Optimized Surface Markers for the Prospective Isolation of High-Quality hiPSCs using Flow Cytometry Selection

Abstract

hiPSC derivation and selection remains inefficient; with selection of high quality clones dependent on extensive characterization which is not amenable to high-throughput (HTP) approaches. We recently described the use of a cocktail of small molecules to enhance hiPSC survival and stability in single cell culture and the use of flow cytometry cell sorting in the HTP-derivation of hiPSCs. Here we report an enhanced protocol for the isolation of bona fide hiPSCs in FACS-based selection using an optimized combination of cell surface markers including CD30. Depletion of CD30+ cells from reprogramming cultures almost completely abolished the NANOG and OCT4 positive sub-population, suggesting it is a pivotal marker of pluripotent cells. Combining CD30 to SSEA4 and TRA-1-81 in FACS greatly enhanced specificity and efficiency of hiPSC selection and derivation. The current method allows for the efficient and automated, prospective isolation of high-quality hiPSC from the reprogramming cell milieu.

Myogenic Differentiation of Muscular Dystrophy-Specific Induced Pluripotent Stem Cells for Use in Drug Discovery

“…Human iPSCs were cultured on Matrigel‐coated plates (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com) in iPSC growth medium containing Dulbecco's modified Eagle's medium (DMEM)/F12 (Mediatech, Manassas, VA, http://www.cellgro.com), 20% vol/vol knockout serum replacement (SR; Life Technologies, Rockville, MD, http://www.lifetech.com), 1% vol/vol nonessential amino acids (Mediatech), 2 mM l ‐glutamine (Mediatech), 100 mM β‐mercaptoethanol (Life Technologies), and 10 ng/ml basic fibroblast factor (bFGF; Life Technologies). To enable single‐cell feeder‐free culture of iPSCs, growth media was supplemented with small molecule 4 (SMC4) cocktail, as previously described [11, 12]. SMC4 media additive contains 0.4 µM PD0325901 (Biovision, Milpitas, CA, http://www.biovision.com), 1 µM CHIR99021 (Biovision), 5 µM Thiazovivin (synthesized at Fate Therapeutics), and 2 µM SB431542 (Biovision).…”

“…Fibroblasts derived from patients with Duchenne muscular dystrophy (DMD) (GM05169 and GM05112) and Becker muscular dystrophy (BMD) (GM05081 and GM02299) were obtained from the Coriell Institute for Medical Research (Camden, NJ, http://www.coriell.org). Generation and feeder‐free culture of human iPSCs were performed as described previously [11, 12]. Briefly, the fibroblasts were infected with a lentivirus containing a polycistronic cassette expressing OCT4, SOX2, and KLF4 in the presence of 4 µg/ml polybrene (Millipore, Billerica, MA, http://www.millipore.com) and transferred to 37°C and 5% CO 2 for 8–12 hours.…”

Conjunctiva derived mesenchymal stem cell (CJMSCs) as a potential platform for differentiation into corneal epithelial cells on bioengineered electrospun scaffolds

“…The cells were pelleted, washed twice with PBS, and fixed with 1% paraformaldehyde in PBS. After fixation, FACS analysis was performed on a FACSCalibur flowcytometer (Attune Acoustic‐Focusing Cytometer, Life Technologies, USA) Flowjo software was used to analyze the data …”

“…These cells provide an alternative source for regeneration of corneal epithelial layers, which are responsible for depleting the most frequent and severe causes of corneal blindness. Based on the best of our knowledge, there is no evidence for investigating the role of CJMSCs in corneal epithelial tissue engineering …”

Generation of integration‐free induced pluripotent stem cells from blood‐derived cells isolated from patient with severe haemophilia A

“…iPSC clones were characterized by a combination of surface and intracellular markers;however, as the surface proteins may be expressed even by partially reprogrammed cells, intracellular molecule analysis is the iPSC clone such as the cell-to-cell genetic variations, expression of genes from somatic cell, residual expression of reprogramming factors and the low efficiency of the reprogramming process 8. HA-iPSC formed EB, three-dimensional spherical structures formed in vitro from pluripotent cells, which allows for differentiation of the three germ lineages (Figure 1D).The teratomas were formed 48 days after the injection of HA-iPSC clone 6, and when analysed by immunohistochemical staining, we identified cells from endoderm, ectoderm and mesoderm(Figure 2-I).…”