Abstract
Functional dedifferentiation of lineage-committed cells toward pluripotency may have a great potential in regenerative medicine. Reversine has been shown to induce dedifferentiation of multiple terminally differentiated mesodermal origin cells, which are capable of being directed to differentiate into other cell types within mesodermal lineages. However, the possibilities of these cells to give rise to other lineages have not been examined. Here we show that large scale gene expression profiling of reversine-treated C2C12 myoblasts identifies a subset of up-regulated genes involved in specification of neuroectodermal as well as mesodermal lineages. Reversine treatment leads to up-regulation of priming genes of neuroectodermal lineages, such as Ngn2, Nts, Irx3, Pax7, Hes1, and Hes6, through active histone modifications in the promoter regions of these genes. Additionally, reversine increases the expression of markers for other cell types of mesodermal lineages, Ogn and apoE, via inducing active histone modifications, while down-regulating the myogenic basic helix-loop-helix factor, MyoD, via repressive histone modifications. Consistent with up-regulation of these genes, reversine-treated C2C12 myoblasts redifferentiate into neural as well as mesodermal lineages, under appropriate stimuli. Taken together, these results indicate that reversine induces a multipotency of C2C12 myoblasts via inducing a specific combination of active histone modifications. Collectively, our findings provide a mechanistic rationale for the application of reversine to dedifferentiation of somatic cells.
Highlights
¶ To whom correspondence should be addressed: Address correspondence to: Jeung-Whan Han, Department of Biochemistry and Molecular Biology, College of Pharmacy Sungkyunkwan University, Suwon 440-746, Korea, Tel: 82-31-290-7716, Fax: Functional dedifferentiation of lineage- dedifferentiation of somatic cells
To evaluate the extent of transcriptional alterations elicited by reversine, and examine the possibility of reversine-treated C2C12 myoblasts to redifferentiate to other lineage cells than mesodermal-lineage cells, global gene expression levels were determined by an AB
As an alternative, a synthetic small molecule, reversine has been demonstrated to induce dedifferentiation of terminally differentiated murine myoblasts, and primary murine and human dermal fibroblasts, which can increase their plasticity toward mesodermallineage cells [15]
Summary
Cell Culture and Transfection - The C2C12 cell line, which is mouse myoblasts, was cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories, Logan, UT) and 1% penicillin/streptomycin (Gibco). C2C12 cells were plated at a seeding density of 5X104/10cm cell culture plates in 15% FBS and antibiotics and day, transfection was performed with Hes RNAi or control RNA (Invitrogen) as indicated in the text. C17.2 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% FBS and 5% horse serum (growth medium: GM) and passaged at the density of 50% confluence every 2 days. Reagents and Antibodies - Reversine was obtained from Calbiochem (La Jolla, CA) or prepared by the known procedures [14]. For Q-PCR, template cDNAs were reverse-transcribed from one microgram of total RNA using oligo-dT primer and SuperScript II reverse transcriptase (Invitrogen).
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