Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency

Abstract

BackgroundPluripotent stem cells (PSCs) offer immense potential as a source for regenerative therapies. The teratoma assay is widely used in the field of stem cells and regenerative medicine, but the cell composition of teratoma is still elusive.MethodsWe utilized PSCs expressing enhanced green fluorescent protein (EGFP) under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo. OCT4-MES (mouse embryonic stem cells) were isolated from the blastocysts of 3.5-day OCT4-EGFP mice (transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter) embryos, and TG iPS 1-7 (induced pluripotent stem cells) were generated from mouse embryonic fibroblasts (MEFs) from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. These pluripotent cells were characterized according to their morphology and expression of pluripotency markers. Their differentiation ability was studied with in vivo teratoma formation assays. Further differences between pluripotent cells were examined by real-time quantitative PCR (qPCR).ResultsThe results showed that several OCT4-expressing PSCs escaped differentiation inside of teratomas, and these escaped cells (MES-FT, GFP-positive cells separated from OCT4-MES-derived teratomas; and iPS-FT, GFP-positive cells obtained from teratomas formed by TG iPS 1-7) retained their pluripotency. Interestingly, a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT (MES-ST, GFP-positive cells isolated from MES-FT-derived teratomas; iPS-ST, GFP-positive cells obtained from teratomas formed by iPS-FT) were still pluripotent, as shown by alkaline phosphatase (AP) staining, immunofluorescent staining and PCR. MES-FT, iPS-FT, MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation, such as Dazl, Stella and Stra8.ConclusionsIn summary, a small number of PSCs escaped differentiation inside of teratomas, and these cells maintained pluripotency and partially developed towards germ cells. Both escaped PSCs and germ cells present a risk of tumor formation. Therefore, medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.

Cancer cells as a new source of induced pluripotent stem cells

“…OSKM factors could provide a condition to promote tumor development on their own because these factors are overexpressed in different cancer cells in a parallel manner [ 32 ]. This is especially true for c-Myc [ 4 , 47 ] and Klf-4 [ 4 ]. A method was adopted by Alvarez-Palomo et al that used synthetic mRNA of some Yamanaka factors along with Cyclin D1 transfection to repair DNA lesions during reprogramming.…”

“…The plasticity of pluripotency is the ability of stem cells to differentiate into three germ layers, including the endoderm, mesoderm, and ectoderm. Pluripotent stem cells express specific pluripotency markers and form embryoid bodies in vitro and teratomas in vivo [ 3 , 4 ].…”

Direct in vivo application of induced pluripotent stem cells is feasible and can be safe

“…Because it is impossible to test whether each single iPSC has differentiated, the bioengineered tissue may contain quiescent undifferentiated or partially differentiated iPSCs due to epigenetic memory as well as inefficient purification. A recent study showed that teratoma generated from pluripotent stem cells contained quiescent stem cells 13. iPSC-derived neural progenitor cells promoted functional and structural recovery of spinal cord injury with no tumor formation but undifferentiated cells still existed 5 14 or 16 15 weeks later.…”

“…Although actively growing iPSCs can be detected, it is not feasible to confirm there is not a single quiescent iPSC present in the differentiated cells. Lineage-specific cells derived from iPSCs are likely contaminated with a small number of undifferentiated cells 13. To date, it is not reported that the local injection of ES- or iPS-derived lineage-specific cells form teratomas 20, 22.…”