A Multistep Workflow to Evaluate Newly Generated iPSCs and Their Ability to Generate Different Cell Types

Abstract

Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality-control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a multistep workflow to assess our newly generated iPSCs. Our workflow tests four benchmarks: cell growth, genomic stability, pluripotency, and the ability to form the three germline layers. We also outline a simple test for assessing cell growth and highlight the need to compare different growth media. Genomic integrity in the human iPSCs is analyzed by G-band karyotyping and a qPCR-based test for the detection of common karyotypic abnormalities. Finally, we confirm that the iPSC lines can differentiate into a given cell type, using a trilineage assay, and later confirm that each iPSC can be differentiated into one cell type of interest, with a focus on the generation of cortical neurons. Taken together, we present a multistep quality-control workflow to evaluate newly generated iPSCs and detail the findings on these lines as they are tested within the workflow.

Keywords: cortical neurons; genomic integrity; human-induced pluripotent stem cells; neural progenitor cells; quality control; trilineage differentiation.

Figure 1

Multistep workflow for phenotyping of hiPSCs. Schematic representation of a multistep QC workflow for newly generated iPSCs to monitor morphology and proliferation, genomic integrity, pluripotency, and ability to form cells of the three germ layers.

Figure 2

HiPSC growth and proliferation under different conditions. (A) Representative daily morphology of hiPSCs maintained in mTeSR1 or E8 media. Both cell lines show smooth-edged, tightly packed cells with a large nucleus-to-cytoplasm ratio. AJG001-C4 cells grow slightly better in mTeSR1 media, while 3448 proliferate better in E8. (B) CV assay of representative cell lines grown in mTeSR1 and E8. AJC001-5 grows well in both media. AJG001-C4 grows better in mTeSR1 media, while AJD002-3 cells proliferate best in E8. (C) Quantification of the hiPSCs’ survival and growth in different media. The CV stain was dissolved in methanol and optical density was measured at 540 nm (OD540). The mean and the standard deviation are from six replicates from two independent experiments.

Figure 3

Expression of pluripotency markers in hiPSCs. (A) Phase contrast images and ICC for pluripotency markers SSEA-4, OCT3/4, NANOG, and Tra-1-60, with Ho342 nucleic acid counterstain. (B) Quantification of OCT3/4-positive cells or SSEA-4-positive cells shown in (A). (C) qPCR for mRNA expression of pluripotency genes POU5F1, NANOG, c-MYC, KLF4, SOX2, and ZFP42 in hiPSCs compared to H9 ESCs. mRNA expression in H9 was set as 1.0. The mean and SD are from technical triplicates from three independent experiments.

Figure 4

Genomic integrity analysis of hiPSCs. (A) Karyotyping and G-band analyses showing examples of one normal (left) and one abnormal hiPSC karyotype (right). An apparently balanced translocation between the long (q) arm of chromosome X and the short (p) arm of chromosome 2 is present in TD10 (red arrow). (B) qPCR-based genetic analysis to detect small chromosome abnormalities in all hiPSC lines. The copy numbers of chr1q, chr8q, chr10p, chr12p, chr17q, chr18q, chr20q, and chrXp were normalized to chr4p expression set at 2. Error bars show standard deviation from technical triplicates from two independent experiments. There are no abnormalities within critical regions in our cell lines, while NCRM1 and KYOU-DXR0109 have amplification of chr20q (indicated by blue arrows). TD03 shows a slight increase in chr20q (indicated with #), however this is not above the threshold of expected variation.

Figure 5

Differentiation of hiPSCs into three germ layers. (A) Representative phase contrast images of EB formation (left) and differentiation into three germ layers by ICC with the ectoderm (PAX6), mesoderm (SMA), and endoderm (VIM) markers (indicated above images). Nuclei are counterstained with Ho342. (B) qPCR for mRNA expression of three germ layer genes. Quantification of expression of the ectoderm (PAX6, NCAM1), mesoderm (MIXL1), and endoderm (VIM) markers in hiPSCs compared to H9 ESC. mRNA expression in H9 was set as 1.0. The mean and SD are from technical triplicates from three independent experiments.

Figure 6

Differentiation of hiPSCs into forebrain cortical neurons. (A) Schematic representation of the in vitro differentiation protocol used to generate cortical neurons from hiPSCs. Boxes indicate differentiation state. This protocol was performed in 11 independent lines, with all lines performing similarly. (B) qPCR for mRNA expression levels of neuronal markers MAP2, NCAM1, and TUBB3 in NPC and 4-week-old differentiated cortical neurons compared to IPSCs. mRNA expression in iPSCs was set as 1.0. The mean and SD are from technical triplicates from two independent experiments. (C) Expression of cortical layer markers FOXP1 and SATB2 by qPCR analysis of 4-week-old differentiated cortical neurons compared to iPSCs. mRNA expression in iPSCs was set as 1.0. The mean and SD are from technical triplicates from two independent experiments.

Figure 7

Immunocytochemistry staining of hiPSC-derived cortical neurons. (A) Representative images of ICC-stained cortical neurons from AJG001-C4, NCRM1, AJD002-3, and TD03 after 4 weeks of neuronal differentiation. Cells were stained with MAP2 and Tuj1 neuronal markers, and cortical neuron markers Brn2 and Tbr1. Nuclei were counterstained with Ho342. (B) Quantification of MAP2-, Tuj1-, Brn2-, or Tbr1-positive cells in (A).