Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling

Kadari, Asifiqbal; Mekala, SubbaRao; Wagner, Nicole; Malan, Daniela; Köth, Jessica; Döll, Katharina; Stappert, Laura; Eckert, Daniela; Peitz, Michael; Matthes, Jan; Sasse, Philipp; Herzig, Stefan; Brüstle, Oliver; Ergün, Süleyman; Edenhofer, Frank

doi:10.1007/s12015-014-9564-6

Cited by 56 publications

(65 citation statements)

References 44 publications

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“…154 A 4-day treatment of differentiating hiPSCs with lactate-containing/glucosedevoid culture medium resulted not only in improved differentiation efficiency (95% cTNT + cells vs. 63% for control cultures) but also in the reduction of line-to-line variability (ranging from 33% to 92% of cTNT + cells for five hiPSC lines). 155 Similar enrichment of hPSC-derived cardiomyocytes has been demonstrated in spinner flask cultures. 156 Another vexing issue is the phenotypic immaturity of hPSC-derived cardiomyocytes including the lack of fully formed sacromeric elements and multinucleation, and their suboptimal Ca + 2 handling.…”

Section: In Vitro Methods For Cardiac Differentiation Of Hpscsmentioning

confidence: 55%

Signaling Pathways and Gene Regulatory Networks in Cardiomyocyte Differentiation

Parikh

Blanton

et al. 2015

Tissue Engineering Part B: Reviews

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Strategies for harnessing stem cells as a source to treat cell loss in heart disease are the subject of intense research. Human pluripotent stem cells (hPSCs) can be expanded extensively in vitro and therefore can potentially provide sufficient quantities of patient-specific differentiated cardiomyocytes. Although multiple stimuli direct heart development, the differentiation process is driven in large part by signaling activity. The engineering of hPSCs to heart cell progeny has extensively relied on establishing proper combinations of soluble signals, which target genetic programs thereby inducing cardiomyocyte specification. Pertinent differentiation strategies have relied as a template on the development of embryonic heart in multiple model organisms. Here, information on the regulation of cardiomyocyte development from in vivo genetic and embryological studies is critically reviewed. A fresh interpretation is provided of in vivo and in vitro data on signaling pathways and gene regulatory networks (GRNs) underlying cardiopoiesis. The state-of-the-art understanding of signaling pathways and GRNs presented here can inform the design and optimization of methods for the engineering of tissues for heart therapies.

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Section: In Vitro Methods For Cardiac Differentiation Of Hpscsmentioning

confidence: 55%

Signaling Pathways and Gene Regulatory Networks in Cardiomyocyte Differentiation

Parikh

Blanton

et al. 2015

Tissue Engineering Part B: Reviews

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“…At 80-90% confluency, cardiac differentiation was initiated as described by Kadari et al (2015). For the 1st day, cells were cultured with 25 ng/ml BMP4 (Gibco) and 5 μM CHIR99021 (Axon Medchem, Groningen, Netherlands) added to a cardiac basal medium comprising RPMI1640 (Gibco), 1× B27 (Gibco), 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol (Gibco), and 50 μg/ml ascorbic acid (Sigma-Aldrich).…”

Section: Directed Differentiation Into Cardiomyocytelike Cellsmentioning

confidence: 99%

“…Thus, whether the hiPSCs grown in bioreactors could be directed to differentiate into cardiomyocytes were assessed following a previously published protocol (Kadari et al, 2015). After 8 days of differentiation, the cells changed morphology and started to beat spontaneously; after an additional 3 days, beating sheets of cells could be seen (Video S1).…”

Section: Quantification Of Spinner and Bioreactor Aggregatesmentioning

confidence: 99%

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single‐use bioreactors

Kwok

Ueda

Kadari

et al. 2017

J Tissue Eng Regen Med

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The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell-based therapies and cell-loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400 ml. The simple and robust two-step process reported here first generates hiPSC aggregates of 324 ± 71 μm diameter in 7 days in 125 ml spinner flasks (100 ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000 ml bioreactors (1000 ml volume), finally yielding hiPSC aggregates of 198 ± 58 μm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40 days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16-fold increase in hiPSC quantity at the 100 ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000 ml scale, an additional 10-fold increase was achieved. Taken together, 16 × 10 6 hiPSCs were expanded into 2 × 10 9 hiPSCs in 14 days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell-based therapies and brings their clinical potential closer to fruition.

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“…However, hiPSC-CM migration into the scaffold was barely detected. The contraction of the cell clusters and the presence of the mature cardiac marker cTnT [20] strengthens the compatibility of carbon fiber meshes for cardiac cells. To prove that the porous carbon fiber scaffolds can be embedded into a relevant target tissue, the scaffolds were used in a tissue engineering approach.…”

Section: Figurementioning

confidence: 72%

Flexible tissue-like electrode as a seamless tissue-electronic interface

et al. 2017

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Abstract:Current implantable electrodes facilitate only a low cellular infiltration impairing the long-term integration into the host's tissue. To accomplish a seamless electronic-tissue interface, conductive three-dimensional (3D) scaffolds were generated by carbonization of electro-spun fiber meshes. When introducing NaCl particles as porogens, tailored tissue-like electrodes were generated. Characterization of the porous 3D fiber electrodes demonstrated improved material and electrical characteristics compared to standard carbon fiber meshes or flat gold surfaces. The feasibility of the porous 3D electrodes was assessed by cell culture experiments, confirming the migration of cells into the electrode and the formation of contracting cardiomyocyte clusters. Finally, a complex cardiac co-culture system proved the integration of the tissue into the 3D electrode in long-term culture of 7 weeks. These results strengthen the development of tissue-like 3D scaffolds as alternative to two-dimensional (2D) electrodes. The main challenge in the development of implantable electrodes is to improve the currently limited long-term tissue integration that induces adverse effects such as the encapsulation of the implant [1], [2]. This drawback is caused by differences in elasticity between the host tissue and the electrode, an insufficient electrode flexibility to follow tissue deformation as well as surface properties impairing the infiltration of the electrode with cells [3], [4]. Therefore, concepts for the generation of flexible electrodes were developed, consisting of microscopic conducting paths embedded in flexible silicone or SU-8 photoresist [5], [6]. However, the infiltration of cells in the electrodes is still limited, although Bryers et al. [7] showed that the presence of interconnected pores, which facilitate cell infiltration in an implant, significantly reduces the foreign body reaction. Thus, an improved approach may be a seamless 3D tissue-electronic interface composed of conductive nanofibers that are embedded in the tissue. A common method for the fabrication of 3D nanofiber scaffolds is electrospinning, producing meshes in the regime of extracellular matrix fibers [8]. For example, by spinning polyacrylonitrile (PAN) with a subsequent carbonization, highly conductive nanofiber scaffolds with biocompatible properties were generated [9]. Disadvantage of electro-spun scaffolds is the small mesh size that impairs cell infiltration of the scaffold. The generation of electro-spun porous scaffolds by introducing porogens like sugar or NaCl into a non-conductive nanofiber scaffold during the spinning process has already been reported by different groups [10], [11]. Although the highly flexible polymer fibers demonstrated the infiltration of high cell numbers, the obtained pores were susceptible to collapse. Moreover, these porous polymer scaffolds are not applicable as electrodes due to their insulating properties. In our study, we fabricated conductive porous fiber scaffolds. Therefore, we combined conductive nan...

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Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling

Cited by 56 publications

References 44 publications

Signaling Pathways and Gene Regulatory Networks in Cardiomyocyte Differentiation

Signaling Pathways and Gene Regulatory Networks in Cardiomyocyte Differentiation

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single‐use bioreactors

Flexible tissue-like electrode as a seamless tissue-electronic interface

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