The outstanding migration and differentiation capacities of neural crest cells (NCCs) have fascinated scientists since Wilhelm His described this cell population in 1868. Today, after intense research using vertebrate model organisms, we have gained considerable knowledge regarding the origin, migration and differentiation of NCCs. However, our understanding of NCC development in human embryos remains largely uncharacterized, despite the role the neural crest plays in several human pathologies. Here, we report for the first time the expression of a battery of molecular markers before, during, or following NCC migration in human embryos from Carnegie Stages (CS) 12 to 18. Our work demonstrates the expression of Sox9, Sox10 and Pax3 transcription factors in premigratory NCCs, while actively migrating NCCs display the additional transcription factors Pax7 and AP-2α. Importantly, while HNK-1 labels few migrating NCCs, p75NTR labels a large proportion of this population. However, the broad expression of p75NTR – and other markers - beyond the neural crest stresses the need for the identification of additional markers to improve our capacity to investigate human NCC development, and to enable the generation of better diagnostic and therapeutic tools.

The use of fetal hematopoietic stem cells for in utero transplantation to create permanent hematochimerism represents a new concept in fetal therapy. In one fetus with alpha-thalassemia, one with sickle cell anemia, and one with beta-thalassemia, we have transplanted fetal liver cells obtained from legal abortions in gestational weeks 6-11. The fetus with alpha-thalassemia was transplanted twice during pregnancy, in the 15th (20.4 x 10(8) cells/kg) and in the 31st weeks of gestation (1.2 x 10(8) cells/kg), and is now two years of age. One fetus with sickle cell anemia received its transplant in the 13th week of gestation (16.7 x 10(8) cells/kg), and is now one year old. The fetus with beta-thalassemia was transplanted in 18th week (8.6 x 10(8) cells/kg), and is now three months old. Engraftment was evaluated by chromosomal analysis (sex chromosomes), red cell phenotyping, HLA class I and II typing, and PCR (polymerase chain reaction) for Y chromosome-specific sequences and DNA polymorphisms in cord and peripheral blood. The children with alpha- and beta-thalassemia underwent bone marrow aspirations at 3 and 7 months of age, respectively. In neither of these cases were we able to detect convincing evidence of stem cell engraftment. Thus, the administration of fetal stem cells to fetal recipients after the 12th week of gestation did not result in permanent hematochimerism. It remains to be determined whether the engraftment process can be promoted by earlier transplantations and/or higher cell doses.

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In vitro expanded neural precursor cells (NPCs) may provide a stable source for cell therapy. In search of the optimal cell source for spinal cord repair, we investigated influences of gestational age, regional heterogeneity, and long-term in vitro propagation. The cellular content of neurosphere cultures prior to and after in vitro differentiation was studied by immunocytochemistry and flow cytometry. Human forebrain and spinal cord NPCs deriving from first-trimester tissue were cultured as neurospheres in the presence of epidermal growth factor, basic fibroblast growth factor, and ciliary neurotrophic factor. Proteins characteristic for embryonic stem cells, i.e., Tra-1-60, Tra-1-81, and SSEA-4, were present in approximately 0.5% of the cells in donor tissues and neurospheres. The proportions of nestin- and proliferating cell nuclear antigen-immunoreactive (IR) cells were also maintained, whereas the CD133-IR population increased in vitro. Glial fibrillary acidic protein-IR cells increased in number, and in contrast the fraction of beta-tubulin III-IR cells decreased, at and beyond passage 5 in spinal cord but not forebrain cultures. However, dissociated and in vitro-differentiated forebrain- and spinal cord-derived neurospheres generated similar proportions of neurons, astrocytes, and oligodendrocytes. Gestational age of the donor tissue, which ranged from 4.5 to 12 weeks for forebrain and from 4.5 to 9.5 weeks for spinal cord, did not affect the proportion of cells with different phenotypes in culture. Thus, cellular composition of human neurosphere cultures differs as a result of long-term in vitro propagation and regional heterogeneity of source tissue, despite expansion under equal culture conditions. This could in turn imply that human spinal cord and forebrain NPCs present different repair potentials in in vivo settings.