Biomedical subjects

Alexey V Terskikh

Publications and source records attributed to Alexey V Terskikh.

9 recordsLinked to original sources

PubMed2006

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells.

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.

Animals↗

PubMed2006

Genetically encoded fluorescent indicator for intracellular hydrogen peroxide.

We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H(2)O(2) occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (DeltaPsi) and a change in cell shape. We also observed local bursts in mitochondrial H(2)O(2) production during DeltaPsi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H(2)O(2) increase upon physiological stimulation. Using HyPer we detected temporal increase in H(2)O(2) in the cytoplasm of PC-12 cells stimulated with nerve growth factor.

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PubMed2006

Mammalian stem cells.

Stem cells are quickly coming into focus of much biomedical research eventually aiming at the therapeutic applications for various disorders and trauma. It is important, however, to keep in mind the difference between the embryonic stem cells, somatic stem cells and somatic precursor cells when considering potential clinical applications. Here we provide the review of the current status of stem cell field and discuss the potential of therapeutic applications for blood and Immune system disorders, multiple sclerosis, hypoxic-ischemic brain injury and brain tumors. For the complimentary information about various stem cells and their properties we recommend consulting the National Institutes of Health stem cell resources (http://stemcells.nih.gov/info/basics).

Adult↗

PubMed2005

Simple and efficient isolation of hematopoietic stem cells from H2K-zFP transgenic mice.

We have generated a transgenic mouse line that allows for simple and highly efficient enrichment for mouse hematopoietic stem cells (HSCs). The transgene expresses a green fluorescent protein variant (zFP) under the control of H2Kb promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs express exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2K-zFP mice. Up to 90% of zFP(bright) cells are also c-kit(high), Sca-1(high), Lin(neg), Flk-2(neg), which is a bona fide phenotype for long-term HSCs. Double-sorted zFP(bright) HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2K-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs without using fluorescence-conjugated monoclonal antibodies. This approach will greatly facilitate gene transfer, including short interfering RNA for gene knockdown, into HSCs and, consequently, into all other hematopoietic lineages.

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PubMed2005

Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation.

Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.

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PubMed2005

Long-term persistence of a nonintegrated lentiviral vector in mouse hematopoietic stem cells.

OBJECTIVE: Lentiviral transduction is an established method for efficiently modifying the gene expression program of primary cells, but the ability of the introduced construct to persist as an episome has not been well studied. MATERIAL AND METHODS: Here we investigated this issue in lethally irradiated female mice injected with 300 or 3000 doubly sorted male lin(neg), Sca-1(high), c-kit(high), Thy-1.1(low) mouse bone marrow cells that had been exposed in vitro to self-inactivating lentivirus vector encoding a green fluorescence protein (GFP) cDNA. Seven to sixteen months later, bone marrow cells from primary mice were injected into secondary female recipients and another 8 months later into tertiary female recipients. Integration study was performed on individual spleen colonies by Southern blot analysis. Inverse polymerase chain reaction (PCR) and sequence of amplified vector-derived DNA was used to verify Southern blot results. RESULTS: Spleen colony-forming cell study revealed that a small fraction of the spleen colonies contained integrated provirus as shown by Southern blot analysis. Unexpectedly, many spleen colonies were found to contain a nonintegrated episomal form of the provirus, which was confirmed by an inverse PCR analysis. In some of the spleen colonies containing only the episomal form, GFP-expressing cells were also detected. Lentiviral sequences were present in hematopoietic tissues of primary mice but not in other tissues. CONCLUSIONS: These results demonstrate that lentiviral vectors produce episomal circles in hematopoietic stem cells that can be transferred through many cell generations and expressed in their progeny.

Animals↗

PubMed2003

Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations.

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.

Animals↗

PubMed2003

Gene expression analysis of purified hematopoietic stem cells and committed progenitors.

Lifelong self-renewal is a unique property of somatic stem cells. Recently, several primitive multipotent yet committed (non-self-renewing) hematopoietic progenitor populations were identified in mouse bone marrow. We have characterized the expression of 1200 selected mouse genes using the Atlas cDNA array in highly purified hematopoietic stem cells (HSCs) and 6 closely related progenitor populations: common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythrocyte progenitors (MEPs), common lymphoid progenitors (CLPs), and pro-T and pro-B cells. Cluster analysis revealed that nearly half of all differentially expressed transcripts are associated with HSCs, supporting the notion of an active transcriptional status of HSCs. Genes found enriched in the HSC cluster encompass many developmentally regulated genes, some previously associated with HSC self-renewal. In contrast, genes that are enriched in committed progenitors are mostly associated with hematopoietic differentiation, immune regulation, and metabolism. Thus, the transition from HSCs toward committed progenitors correlates with the down-regulation of a large number of HSC-associated genes and progressive up-regulation of a limited number of lineage-specific genes. These genetic analyses revealed both quantitative and qualitative differences between the transcripts associated with HSCs versus downstream progenitors and produced a list of the candidate genes, potentially involved in HSC self-renewal.

Animals↗

PubMed2001

Analysis of DsRed Mutants. Space around the fluorophore accelerates fluorescence development.

Earlier mutagenesis of the red fluorescent protein drFP583, also called DsRed, resulted in a mutant named Fluorescent Timer (Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao, X., Lukyanov, S., Matz, M., Kim, S., Weissman, I., and Siebert, P. (2000) Science 290, 1585--1588). Further mutagenesis generated variants with novel and improved fluorescent properties. The mutant called AG4 exhibits only green fluorescence. The mutant, called E5up (V105A), shows complete fluorophore maturation, eventually eliminating residual green fluorescence present in DsRed. Finally, the mutant, called E57 (V105A, I161T, S197A), matures faster than DsRed as demonstrated in vitro with purified protein and in vivo with recombinant protein expressed in Escherichia coli and Xenopus leavis. Comparative analysis of the mutants in the context of the crystal structure of DsRed suggests that mutants with free space around the fluorophore mature faster and more completely.

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