In Vitro Grown Micro-Tissues for Cardiac Cell Replacement Therapy in Vivo

Sahito, Raja Ghazanfar Ali; Sheng, Xiaowu; Maaß, Martina; Mikhael, Nelly; Hamad, Sarkawt; Heras-Bautista, Carlos O; Derichsweiler, Daniel; Spitkovsky, Dimitry; Suhr, Frank; Khalil, Markus; Brockmeier, Konrad; Halbach, Marcel; Šarić, Tomo; Hescheler, Jürgen; Krausgrill, Benjamin; Pfannkuche, Kurt

doi:10.33594/000000092

Cited by 5 publications

(4 citation statements)

References 33 publications

(43 reference statements)

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“…For iPSC-CMs, it was reported that transplanted human iPSC-CMs could improve myocardial functions and alleviate ventricular remodeling in rat hearts ( Guan et al, 2020 ). Another study used iPSC-CMs and mesenchymal stem cells to generate micro-tissues for cardiac cell replacement therapy in vivo ( Sahito et al, 2019 ). These findings raised the possibilities that gene-edited patient-specific iPSC-CMs can be used as cell resources for intramyocardial transplantation to repair the structural or functional defect in FC patients.…”

Section: Discussionmentioning

confidence: 99%

Dual DNA Transfection Using 1,6-Hexanedithiol-Conjugated Maleimide-Functionalized PU-PEI600 For Gene Correction in a Patient iPSC-Derived Fabry Cardiomyopathy Model

Chien

Lin

et al. 2021

Front. Cell Dev. Biol.

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Non-viral gene delivery holds promises for treating inherited diseases. However, the limited cloning capacity of plasmids may hinder the co-delivery of distinct genes to the transfected cells. Previously, the conjugation of maleimide-functionalized polyurethane grafted with small molecular weight polyethylenimine (PU-PEI600-Mal) using 1,6-hexanedithiol (HDT) could promote the co-delivery and extensive co-expression of two different plasmids in target cells. Herein, we designed HDT-conjugated PU-PEI600-Mal for the simultaneous delivery of CRISPR/Cas9 components to achieve efficient gene correction in the induced pluripotent stem cell (iPSC)-derived model of Fabry cardiomyopathy (FC) harboring GLA IVS4 + 919 G > A mutation. This FC in vitro model recapitulated several clinical FC features, including cardiomyocyte hypertrophy and lysosomal globotriaosylceramide (Gb3) deposition. As evidenced by the expression of two reporter genes, GFP and mCherry, the addition of HDT conjugated two distinct PU-PEI600-Mal/DNA complexes and promoted the co-delivery of sgRNA/Cas9 and homology-directed repair DNA template into target cells to achieve an effective gene correction of IVS4 + 919 G > A mutation. PU-PEI600-Mal/DNA with or without HDT-mediated conjugation consistently showed neither the cytotoxicity nor an adverse effect on cardiac induction of transfected FC-iPSCs. After the gene correction and cardiac induction, disease features, including cardiomyocyte hypertrophy, the mis-regulated gene expressions, and Gb3 deposition, were remarkably rescued in the FC-iPSC-differentiated cardiomyocytes. Collectively, HDT-conjugated PU-PEI600-Mal-mediated dual DNA transfection system can be an ideal approach to improve the concurrent transfection of non-viral-based gene editing system in inherited diseases with specific mutations.

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Section: Discussionmentioning

confidence: 99%

Dual DNA Transfection Using 1,6-Hexanedithiol-Conjugated Maleimide-Functionalized PU-PEI600 For Gene Correction in a Patient iPSC-Derived Fabry Cardiomyopathy Model

Chien

Lin

et al. 2021

Front. Cell Dev. Biol.

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“…The cells were cultured in complete DMEM (4.5 g) glucose l −1 with 15% fetal calf serum (FCS) (Sigma-Aldrich, Taufkirchen, Germany). MSCs were prepared as described previously [8] and cultured in complete DMEM containing (1 g) glucose l −1 (Thermo Fisher Scientific) and 15% FCS. For a generation of miPS-CMs, TαP4 [28] cultivated on mitomycin inactivated embryonic feeder cells in Iscove's modified Dulbecco's medium (IMDM) supplemented with 15% FCS and 1000 units ml −1 leukemia inhibitory factor (ORF Genetics, Reykjavik, Iceland).…”

Section: Cell Culture and Generation Of Mips Cell-derived Cmsmentioning

confidence: 99%

“…Although 2D cultures can be used to address uncounted scientific questions the interaction of cells in a 3D environment opens new avenues in many applications. In cardiac cell therapy, it was shown early that single PSC-derived CMs (PSC-CMs) hardly engraft in the diseased murine tissue while the addition of connective tissue cells substantially enhances the cell engraftment in vitro and in vivo [2,8,9]. Based on such findings, we have developed a simple method of generating microtissues from mesenchymal stem cells (MSCs) and murine iPSC-CMs (miPSC-CMs) resulting in >10-fold enhanced early engraftment of PSC-CMs.…”

Section: Introductionmentioning

confidence: 99%

“…The above-mentioned applications have in common that microtissues are mainly generated by nonscalable methods. A simple but rather uncontrolled method is the generation of cell patches composed of one or multiple cell types from thermo-responsible polymer surfaces [8]. More defined methods use hanging drop cultures to combine cells in individual drops of cell suspension hanging at the lid of a petri dish for one to two days.…”

Section: Introductionmentioning

confidence: 99%

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Engineering of cardiac microtissues by microfluidic cell encapsulation in thermoshrinking non-crosslinked PNIPAAm gels

et al. 2022

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Multicellular agglomerates in form of irregularly shaped or spherical clusters can recapitulate cell-cell interactions and are referred to as microtissues. Microtissues gain increasing attention in several fields including cardiovascular research. Cardiac microtissues are evolving as excellent model systems for drug testing in vitro (organ-on-a-chip), are used as tissue bricks in 3D printing processes and pave the way for improved cell replacement therapies in vivo. Microtissues are formed for example in hanging drop culture or specialized microwell plates; truly scalable methods are not yet available. In this study, a novel method of encapsulation of cells in Poly-N-isopropylacrylamid (PNIPAAm) spheres is introduced. Murine induced pluripotent stem cell-derived cardiomyocytes (CMs) and bone marrow-derived mesenchymal stem cells (MSCs) were encapsulated in PNIPAAm by raising the temperature of droplets formed in a microfluidics setup above the lower critical solute temperature (LCST) of 32°C. PNIPAAM precipitates to a water-insoluble physically linked gel above the LCST and shrinks by the expulsion of water, thereby trapping the cells in a collapsing polymer network and increasing the cell density by one order of magnitude. Within 24 hours, stable cardiac microtissues were first formed and later released from their polymer shell by washout of PNIPAAm at temperatures below the LCST. Rhythmically contracting microtissues showed homogenous cell distribution, age-dependent sarcomere organizations and action potential generation. The novel approach is applicable for microtissue formation from various cell types and can be implemented into scalable workflows.

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Poly(acrylamide) Spheroids with Tunable Elasticity for Scalable Cell Culture Applications

Pieroth

Heras-Bautista

Hamad

et al. 2022

Macro Chemistry & Physics

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Many biological tissues resemble hydrogels and display Young's moduli below 50 kPa, corresponding to most cell types for the natural environmental conditions to grow. Contrastingly, conventional cell culture usually involves rigid substrates resulting in stiff priming effects, which are of increasing concern when it comes to scalable culturing of adhesive cells for regenerative purposes. As a solution to this problem, the employment of synthetic poly(acryl amide) (PAAm)-based hydrogel beads with tissue-matched mechanical properties are proposed as soft matrix for culture modes suitable for tidal bioreactor culture. Herein, technology is described to generate spherical, mm-scaled PAAm hydrogel beads with adjustable, soft elastic properties that are produced by a continuous microfluidic approach. A simple and robust method is demonstrated to functionalize the spheroids with protein ligands, and the suitability of the matrix for cell cultivation is successfully demonstrated with three different cell types (murine mesenchymal stem cells, renal carcinoma cells, and human induced pluripotent stem cells) in model experiments and in a tidal bioreactor system. This versatile approach will pave the way toward novel cell culture systems based on bioreactors that allow scalable, soft carrier-based expansion of cells on matrices with tissue-matched elasticity.

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In Vitro Grown Micro-Tissues for Cardiac Cell Replacement Therapy in Vivo

Abstract: This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission.

Cited by 5 publications

References 33 publications

Dual DNA Transfection Using 1,6-Hexanedithiol-Conjugated Maleimide-Functionalized PU-PEI600 For Gene Correction in a Patient iPSC-Derived Fabry Cardiomyopathy Model

Dual DNA Transfection Using 1,6-Hexanedithiol-Conjugated Maleimide-Functionalized PU-PEI600 For Gene Correction in a Patient iPSC-Derived Fabry Cardiomyopathy Model

Engineering of cardiac microtissues by microfluidic cell encapsulation in thermoshrinking non-crosslinked PNIPAAm gels

Poly(acrylamide) Spheroids with Tunable Elasticity for Scalable Cell Culture Applications

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