Generation of hiPSTZ16 (ISMMSi003-A) cell line from normal human foreskin fibroblasts

Article history: Received 29 November 2017 Received in revised form 30 November 2017 Accepted 30 November 2017 Available online 2 December 2017 Human foreskin fibroblasts from a commercial sourcewere reprogrammed into induced pluripotent stem cells to establish a clonal stem cell line, hiPSTZ16 (ISMMSi003-A). These cells show a normal karyotype and full differentiation potential in teratoma assays. The described cells provide a useful resource in combination with other iPS cell lines generated from normal human foreskin fibroblasts to study sourceand reprogramming methodindependent effects in downstream applications. © 2017 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/).

Human foreskin fibroblasts from a commercial source were reprogrammed into induced pluripotent stem cells to establish a clonal stem cell line, hiPSTZ16 (ISMMSi003-A). These cells show a normal karyotype and full differentiation potential in teratoma assays. The described cells provide a useful resource in combination with other iPS cell lines generated from normal human foreskin fibroblasts to study source-and reprogramming methodindependent effects in downstream applications. ©

Resource details
Human foreskin fibroblast (HFF) cells (CCD1079Sk) were reprogrammed into the induced pluripotent stem (iPS) cell state through retroviral delivery (Takahashi et al. 2007, Lowry et al. 2008 of five reprogramming factors (OCT4, SOX2, NANOG, KLF4 and c-MYC). The established hiPSTZ16 (ISMMSi003-A) cells showed human embryonic stem cell-like morphology in phase contrast microscopy ( Fig. 1A), and nuclear expression of the pluripotency marker OCT4 as detected by immunofluorescence (IF) staining (Fig. 1B). Additionally, flow cytometric (FC) analyses confirmed that more than 92% of cells were OCT4 + /SSEA4 + double positive (Fig. 1C). The cells were karyotypically normal at passage 6 (Fig. 1D) and 16 (not shown) as determined by G-banding with a band resolution of 400-450. Furthermore, their short tandem repeat (STR) profile was identical to the one of their parental HFF cells (Fig. 1E). Finally, teratoma formation demonstrated the potential of hiPSTZ16 (ISMMSi003-A) cells to differentiate into cell types of all three germ layers (Fig. 1F) as we were able to detect ectoderm-(left), mesoderm-(middle) and endoderm-like (right) structures in H&E stained teratoma sections.

Immunofluorescence staining
Cells were plated on matrigel, grown to the desired density, fixed in 2% formaldehyde in DPBS for 1 h, washed three times with DPBS, blocked in 1% sodium azide and 2% FBS in DPBS, and then incubated with the primary anti-OCT4 antibody (Table 2) or the corresponding isotype control (Table 2) diluted in blocking solution (1:100) for 30 min at RT. Cells were washed twice in DBPS, incubated in a 1:1000 dilution of the secondary goat anti-mouse-AF488 antibody (Table 2), washed as before, and nuclei were stained with 1 μg/ml DAPI in DPBS for 1 min.

STR, h-IMPACT analysis and G-banding
STR and h-Impact III test (including mycoplasm testing) were performed by Radil (Idexx). Karyotyping by G-banding was done by the Texas Children's Cancer Center Core (Houston, TX) at a band resolution of 400-450.

Teratoma formation assay
Cells were collected after collagenase treatment and approximately 2 × 10 6 cells in 100 μl PBS were injected into the hind leg muscle of SCID mice (Charles River strain 250). Teratoma were harvested after 8 weeks and fixed in 10% Formalin for 24 h. The fixed tissue was embedded in paraffin and 20 μm sections were stained with hematoxylin and eosin (H&E) by the Histology Core at Baylor College of Medicine (Houston, TX).