Search

Telomeres play an important role in the maintenance of genomic stability/integrity and are synthesized by the RNA-dependent polymerase telomerase. Progressive telomere shortening contributes to both in vitro and in vivo aging, and telomere length dynamics and telomerase expression profile in human tissues during extrauterine life have been well characterized. However, little is known about these changes in the early stage of gestation. In the present study, we determined telomere length and the expression of telomerase core units (telomerase reverse transcriptase, hTERT, and telomerase RNA component, hTERC) in human fetus tissues from 6 to 11 weeks of gestational age. A sharp decline in telomere length occurred between 6 and 7 weeks of gestational age, and a relatively stable or slightly shortened telomere length was thereafter maintained until birth. The inverse correlation between TERT or TERC expression and gestational age was steadily observed in these fetus tissues. Taken together, there is a rapid reduction followed by a slow erosion of telomere length in human fetus from gestational age 6-11 weeks, while hTERT and hTERC expression decreases steadily during this period. The present findings not only contribute to better understandings of telomere/telomerase biology in human embryonic development, but also are implicated in telomere/telomerase-related diseases or problems.

We report the cystathionine-β synthase (CBS) gene expression pattern during early human embryogenesis (3 to 6 weeks post conception) by in situ hybridization and in fetal and adult tissue by Northern Blot analysis. Probes were chosen to recognize either the common sequence to all known CBS mRNAs or the sequences of two different major exons 1 issued of we have previously identified. We demonstrate by in situ hybridization that CBS is continuously expressed from the earliest stages studied (22 days post conception) during embryogenesis in the tissues of developing embryos which will after birth present clinical abnormalities in homocystinuria patients. It is expressed at an especially high level in the neural and cardiac systems until the liver primordium appears. In embryonic central nervous system, the whole neural tube and primary brain vesicles are labeled. Secondary brain vesicles labeling are dependent on the neuroepithelium differentiation. The ventricular layer of the rhombencephalon, cranial nerve nuclei and then after cerebellar cortex derived from rhombencephalon ventricular layer are strongly labeled. Thalamus and other derivatives of the diencephalon plate, the neuroblastic layer of the retina, lens and dorsal root ganglia are labeled. After 35 days post conception, CBS mRNAs was detected in endocardial cells and in cells derived from the neural crest of the heart and in particular developing mesodermic regions such as the primitive hepatocytes of the liver, mesonephros vesicles, various endocrine glands and developing bones. We could not detect tissue specificity of different probes at this embryonic stage. Northern blot analysis consistently detected mRNA species in fetal 25 weeks post conception brain, liver and kidney. The common cDNA probe revealed the 2.5 and 3.7 kb mRNA species from brain, liver and kidney. The exon 1b probe detected only the 2.5 kb mRNA and the exon 1c probe the 3.7 kb mRNA in these three tissues. In adult tissue, the 1b probe detected only the 2.5 kb mRNA and the 1c probe only the 3.7 kb mRNA in the liver.

The amyloid-beta precursor protein (AbetaPP) is a ubiquitously expressed adhesion and neuritogenic protein whose processing has previously been shown to be regulated by reproductive hormones including the gonadotropin luteinizing hormone (LH) in human neuroblastoma cells. We report for the first time the expression of AbetaPP in human embryonic stem (hES) cells at the mRNA and protein levels. Using N- and C-terminal antibodies against AbetaPP, we detected both the mature and immature forms of AbetaPP as well as truncated variants ( approximately 53kDa, 47kDa, and 29kDa) by immunoblot analyses. Expression of AbetaPP is regulated by both the stemness of the cells and pregnancy-associated hormones. Addition of human chorionic gonadotropin, the fetal equivalent of LH that is dramatically elevated during pregnancy, markedly increased the expression of all AbetaPP forms. These results indicate a critical molecular signaling link between the hormonal environment of pregnancy and the expression of AbetaPP in hES cells that is suggestive of an important function for this protein during early human embryogenesis prior to the formation of neural precursor cells.

Growing and differentiating human stem cells in vitro can provide access to study the molecular mechanisms that control cellular development in a manner pertinent to human embryogenesis. To fully understand such processes, however, it is important to recreate culture conditions that most closely relate to those in living tissues. As step in this direction, we have developed a robust three-dimensional cell culture system using inert highly porous solid matrices manufactured from polystyrene that can be routinely used to study the differentiation of human pluripotent stem cell-derived neurons in vitro. Neurite outgrowth was significantly enhanced when neurons were grown in a three-dimensional environment compared to traditional flat surfaces and resulted in the formation of extensive neural networks. These data suggest that the topography within the culture environment can significantly alter cell development and will therefore be an important feature when investigating the potential of human stem cells.

The C2H2 zinc-finger motif found in many transcription factors is thought to be important for nucleic acid binding and/or dimerization. Here, we have identified and characterized a novel zinc-finger gene named ZNF323 using degenerate primers from an early human embryo heart cDNA library. The predicted protein contains six different C2H2 type zinc fingers and a SCAN box. ZNF323 maps to chromosome 6p22.1–22.3. The expression levels were different during different development stages of human embryo between 15 and 23 weeks. Northern blot analysis shows that a 3.2-kb transcript specific for ZNF323 was expressed at high levels in the lung, liver, and kidney, while weakly expressed in intestine, brain, muscle, cholecyst, heart, and pancreas. In adult tissues, ZNF323 is expressed at high levels in liver and kidney, weakly in lung, pancreas, brain, placenta, muscle, and heart. Taken together, these results indicate that ZNF323 is a member of the zinc-finger transcription factor family and may be involved in the development of multiple embryonic organs.

Human embryonal carcinoma cells could serve as a useful model system for analysis of early human development. A limited number of human embryonal carcinoma cell lines have been generated from in vivo tumors. We report here that α-difluoromethylornithine, a specific enzyme-activated inhibitor of ornithine decarboxylase activity, can induce differentiation in human embryonal carcinoma cells. The differentiated phenotype could be distinguished from undifferentiated cells by altered cellular morphology, biochemical and cell surface antigenic properties. These results suggest that alterations in the intracellular levels of polyamines may play a role in human embryonal carcinoma cell differentiation, and possibly human embryogenesis.