Derivation of iPSC lines from three young healthy donors of Caucasian origin (NUIGi035-A; NUIGi036-A; NUIGi037-A)

The induced pluripotent stem cell (iPSC) technology has offered an unprecedented opportunity for disease modelling and drug discovery. Here we used non- integrating Sendai viral method and derived iPSCs

The induced pluripotent stem cell (iPSC) technology has offered an unprecedented opportunity for disease modelling and drug discovery. Here we used nonintegrating Sendai viral method and derived iPSCs from three young healthy Caucasian donors. All iPSCs expressed pluripotency markers highly and could be differentiated into three germ lineages. They possess normal karyotype which was confirmed by whole genome SNP array. The availability of the healthy control iPSCs offers an opportunity for phenotypic comparison and genome editing for a variety of diseases.

Resource Table:
Unique stem cell lines identifier

Resource utility
The iPSCs described here are derived from three healthy Caucasian donors of age 16-21, offering essential controls for phenotypic comparison in various diseases. They also provide age-and ethnic originmatched resources for genome editing and creation of isogenic iPSC lines with known genetic defects implicated in different conditions.

Resource details
The discovery of the induced pluripotent stem cell (iPSC) technology, which can reprogram somatic cells into embryonic stem cell-like cells that can be differentiated into almost any cell types in the body, has offered an unprecedented opportunity for disease modelling and drug discovery (Takahashi et al., 2007 Nov 30). However, the heterogeneity of iPSCs is one of the major challenges associated with iPSC disease modelling (Cahan and Origins, 2013;Ortmann and Vallier, 2017), as not all iPSC lines from an individual may have equivalent levels of pluripotency marker gene expression and/or same capacity to differentiate into desired cell types. The exact causes of the heterogeneity are unknown, but the ethnic origin, age, parental tissue type, genetic mosaicism and epigenetics may all contribute to the clonal variability. It is often recommended to use genome editing technologies such as CRISPR/Cas9 to rescue the genetic defects in disease iPSCs and/ or to create isogenic mutant iPSCs from healthy iPSCs (Shinnawi et al., 2019;Wang et al., 2017), and standardized iPSC controls are thus essential for phenotypic characterization.
In this study, we recruited three young healthy Caucasian donors: 02 V was a 20-year female undergraduate, 03 V was a 21-year male undergraduate and 05C was a 16-year high school boy. None of them have a significant family or own history of illness. Here we derived iPSCs from their dermal fibroblasts at P4-5, using non-integrating Sendai viral vectors expressing OCT4, SOX2, KLF4 and C-MYC. They displayed typical embryonic stem cell-like morphology and grew in tightly packed colonies with no obvious cell boundary, but a small cell body and a large nucleus/cytoplasm ratio (Fig. 1A). Their pluripotency were confirmed by high alkaline phosphatase activity (Fig. 1B), positive immunoreactivity to the pluripotency markers OCT4, SSEA4, SOX2 and TRA-1-81 ( Fig. 1C), and abundant mRNA expression of endogenous OCT4, SOX2 and NANOG genes (Fig. 1E). They were able to form embryoid bodies and spontaneously differentiate into cells of three embryonic germ layers, with positive immunoreactivity for endoderm marker α-fetoprotein (AFP), mesoderm maker α-smooth muscle actin (α-SMA), and ectodermal marker βIII-tubulin (TUJ1) (Fig. 1D).
We carried out whole genome SNP array and confirmed no gross chromosomal abnormality or large CNV of concern (Supplementary figure 1, Table 1). The iPSCs were verified free of transgene integration (Fig. 1F), or mycoplasma contamination (Fig. 1G), by RT-PCR and PCR, respectively. The full characterization can be viewed from Table 2 and Fig. 1. The iPSCs from three healthy Caucasian youngsters may offer desirable controls for phenotypic comparison and for genome editing.

Pluripotency validation
Alkaline phosphatase staining was performed using Alkaline Phosphatase Staining Kit II (Stemgent). For immunofluorescence staining, iPSCs were fixed in 4% PFA for 20 min, permeabilized with 0.1% Triton X-100 (Sigma) for 15 min, and blocked for 1 h in 1% BSA-DPBS, and incubated with primary antibodies against OCT4, SSEA4, SOX2 or TRA-1-81 (Table 2) at 4 • C overnight. Alexa Fluor 488-or 555-conjugated secondary antibodies (Cell Signaling Technology) were then applied to visualize cells, and cell nuclei were labelled with Hoechst 33,342 (Life Technologies). Slides were imaged using a Confocal Microscope (Olympus FluoView 1000 system). To detect expression of endogenous pluripotency genes, qRT-PCR was performed with the StepOne Plus Real Time PCR System using Fast SYBR™ Green Master Mix (Applied Biosystems) and specific primers listed in Table 3. The expression levels of OCT4, SOX2 and NANOG were adjusted with an internal control (GAPDH), and then converted to log2 fold of expression over fibroblast mRNA as a negative control.

Karyotyping
The molecular karyotype was analyzed with 990 k SNP array by Beijing Hyslar Biotech Limited Corporation (Beijing, China). SNP data was analyzed by Axiom Analysis software (ThermoFisher, USA) which generated LogR ratio and B allele plots, using 83 samples to create an internal control. IGV software was used then to examine the molecular karyotyping of fibroblasts and derived iPSC lines.

Transgene-free confirmation
Total RNA was extracted from iPSCs using RNeasy Mini Kit (Qiagen), and reversely transcribed to cDNA with sensiFAST cDNA Synthesis Kit (Sigma-Aldrich). The ssDNAs were diluted 1:10 and PCR analysis was performed using TopTaq® Master Mix (Qiagen) under standard conditions using a set of commercially supplied transgene-specific primers (Table 3).

Mycoplasma detection
Absence of mycoplasma contamination was confirmed by PCR using the MycoSensor PCR Assay Kit (Agilent), following manufacturer's instructions.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.