Sergey L. Kiselev

Induction of pluripotency in human endothelial cells resets epigenetic profile on genome scale.

Reprogramming of a limited number of human cell types has been achieved through ectopic expression of four transcription factors to yield induced pluripotent stem (iPS) cells that closely resemble human embryonic stem cells (ESCs). Here, we determined functional and epigenetic properties of iPS cells generated from human umbilical vein endothelial cells (HUVEC) by conventional method of direct reprogramming. Retroviral overexpression of four transcription factors resets HUVEC to the pluripotency. Human endothelial cell-derived iPS (endo-iPS) cells were similar to human ESCs in morphology, gene expression, in vitro and in vivo differentiation capacity. Endo-iPS cells were efficiently differentiated in vitro into endothelial cells. Using genome-wide methylation profiling we show that promoter elements of endothelial specific genes were methylated following reprogramming whereas pluripotency-related gene promoters were hypomethylated similar to levels observed in ESCs. Genome-wide methylation analysis of CpG sites located in the functional regions of over than 14,000 genes indicated that human endo-iPS cells were highly similar to human ES cells, although differences in methylation levels of 46 genes were found. Overall CpG methylation of promoter regions in the pluripotent cells was higher than in somatic. We also show that during reprogramming female human endo-iPS cells exhibited reactivation of the somatically silenced X chromosome. Our findings demonstrate that iPS cells can be generated from human endothelial cells and reprogramming resets epigenetic status of endothelial cells to pluripotency.

Human umbilical cord blood cells transfected with VEGF and L1CAM do not differentiate into neurons but transform into vascular endothelial cells and secrete neuro-trophic factors to support neuro-genesis—a novel approach in stem cell therapy

Genetically modified mono-nuclear cell fraction from human umbilical cord blood (HUCB) expressing human vascular endothelial growth factor (VEGF) and mouse neural L(1) cell adhesion molecule (L(1)CAM) were used for gene-stem cell therapy of transgenic (G)93(A) mice adopted as an animal amyotrophic lateral sclerosis (ALS) model. We generated non-viral plasmid constructs, expressing human VEGF(165) (pcDNA-VEGF) and mouse neural L(1) cell adhesion molecule (pcDNA-mL(1)CAM). Mono-nuclear fraction of HUCB cells were transiently transfected by electro-poration with a mixture of expression plasmids (pcDNA-VEGF+pcDNA-mL(1)CAM). Sixteen transgenic female and male mice were randomly assigned to three groups: (1) transplantation of genetically modified HUCB cells expressing L(1) and VEGF (n=6), (2) transplantation of un-transfected HUCB cells (n=5), and (3) control group (n=5). In first two experimental groups 1x10(6) cells were injected retro-orbitally in pre-symptomatic 22-25-week-old (G)93(A) mice. Our results demonstrate that HUCB cells successfully grafted into nervous tissue of ALS mice and survived for over 3 months. Therefore, genetically modified HUCB cells migrate in the spinal cord parenchyma, proliferate, but instead of transforming into nerve cells, they differentiate into endothelial cells forming new blood vessels. We propose that: (A) expression of mouse neural L(1)CAM is responsible for increased homing and subsequent proliferation of transplanted cells at the site of neuro-degeneration, (B) expression of human VEGF directs HUCB cell differentiation into endothelial cells, and (C) neuro-protective effect may stem from the delivery of various neuro-trophic factors from newly formed blood vessels.

Manifestation of Huntington's disease pathology in human induced pluripotent stem cell-derived neurons

BackgroundHuntington’s disease (HD) is an incurable hereditary neurodegenerative disorder, which manifests itself as a loss of GABAergic medium spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. There is no cure for HD, existing pharmaceutical can only relieve its symptoms.ResultsHere, induced pluripotent stem cells were established from patients with low CAG repeat expansion in the huntingtin gene, and were then efficiently differentiated into GABA MS-like neurons (GMSLNs) under defined culture conditions. The generated HD GMSLNs recapitulated disease pathology in vitro, as evidenced by mutant huntingtin protein aggregation, increased number of lysosomes/autophagosomes, nuclear indentations, and enhanced neuronal death during cell aging. Moreover, store-operated channel (SOC) currents were detected in the differentiated neurons, and enhanced calcium entry was reproducibly demonstrated in all HD GMSLNs genotypes. Additionally, the quinazoline derivative, EVP4593, reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted neuroprotective effects during cell aging.ConclusionsOur data is the first to demonstrate the direct link of nuclear morphology and SOC calcium deregulation to mutant huntingtin protein expression in iPSCs-derived neurons with disease-mimetic hallmarks, providing a valuable tool for identification of candidate anti-HD drugs. Our experiments demonstrated that EVP4593 may be a promising anti-HD drug.

Cerd4, third member of the d4 gene family: expression and organization of genomic locus

Abstract. Two members of the d4 family of presumptive transcription modulators, neuro-d4 (Neud4) and ubi-d4/Requiem (Req), have been characterized previously. We cloned and characterized the third member of this gene family, cer-d4 (Cerd4), from chicken and mouse cDNA libraries. The expression patterns of Cerd4 gene in both species are similar and more restricted than expression patterns of other two d4 genes. The main sites of Cerd4 expression are retina and cerebellum, where multiple transcripts could be detected. Two major types of Cerd4 proteins are a full-length isoform possessing all domains characteristic to the d4 family and truncated XZ isoform without C-terminal tandem of PHD fingers. The developmental kinetics of expression of these isoforms is different. The intron/exon structure of human Cerd4 gene is similar to that of neuro-d4 and ubi-d4/Requiem genes, but most introns of Cerd4 gene are much larger than the corresponding introns of the other two genes.

The Rab5 effector Rabaptin‐5 and its isoform Rabaptin‐5δ differ in their ability to interact with the small GTPase Rab4

Rabaptin‐5 is an effector for the small GTPase Rab5, a regulator of the early steps in endocytosis. In addition, Rabaptin‐5 interacts with the small GTPase Rab4 that has been implicated in recycling from early endosomes to the cell surface. Recently we have identified a ubiquitous transcript encoding the Rabaptin‐5 isoform, Rabaptin‐5δ. To evaluate the interaction properties of Rabaptin‐5δ with the small GTPases Rab4 and Rab5, we have applied protein interaction assays using the yeast two‐hybrid system and a glutathione S‐transferase pull‐down assay. We found that unlike Rabaptin‐5, that interacts with both GTPases in GTP‐bound conformations, Rabaptin‐5δ interacts only with GTP‐bound Rab5, and does not interact with Rab4, presumably due to a disrupted Rab4 binding site. Immunofluorescence microscopy analysis carried out to address the localization of Rabaptin‐5δ relative to GTP‐bound Rab4 and Rab5 in BHK‐21 cells supported these data. Our data suggests that while Rabaptin‐5 was proposed to act as a molecular linker between Rab5 and Rab4, to coordinate endocytic and recycling traffic, Rabaptin‐5δ is involved only in the Rab5‐driven events.

Resistance to tumor necrosis factor induced apoptosis in vitro correlates with high metastatic capacity of cells in vivo.

TNF is one of the cytokines secreted by the cells of the immune system. Our data demonstrate that those cell lines lacking capability to form metastatic tumors in vivo are susceptible to TNF induced apoptosis in vitro. However, cell lines with high metastatic potential are resistant to TNF in vitro. Furthermore, the same cell lines were resistant to cytolytic action of other cytotoxic proteins secreted by LAK cells. Our data showed that TNF resistance in vitro correlates with the increased level of transcription factor NF-kappaB. This finding may provide a tool to improve current protocols of immunotherapy and insights to how tumor cells are or are not killed by LAK cells.

Current Progress and Potential Practical Application for Human Pluripotent Stem Cells

Pluripotent stem cells are able to give rise to all cell types of the organism. There are two sources for human pluripotent stem cells: embryonic stem cells (ESCs) derived from surplus blastocysts created for in vitro fertilization and induced pluripotent stem cells (iPSCs) generated by reprogramming of somatic cells. ESCs have been an area of intense research during the past decade, and two clinical trials have been recently approved. iPSCs were created only recently, and most of the research has been focused on the iPSC generation protocols and investigation of mechanisms of direct reprogramming. The iPSC technology makes possible to derive pluripotent stem cells from any patient. However, there are a number of hurdles to be overcome before iPSCs will find a niche in practice. In this review, we discuss differences and similarities of the two pluripotent cell types and assess prospects for application of these cells in biomedicine.

Apoptotic cleavage of rabaptin-5-like proteins and a model for rabaptin-5 inactivation in apoptosis.

Intracellular membrane transport from the plasma membrane is one of the processes affected in apoptotic cells. Apoptotic inhibition of endosomal transport occurs due to cleavage of Rabaptin-5, an effector of small GTPase Rab5, which results in inhibition of early endosome fusion. Recently several novel Rabaptin-5-like proteins were identified. We investigated whether Rabaptin-5-like proteins, Rabaptin-5? and Rabaptin-5?, are also cleaved in apoptosis and found that both proteins are cleaved in apoptotic cell extracts by caspase-3-related proteases. This suggests that functional inactivation of these proteins is necessary for apoptotic cell death. We also mapped a novel, N-terminal, putative Rab5 binding site in Rabaptin-5-like proteins, which becomes physically separated from the previously known C-terminal Rab5 binding site after apoptotic cleavage of these proteins. Presence of the second Rab5 binding site provides a new insight into Rabaptin-5 function in early endosome fusion and a mechanistic model for functional inactivation of Rabaptin-5 in apoptosis.

Subcellular localization of MAK-V/Hunk protein kinase expressed in COS-1 cells

MAK‐V/Hunk is a MARK/Par‐1‐related protein kinase, whose function is unknown. We studied the subcellular localization of MAK‐V/Hunk in COS‐1 cells by immunofluorescence. It has a nucleocytoplasmic distribution and is localized to the centrosome, as indicated by co‐localization with γ‐tubulin. A putative kinase‐deficient mutant, with a mutation in the invariant lysine residue in the catalytic domain, was not targeted to the nucleus or centrosome. These results suggest that the nuclear and centrosomal targeting of MAK‐V/Hunk is specific, and is likely to be coupled to its catalytic activity.

Reactivation of Х chromosome upon reprogramming leads to changes in the replication pattern and 5hmC accumulation

Once set, the inactive status of the X chromosome in female somatic cells is preserved throughout subsequent cell divisions. The inactive status of the X chromosome is characterized by many features, including late replication. In contrast to induced pluripotent stem cells (iPSCs) in mice, the X chromosome in human female iPSCs usually remains inactive after reprogramming of somatic cells to the pluripotent state, although recent studies point to the possibility of reactivation of the X chromosome. Here, we demonstrated that, during reprogramming, the inactive X chromosome switches from late to synchronous replication, with restoration of the transcription of previously silenced genes. This process is accompanied by accumulation of a new epigenetic mark or intermediate of the DNA demethylation pathway, 5-hydroxymethylcytosine (5hmC), on the activated X chromosome. Our results indicate that the active status of the X chromosome is better confirmed by early replication and the reappearance of 5hmC, rather than by appearance of histone marks of active chromatin, removal of histone marks of inactive chromatin, or an absence of XIST coating.