treatment of orthopedic disorders and cardiovascular diseases to autoimmune diseases and even cancer. However, while more than 27 hMSC-derived therapeutics are currently commercially available, hiPSC-based therapeutics have yet to complete the regulatory approval process. Based on a review of the current commercially available hMSC-derived therapeutic products and upcoming hiPSC-derived products in phase 2 and 3, this paper compares the cell therapy manufacturing process between these two cell types. Moreover, the similarities as well as differences are highlighted and the resulting impact on the production process discussed. Here, emphasis is placed on (i) hMSC and hiPSC characteristics, safety, and ethical aspects, (ii) their morphology and process requirements, as well as (iii) their 2- and 3-dimensional cultivations in dependence of the applied culture medium and process mode. In doing so, also downstream processing aspects are covered and the role of single-use technology is discussed.
Key points
• Mesenchymal and induced pluripotent stem cells exhibit distinct behaviors during cultivation
• Single-use stirred bioreactor systems are preferred for the cultivation of both cell types
• Future research should adapt and modify downstream processes to available single-use devices
... However, the proposed albumin-free protocols have not been optimized or explored in depth, and CDM3-and RPMI/B27-based protocols dominate the landscape of hiPSC-derived cardiomyocyte (hiPSC-CM) differentiation due to their reliability, yield, and cost. The majority of recent developments have focused on small improvements to these protocols, such as with the addition of transferrin to the CDM3 basal medium (Zhang et al., 2021a), and on increased scalability via the use of bioreactors either with CDM3 (Manstein et al., 2021a(Manstein et al., , 2021b or RPMI/B27 (Laco et al., 2020) systems. Similarly, the re-modulation of the Wnt/b-catenin pathway at different time points has been proposed to increase the yield of cardiomyocytes in an RPMI/B27-based differentiation (Buikema et al., 2020;Maas et al., 2021;Sharma et al., 2018). ...
The methods for the culture and cardiomyocyte differentiation of human embryonic stem cells, and later human induced pluripotent stem cells (hiPSC), have moved from a complex and uncontrolled systems to simplified and relatively robust protocols, using the knowledge and cues gathered at each step. HiPSC-derived cardiomyocytes have proven to be a useful tool in human disease modelling, drug discovery, developmental biology, and regenerative medicine. In this protocol review, we will highlight the evolution of protocols associated with hPSC culture, cardiomyocyte differentiation, sub-type specification, and cardiomyocyte maturation. We also discuss protocols for somatic cell direct reprogramming to cardiomyocyte-like cells.
... Importantly, RepMC can produce iPSCs with high differentiation potential forming all three germ layers and further demonstrated by the formation of functional cardiomyocytes 25 and erythroblasts. 27,28 It is worth noting that there is variability between the differentiation efficiencies of different clones ( Figure 6). ...
Objectives:
Induced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges.
Materials and methods:
Five sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures.
Results:
Improvement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50-fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β-catenin, E-cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ-layers, cardiomyocytes and haematopoietic stem cells were further demonstrated.
Conclusions:
Our method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.
... 38 hPSCs, and it has been suggested that these hPSC-CMs show various characteristics because these methods have different points in the culture system 2D or 3D and combination of recombinant proteins and small molecules (Table 2). 4,[39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54] As there is a concern about a large number of variations in hPSC-CMs for transplantation, 55 it is desirable to induce large numbers of hPSC-CMs at the same time and develop non-invasive methods to prepare hPSC-CMs only. One of the concerns with regard to mass culture in 2D culture is that there are fluctuations in differentiation efficiency, resulting from starting cell density and cell confluency. ...
Basic research on human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) for cardiac regenerative therapy is one of the most active and complex fields to achieve this alternative to heart transplantation and requires the integration of medicine, science, and engineering. Mortality in patients with heart failure remains high worldwide. Although heart transplantation is the sole strategy for treating severe heart failure, the number of donors is limited. Therefore, hPSC-derived CM (hPSC-CM) transplantation is expected to replace heart transplantation. To achieve this goal, for basic research, various issues should be considered, including how to induce hPSC proliferation efficiently for cardiac differentiation, induce hPSC-CMs, eliminate residual undifferentiated hPSCs and non-CMs, and assess for the presence of residual undifferentiated hPSCs in vitro and in vivo. In this review, we discuss the current stage of resolving these issues and future directions for realizing hPSC-based cardiac regenerative therapy.
... 8 While incorporating cells within biocompatible scaffolds has been shown to increase cell retention, the implantation cell culture systems can achieve high cell yields, allowing the mass production of cells for cell therapy and tissue engineering approaches. [16][17][18][19] Additionally, the resulting cell-laden microcarriers constitute an optimal platform for cell delivery. ...
Cell-based therapies require a large number of cells, as well as appropriate methods to deliver the cells to damaged tissue. Microcarriers provide an optimal platform for large scale cell culture while also improving cell retention during cell delivery. However, this technology still presents significant challenges due to low throughput fabrication methods and an inability of the microcarriers to recreate the properties of human tissue. This work proposes, for the first time, the use of methacryloyl platelet lysates (PLMA), a photocrosslinkable material derived from human platelet lysates, to produce porous microcarriers. Initially, high quantities of PLMA/alginate core-shell microcapsules are produced using coaxial electrospray. Subsequently, the microcapsules are collected, irradiated with UV light, washed, and freeze-dried yielding PLMA microsponges. These microsponges are able to support the adhesion and proliferation of human adipose-derived stem cells (hASCs), while also displaying potential in the assembly of autologous microtissues. Cell-laden microsponges were shown to self-organize into aggregates, suggesting possible applications in bottom-up tissue engineering applications.
... To overcome these limitations, we explored the possibility of using ZM, an inhibitor of Aur-A (which controls the cell cycle), in the differentiation stage. The differentiation of CMs is related to the cell cycle, and the Wnt/Gsk3β mechanism in CMs varies depending on the confluence of hiPSCs at the beginning of differentiation [18,42]. Our experimental results confirmed that the cell cycle ratio changed according to the confluence of hiPSCs. ...
Drug-induced cardiotoxicity reduces the success rates of drug development. Thus, the limitations of current evaluation methods must be addressed. Human cardiac organoids (hCOs) derived from induced pluripotent stem cells (hiPSCs) are useful as an advanced drug-testing model; they demonstrate similar electrophysiological functionality and drug reactivity as the heart. How-ever, similar to other organoid models, they have immature characteristics compared to adult hearts, and exhibit batch-to-batch variation. As the cell cycle is important for the mesodermal differentiation of stem cells, we examined the effect of ZM447439, an aurora kinase inhibitor that regulates the cell cycle, on cardiogenic differentiation. We determined the optimal concentration and timing of ZM447439 for the differentiation of hCOs from hiPSCs and developed a novel protocol for efficiently and reproducibly generating beating hCOs with improved electrophysiological functionality, contractility, and yield. We validated their maturity through electro-physiological- and image-based functional assays and gene profiling with next-generation sequencing, and then applied these cells to multi-electrode array platforms to monitor the cardio-toxicity of drugs related to cardiac arrhythmia; the results confirmed the drug reactivity of hCOs. These findings may enable determination of the regulatory mechanism of cell cycles underlying the generation of iPSC-derived hCOs, providing a valuable drug testing platform.
... Three-dimensional embryoid body (EB)-based protocols utilise aggregates of hPSCs which differentiate in suspension to give beating clusters containing CMs [103,104]. The advantage of this method is its scalability and compatibility with bioreactors [56], but the success of such protocols requires very careful control of the size of the initial hPSC aggregates and are therefore prone to variability among batches and operators [6,45,98]. Furthermore, long-term culture of EBs encourages the formation of extracellular matrix, which hampers dissociation into single cells for experiments. ...
... Genomic abnormalities can affect the expression of lineage-associated genes and thereby impact upon differentiation propensities [30]. Consistent with the above, numerous papers have shown that different hiPSC lines exhibit vastly different differentiation efficiencies using the same protocols [56,57,82]. In a recent population-based study of cardiac toxicity, Burnett et al. commented that only ~ 20% of the iPSC clones from non-diseased human donors could be successfully differentiated into CM-like cells by the commercial supplier [13]. ...
Human pluripotent stem cells (hPSC) self-renew and represent a potentially unlimited source for the production of car-diomyocytes (CMs) suitable for studies of human cardiac development, drug discovery, cardiotoxicity testing, and disease modelling and for cell-based therapies. However, most cardiac differentiation protocols yield mixed cultures of atrial-, ventricular-, and pacemaker-like cells at various stages of development, as well as non-CMs. The proportions and matu-ration states of these cell types result from disparities among differentiation protocols and time of cultivation, as well as hPSC reprogramming inconsistencies and genetic background variations. The reproducible use of hPSC-CMs for research and therapy is therefore limited by issues of cell population heterogeneity and functional states of maturation. A validated method that overcomes issues of cell heterogeneity is immunophenotyping coupled with live cell sorting, an approach that relies on accessible surface markers restricted to the desired cell type(s). Here we review current progress in unravelling heterogeneity in hPSC-cardiac cultures and in the identification of surface markers suitable for defining cardiac identity, subtype specificity, and maturation states.
... However, because the GiWi method is typically used to differentiate two-dimensional (2D) cell sheets in 6-well culture plates (Sharma et al., 2015), it may not be sufficiently scalable to produce the number of hiPSC-CMs needed for high-throughput cardiotoxicity assessments or for clinical applications such as the treatment of acute myocardial infarction (MI), which often results in the loss of ∼1 billion cardiomyocytes (Chong et al., 2014;Kropp et al., 2016;Dunn and Palecek, 2018). Higher yields may be achievable with multilayered/stacked flasks or multicarrier-based systems, but neither of these methods have been fully scaled, and both require materials and reagents that are not readily available (Ting et al., 2014Breckwoldt et al., 2017;Le and Hasegawa, 2019;Chang et al., 2020;Laco et al., 2020). ...
... The GiWi method is among the most efficient strategies for differentiating hiPSCs into cardiomyocytes; however, it may not be sufficiently scalable to produce the billions of hiPSC-CMs needed for treatment of myocardial disease or for highthroughput drug-testing, because it is typically conducted with 2D cell sheets in 6-well culture plates (Sharma et al., 2015). hiPSC-CMs can also be produced in suspension culture (Shafa et al., 2011;Kempf et al., 2014Kempf et al., , 2015Kempf et al., , 2016Fonoudi et al., 2016;Halloin et al., 2019;Hamad et al., 2019;Chang et al., 2020;Laco et al., 2020;Miwa et al., 2020), which is more compatible with largescale production, and the GiWi-based suspension-differentiation protocol introduced here incorporates a number of other key innovations, such as (1) the use of hiPSC culture media that was designed specifically for 3D culture and supplied via a fed batch reactor, (2) a filtration step before differentiation to reduce the heterogeneity of the spheroid population, (3) partial media changes on day 3 and from day 12 onward, which reduced processing time, and (4) direct incorporation of metabolic purification, which increased the purity of the differentiated hiPSC-CM populations to > 98%. Furthermore, whereas newly differentiated hiPSC-CMs are more phenotypically similar to fetal than to adult cardiomyocytes (Xu et al., 2009;Gupta et al., 2010;Yang et al., 2014;van den Berg et al., 2015), our results suggest that at least some markers for cardiomyocyte maturation tended to be more highly expressed in suspension-differentiated than monolayer-differentiated hiPSC-CMs; this observation is consistent with previous reports that 3D culture conditions appear to promote hiPSC-CM maturity (Correia et al., 2018;Beauchamp et al., 2020;Giacomelli et al., 2020). ...
Human induced-pluripotent stem cells (hiPSCs) can be efficiently differentiated into cardiomyocytes (hiPSC-CMs) via the GiWi method, which uses small-molecule inhibitors of glycogen synthase kinase (GSK) and tankyrase to first activate and then suppress Wnt signaling. However, this method is typically conducted in 6-well culture plates with two-dimensional (2D) cell sheets, and consequently, cannot be easily scaled to produce the large numbers of hiPSC-CMs needed for clinical applications. Cell suspensions are more suitable than 2D systems for commercial biomanufacturing, and suspended hiPSCs form free-floating aggregates (i.e., spheroids) that can also be differentiated into hiPSC-CMs. Here, we introduce a protocol for differentiating suspensions of hiPSC spheroids into cardiomyocytes that is based on the GiWi method. After optimization based on cardiac troponin T staining, the purity of hiPSC-CMs differentiated via our novel protocol exceeded 98% with yields of about 1.5 million hiPSC-CMs/mL and less between-batch purity variability than hiPSC-CMs produced in 2D cultures; furthermore, the culture volume could be increased ∼10-fold to 30 mL with no need for re-optimization, which suggests that this method can serve as a framework for large-scale hiPSC-CM production.
