Cytogenetic instability of dental pulp stem cell lines

Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.INCTBiofabrication InstituteINCT-Biofabrication InstituteCNPqCNPq [573661/2008-1]FAPESP [08/57860-3, 07-58856-7, 07-51227-4, 07-59488-1]FAPESPRede Biofab, IberoAmerican Network of BiofabricationBIOFABCYTEDRede Biofab, Ibero-American Network of Biofabrication-BIOFAB-CYTED [208RT0340]NIH/NIDCR [R01DE016132, R03TW007665]NIH/NIDC

Cytogenetic instability of dental pulp stem cell lines

Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. the heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.INCT-Biofabrication InstituteConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Rede Biofab, Ibero-American Network of Biofabrication-BIOFAB-CYTEDNIH/NIDCRUniv São Paulo, Dept Pathol, Cytogenom Lab, BR-05403000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Plast Surg, Ctr Cellular & Mol Therapy, UNIFESP CTCMol, São Paulo, BrazilINCT Biofabris, Natl Inst Sci & Technol, Biofabricat Inst, São Paulo, BrazilUNIFESP Sao Jose Campos, Sci & Technol Inst Biomed Engn, São Paulo, BrazilUniversidade Federal de São Paulo, Div Endocrinol, São Paulo, BrazilMassachusetts Gen Hosp, Lab Tissue Engn & Organ Fabricat, Boston, MA 02114 USAHarvard Univ, Sch Med, Boston, MA USADept Surg, Boston, MA USATufts Univ, Dept Oral & Maxillofacial Pathol, Boston, MA 02111 USAUniversidade Federal de São Paulo, Dept Plast Surg, Ctr Cellular & Mol Therapy, UNIFESP CTCMol, São Paulo, BrazilUNIFESP Sao Jose Campos, Sci & Technol Inst Biomed Engn, São Paulo, BrazilUniversidade Federal de São Paulo, Div Endocrinol, São Paulo, BrazilCNPq: 573661/2008-1FAPESP: 08/57860-3FAPESP: 07-58856-7FAPESP: 07-51227-4FAPESP: 07-59488-1Rede Biofab, Ibero-American Network of Biofabrication-BIOFAB-CYTED: 208RT0340NIH/NIDCR: R01DE016132NIH/NIDCR: R03TW007665Web of Scienc

Tooth Tissue Engineering: Optimal Dental Stem Cell Harvest Based on Tooth Development

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications.FAPESP[07-58856-7]FAPESP[07-51227-4]FAPESP[07-59488-1]NIH/NIDCR[R01DE016132]NIH/NIDCR[R03TW007665

Zebrafish wnt9b Synteny and Expression During First and Second Arch, Heart, and Pectoral Fin Bud Morphogenesis

Roles for Wnt9b in craniofacial development are indicated by the cleft lip mutant phenotype observed in the A/WySn mouse strain,1 caused by a retrotransposon insertion mutation at the Wnt9b locus. Analyses of the zebrafish Wnt9b ortholog, wnt9b, were pursued to provide insight into early vertebrate craniofacial patterning events mediated by Wnt9b signaling. Zebrafish wnt9b cDNA clones were isolated and found to encode an open reading frame of 358 amino acids, with 68% amino acid identity to mouse Wnt9b and 70% amino acid identity to human WNT9B. Syntenic analyses demonstrated that wnt9b and wnt3 exist as a contiguous pair in amniote vertebrate species, and that these genes are separate in the zebrafish and Takifugu genomes. During the pharyngula period, a time of extensive growth and morphogenesis, zebrafish wnt9b exhibits discrete expression in dorsal and ventral first and second branchial arch tissues, the heart, and pectoral fin buds. These analyses suggest that in zebrafish, as in humans, wnt9b plays distinct roles in directing morphogenetic movements of developing branchial arch elements, and identify the zebrafish as a useful developmental model for the study of human craniofacial cleft lip and palate

Prevention of the Disrupted Enamel Phenotype in Slc4a4-Null Mice Using Explant Organ Culture Maintained in a Living Host Kidney Capsule

Slc4a4-null mice are a model of proximal renal tubular acidosis (pRTA). Slc4a4 encodes the electrogenic sodium base transporter NBCe1 that is involved in transcellular base transport and pH regulation during amelogenesis. Patients with mutations in the SLC4A4 gene and Slc4a4-null mice present with dysplastic enamel, amongst other pathologies. Loss of NBCe1 function leads to local abnormalities in enamel matrix pH regulation. Loss of NBCe1 function also results in systemic acidemic blood pH. Whether local changes in enamel pH and/or a decrease in systemic pH are the cause of the abnormal enamel phenotype is currently unknown. In the present study we addressed this question by explanting fetal wild-type and Slc4a4-null mandibles into healthy host kidney capsules to study enamel formation in the absence of systemic acidemia. Mandibular E11.5 explants from NBCe1-/- mice, maintained in host kidney capsules for 70 days, resulted in teeth with enamel and dentin with morphological and mineralization properties similar to cultured NBCe1+/+ mandibles grown under identical conditions. Ameloblasts express a number of proteins involved in dynamic changes in H+/base transport during amelogenesis. Despite the capacity of ameloblasts to dynamically modulate the local pH of the enamel matrix, at least in the NBCe1-/- mice, the systemic pH also appears to contribute to the enamel phenotype. Extrapolating these data to humans, our findings suggest that in patients with NBCe1 mutations, correction of the systemic metabolic acidosis at a sufficiently early time point may lead to amelioration of enamel abnormalities