Inducing organogenesis in 3D culture is an important aspect of stem cell research. Anterior neural structures have been produced from large embryonic stem cell (ESC) aggregates, but the steps involved in patterning such complex structures have been ill defined, as embryoid bodies typically contained many cell types. Here we show that single mouse ESCs directly embedded in Matrigel or defined synthetic matrices under neural induction conditions can clonally form neuroepithelial cysts containing a single lumen in 3D. Untreated cysts were uniformly dorsal and could be ventralized to floor plate (FP). Retinoic acid posteriorized cysts to cervical levels and induced localize FP formation yielding full patterning along the dorsal/ventral (DV) axis. Correct spatial organization of motor neurons, interneurons, and dorsal interneurons along the DV axis was observed. This system serves as a valuable tool for studying morphogen action in 3D and as a source of patterned spinal cord tissue.

In addition to the chief function of erythropoietin (Epo) in promoting erythropoiesis, some other roles have been found in the brain and uterus. We have reported that signalling pathways of Epo and Epo receptor (EpoR) are involved in the tumourigenesis of ovarian and uterine cancers. To determine whether Epo plays a similar role in other malignancies, we studied the expression of Epo in several malignant human cell lines. We found that 24 malignant human cell lines examined express Epo and EpoR regardless of their origins, types, genetic characteristics and biological properties and secrete a very small amount of Epo individually and that most of them respond to hypoxic stimuli by enhanced secretion of Epo. To determine whether the Epo–EpoR pathway operates in tumours of these cell lines, we transplanted several cell lines into nude mice and confirmed the presence of Epo-responsive sites in xenografts in which the phosphorylation of the STAT5 (signal transducer and activator of transcription) is detectable. Furthermore, in nude mice we blocked the Epo signalling in xenografts of two representative cell lines, stomach choriocarcinoma and melanoma, by i.p. injections of EpoR antagonist and found inhibition of angiogenesis and survival of tumour cells leading to destruction of tumour masses and disturbances of phosphorylation of STAT5. In contrast, Epo mimetic peptide promotes angiogenesis and tumour cell survival. These findings suggest that Epo is indispensable for the growth and viability of malignant tumour and also that the deprivation of Epo signalling may be a promising therapy for human malignancy.

The salamander is the only tetrapod that functionally regenerates all cell types of the limb and spinal cord (SC) and thus represents an important regeneration model, but the lack of gene-knockout technology has limited molecular analysis. We compared transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) in the knockout of three loci in the axolotl and find that CRISPRs show highly penetrant knockout with less toxic effects compared to TALENs. Deletion of Sox2 in up to 100% of cells yielded viable F0 larvae with normal SC organization and ependymoglial cell marker expression such as GFAP and ZO-1. However, upon tail amputation, neural stem cell proliferation was inhibited, resulting in spinal-cord-specific regeneration failure. In contrast, the mesodermal blastema formed normally. Sox3 expression during development, but not regeneration, most likely allowed embryonic survival and the regeneration-specific phenotype. This analysis represents the first tissue-specific regeneration phenotype from the genomic deletion of a gene in the axolotl.

Understanding how the limb blastema is established after the initial wound healing response is an important aspect of regeneration research. Here we performed parallel expression profile time courses of healing lateral wounds versus amputated limbs in axolotl. This comparison between wound healing and regeneration allowed us to identify amputation-specific genes. By clustering the expression profiles of these samples, we could detect three distinguishable phases of gene expression – early wound healing followed by a transition-phase leading to establishment of the limb development program, which correspond to the three phases of limb regeneration that had been defined by morphological criteria. By focusing on the transition-phase, we identified 93 strictly amputation-associated genes many of which are implicated in oxidative-stress response, chromatin modification, epithelial development or limb development. We further classified the genes based on whether they were or were not significantly expressed in the developing limb bud. The specific localization of 53 selected candidates within the blastema was investigated by in situ hybridization. In summary, we identified a set of genes that are expressed specifically during regeneration and are therefore, likely candidates for the regulation of blastema formation.

Limb Regeneration Mirrors Development: Salamanders regenerate the right amount of limb after cutting anywhere along its length. A long-discussed explanation suggests that the regenerating tissue first sets the fingertips as a boundary and then regenerates everything in between. However, Roensch et al. (p. 1375 ) report that the limb regenerates in the opposite order. Similar to the processes followed during development, the regenerating salamander limb first establishes a field of cells with the identity of the cut site, and then cells progressively commit to alternate fates as they grow closer to the tip of the regenerated limb.