Tissue engineering using human embryonic stem cells.

The possibility of using stem cells for tissue engineering has greatly encouraged scientists to design new platforms in the field of regenerative and reconstructive medicine. Stem cells have the ability to rejuvenate and repair damaged tissues and can be derived from both embryonic and adult sources. Among cell types suggested as a cell source for tissue engineering (TE), human embryonic stem cells (hESCs) are one of the most promising candidates. Isolated from the inner cell mass of preimplantation stage blastocysts, they possess the ability to differentiate into practically all adult cell types. In addition, their unlimited self-renewal capacity enables the generation of sufficient amount of cells for cell-based TE applications. Yet, several important challenges are to be addressed, such as the isolation of the desired cell type and gaining control over its differentiation and proliferation. Ultimately, combing scaffolding and bioactive stimuli, newly designed bioengineered constructs, could be assembled and applied to various clinical applications. Here we define the culture conditions for the derivation of connective tissue lineage progenitors, design strategies, and highlight the special considerations when using hESCs for TE applications.

A novel MLH1 mutation harbored as a germ line aberration by a young woman of an HNPCC-like family and exhibited by a CML patient when occurring prior to the initiation of the blast phase concomitant with a c-MYC amplification.

Germ line mutations in the MLH1 and MSH2 genes account for the majority of hereditary nonpolyposis colorectal cancer (HNPCC) families. Here, we describe a family that does not meet the international criteria for HNPCC, of which a young woman harbors a missense mutation (D132H). This novel germ line mutation has not previously been reported. Of the mismatch repair (MMR) genes, MLH1 has been shown to play an important role in hematologic malignancies. The novel mutation was also revealed to be a somatic aberration occurring prior to the initiation of the blast phase in a chronic myelogenous leukemia (CML) patient. Among the possible MLH1 partners involved in signaling MMR or apoptosis is the proto-oncogene c-MYC, which is closely related to cellular proliferation. We further revealed a concomitant c-MYC dramatic amplification in the CML-MLH1-mutation carrier patient, also occurring at the pre-blast phase. Our data contribute further to characterizing the mutational spectrum of the MLH1 gene. Furthermore, given the role of c-MYC and its interaction with MLH1, taken together with the mutational status of both genes revealed at the pre-blast phase in the CML patient, a plausible increased genetic instability might be expected to take place, possibly contributing to blast triggering. Our results may provide additional insight into the complex interplay between the MMR system and other cellular pathways.

The soluble extracellular domain of EphB4 (sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and inhibits tumor growth.

The receptor tyrosine kinase EphB4 and its ligand EphrinB2 play a crucial role in vascular development during embryogenesis. The soluble monomeric derivative of the extracellular domain of EphB4 (sEphB4) was designed as an antagonist of EphB4/EphrinB2 signaling. sEphB4 blocks activation of EphB4 and EphrinB2; suppresses endothelial cell migration, adhesion, and tube formation in vitro; and inhibits the angiogenic effects of various growth factors (VEGF and bFGF) in vivo. sEphB4 also inhibits tumor growth in murine tumor xenograft models. sEphB4 is thus a therapeutic candidate for vascular proliferative diseases and cancer.

A novel case of a CAT to AAT transversion in codon 179 of the p53 gene in a supratentorial primitive neuroectodermal tumor harbored by a young girl. Case report and review of the literature.

BACKGROUND: The most common cytogenetic abnormality encountered in primitive neuroectodermal tumors (PNET)/medulloblastoma is loss of heterozygosity in the region of the short arm of chromosome 17. There is some evidence that supratentorial PNET has different cytogenetic markers than infratentorial PNET/medulloblastoma. Particularly, loss of 17p is more frequent in the latter than in the former. We describe a young girl diagnosed with supratentorial PNET (SPNET). Analysis of the tumor suppressor gene p53 in the tumorous tissue revealed a rare transversion mutational event of CAT to AAT in position 179 of exon 5. To the best of our knowledge, this is the first case of such a transversion at codon 179 in the p53 gene in SPNET. CASE REPORT: A 12-year-old girl was admitted with nausea, headache and vision disturbances. MRI of the brain showed a large space- occupying lesion in the right frontal lobe. Histological examination of the macroscopic resection of the tumor revealed PNET of the brain. Polymerase chain reaction-single strand conformation polymorphism analysis of all p53 exons was performed, and a unique variant of a transversion at codon 179 of exon 5 was revealed. Therapy was started according to the Children's Cancer group protocol (CCG-99702) designated for treatment of high-risk central nervous system embryonal tumors. She received an initial course of chemotherapy, consisting of cyclophosphamide and vincristine for mobilizing and harvesting peripheral blood stem cells (PBSCs). Then she was given craniospinal irradiation (3,600 cGy) with a boost to the tumor bed (1,980 cGy) and three consecutive courses of high-dose chemotherapy with carboplatin, vincristine and thiotepa/cyclophosphamide, with PBSCs support after each course. RESULTS: The patient is in complete remission 17 months after diagnosis, based on the results of physical examination and imaging studies. DISCUSSION: The mutation results in an alteration of the amino acid HIS to ASN. The amino acids surrounding position 175 play an important role in stabilizing the p53/DNA complex. There are only 12 known mutations of the reported type, and the finding of such a rare mutational event in a low-incidence p53 mutation tumor, such as SPNET, might add additional insight into the p53-SPNET relationship in tumorigenesis. Although not widely accepted, it is possible that different mutations of the p53 gene in patients with brain tumors may imply a different ultimate prognosis. In our case, we cannot exclude the fact that transversion of CAT to AAT in position 179 of exon 5 may explain prolonged survival of a patient with good response to therapy.

Maternal mosaicism for a second mutational event--a novel deletion--in a familial adenomatous polyposis family harboring a new germ-line mutation in the alternatively spliced-exon 9 region of APC.

Familial Adenomatous Polyposis (FAP) is an autosomal dominant heritable disorder caused by germ-line mutations in the APC gene. To date, more than 300 germ-line mutations within this gene have been described. Using PCR, SSCP and DNA sequencing, we have identified a new mutation in the alternatively spliced region of exon 9 (1042C-->T), which results in a stop signal. This mutation manifested an aggressive form of FAP with onset of symptoms in one proband at age 17. Our results differ from reported exon 9 mutations in the spliced-out portion of the gene manifesting an attentuated form of FAP (AAPC) [Varesco et al 1994; van der Luijt et al. 1995; Curia et al. 1998; Young et al. 1998]. When analyzing this family, we encountered a mutant FAP gene which had undergone a second mutational event, a deletion. In addition to linkage analysis, both the occurrence of the two exon 9 mutation-carrier siblings, of which one is affected, harboring the same novel deletion in one generation of this family, and its absence in both parents indicates the existence of maternal germ-line mosaicism for cells bearing the latter second mutational event. Our study is only the second report of parental mosaicism in the APC gene.