High‐Efficiency Serum‐Free Feeder‐Free Erythroid Differentiation of Human Pluripotent Stem Cells Using Small Molecules

• Published in: Stem cells translational medicine 2016-10, Vol.5 (10), p.1394-1405
• Main Authors: Olivier, Emmanuel N., Marenah, Lamin, McCahill, Angela, Condie, Alison, Cowan, Scott, Mountford, Joanne C.
• Format: Article
• Language: English
• Subjects: Cell Culture Techniques - methods; Cell Differentiation; Cell Line; Cytokines; Data analysis; Data collection; Efficiency; Embryo cells; Embryos; Erythrocytes - cytology; Erythropoiesis; Hematopoiesis; Humans; Kinases; Methods; Pluripotency; Pluripotent stem cells; Pluripotent Stem Cells - cytology; Small molecule; Stem cells; Studies; Tissue Engineering and Regenerative Medicine; Vascular endothelial growth factor
• ISSN: 2157-6564; 2157-6580
• DOI: 10.5966/sctm.2015-0371

This article describes a highly efficient, fully feeder‐free, serum‐free method for erythroid differentiation of induced pluripotent stem cells and human embryonic stem cells, including a clinical‐grade line, that is amenable to scale‐up and as such will be of significant value for basic and translational studies of hematopoiesis and erythropoiesis. 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. Significance This 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