The contribution of the POU domain, class 5, transcription factor-1 (POU5F1) in maintaining totipotency in human embryonic stem cells (hESCs) has been repeatedly proven. In humans, two isoforms are encoded: POU5F1_iA and POU5F1_iB. So far, no discrimination has been made between the isoforms in POU5F1 studies, and it is unknown which isoform contributes to the undifferentiated phenotype. Using immunocytochemistry, expression of POU5F1_iA and POU5F1_iB was examined in hESCs and all stages of human preimplantation development to look for differences in expression, biological activity, and relation to totipotency. POU5F1_iA and POU5F1_iB displayed different temporal and spatial expression patterns. During human preimplantation development, a significant POU5F1_iA expression was seen in all nuclei of compacted embryos and blastocysts and a clear POU5F1_iB expression was detected from the four-cell stage onwards in the cytoplasm of all cells. The cytoplasmic localization might imply no or other biological functions beyond transcription activation for POU5F1_iB. The stemness properties of POU5F1 can be assigned to POU5F1_iA because hESCs expressed POU5F1_iA but not POU5F1_iB. However, POU5F1_iA is not the appropriate marker to identify totipotent cells, because POU5F1_iA was also expressed in the nontotipotent trophectoderm and was not expressed in zygotes and early cleavage stage embryos, which are assumed to be totipotent. The expression pattern of POU5F1_iA may suggest that POU5F1_iA alone cannot sustain totipotency and that coexpression with other stemness factors might be the key to totipotency.

POU5F1 isoforms show different expression patterns in human embryonic stem cells and preimplantation embryos.

Juvenile myelomonocytic leukemia (JMML) is a malignant hematopoietic disorder of early childhood with excessive proliferation of the myeloid and monocytic lineage. Deregulation of the RAS signal transduction pathway is thought to play a key role in its pathogenesis. We examined peripheral blood or bone marrow cells of 36 children with JMML for activating point mutations in codons 12, 13 and 61 of the NRAS and KRAS proto-oncogenes by allele-specific restriction assay, single-strand conformation polymorphism and/or direct sequencing. Codons 12, 13 and 61 of HRAS were examined in 26 of these patients. We detected RAS mutations in six cases (17%) located at N12 (n = 2), N13 (n = 3) and K13 (n = 1). In addition, we performed clonality studies on different cell lineages in four of these patients applying the RAS mutation, the karyotype and X-chromosome inactivation patterns as clonal markers. Erythroid cells carried mutant RAS, indicating clonal origin. In EBV B cell lines, one of three patients studied harbored a RAS mutation, while the other two patients had polyclonal B cells with wild-type RAS. T lymphocytes were examined in one patient; they were polyclonal and had wild-type RAS. It is likely that JMML is a heterogeneous disease with respect to clonal involvement of different lineages.

RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML)

The transcription factor OCT-4 is regarded as a critical factor in controlling mammalian early embryonic development because of its role in toti-/pluripotency. In human preimplantation embryos, OCT-4 studies are limited to RNA analysis of abnormally developing embryos. This study thoroughly investigated the expression pattern of OCT-4 throughout the human preimplantation development. Expression was examined by single-cell RT-PCR or indirect immunocytochemistry in 36 single oocytes of various maturity and 112 normally developing preimplantation embryos at the level of single blastomeres, morulas, blastocysts, or inner cell mass (ICM) and trophectoderm (TE) samples. Oocytes and cleavage stage embryos revealed a variable OCT-4 expression pattern, concomitant with a pure cytoplasmic localization of the protein. During compaction, the variability in expression faded away indicating embryonic OCT-4 expression and the protein appeared in the nucleus implying biological activity. In blastocysts, OCT-4 transcripts and proteins were present in the ICM and the TE. At protein level, blastocysts displayed different spatial expression patterns within a cell for the splice variants of OCT-4, which may endow them with different functional properties. As OCT-4 transcripts were also found in various differentiated cells, the presence of OCT-4 transcripts or proteins may not be sufficient for identifying undifferentiated cell lines in humans. Further, we suggest to examine the localization of OCT-4 proteins within a cell rather than to look for the presence and/or amount of transcripts.

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Oct-4 mRNA and protein expression during human preimplantation development.

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.

Optimization and evaluation of single-cell whole-genome multiple displacement amplification.

Based on the capacity to transfer α-L-fucose onto type-1 and type-2 synthetic blood group H and sialylated acceptors, a comparison of the α-3-fucosyltransferase activities of different human tissues is shown. Three distinct acceptor specificity patterns are described: (I) myeloid α-3-fucosyltransferase pattern, in which leukocytes and brain enzymes transfer fucose actively onto H type-2 acceptor and poorly onto sialylated N-acetyllactosamine; (II) plasma α-3-fucosyltransferase (EC 2.4.1.152), in which plasma and hepatocyte enzymes transfer, in addition, onto the sialylated N-acetyllactosamine; (III) Lewis α-3/4-fucosyltransferase (EC 2.4.1.65), in which gall-bladder, kidney and milk enzymes transfer, in addition, onto type-1 acceptors. The small amount (<10%) of α-3-fucosyltransferase activity found in the plasma of an α-3-fucosyltransferase-deficient individual had a myeloid-type acceptor pattern, suggesting that this small proportion of the plasma enzyme is derived from leukocytes. In addition to the three acceptor specificity patterns, these enzyme activities can be differentiated by their optimum pH: 8.0–8.7 for the enzymes from myeloid cells and brain, 7.2–8.0 for liver enzymes and 6.0–7.2 for gallbladder enzymes. Milk samples had two α-3-fucosyltransferase activities, the Lewis or α-3/4-fucosyltransferase under control of the Lewis gene and an α-3-fucosyltransferase with plasma acceptor pattern which was independent of the control of the Lewis gene. The apparent affinity for GDP-fucose of the myeloid-like enzyme was weaker than those of the plasma and Lewis-like enzymes. The apparent affinities for H type 2 and sialylated N-acetyllactosamine were stronger for exocrine secretions as compared to the plasma and myeloid enzymes. The plasma type of α-3-fucosyltransferase activity was more sensitive to N-ethylmaleimide and heat inactivation than the samples with myeloid-like α-3-fucosyltransferase activity.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1990.tb19107.x

Acceptor specificity and tissue distribution of three human α-3-fucosyltransferases

A simple method for the preparation of lymphoblastoid cell lines from small amounts (100 microliter) of frozen whole blood is described A success score greater than 90% was obtained for EBV transformations using blood samples which had been collected several months before the infection Due to the simplicity of the technique, up to 80 samples could be processed per day This technique was used to prepared 242 permanent cell lines from 13 large families from Reunion Island showing blood group H deficiency These cell lines are now available for genetic studies

J. Le Pendu

Papers

Use of a Simple Method for the Epstein-Barr Virus Transformation of Lymphocytes from Members of Large Families of Réunion Island