Induction of Stem Cell Like Cells from Mouse Embryonic Fibroblast by Short-Term Shear Stress and Vitamin C

Jaewoon Lim,Shambhavi Pandey,Myung Chul Lee,Seungmin Yeom,Hoon Seonwoo,Sangbae Park,Kyoung Je Jang,Jong Hoon Chung,Pankaj Garg,Jae Eun Kim

doi:10.3390/app11041941

Abstract

Induced pluripotent stem cells (iPSCs) are a good medicine source because of their potential to differentiate into various tissues or cells. However, traditionally, iPSCs made by specific transgenes and virus vectors are not appropriate for clinical use because of safety concerns and risk of tumor development. The goal of this research was to develop an alternative method for reprogramming, using small molecules and external stimuli. Two groups were established: short-term shear stress (STSS) under suspension culture and a combination of short-term shear stress and vitamin C (SSVC) under suspension culture. For STSS, the pipetting was carried out for cells twice per day for 2 min for 14 days in the embryonic stem cell (ES) medium. In the case of SSVC, the procedure was the same as for STSS however, its ES medium included 10 µM of vitamin C. After 14 days, all spheroids were picked and checked for pluripotency by ALP (alkaline phosphatase) assay and immunocytochemistry. Both groups partially showed the characteristics of stem cells but data demonstrated that the spheroids under shear stress and vitamin C had improved stem cell-like properties. This research showed the possibility of external stimuli and small molecules to reprogram the somatic cells without the use of transgenes.

Highlights

In 1957, Waddington suggested the concept of an “epigenetic landscape” which is involved in reprogramming [1]
We alternatively induced stem cell-like cells using external stimuli without integrating OSKM genes required for induced pluripotent stem cells (iPSCs) induction
The MEF spheroids cultured under stress and vitamin C (SSVC) responded stronger with Alkaline Phosphatase (ALP) solution than that of the STSS group

Summary

Introduction

In 1957, Waddington suggested the concept of an “epigenetic landscape” which is involved in reprogramming [1]. Spheroids attached on a 4-well plate were fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) for 15 min, and washed twice with DPBS. The cells in spheroids were permeabilized with DPBS including 0.2% Tween 20 (Amresco, Solon, OH, USA) for 10 min at room temperature. MEF spheroids were incubated with each of the primary antibodies diluted in 10% BSA solution at 4 ◦C overnight. The spheroids were rinsed twice with DPBS and incubated with secondary antibodies diluted in 10% BSA solution at room temperature for 2 h. The MEF spheroids were washed twice with DPBS and 2 mg/mL of 4’,6-diamidino-2phenylindole (DAPI, Sigma-Aldrich, USA) was added for nuclear staining. After 10 min of incubation at room temperature and washing once with DPBS, the spheroids were placed on a slide glass and mounted using aqua poly/mount (Polysciences Inc., Warrington, PA, USA). Images of Oct and SSEA-1 stained spheroids were acquired by LSM710 (Carl Zeiss, Oberkochen, Germany) with Zen 2009 LE, and images of three germ layers stained in vitro were obtained by an ECLIPSE Ti (Nikon, Japan) with Zyla sCMOS (Andor Technology, Belfast, UK) camera and NIS-Elements AR. 4.20 program

Western Blotting

Differentiation to Three Germ Layers In Vitro

Results and Discussion

Conclusions