Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Summary

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Abstract

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate ^1-3. We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged ^4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals ⁶, human newborn fibroblasts, as well as human keratinocytes.

Protocol

1. Virus production and transduction

1. Plate Phoenix Ampho cells at a density of ~7-8x10⁶ per 10 cm plate in 10 ml of DMEM medium (DMEM high glucose, 10% FBS heat inactivated, 2 mM L-glutamine, no antibiotics). Place in the incubator and culture overnight at 37°C, 5% CO₂.
2. The next day transfect Phoenix cells using 12 μg of a vector encoding either Oct3/4, Sox2, Klf4, c-myc, or GFP gene (Addgene plasmids 17217, 17218, 17219, 17220) and 35 μl Fugene 6. Prepare transfection mix in 500 μl of DMEM medium (DMEM high glucose, 2 mM L-glutamine, no FBS and antibiotics). Incubate 20 min. Gently pipette DNA complexes into the 10 cm plate....

Discussion

Studying human diseases, especially neurological and neurodegenerative, has been particularly challenging due to the inaccessibility of adequate human cellular models. The ability to reprogram easily obtainable somatic cells into induced pluripotent stem cells and the potential to differentiate them into diverse cell types opened a possibility to create cellular models of genetic diseases. In addition, iPSCs hold a great promise in the future of regenerative medicine. Therefore, it is essential to develop and optimize re.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen	11965
DMEM/F12	Invitrogen	11330
KSR	Invitrogen	10828
Non-essential aminoacids	Invitrogen	11140
Sodium butyrate	Sigma-Aldrich	B5887
Y27632	Stemgent	04-0012
bFGF	Stemgent	03-0002
Tra-1-81 antibody	Stemgent	09-0069
Oct3/4 antibody	Santa Cruz Biotechnology, Inc.	sc-8628
Nanog antibody	Cell Signaling Technology	4903S
Tra-1-60 antibody	EMD Millipore	MAB4360
Sox2 antibody	Cell Signaling Technology	3579S
SSEA4	EMD Millipore	MAB4304
CF1 MEFs	Globalstem	GSC-6201G
Objective marker	Nikon Instruments	MBW10010
Matrigel	BD Biosciences	354277
mTeSR1	Stem Cell Technologies	05850
β-mercapt–thanol	Sigma-Aldrich	M7522
Fugene 6	Roche Group	11814443001
polybrene	Sigma-Aldrich	H9268
Object marker	Nikon Instruments	MBW10010