generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a modified rna system

Generation of human induced pluripotent stem cell lines fromhuman dermal fibroblasts using a modified RNA system Kyung-Ok Uhm, Gue Youn Go, So-Jung Kim, Eun Hee Jo, Hye Young Choi, Young

Generation of human induced pluripotent stem cell lines from

human dermal fibroblasts using a modified RNA system

Kyung-Ok Uhm, Gue Youn Go, So-Jung Kim, Eun Hee Jo, Hye

Young Choi, Young Sam Im, Hye-Yeong Ha, Jung-Hyun Kim,

Soo Kyung Koo

To appear in: Stem Cell Research

Received date: 20 December 2016

Revised date: 10 February 2017

Accepted date: 20 February 2017

Please cite this article as: Kyung-Ok Uhm, Gue Youn Go, So-Jung Kim, Eun Hee Jo, Hye Young Choi, Young Sam Im, Hye-Yeong Ha, Jung-Hyun Kim, Soo Kyung Koo , Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a modified RNA system The address for the corresponding author was captured

as affiliation for all authors Please check if appropriate Scr(2017), doi: 10.1016/ j.scr.2017.02.009

This is a PDF file of an unedited manuscript that has been accepted for publication As

a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before

it is published in its final form Please note that during the production process errors may

be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Generation of human induced pluripotent stem cell lines from

human dermal fibroblasts using a modified RNA system

Kyung-Ok Uhm, Gue Youn Go, So-Jung Kim, Eun Hee Jo, Hye Young Choi, Young Sam Im, Hye-Yeong Ha, Jung-Hyun Kim, Soo Kyung* Koo skkoo@nih.go.kr

Division of Intractable Disease, Center for Biomedical Sciences, Korea National Institute of Health, Chungcheongbuk-do, South Korea

*

Corresponding author at: Soo Kyung Koo, Ph.D

Address: 202, Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, 28160, Republic of Korea

Stem Cell Research: Lab Resource

Abstract: We generated human induced pluripotent stem cells (hFmiPS1 and hFmiPS2) from

the dermal fibroblasts of a donor using a modified RNA-based gene delivery method According to GTG-banding analysis, the generated hFmiPS1 line has a cytogenetic abnormality (46,XY, t(1;4)(q21;q25)) that is distinct from that of the donor, whereas hFmiPS2 has a normal karyotype (46,XY) These cell lines can be useful as a model for characterizing the hiPSCs generated by a non-viral and non-integrative system, or as a chromosomal balanced translocation model These two cell lines are registered and available from the National Stem Cell Bank, Korea National Institute of Health

Resource Table:

Name of Stem Cell line hFmiPS1 and hFmiPS2

Institution Korea National Institute of Health

Person who created resource Kyung-Ok Uhm

Contact person and email Soo Kyung Koo, skkoo@nih.go.kr

ACCEPTED MANUSCRIPT

Date archived/stock date hFmiPS1: April, 2014

hFmiPS2: November, 2015

Type of resource Biological reagent: Human induced pluripotent stem cell

(iPSC); derived from dermal fibroblasts of an adult male donor (ScienCell, 2320)

Key transcription factors hOct4, hSox2, hKlf4, hc-MYC, hLin28 (mRNA Reprogramming

kit, Stemgent) Authentication Identity and purity of cell line confirmed: expression of

pluripotency and differentiation genes by real-time PCR, ICC,

EB formation, STR analysis, karyotyping by GTG-banding Link to related literature /

Information in public databases http://kscr.nih.go.kr

http://hpscreg.eu

Ethics Institutional Review Board approval obtained

(2013-06EXP-06-R, 2014-10CON-04-1C-A)

1 Resource Details

 Human dermal fibroblasts (ScienCell, 2320) of a male donor were induced to human

pluripotent stem cells (hiPSCs) using modified RNA based integration-free vectors

 Karyotypes of generated hiPSCs, hFmiPS1 (46,XY,t(1;4)(q21;q25)) and hFmiPS2

(46,XY) were analyzed by GTG-banding (Figure 1B)

 Pluripotency of hFmiPS1 and hFmiPS2 was characterized by immunofluorescence

staining markers for Oct4, SSEA4, Tra-1-60, and Tra-1-81 (Figure 1B)

 Differentiation capacity of hFmiPS1 and hFmiPS2 into all three germ layers was

confirmed by real-time PCR using germ layer specification markers for Pax6

ACCEPTED MANUSCRIPT

(ectoderm), AFP (endoderm), and T (mesoderm) in hiPSCs-derived embryoid bodies (EB) (Figure 1C-D, Table 1)

 Teratoma-derived from hFmiPS1 with chromosomal abnormality presented all three germ layers, and there was no teratocarcinoma (Figure 1E)

 All 16 allele loci of hiFmiPS1 and hFmiPS2 are consistent with those of donor cells, as confirmed by STR analysis (Table 2)

2 Materials and Methods

2.1 Donor cells

Human dermal fibroblasts were obtained from a male donor (ScienCell, 2320) The cells were maintained in Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum (Gibco) and grown at 37 °C in a 5% CO2 atmosphere

2.2 Reprogramming and hiPSC maintenance

Obtained dermal fibroblasts were transduced using mRNA Reprogramming kit according to the manufacturer’s suggested protocol (Stemgent, 00-0071) (Warren et al., 2010) Clones were picked and cultivated onto feeder STO (ATCC, CRL1503) cells with DMEM/F12 media

(Gibco) supplemented with 20% (v/v) Knock-out™ Serum Replacement (Gibco), 0.1 mM NEAA (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), and 1 % (v/v) antibiotic-antimycotic

(Gibco) with basic fibroblast growth factor (bFGF) (4 ng/ml) The culture medium was changed every once per day

2.3 Immunofluorescence staining

Cells were fixed with 4% paraformaldehyde (Wako) for 20 min, blocked with 5% (v/v) goat serum in 0.25% (v/v) Triton X, and incubated with primary antibodies for Oct4 (FITC, Santa

Cruz, 1:200), SSEA4 (FITC, Millipore, 1:200), Tra-1-60 (FITC, Millipore, 1:200), and Tra-1-81 (FITC, Millipore, 1:200) Images were acquired using a fluorescence microscope

2.4 Real-time PCR analysis

Real-time PCR was performed using the TaqMan® Gene Expression Master Mix (Applied Biosystems) The total RNA of generated hiPSCs was isolated using the RNeasy Mini Kit (Qiagen), and cDNA was synthesized by reverse transcription using RNA to cDNA EcoDry

ACCEPTED MANUSCRIPT

quantitative gene expression data were normalized to the expression levels of GAPDH The

stem cell (hESC), was determined

2.5 In vitro differentiation into three germ layers

To test the capacity to differentiate into three germ layers, we induced embryoid bodies (EB) using hiPSCs harvested by dispase (Gibco) EB were cultivated on a petri dish in hiPSCs culture medium without bFGF for 14 days The culture medium for hiPSCs without bFGF was changed every other day

2.6 Teratoma formation

To test the in vivo differentiation of hFmiPS1, teratoma formation was performed using

hFmiPS1 suspended at 5 x 106 cells/ml in 50% (v/v) hiPSCs culture medium and 50% (v/v)

Matrigel (BD) Next, 200 ㎕ of suspended cells was injected subcutaneously into the dorsal flank of NOD-SCID mice anesthetized with diethyl ether Twelve weeks after injection, tumors

were surgically dissected from the mice, fixed in PBS containing 4% (v/v) formaldehyde, and

embedded in paraffin Tissue sections were stained with hematoxylin and eosin (H&E) 2.7 Karyotyping

Generated hiPSCs were karyotyped by standard cytogenetic procedures using the GTG-band method The cultured cells were treated for 45 min with colcemid (Thermo Fisher Scientific), harvested in fixative (acetic acid:methanol, 1:3), and the metaphase slides were prepared After Giemsa-Trypsin banding, we karyotyped hiPSCs The karyotype was analyzed according

to the International System for Human Cytogenetic Nomenclature

2.8 STR analysis

Short Tandem Repeat (STR) analysis was performed on generated hiPSCs and donor cells using the PowerPlex®16 System (Promega) with detection of 16 loci D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PentaD, vWA, D8S1179, TPOX, FGA, and Amelogenin

Figures and Supplementary tables

Figure 1 Morphologies of donor cells and generated hiPSCs, pluripotent marker expression, and karyotype (A) Phase contrast images of donor cells, HDF, hFmiPS1, and hFmiPS2 (B)

ACCEPTED MANUSCRIPT

Immunofluorescence staining of pluripotent markers Oct4, SSEA4, Tra-1-60, and Tra-1-81 and karyotypes of hFmiPS1 and hFmiPS2 Resolution of karyotype: 550 bands Scale bar: 200 ㎛

(C) In vitro differentiation into three germ layers of hiPSCs Phase contrast images of EB

derived from hFmiPS1 or hFmiPS2 and (D) real-time PCR result using germ layer-specific makers, Pax6 (ectoderm), AFP (endoderm), and T (mesorderm) All quantitative gene expression data were normalized to the expression levels of GAPDH The relative fold changes (2-△△Ct) in mRNA expression of hiPSCs by that of H9, human embryonic stem cells (hESC), was calculated (E) All three germ layers are shown in teratoma derived from hFmiPS1 with H&E staining Scale bar: 200 ㎛

Supplimentary table 1 List of primers for three germ layer specification

Supplimentary table 2 Result of STR analysis

Acknowledgment

This work was supported by the Korea National Institute of Health (grant number 2016-NG61002-00, 2016)

References

Warren, L., Manos, P.D., Ahfeldt, T., Loh, Y.H., Li, H., Lau, F., Ebina, W., Mandel, P.K., Smith, Z.D., Meissner, A., Daley, D.Q., Brack, A.S, Collin, J.J., Cowan, C., Schlaeger, T.M., Rossi, D.J.,

2010 Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA Cell Stem Cell 7:618

ACCEPTED MANUSCRIPT

Figure 1