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Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes.

ABSTRACT: We previously established a method for a directed differentiation of human pluripotent stem cells into classical brown adipocytes (BA) by forming aggregates via massive floating culture in the presence of a specific cytokine cocktail. However, use of recombinant cytokines requires significant cost. Moreover, an enforced differentiation by exogenously added cytokines may amend skewed differentiation propensity of patient's pluripotent stem cells, providing unsatisfactory disease models. Therefore, an exogenous cytokine-free method, where cytokines required for differentiation are provided in an auto/paracrine manner mimicking natural developmental process, is beneficial. Here we show that, if human pluripotent stem cells are cultured as size-controlled spheroids (100-120 µm radius, 2000-2500 cells/spheroid) in a mutually segregated manner with half-change of the medium every other day, they differentiate into classical BA via an authentic MYF5-positive myoblast route in the absence of exogenous cytokines. Differentiated BA exerted thermogenic activity in transplanted mice in response to beta-adrenergic receptor agonist stimuli. The cytokine-free differentiation method has further advantages in exploring BATokines, BA-derived physiologically active substances. Indeed, we have found that BA produces an unknown small (<1000 Da), highly hydrophilic molecule that augments insulin secretion from pancreatic beta cells. Our upgraded technique will contribute to an advancement of stem cell study for diverse purposes.

SUBMITTER: Oka M

PROVIDER: S-EPMC6523334 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes.

Oka Masako M Kobayashi Norihiko N Matsumura Kazunori K Nishio Miwako M Saeki Kumiko K

Cells 20190424 4

We previously established a method for a directed differentiation of human pluripotent stem cells into classical brown adipocytes (BA) by forming aggregates via massive floating culture in the presence of a specific cytokine cocktail. However, use of recombinant cytokines requires significant cost. Moreover, an enforced differentiation by exogenously added cytokines may amend skewed differentiation propensity of patient's pluripotent stem cells, providing unsatisfactory disease models. Therefore ...[more]

PMID: 31022954

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Project description:: This article describes a good manufacturing practice (GMP)-compatible, feeder-free and serum-free method to produce large numbers of erythroid cells from human pluripotent stem cells (hPSCs), either embryonic or induced. This multistep protocol combines cytokines and small molecules to mimic and surpass the early stages of development. It produces, without any selection or sorting step, a population of cells in which 91.8% ± 5.4% express CD34 at day 7, 98.6% ± 1.3% express CD43 at day 10, and 99.1% ± 0.95% of cells are CD235a positive by day 31 of the differentiation process. Moreover, this differentiation protocol supports extensive expansion, with a single hPSC producing up to 150 hematopoietic progenitor cells by day 10 and 50,000-200,000 erythroid cells by day 31. The erythroid cells produced exhibit a definitive fetal hematopoietic type, with 90%-95% fetal globin and variable proportion of embryonic and adult globin at the protein level. The presence of small molecules during the differentiation protocol has quantitative and qualitative effects; it increases the proportion of adult globin and decreases the proportion of embryonic globin. Given its level of definition, this system provides a powerful tool for investigation of the mechanisms governing early hematopoiesis and erythropoiesis, including globin switching and enucleation. The early stages of the differentiation protocol could also serve as a starting point for the production of endothelial cells and other hematopoietic cells, or to investigate the production of long-term reconstituting hematopoietic stem cells from hPSCs.SignificanceThis differentiation protocol allows the production of a large amount of erythroid cells from pluripotent stem cells. Its efficiency is compatible with that of in vitro red blood cell production, and it can be a considerable asset for studying developmental erythropoiesis and red blood cell enucleation, thereby aiding both basic and translational research. In addition to red cells, the early stages of the protocol could also be used as a starting point for the large-scale production of other hematopoietic cell types, including the ultimate goal of generating long-term reconstituting hematopoietic stem cells.

Dataset Information

Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes.

Publications

Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes.

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