Sustained embryoid body formation and culture in a non-laborious three dimensional culture system for human embryonic stem cells

Stenberg, Johan; Elovsson, Maria; Strehl, Raimund; Kilmare, Eva; Hyllner, Johan; Lindahl, Anders

doi:10.1007/s10616-011-9344-y

Cited by 16 publications

(19 citation statements)

References 26 publications

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“…Several hydrogel materials-such as alginate (24,27), agarose (28), and hyaluronic acid (29)-have been previously investigated for hPSC expansion or differentiation. However, to date thermoreversible materials have not been studied in hPSC culture, despite the fact that they offer a number of promising features for GMP-compatible, large-scale culture.…”

Section: Resultsmentioning

confidence: 99%

“…The study revealed that (i) the hydrogel promoted high expansion rates and prevented cell agglomeration ( We also assessed whether other, nonthermoreversible materials systems-including UV cross-linked hyarulonic acid (29), agarose (28), and alginate hydrogel (24)-could support high level hPSC expansion. We generated and used these materials as described previously, in conjunction with E8 and RI.…”

Section: Resultsmentioning

confidence: 99%

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A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

Lei

Schaffer

2013

Proc. Natl. Acad. Sci. U.S.A.

285

312

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Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human-or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 10 72 -fold expansion over 280 d), yield (∼2.0 × 10 7 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 10 7 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.H uman pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) (1) and induced pluripotent stem cells (iPSCs) (2), have the capacities for indefinite in vitro expansion and differentiation into all cell types within adults (3). They therefore represent highly promising cell sources for numerous biomedical applications, such as cell replacement therapies (4, 5), tissue and organ engineering (6), and pharmacology and toxicology screens (7,8). However, these applications require large numbers of cells of high quality (4, 6-8). For instance, ∼10 5 surviving dopaminergic (DA) neurons, ∼10 9 cardiomyocytes, or ∼10 9 beta cells are likely required to treat a patient with Parkinson disease (PD), myocardial infarction (MI), or type I diabetes, respectively (9). Additionally, far more cells are needed initially because both in vitro cell culture yields and subsequent in vivo survival of transplanted cells are typically very low. As examples of the latter, only ∼6% of transplanted dopaminergic neurons or ∼1% of injected cardiomyocytes reportedly survive in rodent models several months after transplantation (10, 11). Furthermore, there are large patient populations with degenerative diseases or organ failure (9), including over 1 million people with PD, 1-2.5 million with type I diabetes, and ∼8 million with MI in the United States alone (12). Large numbers of cells are also necessary for applications such as tissue engineering, where for ex...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

Lei

Schaffer

2013

Proc. Natl. Acad. Sci. U.S.A.

285

312

View full text Add to dashboard Cite

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“…To eliminate these problems, hESC single-cell suspension was encapsulated/cultured in a 3D semisolid matrix, such as a hydrogel, to physically isolate individual cells as well as provide a niche-like milieu for EB formation from individual hESCs [48]. When compared with the regular suspension culture in Petri dishes, EB formation in the agarose-based 3D environment…”

Section: Conventional Methodsmentioning

confidence: 99%

“…5). Common problems of the semisolid culture system for EB formation include low EB yield due to the intrinsic instability of single hiPSCs during prolonged culture, the hindrance of mass transport necessary for effective soluble factor treatments, as well as the complicated process for differentiated cell retrieval [48]. It remains interesting to see whether the 3D semisolid culture would improve the yield in lineage-specific terminal differentiation of the hEBs.…”

Section: Figmentioning

confidence: 99%

Engineering Strategies for the Formation of Embryoid Bodies from Human Pluripotent Stem Cells

Pettinato

Wen

Zhang

2015

Stem Cells and Development

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Human pluripotent stem cells (hPSCs) are powerful tools for regenerative therapy and studying human developmental biology, attributing to their ability to differentiate into many functional cell types in the body. The main challenge in realizing hPSC potential is to guide their differentiation in a well-controlled manner. One way to control the cell differentiation process is to recapitulate during in vitro culture the key events in embryogenesis to obtain the three developmental germ layers from which all cell types arise. To achieve this goal, many techniques have been tested to obtain a cellular cluster, an embryoid body (EB), from both mouse and hPSCs. Generation of EBs that are homogeneous in size and shape would allow directed hPSC differentiation into desired cell types in a more synchronous manner and define the roles of cell-cell interaction and spatial organization in lineage specification in a setting similar to in vivo embryonic development. However, previous success in uniform EB formation from mouse PSCs cannot be extrapolated to hPSCs possibly due to the destabilization of adherens junctions on cell surfaces during the dissociation into single cells, making hPSCs extremely vulnerable to cell death. Recently, new advances have emerged to form uniform human embryoid bodies (hEBs) from dissociated single cells of hPSCs. In this review, the existing methods for hEB production from hPSCs and the results on the downstream differentiation of the hEBs are described with emphases on the efficiency, homogeneity, scalability, and reproducibility of the hEB formation process and the yield in terminal differentiation. New trends in hEB production and directed differentiation are discussed.

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“…The culture on 3-D scaffolds is amenable to scale up of stem cell production while recreating the stem cell niches to maintain the cellular functions (Li et al 2003;Abranches et al 2007;Singh et al 2010;Stenberg et al 2011). Scaffold or substrate biomaterials provide additional mechanical signaling to stem cells.…”

Section: Modulation Of Stem Cell Behavior Through 3-d Culture Configumentioning

confidence: 99%

Stem cell bioprocess engineering towards cGMP production and clinical applications

Sart

Schneider

et al. 2014

Cytotechnology

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Stem cells, including mesenchymal stem cells and pluripotent stem cells, are becoming an indispensable tool for various biomedical applications including drug discovery, disease modeling, and tissue engineering. Bioprocess engineering, targeting large scale production, provides a platform to generate a controlled microenvironment that could potentially recreate the stem cell niche to promote stem cell proliferation or lineage-specific differentiation. This survey aims at defining the characteristics of stem cell populations currently in use and the present-day limits in their applications for therapeutic purposes. Furthermore, a bioprocess engineering strategy based on bioreactors and 3-D cultures is discussed in order to achieve the improved stem cell yield, function, and safety required for production under current good manufacturing practices.

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Sustained embryoid body formation and culture in a non-laborious three dimensional culture system for human embryonic stem cells

Cited by 16 publications

References 26 publications

A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

Engineering Strategies for the Formation of Embryoid Bodies from Human Pluripotent Stem Cells

Stem cell bioprocess engineering towards cGMP production and clinical applications

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