Veerakumar Balasubramaniyan

Differentiation of Neural Stem Cells into Oligodendrocytes : Involvement of the Polycomb Group Protein Ezh2

The mechanisms underlying the regulation of neural stem cell (NSC) renewal and maintenance of their multipotency are still not completely understood. Self‐renewal of stem cells in general implies repression of genes that encode for cell lineage differentiation. Enhancer of zeste homolog 2 (Ezh2) is a Polycomb group protein involved in stem cell renewal and maintenance by inducing gene silencing via histone methylation and deacetylation. To establish the role of Ezh2 in the maintenance and differentiation of NSCs, we have examined the expression of Ezh2 in NSCs isolated from embryonic (embryonic day 14) mice during proliferation and differentiation in vitro. Our results show that Ezh2 is highly expressed in proliferating NSCs. In accordance with its suggested role as a transcription repressor, the expression of Ezh2 decreased when the NSCs differentiated into neurons and was completely suppressed during differentiation into astrocytes. Surprisingly, Ezh2 remained highly expressed in NSCs that differentiated into an oligodendrocytic cell lineage, starting from oligodendrocyte precursor cells (OPCs) up to the immature (premyelinating) oligodendrocyte stage. To further establish the role of Ezh2 in NSC differentiation, we silenced and induced overexpression of the Ezh2 gene in NSCs. High levels of Ezh2 in differentiating NSCs appeared to be associated with an increase in oligodendrocytes and a reduction in astrocytes, whereas low levels of Ezh2 led to completely opposite effects. The increase in the number of oligodendrocytes induced by enhanced expression of Ezh2 could be ascribed to stimulation of OPC proliferation although stimulation of oligodendrocyte differentiation cannot be excluded.

Olig2 overexpression induces the in vitro differentiation of neural stem cells into mature oligodendrocytes

Differentiation induction of neural stem cells (NSCs) into oligodendrocytes during embryogenesis is the result of a complex interaction between local induction factors and intracellular transcription factors. At the early stage of differentiation, in particular, the helix‐loop‐helix transcription factors Olig1 and Olig2 have been shown to be essential for oligodendrocyte lineage determination. In view of the possible application of NSCs as a source for remyelinating cell transplants in demyelinating diseases (e.g., multiple sclerosis), in vitro procedures need to be developed to drive the oligodendrocyte differentiation process. Mere culture in medium supplemented with major embryonic oligodendrogenic induction factors, such as Sonic hedgehog, results in oligodendrocyte differentiation of only about 10% of NSCs. We previously showed that induction of Olig1 expression by gene transfection could indeed initiate the first stage of oligodendrocyte differentiation in NSCs, but appeared to be unable to generate fully mature, functional oligodendrocytes. In this study, we transfected NSCs isolated from the embryonic mouse brain with the Olig2 gene and found that the introduced overexpression of Olig2 could induce the development of fully mature oligodendrocytes expressing the transcription factor Nkx2.2 and all major myelin‐specific proteins. Moreover, Olig2‐transfected NSCs, in contrast to nontransfected NSCs, developed into actively remyelinating oligodendrocytes after transplantation into the corpus callo‐sum of long‐term cuprizonefed mice, an animal model for demyelination. Our results show that transfection of genes encoding for oligodendrogenic transcription factors can be an efficient way to induce the differentiation of NSCs into functional oligodendrocytes.

Differentiation of Induced Pluripotent Stem Cells Into Functional Oligodendrocytes

The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell‐based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in‐vitro (co‐culture with DRG neurons) as well as in‐vivo (after implantation in the demyelinated corpus callosum of cuprizone‐treated mice). We report the importance of complete purification of the iPS‐derived OPC suspension to prevent the contamination with teratoma‐forming iPS cells.

Oligodendrocyte differentiation and implantation: new insights for remyelinating cell therapy

PURPOSE OF REVIEWnRecent research on oligodendrocyte development has yielded new insights on the involvement of morphogens and differentiation factors in oligodendrogenesis. This knowledge has improved strategies to control neural stem cell-derived oligodendrocyte differentiation and functional maturation in vitro. In this review, we highlight the current knowledge on oligodendrocyte differentiation and discuss the novel possibilities of neural stem cell-derived oligodendrocytes for graft-based remyelination therapy, for example, for multiple sclerosis.nnnRECENT FINDINGSnDetailed insight into the cellular and molecular processes of embryonic and adult oligodendrogenesis has extended considerably in the past 2 years. Application of extrinsic factors and manipulation of intrinsic factors in neural stem cells have yielded convincing oligodendrocyte differentiation strategies. In addition, the recent groundbreaking developments regarding induced pluripotent stem cells generated from easily accessible somatic cells seem to offer an almost inexhaustible source for transplantable, autologous neural stem cells. Moreover, new approaches to optimize the implantation site for oligodendrocyte survival and functionality have improved the feasibility of stem cell-based oligodendrocyte replacement therapy.nnnSUMMARYnLoss of myelin in demyelinating diseases is only partly restored by endogenous oligodendrocyte precursor cells. Application of optimally functional, neural stem cell-derived oligodendrocyte precursors at the lesion site has become a realistic therapeutic approach to promote the remyelination process.

Transient Expression of Olig1 Initiates the Differentiation of Neural Stem Cells into Oligodendrocyte Progenitor Cells

In order to develop an efficient strategy to induce the in vitro differentiation of neural stem cells (NSCs) into oligodendrocyte progenitor cells (OPCs), NSCs were isolated from E14 mice and grown in medium containing epidermal growth factor and fibroblast growth factor (FGF). Besides supplementing the medium with oligodendrogenic factors such as Sonic Hedgehog (Shh), FGF‐2, and PDGF, we attempted to initiate the gene transcription program for OPC differentiation by transfection of the Olig1 gene, a transcription factor known to be involved in the induction of oligodendrocyte lineage formation during embryogenesis. Whereas addition of Shh, FGF‐2, and PDGF could induce OPC differentiation in 12% of the NSCs, the transient expression of Olig1 by use of Nucleofector gene transfection initiated OPC differentiation in 55% of the NSCs. Our results show that nonviral transfection of genes encoding for oligodendrogenic transcription factors may be an efficient way to initiate the in vitro differentiation of NSCs into OPCs.

Commentary: Periostin (POSTN) Regulates Tumor Resistance to Antiangiogenic Therapy in Glioma Models

Glioblastoma (GBM) are highly vascularized, invasive brain tumors with a dismal prognosis. Despite surgical de-bulking of the tumor mass followed by concomitant intensive chemo/radiotherapy, GBM patients exhibit poor survival rates (REF). The hallmark presence of microvascular proliferation in GBM has attracted vast interest in the use of antiangiogenic therapies. Bevacizumab a humanized recombinant monoclonal antibody against VEGF-A was approved by the FDA for the treatment of recurrent GBM. However, several recent studies have reported the lack of a survival benefit suggesting tumors have both intrinsic and acquired resistance to anti-VEGF therapy1,2. Studies suggest mesenchymal transition and hypoxia signaling as two major pathways associated with the development of resistance to anti-VEGF therapy. Recently, we reported that the glioma treated with bevacizumab have higher periostin (POSTN) expression than control tumors in a murine glioma tumor models3. In this commentary, we review our recent findings as well as the role of POSTN in antiangiogenic therapy resistance in glioma.