Prolonged proteasome inhibition cyclically upregulates Oct3/4 and Nanog gene expression, but reduces induced pluripotent stem cell colony formation.

There is ample evidence that the ubiquitin-proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover, proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells, acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein, we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4, and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog, but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion, our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however, efficient colony formation requires proteasome activity. Therefore, discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells.

Silencing histone deacetylase-specific isoforms enhances expression of pluripotency genes in bovine fibroblasts.

Histone deacetylases (HDACs) catalyze deacetylation of histones that results in altered transcriptional activity. Inhibitors of HDACs have been shown to induce transcriptional changes that contribute positively to reprogramming somatic cells either by nuclear transfer or inducing a pluripotent state. However, the exact molecular mechanisms whereby HDAC inhibitors function and the specificity of the HDAC isoforms in cell reprogramming are not yet fully understood. Herein, we report the ability of individual isoform-specific HDACs to modulate endogenous expression of pluripotency-associated genes in bovine somatic cells. This in vitro study showed that a transient selective depletion of HDACs resulted in elevated mRNA levels of Oct-4, Sox2, and Nanog. In particular, we found that inhibition of specific HDAC isoforms using small interfering (si) RNA significantly increased expression of Nanog, a key factor required for totipotency induced by somatic cell nuclear transfer and for maintaining pluripotency in embryonic and induced pluripotent stem cells. Our study suggests that this gene might be the most susceptible to HDAC activity inhibition. Moreover, a regulatory role of the class III HDAC, SIRT3, on an Oct4-Sox2-Nanog transcriptional network was revealed. We observed the upregulation of pluripotency-related genes by depletion of SIRT3. SIRT3 is localized to mitochondria and is associated with energy metabolism processes, suggesting metabolic changes may be linked to reprogramming in bovine fibroblasts. In conclusion, we show that targeting selective HDACs can potentially be useful to enhance reprogramming and that sirtuins may play a pivotal role in somatic cell reprogramming by upregulating an Oct4-Sox2-Nanog transcriptional network. Dedifferentiating donor somatic cells by upregulating developmentally important genes through specific knockdown of epigenetic targets, in particular HDACs, may provide a path to improving livestock cloning and the in vitro production of pluripotent cells.

Cell-surface expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) in heterogeneous cultures of marrow-derived mesenchymal stem cells.

Cellular heterogeneity of mesenchymal stem cells (MSCs) impedes their use in regenerative medicine. The objective of this research is to identify potential biomarkers for the enrichment of progenitors from heterogeneous MSC cultures. To this end, the present study examines variation in expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) on the surface of MSCs derived from human bone marrow in response to culture conditions and among cell populations. Multipotent cells isolated from heterogeneous MSC cultures exhibit a greater than three-fold increase in surface expression for NG2 and greater than two-fold increase for CD146 as compared with parental and lineage-committed MSCs. For both antigens, surface expression is downregulated by greater than or equal to six-fold when MSCs become confluent. During serial passage, maximum surface expression of NG2 and CD146 is associated with minimum doubling time. Upregulation of NG2 and CD146 during loss of adipogenic potential at early passage suggests some limits to their utility as potency markers. A potential relationship between proliferation and antigen expression was explored by sorting heterogeneous MSCs into rapidly and slowly dividing groups. Fluorescence-activated cell sorting revealed that rapidly dividing MSCs display lower scatter and 50% higher NG2 surface expression than slowly dividing cells, but CD146 expression is comparable in both groups. Heterogeneous MSCs were sorted based on scatter properties and surface expression of NG2 and CD146 into high (HI) and low (LO) groups. Sc(LO)NG2(HI) and Sc(LO)NG2(HI)CD146(HI) MSCs have the highest proliferative potential of the sorted groups, with colony-forming efficiencies that are 1.5-2.2 times the value for the parental controls. The Sc(LO) gate enriches for rapidly dividing cells. Addition of the NG2(HI) gate increases cell survival to 1.5 times the parental control. Further addition of the CD146(HI) gate does not significantly improve cell division or survival. The combination of low scatter and high NG2 surface expression is a promising selection criterion to enrich a proliferative phenotype from heterogeneous MSCs during ex vivo expansion, with potentially numerous applications.

Metallo-phthalocyanine near-IR fluorophores: oligonucleotide conjugates and their applications in PCR assays.

Water soluble, metallo-pthalocyanine (MPc) near-IR fluorophores were designed, synthesized, and evaluated as highly stable and sensitive reporters for fluorescence assays. Their conjugation to oligonucleotides was achieved via succinimidyl ester-amino coupling chemistry with the conditions for conjugation extensively examined and optimized. In addition, various conjugate purification and isolation techniques were evaluated as well. Results showed that under proper conditions and following purification using reverse-phase ion-pair chromatography, labeling efficiencies near 80% could be achieved using ZnPc (Zn phthalocyanine) as the labeling fluorophore. Absorption and fluorescence spectra accumulated for the conjugates indicated that the intrinsic fluorescence properties of the MPc's were not significantly altered by covalent attachment to oligonucleotides. As an example of the utility of MPc reporters, we used the MPc-oligonucleotide conjugates as primers for PCR (polymerase chain reaction) amplifications with the products sorted via electrophoresis and detected using near-IR fluorescence (lambda ex = 680 nm). The MPc dyes were found to be more chemically stable under typical thermal cycling conditions used for PCR compared to the carbocyanine-based near-IR reporter systems typically used and produced single and narrow bands in the electrophoretic traces, indicative of producing a single PCR product during amplification.