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1.
Stem Cell Res Ther ; 15(1): 89, 2024 Mar 25.
Article in English | MEDLINE | ID: mdl-38528578

ABSTRACT

BACKGROUND: Human pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown. METHODS AND RESULTS: STBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs' pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls. CONCLUSIONS: Together, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale.


Subject(s)
Cell Culture Techniques , Pluripotent Stem Cells , Humans , Cell Culture Techniques/methods , Pluripotent Stem Cells/metabolism , Cell Differentiation , Bioreactors , Cryopreservation
3.
Nat Biotechnol ; 39(6): 737-746, 2021 06.
Article in English | MEDLINE | ID: mdl-33558697

ABSTRACT

Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice.


Subject(s)
Heart/embryology , Intestines/embryology , Organoids/embryology , Body Patterning , Embryonic Development , Gene Knockdown Techniques , Green Fluorescent Proteins/genetics , Hepatocyte Nuclear Factor 4/genetics , Homeobox Protein Nkx-2.5/genetics , Humans , SOXB1 Transcription Factors/genetics , SOXF Transcription Factors/genetics , Sequence Analysis, RNA
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