Understanding gene expression profiles during early human pancreas development is limited by comparison to studies in rodents. In this study, from the inception of pancreatic formation, embryonic pancreatic epithelial cells, approximately half of which were proliferative, expressed nuclear PDX1 and cytoplasmic CK19. Later, in the fetal pancreas, insulin was the most abundant hormone detected during the first trimester in largely nonproliferative cells. At sequential stages of early fetal development, as the number of insulin-positive cell clusters increased, the detection of CK19 in these cells diminished. PDX1 remained expressed in fetal beta cells. Vascular structures were present within the loose stroma surrounding pancreatic epithelial cells during embryogenesis. At 10 weeks post-conception (w.p.c.), all clusters containing more than ten insulin-positive cells had developed an intimate relationship with these vessels, compared with the remainder of the developing pancreas. At 12-13 w.p.c., human fetal islets, penetrated by vasculature, contained cells independently immunoreactive for insulin, glucagon, somatostatin and pancreatic polypeptide (PP), coincident with the expression of maturity markers prohormone convertase 1/3 (PC1/3), islet amyloid polypeptide, Chromogranin A and, more weakly, GLUT2. These data support the function of fetal beta cells as true endocrine cells by the end of the first trimester of human pregnancy.

Based on evidence suggesting similarities to human embryonic stem cells, human embryonic germ (hEG) cells have been advocated as an alternative pluripotent stem cell resource but have so far received limited attention. To redress this imbalance, human fetal gonads were collected for the isolation and culture of primordial germ cells at 7-9 weeks postconception. We provide evidence for the derivation, culture, and differentiation of hEG cells in vitro. This evidence includes the expression of markers characteristic of pluripotent cells, the retention of normal XX or XY karyotypes, and the demonstration of pluripotency, as suggested by the expression of markers indicative of differentiation along the three germ lineages (ectoderm, mesoderm, and endoderm) and an associated loss of pluripotent markers. In assessing this differentiation, however, we also demonstrate a hitherto unacknowledged overlap in gene expression profiles between undifferentiated and differentiated cell types, highlighting the difficulty in ascribing cell lineage by gene expression analyses. Furthermore, we draw attention to the problems inherent in the management of these cells in prolonged culture, chiefly the difficulty in preventing spontaneous differentiation, which hinders the isolation of pure, undifferentiated clonal lines. While these data advocate the pursuit of pluripotent hEG cell studies with relevance to early human embryonic development, culture limitations carry implications for their potential applicability to ambitious cell replacement therapies.

NANOG, POU5F1, and SOX2 are required by the inner cell mass of the blastocyst and act cooperatively to maintain pluripotency in both mouse and human embryonic stem cells. Inadequacy of any one of them causes loss of the undifferentiated state. Mouse primordial germ cells (PGCs), from which pluripotent embryonic germ cells (EGCs) are derived, also express POU5F1, NANOG, and SOX2. Thus, a similar expression profile has been predicted for human PGCs. Here we show by RT-PCR, immunoblotting, and immunohistochemistry that human PGCs express POU5F1 and NANOG but not SOX2, with no evidence of redundancy within the group B family of human SOX genes. Although lacking SOX2, proliferative human germ cells can still be identified in situ during early development and are capable of culture in vitro. Surprisingly, with the exception of FGF4, many stem cell-restricted SOX2 target genes remained detected within the human SOX2-negative germ cell lineage. These studies demonstrate an unexpected difference in gene expression between human and mouse. The human PGC is the first primary cell type described to express POU5F1 and NANOG but not SOX2. The data also provide a new reference point for studies attempting to turn human stem cells into gametes by normal developmental pathways for the treatment of infertility.

Visceral left-right asymmetry occurs in all vertebrates, but the inversion of embryo turning (inv) mouse, which resulted following a random transgene insertion, is the only model in which these asymmetries are consistently reversed. We report positional cloning of the gene underlying this recessive phenotype. Although transgene insertion was accompanied by neighbouring deletion and duplication events, our YAC phenotype rescue studies indicate that the mutant phenotype results from the deletion. After extensively characterizing the 47-kb deleted region and flanking sequences from the wild-type mouse genome, we found evidence for only one gene sequence in the deleted region. We determined the full-length 5.5-kb cDNA sequence and identified 16 exons, of which exons 3-11 were eliminated by the deletion, causing a frameshift. The novel gene specifies a 1062-aa product with tandem ankyrin-like repeat sequences. Characterization of complementing and non-complementing YAC transgenic families revealed that correction of the inv mutant phenotype was concordant with integration and intact expression of this novel gene, which we have named inversin (Invs).