Human trophectoderm apposition is regulated by interferon γ-induced protein 10 (IP-10) during early implantation.

INTRODUCTION: The first step in human implantation is the attraction of the blastocyst to the endometrium. We aimed to study attraction of the human blastocyst to the endometrium, and how this process is accomplished by chemokines secreted by the endometrium. MATERIALS AND METHODS: Blastocyst trophectoderm cells and other trophoblast lineage cells were subjected to attraction assays by IP-10 and other chemokines using transwell migration and chemotaxis assays. Chemokine expression and secretion were investigated using immunohistochemistry, ELISA, FACS analysis, and RT-PCR on material from flushing of the uterine cavity in endometrial biopsies. Chemokine receptor expression by blastocyst trophectoderm following PGD biopsy, trophectoderm derived from hES, placental villi, and other trophoblast lineage cells were characterized by the same methods. RESULTS: IP-10 dramatically attracted trophectoderm derived from hES cells and other lineages by interaction with CXCR3 chemokine receptors, as shown by both chemotaxis and transwell migration. High levels of IP-10 were detected throughout the menstrual cycle at flushing of the uterine cavity. Immunohistochemistry, FACS analysis, and RT-PCR of endometrial biopsy detected IP-10 in glandular and stromal cells of the endometrium. High levels of IP-10 were detected in condition medium of the endometrial stromal and glandular cells. Of all of the chemokine/chemokine receptor combinations examined, the IP-10/CXCR3 interaction was the only cytokine that was significantly elevated. DISCUSSION: While they await the wandering blastocyst, IP-10 is produced by many cells of the endometrium, but not by endometrial natural killer cells. CONCLUSION: Endometrial IP-10 may specifically attract human blastocyst trophectoderm cells early in implantation.

Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.

BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.

Neural progenitors from human embryonic stem cells.

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.

Stomatocytosis is absent in "stomatin"-deficient murine red blood cells.

To examine the relationship between erythrocyte membrane protein 7. 2b deficiency and the hemolytic anemia of human hereditary stomatocytosis, we created 7.2b knock-out mice by standard gene targeting approaches. Immunoblots showed that homozygous knock-out mice completely lacked erythrocyte protein 7.2b. Despite the absence of protein 7.2b, there was no hemolytic anemia and mouse red blood cells (RBCs) were normal in morphology, cell indices, hydration status, monovalent cation content, and ability to translocate lipids. The absence of the phenotype of hereditary stomatocytosis implies that protein 7.2b deficiency plays no direct role in the etiology of this disorder and casts doubt on the previously proposed role of this protein as a mediator of cation transport in RBC.

Genomic organization and 5'-flanking DNA sequence of the murine stomatin gene (Epb72).

Stomatin is a poorly understood integral membrane protein that is absent from the erythrocyte membranes of many patients with hereditary stomatocytosis. This report describes the cloning of the murine stomatin chromosomal gene, determination of its genomic structure, and characterization of the 5'-flanking genomic DNA sequences. The stomatin gene is encoded by seven exons spread over approximately 25 kb of genomic DNA. There is no concordance between the exon structure of the stomatin gene and the locations of three domains predicted on the basis of protein structure. Inspection of the 5'-flanking DNA sequences reveals features of a TATA-less housekeeping gene promoter and consensus sequences for a number of potential DNA-binding proteins.

Identification of the hereditary pyropoikilocytosis carrier state.

We evaluated the hematologic, rheologic, and biochemical features of erythrocytes obtained from 10 relatives of a 5-yr-old black female with hereditary pyropoikilocytosis (HPP) and severe hemolytic anemia. Erythrocyte morphology was normal in the father and five other relatives, but ghost mechanical fragility and drug-induced red cell endocytosis were increased, as was the percentage of spectrin dimers noted on 3.2% nondenaturing PAGE of spectrin extracts. Identical changes were also noted in the mother and her sister, whose erythrocytes were elliptocytic and exhibited morphological changes upon heating to 45 degrees-48 degrees C (normal 49 degrees). The two other family members were normal in every respect. SDS-PAGE analysis of membrane proteins demonstrated diminished amounts of spectrin in HPP erythrocytes, but was normal in other family members. A diffuse band (mol wt 575,000-665,000), composed entirely of spectrin, was apparent adjacent to the dimer region on nondenaturing PAGE of spectrin extracts from the propositus, mother, and aunt. In this family, HPP appears to have resulted from compound heterozygosity for two distinct genetic abnormalities (reflected by the differences between elliptocytic and nonelliptocytic carriers). Although the membrane abnormalities in carriers did not result in hemolytic anemia, they were of sufficient magnitude to allow the detection of the carrier state.