Simultaneous Detection of FISH Signals and Bromo-Deoxyuridine Incorporation in Fixed Tissue Cultured Cells

FISH (Fluorescence in situ hybridization) is a powerful technique that detects and localises specific DNA sequences on metaphase chromosomes, interphase nuclei or chromatin fibres. When coupled to BrdU (5-Bromo 2-deoxy-uridine) labeling of newly replicated DNA, the replication properties of different DNA sequences can be analysed. However, the technique for the detection of BrdU incorporation is time consuming, and relies on acidic pH buffer treatments, that prevent use of pH sensitive fluorochromes such as FITC (Fluoro-isothiocianate) during FISH. In this work, we describe a simplified protocol that allows the simultaneous detection of FISH signals and BrdU incorporation. Since the technique does not involve paraformaldehyde for cell fixation, or formamide for denaturation of the target DNA and in post-hybridisation washes, it represents a safer alternative to classical FISH techniques

An Improved Technique for Chromosomal Analysis of Human ES and iPS Cells

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this, time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive ‘passaging’. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis, including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique, in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work, and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality, fast chromosomal analysis of human ES and iPS cells

Comparison of efficiency of simultaneous detection versus standard technique.

<p>Comparison of efficiency of simultaneous detection versus standard technique.</p

BrdU and FISH signal detection.

<p>a) Human AG6-1 chromosome metaphase spread. The probe, chromosome 17 alphoid DNA, identified the HAC (white arrow) and the endogenous chromosome 17. b) Replicating murine LAMF4-5 cell, containing the HAC derived from chromosome 17, probed with chromosome 17 alphoid DNA. In a) and b) the BrdU signal is in red, while the FISH signal is in green. The chromosomes are counterstained in DAPI, blue. The left panels show the merge of all three channels, while in the other panels the channels have been split, to show BrdU and DAPI, FISH and DAPI, and DAPI only, respectively. c) AG6-1 chromatin fibres. BrdU signal is blue, while the red and green signals correspond to different regions of the HAC DNA (chromosome 17 alphoid DNA in green, vector DNA in red). The top panel shows the merge of all three channels. The mid panel shows the BrdU signal only, while the bottom panel shows the FISH signal.</p

Noncoding Centromeric RNA Expression Impairs Chromosome Stability in Human and Murine Stem Cells

We analyzed the effect of transcribed noncoding RNA centromeric satellites on chromosome segregation in normal human and murine stem and fibrosarcoma cells. The overexpression of different centromeric alphoid DNAs in all cell lines induced a marked increase in chromosome mis-segregation in anaphase. Overexpression of centromeric mouse minor satellite also increased chromosome instability in the murine stem but not in human cells. Analysis of chromosome segregation in vivo showed disturbances in the mitotic progression, which was frequently unresolved. Live cell imaging revealed that overexpression of centromeric satellites resulted in several different chromosomal morphological errors in the cell nuclei. Our findings correlated with other reports that several centromeric noncoding RNAs are detected in different carcinoma cells and their expression resulted in segregation errors. Our study furnishes further insights into a novel source of genomic instability in human and murine cells. It has recently been shown that noncoding centromeric RNAs are present in some form of cancer, and thus, overexpression of several types of centromeric noncoding RNAs may be useful as a specific maker for neoplastic cells

An improved technique for chromosomal analysis of human ES and iPS cells. Stem Cell Rev 2011; 7:471–477

Abstract Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this, time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive &apos;passaging&apos;. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis, including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique, in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work, and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality, fast chromosomal analysis of human ES and iPS cells

Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells

We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications