Generation of a human control iPS cell line (ESi080‐A) from a donor with no rheumatic diseases

, we report the establishment of the human iPS cell line N1-FiPS4F#7 generated from skin cells of a patient with no rheumatic diseases, thus obtaining an appropriate control iPS cell line for researchers working in the field of rheumatic diseases. The reprogramming factors Oct4, Sox2, Klf4 and c-Myc were introduced using a non-integrating reprogramming strategy involving Sendai Virus.

Here, we report the establishment of the human iPS cell line N1-FiPS4F#7 generated from skin cells of a patient with no rheumatic diseases, thus obtaining an appropriate control iPS cell line for researchers working in the field of rheumatic diseases.The reprogramming factors Oct4, Sox2, Klf4 and c-Myc were introduced using a nonintegrating reprogramming strategy involving Sendai Virus.

Resource utility
Although iPSC-lines have been generated from patients with rheumatic diseases, there is still lack of appropriate control lines generated from patients with radiographic information regarding principal joints (knee, hip, hands and/or spine).Therefore, we generated a control iPS cell line from a donor with no rheumatic diseases, as proved radiographically.

Resource details
Dermal fibroblasts were isolated from a skin biopsy of a donor with nor radiographic signs neither symptoms of rheumatic diseases using a protocol previously described (Vangipuram et al., 2013)  T fibroblasts for reprogramming were obtained after two weeks in culture and three cell passages.These fibroblasts at the 3rd passage were reprogrammed by using Sendai virus vectors containing the human reprogramming factors Oct4, Sox2, Klf4 and c-Myc following the instructions of the manufacturer.One week after reprogramming, we found several tight, small cell colonies in feeder-culture that grew quickly and, by day 22 after transduction, showed the typical human ESC-like morphology (Fig. 1 and Table 1), which means compact colonies with defined borders, high nucleus to cytoplasm ratio and prominent nucleoli (Marti et al., 2013).When we picked 10 of these ESClike colonies and passaged the fragments onto fresh feeder cells, most of these subcultures (clones) gave rise to new ESC-like colonies, which showed positivity for alkaline phosphatase activity (AP) (Fig. 1).The clearance of the virus and the exogenous reprogramming factor genes were confirmed by quantitative real time PCR (qRT-PCR) after ten cell culture passages.The endogenous expression of the pluripotency associated transcription factors OCT4, SOX2, KLF4, NANOG and CRIPTO as well as c-MYC was also evaluated by qRT-PCR (Fig. 1 and Table 1).According to these above-mentioned parameters all together with the low expression levels of c-MYC, one clone was selected to establish the iPS cell line.We confirmed by DNA fingerprinting analysis that the selected line shared identity with parental fibroblasts (Fig. 1 and Table 1).Immunofluorescence analysis showed that the line is positive for the intracellular self-renewal makers NANOG, OCT4 and SOX2, and the surface pluripotency-related markers SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 (Fig. 1 and Table 1).The differentiation potential of the selected iPS cell line was confirmed according to the immunofluorescence-based detection of the endoderm marker alpha-Fetoprotein (AFP), the mesoderm marker smooth muscle actin (SMA) and the ectoderm marker beta-Tubulin III (TUJ1) (Fig. 1) after the pluripotent differentiation protocols based on embryoid bodies (EBs) formation.Interestingly, after 2-3 weeks of mesodermal differentiation, spontaneously beating cardiomyocytes were observed in cell culture dishes.The selected line has been adapted to feeder-free culture conditions and karyotype has been checked, showing a partial mosaic chromosomal gain on chromosome 5 in ~40% of the cells at passage 63 (Fig. 1, red arrow and suppl figure 2 and Table 1).Absence of mycoplasma contamination was checked regularly by PCR (suppl Fig. 1 and Table 1).

Isolation and characterization of dermal fibroblasts
A 44 years old woman with no symptoms of rheumatic diseases was selected for the study.Absence of joint damage was confirmed radiographically in hip, knee, hand and spine.Dermal fibroblasts were obtained from a skin biopsy as previously described (Vangipuram et al., 2013).

Alkaline phosphatase analysis
The iPS cell line N1-FiPS4F#7 with more than twenty passages was seeded on a feeder layer plate.When colonies appeared, AP activity was studied using alkaline phosphatase blue membrane substrate solution kit (AB03000, Sigma-Aldrich).

RNA extraction and qRT-PCR analyses
Total RNA from parental fibroblasts at the 4th passage, the iPS cell line at the 10th passage, and parental fibroblasts immediately after reprogramming (positive control for Sendai virus) was extracted using the RNeasy Mini Kit (Qiagen).cDNA synthesis was performed using SuperScriptTM Vilo TM master mix (ThermoFisher Scientific).Expression levels of the endogenous pluripotency associated genes (OCT4, SOX2, KLF4, NANOG and CRIPTO), the oncogene c-MYC, and the silencing of the exogenous reprogramming factor genes and Sendai virus genome were analyzed, in duplicate, by qRT-PCR on the LightCycler 480 Instrument (Roche) using LightCycler 480 SYBR Green I Master (Roche).Primers used for the amplification were previously described (Aasen et al., 2008).

EB formation and in vitro differentiation
Pluripotent differentiation assay was performed through the EB formation protocol.For EB formation, hanging drop method was used (Vangipuram et al., 2013).After 48h, formed EBs were transferred independently to 0.1% gelatin (Millipore)-coated 8 well chamber-slides (Merk), and cultured at 37 °C in a humidified atmosphere with 5% CO 2 and in specific differentiation media (Galera et al., 2016) to stimulate differentiation towards endodermal, mesodermal and ectodermal lineages.

Immunofluorescence analyses
Undifferentiated iPS cells at passages 40-60, and cells sprouted from EBs during pluripotent differentiation were fixed, and immunofluorescence was performed as previously described (Borestrom et al., 2014).Antibodies used are listed in Table 2.
/ © 2019 The Authors.Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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Fig. 1 panel D

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Table 1
Characterization and validation.