PCDAmpl, a new antigen at the interface of the embryonic collecting duct epithelium and the nephrogenic mesenchyme

P CDAmpl, a new antigen at the interface of the embryonic collecting duct epithelium and the nephrogenic mesenchyme. In the neonatal rabbit kidney nephrogenesis is not yet terminated. The ampullar collecting duct epithelium acts as an inducer that generates the nephron anlagen, however, to date the morphogenic mechanisms involved are unknown. A presupposition for successful nephron induction is the close tissue interaction between the basal aspect of the ampullar collecting duct epithelium and the surrounding mesenchyme. To gain new insights in this area we raised monoclonal antibodies (mabs), to identify specific structures localized at the tissue interface. With the generated mab CDAmpl we found an intensive immunohistochemical reaction between the basal aspect of the ampullar collecting duct epithelium and the mesenchyme. The label was most concentrated at the ampullar tip and continuously decreased in the shaft region. In the maturing collecting duct of the neonatal kidney and in the adult renal collecting duct no immunohistochemical reaction was found. The binding pattern of mab CDAmpl is different from that of all known collecting duct cell markers and from antibodies against known basement membrane compounds such as laminin or collagen type IV. Under in vitro conditions immunoreactivity with mab CDAmpl was obtained using embryonic collecting duct epithelia and perfusion culture. The antigen was present in specimens treated with Iscove's modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum. Omittance of serum or hormonal treatment with aldosterone, insulin or vitamin D3 led to the disappearance of the newly detected antigen, while characteristics of the differentiated collecting duct cells were up-regulated. We conclude that the expression of P CDAmpl is a characteristic feature of the embryonic parts of the collecting duct epithelium. It may play a pivotal role during nephron induction

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells

Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation

Traditionally, human embryonic stem cells (hESCs) are propagated by mechanical dissection or enzymatic dissociation into clusters of cells. To facilitate up-scaling and the use of hESC in various experimental manipulations, such as fluorescence-activated cell sorting, electroporation, and clonal selection, it is important to develop new, stable culture systems based on single-cell enzymatic propagation. Here, we show that hESCs, which were derived and passaged by mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. As an indication of the stability of this culture system, we demonstrate that hESCs can be maintained in an undifferentiated, pluripotent, and genetically normal state for up to 40 enzymatic passages. We also demonstrate that a recombinant trypsin preparation increases clonal survival compared with porcine trypsin. Finally, we show that human foreskin fibroblast feeders are superior to the commonly used mouse embryonic fibroblast feeders in terms of their ability to prevent spontaneous differentiation after single-cell passaging. Importantly, the culture system is widely applicable and should therefore be of general use to facilitate reliable large-scale cultivation of hESCs, as well as their use in various experimental manipulations

Derivation of a xeno-free human ES cell line.

Elimination of all animal material during both the derivation and long-term culture of human embryonic stem cells (hESCs) is necessary prior to future application of hESCs in clinical cell therapy. The potential consequences of transplanting xeno-contaminated hESCs into patients, such as an increased risk of graft rejection [STEM CELLS 2006;24:221229] and the potential transfer of nonhuman pathogens, make existing hESC lines unsuitable for clinical applications. To avoid xeno-contamination during derivation and culture of hESCs, we first developed a xeno-free medium supplemented with human serum, which supports long-term (> 50 passages) culture of hESCs in an undifferentiated state. To enable derivation of new xeno-free hESCs, we also established xeno-free human foreskin fibroblast feeders and replaced immunosurgery, which involves the use of guinea pig complement, with a modified animal-product-free derivation procedure. Here, we report the establishment and characterization (> 20 passages) of a xeno-free pluripotent diploid normal hESC line, SA611

The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking

This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means

Repair of Long Nerve Defects with a New Decellularized Nerve Graft in Rats and in Sheep

Decellularized nerve allografts (DC) are an alternative to autografts (AG) for repairing severe peripheral nerve injuries. We have assessed a new DC provided by VERIGRAFT. The decellularization procedure completely removed cellularity while preserving the extracellular matrix. We first assessed the DC in a 15 mm gap in the sciatic nerve of rats, showing slightly delayed but effective regeneration. Then, we assayed the DC in a 70 mm gap in the peroneal nerve of sheep compared with AG. Evaluation of nerve regeneration and functional recovery was performed by clinical, electrophysiology and ultrasound tests. No significant differences were found in functional recovery between groups of sheep. Histology showed a preserved fascicular structure in the AG while in the DC grafts regenerated axons were grouped in small units. In conclusion, the DC was permissive for axonal regeneration and allowed to repair a 70 mm long gap in the sheep nerve

Derivation of endothelial cells from human embryonic stem cells by directed differentiation: Analysis of microrna and angiogenesis in vitro and in vivo

<b>Objective</b>: To develop an embryoid body-free directed differentiation protocol for the rapid generation of functional vascular endothelial cells derived from human embryonic stem cells (hESCs) and to assess the system for microRNA regulation and angiogenesis.<p></p> <b>Methods and Results</b>: The production of defined cell lineages from hESCs is a critical requirement for evaluating their potential in regenerative medicine. We developed a feeder- and serum-free protocol. Directed endothelial differentiation of hESCs revealed rapid loss of pluripotency markers and progressive induction of mRNA and protein expression of vascular markers (including CD31 and vascular endothelial [VE]-cadherin) and angiogenic growth factors (including vascular endothelial growth factor), increased expression of angiogenesis-associated microRNAs (including miR-126 and miR-210), and induction of endothelial cell morphological features. In vitro, differentiated cells produced nitric oxide, migrated across a wound, and formed tubular structures in both the absence and the presence of 3D matrices (Matrigel). In vivo, we showed that cells that differentiated for 10 days before implantation were efficient at the induction of therapeutic neovascularization and that hESC-derived cells were incorporated into the blood-perfused vasculature of recipient mice.<p></p> <b>Conclusion</b>: The directed differentiation of hESCs is efficient and effective for the differentiation of functional endothelial cells from hESCs.<p></p&gt