Date Published: March 27, 2019
Publisher: Public Library of Science
Author(s): Kathryn R. Bowles, Julia T. C. W., Lu Qian, Benjamin M. Jadow, Alison M. Goate, Wenhui Hu.
Genetic and epigenetic variability between iPSC-derived neural progenitor cells (NPCs) combined with differences in investigator technique and selection protocols contributes to variability between NPC lines, which subsequently impacts the quality of differentiated neuronal cultures. We therefore sought to develop an efficient method to reduce this variability in order to improve the purity of NPC and neuronal cultures. Here, we describe a magnetic activated cell sorting (MACS) method for enriching NPC cultures for CD271-/CD133+ cells at both early (<2–3) and late (>10) passage. MACS results in a similar sorting efficiency to fluorescence activated cell sorting (FACS), while achieving an increased yield of live cells and reduced cellular stress. Furthermore, neurons derived from MACS NPCs showed greater homogeneity between cell lines compared to those derived from unsorted NPCs. We conclude that MACS is a cheap technique for incorporation into standard NPC differentiation and maintenance protocols in order to improve culture homogeneity and consistency.
The reprogramming of adult, fully differentiated peripheral human cells into induced pluripotent stem cells (iPSCs; [1,2]) has been a monumental breakthrough that has allowed investigators across multiple disciplines to conduct research in a diverse array of cell types from numerous individuals, resulting in the development and use of more accurate and appropriate cell culture models of human disease. Given the inaccessibility of live human brains, neuroscience in particular has benefited greatly from the ability to differentiate iPSCs into an array of neuronal and glial subtypes for the investigation and discovery of novel disease-associated phenotypes in 2D and 3D cultures [3–5]. Many protocols have been developed for the generation of iPSC-derived neural cells, most of which pass through a neural progenitor cell (NPC) intermediate before terminal differentiation into neurons or glia [6–15].
iPSC lines derived from multiple individuals, that have undergone independent and varied differentiation processes by diverse protocols, show substantial heterogeneity. We sought to develop a simple, effective method to reduce this variability in order to improve the purity of NPCs and their subsequent neuronal cultures. Sorting NPCs using FACS in order to increase cell line homogeneity has previously been reported and is a highly effective method of addressing this issue [6,9,17]. However, FACS can be very costly and time consuming, with a contamination risk. In addition, we and others have experienced substantial cell stress and death following FACS , resulting in slow proliferation and recovery time of cultures. Here, we demonstrate that MACS is an effective alternative method of enrichment for CD271-/CD133+, which is both faster and more economical than FACS.