Since the discovery of vitamin C, the number of its known biological functions is continually expanding. Both the names ascorbic acid and vitamin C reflect its antiscorbutic properties due to its role in the synthesis of collagen in connective tissues. Ascorbate acts as an electron-donor keeping iron in the ferrous state thereby maintaining the full activity of collagen hydroxylases; parallel reactions with a variety of dioxygenases affect the expression of a wide array of genes, for example via the HIF system, as well as via the epigenetic landscape of cells and tissues. In fact, all known physiological and biochemical functions of ascorbate are due to its action as an electron donor. The ability to donate one or two electrons makes AscH(-) an excellent reducing agent and antioxidant. Ascorbate readily undergoes pH-dependent autoxidation producing hydrogen peroxide (H(2)O(2)). In the presence of catalytic metals this oxidation is accelerated. In this review, we show that the chemical and biochemical nature of ascorbate contribute to its antioxidant as well as its prooxidant properties. Recent pharmacokinetic data indicate that intravenous (i.v.) administration of ascorbate bypasses the tight control of the gut producing highly elevated plasma levels; ascorbate at very high levels can act as prodrug to deliver a significant flux of H(2)O(2) to tumors. This new knowledge has rekindled interest and spurred new research into the clinical potential of pharmacological ascorbate. Knowledge and understanding of the mechanisms of action of pharmacological ascorbate bring a rationale to its use to treat disease especially the use of i.v. delivery of pharmacological ascorbate as an adjuvant in the treatment of cancer. View Publication

The generation of induced pluripotent stem cells (iPSCs) often results in aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster, compromising the ability to generate entirely iPSC-derived adult mice ('all-iPSC mice'). Here, we show that reprogramming in the presence of ascorbic acid attenuates hypermethylation of Dlk1-Dio3 by enabling a chromatin configuration that interferes with binding of the de novo DNA methyltransferase Dnmt3a. This approach allowed us to generate all-iPSC mice from mature B cells, which have until now failed to support the development of exclusively iPSC-derived postnatal animals. Our data show that transcription factor-mediated reprogramming can endow a defined, terminally differentiated cell type with a developmental potential equivalent to that of embryonic stem cells. More generally, these findings indicate that culture conditions during cellular reprogramming can strongly influence the epigenetic and biological properties of the resultant iPSCs. View Publication

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process, but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b, two known vitamin-C-dependent H3K36 demethylases, as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore, we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367, an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming. View Publication

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However, the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound, vitamin C (Vc), enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence, a recently identified roadblock for reprogramming. In addition, Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process. View Publication

Mesenchymal stem cells (MSCs) derived from bone marrow are an important tool in tissue engineering and cell-based therapies because of their multipotent capacity. Majority of studies on MSCs have investigated the roles of growth factors, cytokines, and hormones. Antioxidants such as ascorbic acid can be used to expand MSCs while preserving their differentiation ability. Moreover, ascorbic acid can also stimulate MSC proliferation without reciprocal loss of phenotype and differentiation potency. In this study, we evaluated the effects of ascorbic acid on the proliferation, differentiation, extracellular matrix (ECM) secretion of MSCs. The MSCs were cultured in media containing various concentrations (0-500 microM) of L-ascorbate-2-phosphate (Asc-2-P) for 2 weeks, following which they were differentiated into adipocytes and osteoblasts. Ascorbic acid stimulated ECM secretion (collagen and glycosaminoglycan) and cell proliferation. Moreover, the phenotypes of the experimental groups as well as the differentiation potential of MSCs remained unchanged. The apparent absence of decreased cell density or morphologic change is consistent with the toxicity observed with 5-250 microM concentrations of Asc-2-P. The results demonstrate that MSC proliferation or differentiation depends on ascorbic acid concentration. View Publication

The future use of adult mesenchymal stem cells (MSCs) for human therapies depends on the establishment of preclinical studies with other mammals such as mouse. Surprisingly, purification and characterisation of murine MSCs were only poorly documented. The aim of this study was to purify mouse MSCs from adult bone marrow and to functionally characterise their abilities to differentiate along diverse lineages. Adherent cells from adult C57Bl/6J mouse bone marrow were depleted of granulo-monocytic cells and subsequently allowed to grow on fibronectin-coated dishes in presence of fetal bovine serum and growth factors. The growing fibroblastoid cell population primarily consisted of spindle- and star-shaped cells with significant renewal capacity as they were cultured until 30 passages (about 60 doubling population). We fully demonstrated the MSC phenotype of these cells by inducing them to differentiate along osteoblastic, adipocytic, and chondrocytic pathways. Mouse MSCs (mMSCs) sharing the same morphological and functional characteristics as human MSCs can be successfully isolated from adult bone marrow without previous mouse or bone marrow treatment. Therefore, mMSCs will be an important tool to study the in vivo behaviour and fate of this cell type after grafting in mouse pathology models. View Publication

BACKGROUND Embryonic stem (ES) cells are capable of self-renewal and differentiation into cellular derivatives of all 3 germ layers. In appropriate culture conditions, ES cells can differentiate into specialized cells, including cardiac myocytes, but the efficiency is typically low and the process is incompletely understood. METHODS AND RESULTS We evaluated a chemical library for its potential to induce cardiac differentiation of ES cells in the absence of embryoid body formation. Using ES cells stably transfected with cardiac-specific alpha-cardiac myosin heavy chain (MHC) promoter-driven enhanced green fluorescent protein (EGFP), 880 compounds approved for human use were screened for their ability to induce cardiac differentiation. Treatment with ascorbic acid, also known as vitamin C, markedly increased the number of EGFP-positive cells, which displayed spontaneous and rhythmic contractile activity and stained positively for sarcomeric myosin and alpha-actinin. Furthermore, ascorbic acid induced the expression of cardiac genes, including GATA4, alpha-MHC, and beta-MHC in untransfected ES cells in a developmentally controlled manner. This effect of ascorbic acid on cardiac differentiation was not mimicked by the other antioxidants such as N-acetylcysteine, Tiron, or vitamin E. CONCLUSIONS Ascorbic acid induces cardiac differentiation in ES cells. This study demonstrates the potential for chemically modifying the cardiac differentiation program of ES cells. View Publication

Embryonic stem cells are pluripotent cells derived from the inner cell mass of mouse blastocysts that have been shown to differentiate spontaneously into cell types representing all three germ layers. This study shows that ES cells were induced to differentiate in vitro into mineralized osteoblasts under the influence of ascorbic acid, beta-glycerophosphate and 1alpha,25-OH vitamin D3. The activity of alkaline phosphatase, an early osteoblast marker, was found to be increased around day 12 of culture. Mineralized cells were clearly identified by histochemical staining, which detects mineralized calcium. The major noncollagenous component of bone matrix, osteocalcin, was localized to the mineralized cells by immunofluorescence. The expression of bone-specific genes was analyzed by real-time quantitative PCR. Osteocalcin and bone sialoprotein (BSP) were identified as early as in the fourth week of embryonic stem cell culture, both being characteristic for late stages of osteoblastic differentiation, indicating that at this time of culture the identified cells represent mature" osteoblasts. The osteoblast-specific transcription factor Cbfa1 was induced a few days earlier. The expression of osteopontin and osteonectin� View Publication

Vitamin C (ascorbic acid (AA)) is very popular for its antioxidant properties. Consequently, many other important aspects of this multifaceted molecule are often underestimated or even ignored. In the present paper, we have tried to bring to the foreground some of these aspects, including the peculiarities of the AA biosynthetic pathway in different organisms, the remarkable function of AA as a co-substrate of many important dioxygenases, the role of AA-regenerating enzymes and the known pathways of AA catabolism, as well as the intriguing function of AA in gene expression. View Publication

Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential. View Publication