Engineering Stem Cell Self-organization to Build Better Organoids

Brassard, Jonathan A.; Lütolf, Matthias P.

doi:10.1016/j.stem.2019.05.005

Cited by 250 publications

(237 citation statements)

References 153 publications

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“…Since the development of the first intestinal organoids by Sato and collaborators in 2009 [146], many other models of various tissues have been developed following diverse approaches such as (i) Matrigel scaffold-based, (ii) Embryoid Bodies (EB)-based, and (iii) Air-Liquid Interface (ALI) method [147], and they have been used to model a great range of pathologies, from viral infection to solid cancers, cystic fibrosis, and endometriosis ( Table 5). Even if in the last decade efforts have been made for improving organoids fidelity to the in vivo counterpart [148], there are still several critical aspects that have to be taken into account when evaluating organoids predictability. In particular, there is poor control on the shape, size and cells appropriate organization, causing hurdles in terms of scalability and high-throughput approaches.…”

Section: Ex Vivo Stem Cell-based Systems: Organoidsmentioning

confidence: 99%

Harnessing the Potential of Stem Cells for Disease Modeling: Progress and Promises

Argentati

Tortorella

Bazzucchi

et al. 2020

JPM

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Ex vivo cell/tissue-based models are an essential step in the workflow of pathophysiology studies, assay development, disease modeling, drug discovery, and development of personalized therapeutic strategies. For these purposes, both scientific and pharmaceutical research have adopted ex vivo stem cell models because of their better predictive power. As matter of a fact, the advancing in isolation and in vitro expansion protocols for culturing autologous human stem cells, and the standardization of methods for generating patient-derived induced pluripotent stem cells has made feasible to generate and investigate human cellular disease models with even greater speed and efficiency. Furthermore, the potential of stem cells on generating more complex systems, such as scaffold-cell models, organoids, or organ-on-a-chip, allowed to overcome the limitations of the two-dimensional culture systems as well as to better mimic tissues structures and functions. Finally, the advent of genome-editing/gene therapy technologies had a great impact on the generation of more proficient stem cell-disease models and on establishing an effective therapeutic treatment. In this review, we discuss important breakthroughs of stem cell-based models highlighting current directions, advantages, and limitations and point out the need to combine experimental biology with computational tools able to describe complex biological systems and deliver results or predictions in the context of personalized medicine.Since their establishment, cell cultures have been proven to be the first and most powerful tool for the in vitro investigation of cell biology, from basic research to more complex translational approaches [6]. Classical two-dimensional (2D)-cultures usually consist of a unique type of cells (primary or continuous cultures) that grow in tissue culture polystyrene as adherent monolayers or in floating suspension, both in culture media that provide essential nutrients and growth factors. 2D-strategies are widely used to obtain a large number of cells, thus allowing the in vitro study of molecular mechanisms and development of metabolic assays [7-9]. However, due to their inability to recreate the in vivo complexity of the biological environment, they are not suitable for accurate disease modeling. These limitations have been overcome by the development of three-dimensional (3D)-cell cultures systems [10]. The rationale design of 3D-cell cultures is to harvest cells in microstructures that resemble tissues or organs shape and organization, thus allowing better cell-to-cell/cell-environment contacts and signaling crosstalk [11][12][13][14][15], essential events for tissues correct development and functioning [14,15]. The 3D-cell culture strategy is articulated in many different methods and techniques that can be grouped in scaffold-based or non-scaffold based platforms, each with particular advantages and disadvantages that make them useful for different applications [10,[16][17][18][19]. 3D-cell culture strategies are supported by the in silico...

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Section: Ex Vivo Stem Cell-based Systems: Organoidsmentioning

confidence: 99%

Harnessing the Potential of Stem Cells for Disease Modeling: Progress and Promises

Argentati

Tortorella

Bazzucchi

et al. 2020

JPM

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“…Note that N-termini also contain 6xHis sequences. B: SDS-PAGE of crude extracts (1,2,4) and purified ColBD-Twitch (3) and CBD-Twitch (5) proteins. Mw standards are shown on the right.…”

Section: Figure 1 Design and Initial Evaluation Of The Cellulose-andmentioning

confidence: 99%

“…Extracellular cues become increasingly important to understand tissue organization and development. For instance, building-up the tissue from the stem cell-derived organoids demands a deep understanding of the timedependent changes of biophysical parameters experienced during growth and differentiation [1][2][3]. In addition to the physical tension [4] and the extracellular matrix itself [5], changing cell density, composition and organization can also affect gradients of O2 (hypoxia) [6,7], distribution and diffusion of reactive oxygen species, peptides and growth factors, pH and other ions such as Ca 2+ [8,9].…”

Section: Introductionmentioning

confidence: 99%

Extracellular Ca²⁺-sensitive fluorescent protein biosensor based on a collagen-binding domain

Okkelman

McGarrigle

O’Carroll

et al. 2020

Preprint

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The importance of extracellular gradients of biomolecules becomes increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, dynamics of extracellular calcium concentration is rarely studied. Here, we present low affinity Ca 2+ biosensor based on Twitch-2B fluorescent protein fused with the celluloseand collagen-binding peptides. These recombinant chimeric proteins can bind cellulose and collagen scaffolds and enable for scaffold-based biosensing of Ca 2+ in proximity of live 3D tissue models. We found that the Twitch-2B mutant is compatible with intensity-based ratiometric and fluorescence lifetime imaging microscopy (FLIM) measurement formats, under one-and twophoton excitation modes. Furthermore, the donor fluorescence lifetime of ColBD-Twitch displays response to [Ca 2+ ] over a range of ~2-2.5 ns, making it attractive biosensor for multiplexed FLIM microscopy assays. To evaluate performance of this biosensor in physiological measurements, we applied ColBD-Twitch to the live Lgr5-GFP mouse intestinal organoid culture and measured its responses to the changes in extracellular Ca 2+ upon chelation with EGTA. When we combined it with spectrally resolved FLIM of lipid droplets using Nile Red dye, we observed changes in cytoplasmic and basal membrane-associated lipid droplet composition in response to the extracellular Ca 2+ depletion, suggesting that intestinal epithelium can respond to and compensate such treatment. Altogether, our results demonstrate ColBD-Twitch as a prospective Ca 2+ sensor for multiplexed FLIM analysis in a complex 3D tissue environment. IntroductionExtracellular cues become increasingly important to understand tissue organization and development. For instance, building-up the tissue from the stem cell-derived organoids demands a deep understanding of the timedependent changes of biophysical parameters experienced during growth and differentiation [1][2][3]. In addition to the physical tension [4] and the extracellular matrix itself [5], changing cell density, composition and organization can also affect gradients of O2 (hypoxia) [6,7], distribution and diffusion of reactive oxygen species, peptides and growth factors, pH and other ions such as Ca 2+ [8,9]. A number of reports have indicated the role of extracellular Ca 2+ as one the primary signals that influence cell function [10]: thus, regulation of extracellular Ca 2+ have been shown to be relevant to gastrointestinal tract function [11,12], bone marrow [13], brain and CNS function [14, 15], smooth and skeletal muscles [16], wound healing [17], plasma membrane repair [18], engineered organoid-like tissues [19] and microbial biofilm formation [20]. Every mammalian cell relies on the Ca 2+ homeostasis, which in turn is tightly regulated through the activities of channels, ATPases, Na + / Ca 2+ exchanger, cytosolic Ca 2+ -binding proteins and various depots such as endoplasmic reticulum, secretory vesicles and mitochondria [21][22][23][24]. Calcium influx and efflux processes thr...

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“…For example, human gastruloids, which are ESC-derived aggregates that stimulate gastrulation-like events, disappear on small micropatterns, because of too little tissue to induce self-organization. 38 Second, the maturity of in vitro self-organized tissue could be enhanced perfusing vasculature to release endogenous signals and provide external factors. 39 Third, mass transport problems should be prevented by enhancing perfusion and increasing the concentrations of oxygen and nutrients.…”

Section: Limitations Of Self-organizationmentioning

confidence: 99%

Building Complex Life Through Self-Organization

Sthijns¹,

LaPointe²,

Blitterswijk³

2019

Tissue Engineering Part A

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Cells are inherently conferred with the ability to self-organize into the tissues and organs comprising the human body. Self-organization can be recapitulated in vitro and recent advances in the organoid field are just one example of how we can generate small functioning elements of organs. Tissue engineers can benefit from the power of self-organization and should consider how they can harness and enhance the process with their constructs. For example, aggregates of stem cells and tissue-specific cells benefit from the input of carefully selected biomolecules to guide their differentiation toward a mature phenotype. This can be further enhanced by the use of technologies to provide a physiological microenvironment for self-organization, enhance the size of the constructs, and enable the long-term culture of self-organized structures. Of importance, conducting selforganization should be limited to fine-tuning and should avoid over-engineering that could counteract the power of inherent cellular self-organization.Self-organization is a powerful innate feature of cells that can be fine-tuned but not over-engineered to create new tissues and organs.

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Engineering Stem Cell Self-organization to Build Better Organoids

Cited by 250 publications

References 153 publications

Harnessing the Potential of Stem Cells for Disease Modeling: Progress and Promises

Harnessing the Potential of Stem Cells for Disease Modeling: Progress and Promises

Extracellular Ca²⁺-sensitive fluorescent protein biosensor based on a collagen-binding domain

Building Complex Life Through Self-Organization

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Engineering Stem Cell Self-organization to Build Better Organoids

Cited by 250 publications

References 153 publications

Harnessing the Potential of Stem Cells for Disease Modeling: Progress and Promises

Harnessing the Potential of Stem Cells for Disease Modeling: Progress and Promises

Extracellular Ca2+-sensitive fluorescent protein biosensor based on a collagen-binding domain

Building Complex Life Through Self-Organization

Contact Info

Product

Resources

About

Extracellular Ca²⁺-sensitive fluorescent protein biosensor based on a collagen-binding domain