Christoph Hansis

Cell differentiation in the preimplantation human embryo.

This brief paper analyses current knowledge on gene expression in individual blastomeres of preimplantation mammalian embryos. Initially, current knowledge on axes and cleavage planes in mammalian eggs and embryo blastomeres is described, together with gene and system homologies with flies and nematodes, and their influence on differentiation. Stress is placed on the need to study individual blastomeres, and even specific components within blastomeres. Examples of published work concentrate on the possible allocation of a single founder blastomere for trophectoderm, which contains large amounts of maternal leptin, STAT3 and other proteins positioned at the animal pole. The recent discovery that single human blastomeres in cleaving embryos contain high levels of HCGbeta mRNA and LHbeta mRNA suggests these are also trophectoderm foundation cells. It is now essential to discover if the maternal proteins leptin/STAT3 and maternal/embryonic HCGbeta transcripts locate to the same blastomere. Problems in jointly identifying maternal proteins and embryonic and maternal transcripts for specific proteins within one cell, and the nature of early cell allocation in mouse and human embryo, are discussed.

Candidate lineage marker genes in human preimplantation embryos

Cell allocation and subsequent lineage commitment in the human embryo may be established as early as in the unfertilized oocyte. This phenomenon might be the result of subtle differences of gene expression and protein distribution. To assess whether gene expression profiling by reverse transcription-polymerase chain reaction could be a suitable tool for the detection of cell allocation and lineage commitment, the expression pattern of the putative inner cell mass marker gene Oct-4 and the trophectodermal marker genes beta-HCG and beta-LH were correlated in individual blastomeres of preimplantation human embryos. In 2- to 5-cell stage embryos, expression of beta-HCG and Oct-4 mRNA was negatively correlated in all blastomeres with statistical significance, suggesting that cell allocation can be assessed by those markers at early stages. In 7- to 10-cell stage embryos, expression of beta-LH and Oct-4 mRNA was negatively correlated in some blastomeres without statistical significance, suggesting that more experiments are necessary to decide if lineage commitment can be assessed in some cells by those markers at later stages.

Assessment of β-HCG, β-LH mRNA and ploidy in individual human blastomeres

In human embryos, blastomeres differentiate into trophectoderm (TE) cells and inner cell mass (ICM) cells of blastocysts. Although morphologically indistinguishable, blastomeres at early cleavage stages are likely to undergo changes on a molecular level that make them destined to become ICM or TE cells. While the transcription factor Oct-4 might serve as a marker for totipotent ICM cells, human chorionic gonadotrophin might be used as the equivalent for TE cells. This study reports a reverse transcription-polymerase chain reaction procedure to assess human β-HCG mRNA concentrations as well as ploidy in individual blastomeres from normally and abnormally fertilized human embryos. β-HCG mRNA was detected in both euploid and aneuploid cells and in oocytes. Surprisingly, β-LH mRNA was also detected in some euploid blastomeres. In regard to preimplantation genetic diagnosis, assessment of expression levels of β-HCG and Oct-4 mRNA in individual biopsied cells might serve as a tool to identify embryogenic blastomeres in combination with testing for chromosome and single gene abnormalities.

Derivation of novel genetically diverse human embryonic stem cell lines.

Human embryonic stem cells (hESCs) have the potential to revolutionize many biomedical fields ranging from basic research to disease modeling, regenerative medicine, drug discovery, and toxicity testing. A multitude of hESC lines have been derived worldwide since the first 5 lines by Thomson et al. 13 years ago, but many of these are poorly characterized, unavailable, or do not represent desired traits, thus making them unsuitable for application purposes. In order to provide the scientific community with better options, we have derived 12 new hESC lines at New York University from discarded genetically normal and abnormal embryos using the latest techniques. We examined the genetic status of the NYUES lines in detail as well as their molecular and cellular features and DNA fingerprinting profile. Furthermore, we differentiated our hESCs into the tissues most affected by a specific condition or into clinically desired cell types. To our knowledge, a number of characteristics of our hESCs have not been previously reported, for example, mutation for alpha thalassemia X-linked mental retardation syndrome, linkage to conditions with a genetic component such as asthma or poor sperm morphology, and novel combinations of ethnic backgrounds. Importantly, all of our undifferentiated euploid female lines tested to date did not show X chromosome inactivation, believed to result in superior potency. We continue to derive new hESC lines and add them to the NIH registry and other registries. This should facilitate the use of our hESCs and lead to advancements for patient-benefitting applications.

Analysis of Oct-4 Expression and Ploidy in Individual Human Blastomeres

Oct-4, a decisive factor that maintains totipotency in murine embryonic and germ cells, is exclusively expressed in such cells. In mice, different levels of oct-4 expression in blastomeres predict development towards inner cell mass (ICM) (high oct-4) or trophectoderm (TE) (low oct-4). To address whether the mouse model also applies to human embryos, the cytoplasm of individual human blastomeres from normally and abnormally fertilized embryos was tested for Oct-4 expression by reverse transcription-polymerase chain reaction (RT-PCR). The nuclei of the same blastomeres were subjected to fluorescence in-situ hybridization (FISH) to determine ploidy. A significant difference in Oct-4 mRNA levels was revealed between blastomeres. The distribution of blastomeres with high Oct-4 levels varied according to the cleavage stage of the embryo: the more blastomeres, the lower the percentage with high Oct-4 levels. Aneuploid blastomeres did not exhibit lower Oct-4 mRNA levels than diploid ones. Thus, differential Oct-4 expression in individual human blastomeres appears to direct cells towards the ICM or TE lineages without regard to chromosomal status. Oct-4 might be used as a marker in preimplantation genetic diagnosis to identify embryogenic blastomeres.