Physioxia improves the selectivity of hematopoietic stem cell expansion cultures

Igarashi, Kyomi J.; Kuciński, Iwo; Chan, Yan Yi; Tan, Tze-Kai; Khoo, Hwei Minn; Kealy, David; Bhadury, Joydeep; Hsu, Ian; Ho, Pui Yan; Niizuma, Kouta; Hickey, John W.; Nolan, Garry P.; Bridge, Katherine S.; Czechowicz, Agnieszka; Göttgens, Berthold; Nakauchi, Hiromitsu; Wilkinson, Adam C.

doi:10.1182/bloodadvances.2023009668

Cited by 11 publications

(6 citation statements)

References 41 publications

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“…Genetically engineered autologous HSCs could be delivered in the 2 nd BMT to correct a metabolic or cellular disease ( 13 ). The recent advancement in the ex vivo expansion of HSCs ( 39 – 41 ), including human HSCs ( 42 , 43 ), are particularly relevant in this context.…”

Section: Discussionmentioning

confidence: 99%

Second bone marrow transplantation into regenerating hematopoiesis enhances reconstitution of immune system

Faltusová,

Báječný,

Heizer

et al. 2024

Front. Immunol.

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In bone marrow transplantation (BMT), hematopoiesis-reconstituting cells are introduced following myeloablative treatment, which eradicates existing hematopoietic cells and disrupts stroma within the hematopoietic tissue. Both hematopoietic cells and stroma then undergo regeneration. Our study compares the outcomes of a second BMT administered to mice shortly after myeloablative treatment and the first BMT, with those of a second BMT administered to mice experiencing robust hematopoietic regeneration after the initial transplant. We evaluated the efficacy of the second BMT in terms of engraftment efficiency, types of generated blood cells, and longevity of function. Our findings show that regenerating hematopoiesis readily accommodates newly transplanted stem cells, including those endowed with a robust capacity for generating B and T cells. Importantly, our investigation uncovered a window for preferential engraftment of transplanted stem cells coinciding with the resumption of blood cell production. Repeated BMT could intensify hematopoiesis reconstitution and enable therapeutic administration of genetically modified autologous stem cells.

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Section: Discussionmentioning

confidence: 99%

Second bone marrow transplantation into regenerating hematopoiesis enhances reconstitution of immune system

Faltusová,

Báječný,

Heizer

et al. 2024

Front. Immunol.

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“…O2 tension in physioxia can vary greatly (0.1-4.5% O2) depending on experimental set-up, engendering reproducibility concerns. These findings are a major concern for physioxic culture of stem cell expansion and differentiation [55][56][57] , and controlling pericellular O2 tension may improve our understanding of these processes.…”

Section: Discussionmentioning

confidence: 99%

Controlling pericellular oxygen tension in cell culture reveals distinct breast cancer responses to low oxygen tensions

Rogers,

Colombani,

Khan

et al. 2023

Preprint

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Oxygen (O2) tension plays a key role in tissue function and pathophysiology. O2-controlled cell culture, in which the O2concentration in an incubator’s gas phase is controlled, is an indispensable tool to study the role of O2in vivo. For this technique, it is presumed that the incubator setpoint is equal to the O2tension that cells experience (i.e., pericellular O2). We discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0.0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2consumption is the driving factor. RNA-seq revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to anoxic exposure followed by rapid reoxygenation. To better understand the relationship between incubator gas phase and pericellular O2tensions, we developed a reaction-diffusion model that predicts pericellular O2tensiona priori. This model revealed that the effect of cellular O2consumption is greatest in smaller volume culture vessels (e.g.,96-well plate). By controlling pericellular O2tension in cell culture, we discovered that MCF7 cells have stronger glycolytic and glutamine metabolism responses in anoxiavs.hypoxia. MCF7 also expressed higher levels ofHIF2A,CD73,NDUFA4L2,etc.and lower levels ofHIF1A,CA9,VEGFA, etc.in response to hypoxiavs.anoxia. Proteomics revealed that 4T1 cells had an upregulated epithelial-to-mesenchymal transition (EMT) response and downregulated reactive oxygen species (ROS) management, glycolysis, and fatty acid metabolism pathways in hypoxiavs.anoxia. Collectively, these results reveal that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable to model hypoxia. We demonstrate that controlling atmospheric O2tension in cell culture incubators is insufficient to control O2in cell culture and introduce the concept ofpericellular O2-controlled cell culture.

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“…Ai14 fibroblasts and mouse HSCs were generated from Ai14 mice as previously described 23, 24, 25 . Ai9 293T cells were generated through the knock in of the Ai9 reporter construct into the AAVS1 locus by homology dependent repair and puromycin selection (2 ug/ml).…”

Section: Methodsmentioning

confidence: 99%

Secreted Particle Information Transfer (SPIT) – A Cellular Platform forIn VivoGenetic Engineering

Charlesworth,

Homma,

Suchy

et al. 2024

Preprint

Self Cite

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A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for in vivo genetic engineering, Secreted Particle Information Transfer (SPIT) that utilizes human cells as delivery vectors for in vivo genetic engineering. We demonstrate the application of SPIT for cell-cell delivery of Cre recombinase and CRISPR-Cas9 enzymes, we show that genetic logic can be incorporated into SPIT and present the first demonstration of human cells as a delivery platform for in vivo genetic engineering in immunocompetent mice. We successfully applied SPIT to genetically modify multiple organs and tissue stem cells in vivo including the liver, spleen, intestines, peripheral blood, and bone marrow. We anticipate that by harnessing the large packaging capacity of a human cell's nucleus, the ability of human cells to engraft into patients' long term and the capacity of human cells for complex genetic programming, that SPIT will become a paradigm shifting approach for in vivo genetic engineering.

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Physioxia improves the selectivity of hematopoietic stem cell expansion cultures

Cited by 11 publications

References 41 publications

Second bone marrow transplantation into regenerating hematopoiesis enhances reconstitution of immune system

Second bone marrow transplantation into regenerating hematopoiesis enhances reconstitution of immune system

Controlling pericellular oxygen tension in cell culture reveals distinct breast cancer responses to low oxygen tensions

Secreted Particle Information Transfer (SPIT) – A Cellular Platform forIn VivoGenetic Engineering

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