Genetic selection of sox1GFP‐expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation

Abstract

Because of their ability to proliferate and to differentiate into diverse cell types, embryonic stem (ES) cells are a potential source of cells for transplantation therapy of various diseases, including Parkinson's disease. A critical issue for this potential therapy is the elimination of undifferentiated cells that, even in low numbers, could result in teratoma formation in the host brain. We hypothesize that an efficient solution would consist of purifying the desired cell types, such as neural precursors, prior to transplantation. To test this hypothesis, we differentiated sox1-green fluorescent protein (GFP) knock-in ES cells in vitro, purified neural precursor cells by fluorescence-activated cell sorting (FACS), and characterized the purified cells in vitro as well as in vivo. Immunocytofluorescence and RT-PCR analyses showed that this genetic purification procedure efficiently removed undifferentiated pluripotent stem cells. Furthermore, when differentiated into mature neurons in vitro, the purified GFP + cell population generated enriched neuronal populations, whereas the GFP -population generated much fewer neurons. When treated with dopaminergic inducing signals such as sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF8), FACS-purified neural precursor cells responded to these molecules and generated dopaminergic neurons as well as other neural subtypes. When transplanted, the GFP + cell population generated well contained grafts containing dopaminergic neurons, whereas the GFP -population generated significantly larger grafts (about 20-fold) and frequent tumor-related deaths in the transplanted animals. Taken together, our results demonstrate that genetic purification of neural precursor cells using FACS isolation can effectively remove unwanted proliferating cell types and avoid tumor formation after transplantation.

Defining the Integration Capacity of Embryonic Stem Cell-Derived Photoreceptor Precursors

“…In transplantations to the brain, selection of Sox1.GFP þ cells and culture prior to transplantation attenuated tumor formation [40]. Here, we found that despite selecting Rax.GFP þ retinal progenitors prior to transplantation, there was still a risk of tumor formation from ESC-derived cultures.…”

Bcl2, a transcriptional target of p38α, is critical for neuronal commitment of mouse embryonic stem cells

“…8 The expression of p38a-regulated targets is correlated with the onset of RA-induced neuronal differentiation. To determine whether expression levels of these p38a targets were correlated with the establishment of ESderived neurons, we have analyzed their expression profiles in the E14TG2a-derived sox1-targeted GFP ES cells, known to differentiate efficiently in functional neurons expressing b3-TUBULIN/TUJ1 and MAP2 (microtubule-associated protein 2) proteins under RA treatment (Li et al, 10 Ying et al, 30 Chung et al 31 and O Feraud, unpublished results).…”

ES cell-derived renewable and functional midbrain dopaminergic progenitors

“…This approach has recently been used to generate Purkinje cell progenitors or floor plate cells (28,29). Previously, we purified Sox1 + NPs from differentiating ES cells, but these cell populations inefficiently generated DA neurons (30), possibly due to the nature of mDA NPs as glia-like floor plate cells rather than Sox1 + NPs (4). These findings led us to hypothesize that it is critical to use developmentally appropriate marker(s) to identify and purify functional and authentic subtype-specific NPs.…”