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Open accessJournal ArticleDOI: 10.1002/SCTM.20-0453

High density bioprocessing of human pluripotent stem cells by metabolic control and in silico modeling

04 Mar 2021-Stem Cells Translational Medicine (John Wiley & Sons, Ltd)-Vol. 10, Iss: 7, pp 1063-1080
Abstract: To harness the full potential of human pluripotent stem cells (hPSCs) we combined instrumented stirred tank bioreactor (STBR) technology with the power of in silico process modeling to overcome substantial, hPSC-specific hurdles toward their mass production. Perfused suspension culture (3D) of matrix-free hPSC aggregates in STBRs was applied to identify and control process-limiting parameters including pH, dissolved oxygen, glucose and lactate levels, and the obviation of osmolality peaks provoked by high density culture. Media supplements promoted single cell-based process inoculation and hydrodynamic aggregate size control. Wet lab-derived process characteristics enabled predictive in silico modeling as a new rational for hPSC cultivation. Consequently, hPSC line-independent maintenance of exponential cell proliferation was achieved. The strategy yielded 70-fold cell expansion in 7 days achieving an unmatched density of 35 × 106 cells/mL equivalent to 5.25 billion hPSC in 150 mL scale while pluripotency, differentiation potential, and karyotype stability was maintained. In parallel, media requirements were reduced by 75% demonstrating the outstanding increase in efficiency. Minimal input to our in silico model accurately predicts all main process parameters; combined with calculation-controlled hPSC aggregation kinetics, linear process upscaling is also enabled and demonstrated for up to 500 mL scale in an independent bioreactor system. Thus, by merging applied stem cell research with recent knowhow from industrial cell fermentation, a new level of hPSC bioprocessing is revealed fueling their automated production for industrial and therapeutic applications.

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Open accessJournal ArticleDOI: 10.3390/IJMS22083890
Abstract: Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics.

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3 Citations



Open accessJournal ArticleDOI: 10.1002/SCTM.21-0161
Abstract: Diabetes is a debilitating disease characterized by high blood glucose levels. The global prevalence of this disease has been projected to reach 700 million adults by the year 2045. Type 1 diabetes represents about 10% of the reported cases of diabetes. Although islet transplantation can be a highly effective method to treat type 1 diabetes, its widespread application is limited by the paucity of cadaveric donor islets. The use of pluripotent stem cells as an unlimited cell source to generate insulin-producing cells for implant is a promising alternative for treating diabetes. However, to be clinically relevant, it is necessary to manufacture these stem cell-derived cells at sufficient scales. Significant advances have been made in differentiation protocols used to generate stem cell-derived cells capable of reversing diabetes in animal models and for testing in clinical trials. We discuss the potential of both stem cell-derived pancreatic progenitors and more matured insulin-producing cells to treat diabetes. We discuss the need for rigorous bioprocess parameter optimization and identify some critical process parameters and strategies that may influence the critical quality attributes of the cells with the goal of facilitating scalable manufacturing of human pluripotent stem cell-derived pancreatic endocrine cells.

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Topics: Stem cell (61%), Induced pluripotent stem cell (60%), Progenitor cell (55%) ... show more

1 Citations


Open accessJournal ArticleDOI: 10.3390/PHARMACEUTICS13071097
18 Jul 2021-Pharmaceutics
Abstract: In this work, a method for the preparation of the highly lipophilic labeling synthon [89Zr]Zr(oxinate)4 was optimized for the radiolabeling of liposomes and human induced pluripotent stem cells (hiPSCs). The aim was to establish a robust and reliable labeling protocol for enabling up to one week positron emission tomography (PET) tracing of lipid-based nanomedicines and transplanted or injected cells, respectively. [89Zr]Zr(oxinate)4 was prepared from oxine (8-hydroxyquinoline) and [89Zr]Zr(OH)2(C2O4). Earlier introduced liquid-liquid extraction methods were simplified by the optimization of buffering, pH, temperature and reaction times. For quality control, thin-layer chromatography (TLC), size-exclusion chromatography (SEC) and centrifugation were employed. Subsequently, the 89Zr-complex was incorporated into liposome formulations. PET/CT imaging of 89Zr-labeled liposomes was performed in healthy mice. Cell labeling was accomplished in PBS using suspensions of 3 × 106 hiPSCs, each. [89Zr]Zr(oxinate)4 was synthesized in very high radiochemical yields of 98.7% (96.8% ± 2.8%). Similarly, high internalization rates (≥90%) of [89Zr]Zr(oxinate)4 into liposomes were obtained over an 18 h incubation period. MicroPET and biodistribution studies confirmed the labeled nanocarriers' in vivo stability. Human iPSCs incorporated the labeling agent within 30 min with ~50% efficiency. Prolonged PET imaging is an ideal tool in the development of lipid-based nanocarriers for drug delivery and cell therapies. To this end, a reliable and reproducible 89Zr radiolabeling method was developed and tested successfully in a model liposome system and in hiPSCs alike.

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Topics: Liposome (51%), Biodistribution (51%)

Open accessPosted ContentDOI: 10.1101/2021.09.19.460940
29 Sep 2021-bioRxiv
Abstract: Drug-inducible suicide systems may help to minimize risks of cellular therapies due to the tumor forming potential of human induced pluripotent stem cells (hiPSCs). Recent research challenged the usefulness of such systems since rare drug-resistant subclones were observed that showed elimination or silencing of the transgene. We have introduced a drug-inducible Caspase9 suicide system (iCASP9) into the AAVS1 safe harbor locus of hiPSCs. In these cells, apoptosis could be efficiently induced in vitro. In mice, drug treatment generally led to rapid elimination of teratomas, but individual animals subsequently formed tumor tissue from monoallelic iCASP9 hiPSCs. Very rare drug-resistant subclones of monoallelic iCASP9 hiPSCs appeared in vitro with frequencies of ~ 3x10-8. Transgene elimination, presumably via Loss of Heterozygosity (LoH), or methylation of the CAG promoter but not methylation of the PPP1R12C locus were identified as underlying mechanisms. In contrast, we never observed any escapees from biallelic iCASP9 hiPSCs, even after treatment of up to 0.8 billion hiPSCs. In conclusion, biallelic integration of an iCASP9 system in the AAVS1 locus may substantially contribute to the safety level of iPSC-based therapies, which should be calculated by relating clonal escapee frequencies to the cell number in tumors of a size that is readily detectable during routine screening procedures.

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75 results found


Journal ArticleDOI: 10.1146/ANNUREV.MI.03.100149.002103
Abstract: The study of the growth of bacterial cultures does not constitute a specialized subject or branch of research: it is the basic method of Microbiology. It would be a foolish enterprise, and doomed to failure, to attempt reviewing briefly a \"subject\" which covers actually our whole discipline. Unless, of course, we considered the formal laws of growth for their own sake, an approach which has repeatedly proved sterile. In the present review we shall consider bacterial growth as a method for the study of bacterial physiology and biochemistry. More precisely, we shall concern ourselves with the quantitative aspects of the method, with the interpretation of quantitative data referring to bacterial growth. Furthermore, we shall considerz exclusively the positive phases of growth, since the study of bacterial \"death,\" i.e., of the negative phases of growth, involves distinct problems and methods. The discussion will be limited to populations considered genetically homogeneous. The problems of mutation and selection in growing cultures have been excellently dealt with in recent review articles by Delbriick (1) and Luria (2). No attempt is made at reviewing the literature on a subject which, as we have just seen, is not really a subject at all. The papers and results quoted have been selected as illustrations of the points discussed.

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3,787 Citations


Journal ArticleDOI: 10.1056/NEJMSR040330
Abstract: This report, first published online on March 3, 2004, discusses the procedures used to develop 17 lines of human embryonic stem cells from the inner cell masses of blastocysts. These cell lines are available to researchers under a Material Transfer Agreement; according to current regulations, the cells cannot be used for research supported by federal funds. These cells are expected to facilitate research on a variety of serious chronic diseases.

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Topics: Stem cell (60%), Embryonic stem cell (56%)

1,024 Citations


Journal ArticleDOI: 10.1038/NRC1713
Abstract: Recent research has highlighted the fundamental role of the tumour's extracellular metabolic microenvironment in malignant invasion. This microenvironment is acidified primarily by the tumour-cell Na(+)/H(+) exchanger NHE1 and the H(+)/lactate cotransporter, which are activated in cancer cells. NHE1 also regulates formation of invadopodia - cell structures that mediate tumour cell migration and invasion. How do these alterations of the metabolic microenvironment and cell invasiveness contribute to tumour formation and progression?

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Topics: Invadopodia (56%), Cell migration (52%), Cancer cell (51%)

737 Citations


Open accessJournal ArticleDOI: 10.1016/J.STEM.2012.10.002
02 Nov 2012-Cell Stem Cell
Abstract: Plasticity in energy metabolism allows stem cells to match the divergent demands of self-renewal and lineage specification. Beyond a role in energetic support, new evidence implicates nutrient-responsive metabolites as mediators of crosstalk between metabolic flux, cellular signaling, and epigenetic regulation of cell fate. Stem cell metabolism also offers a potential target for controlling tissue homeostasis and regeneration in aging and disease. In this Perspective, we cover recent progress establishing an emerging relationship between stem cell metabolism and cell fate control.

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Topics: Cell Fate Control (64%), Tissue homeostasis (60%), Cellular differentiation (58%) ... show more

490 Citations


Open accessJournal ArticleDOI: 10.1161/CIRCULATIONAHA.108.778795
29 Jul 2008-Circulation
Abstract: Background— The recent breakthrough in the generation of induced pluripotent stem (iPS) cells, which are almost indistinguishable from embryonic stem (ES) cells, facilitates the generation of murine disease– and human patient–specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line. Methods and Results— With the use of a standard embryoid body–based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell–derived cardiomyocytes show typical features of ES cell–derived cardiomyocytes. Reverse transcription p...

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Topics: Embryoid body (65%), Embryonic stem cell (63%), Induced pluripotent stem cell (61%) ... show more

489 Citations