OLIG gene targeting in human pluripotent stem cells for motor neuron and oligodendrocyte differentiation

Department of Reproductive Medicine, University of California San Diego, San Diego, California, USA.
Nature Protocol (Impact Factor: 9.67). 05/2011; 6(5):640-55. DOI: 10.1038/nprot.2011.310
Source: PubMed


Pluripotent stem cells can be genetically labeled to facilitate differentiation studies. In this paper, we describe a gene-targeting protocol to knock in a GFP cassette into key gene loci in human pluripotent stem cells (hPSCs), and then use the genetically tagged hPSCs to guide in vitro differentiation, immunocytochemical and electrophysiological profiling and in vivo characterization after cell transplantation. The Olig transcription factors have key roles in the transcription regulatory pathways for the genesis of motor neurons (MNs) and oligodendrocytes (OLs). We have generated OLIG2-GFP hPSC reporter lines that reliably mark MNs and OLs for monitoring their sequential differentiation from hPSCs. The expression of the GFP reporter recapitulates the endogenous expression of OLIG genes. The in vitro characterization of fluorescence-activated cell sorting-purified cells is consistent with cells of the MN or OL lineages, depending on the stages at which they are collected. This protocol is efficient and reliable and usually takes 5-7 months to complete. The genetic tagging-differentiation methodology used herein provides a general framework for similar work for differentiation of hPSCs into other lineages.

Download full-text


Available from: Peng Jiang, Apr 20, 2014
  • Source
    • "Moreover, effective derivation of OPCs and oligodendrocytes from mouse ESCs has been achieved using RA, PDGF-AA, T3 and so on [21], [22]. In contrast, many researchers have attempted human ESC differentiation into OPCs or oligodendrocytes over the past decade, but there are only a few recent reports indicating the successful differentiation of human ESCs and iPSCs into oligodendrocytes using the medium containing RA, Sonic hedgehog (Shh), PDGF-AA and T3 [19], [23], [24]. As for non-human primate ESCs, rhesus monkey ESCs have been successfully differentiated into neural cells by EB formation and neural rosettes, but this method is inefficient to obtain oligodendrocytes [25]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The common marmoset (Callithrix jacchus) is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) derived from mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets.
    Full-text · Article · Nov 2012 · PLoS ONE
  • [Show abstract] [Hide abstract]
    ABSTRACT: Multiple sclerosis is an autoimmune disease of the human central nervous system characterized by immune-mediated myelin and axonal damage, and chronic axonal loss attributable to the absence of myelin sheaths. There are two aspects to the treatment of MS-first, the prevention of damage by suppressing the maladaptive immune system, and second, the long-term preservation of axons by the promotion of remyelination, a regenerative process in which new axons are restored to demyelinated axons. Medicine has made significant progress in the first of these in recent years-there is an increasing number of ever more effective disease-modifying immunomodulatory interventions. However, there are currently no widely used regenerative therapies in MS. Conceptually, there are two approaches to remyelination therapy-transplantation of myelinogenic cells and promotion of endogenous remyelination mediated by myelinogenic cells present within the diseased tissue. In this chapter, in addition to describing why remyelination therapies are important, we review both these approaches, outlining their current status and future developments.
    No preview · Article · Nov 2012 · Progress in brain research
  • Source

    Preview · Chapter · Jan 2012
Show more