Daniela F. Bueno

Stem Cell Proliferation Under Low Intensity Laser Irradiation: A Preliminary Study

Phototherapy with low intensity laser irradiation has shown to be effective in promoting the proliferation of different cells. The aim of this in vitro study was to evaluate the potential effect of laser phototherapy (660 nm) on human dental pulp stem cell (hDPSC) proliferation.

Mesenchymal Stem Cells Derived From Canine Umbilical Cord Vein—A Novel Source for Cell Therapy Studies

The canine model provides a large animal system to evaluate many treatment modalities using stem cells (SCs). However, only bone marrow (BM) protocols have been widely used in dogs for preclinical approaches. BM donation consists of an invasive procedure and the number and differentiation potential of its mesenchymal stem cells (MSCs) decline with age. More recently, umbilical cord was introduced as an alternative source to BM since it is obtained from a sample that is routinely discarded. Here, we describe the isolation of MSCs from canine umbilical cord vein (cUCV). These cells can be obtained from every cord received and grow successfully in culture. Their multipotent plasticity was demonstrated by their capacity to differentiate in adipocytic, chondrocytic, and osteocytic lineages. Furthermore, our results open possibilities to use cUCV cells in preclinical trials for many well-characterized canine model conditions homologs to human diseases.

Human fallopian tube: a new source of multipotent adult mesenchymal stem cells discarded in surgical procedures.

BackgroundThe possibility of using stem cells for regenerative medicine has opened a new field of investigation. The search for sources to obtain multipotent stem cells from discarded tissues or through non-invasive procedures is of great interest. It has been shown that mesenchymal stem cells (MSCs) obtained from umbilical cords, dental pulp and adipose tissue, which are all biological discards, are able to differentiate into muscle, fat, bone and cartilage cell lineages. The aim of this study was to isolate, expand, characterize and assess the differentiation potential of MSCs from human fallopian tubes (hFTs).MethodsLineages of hFTs were expanded, had their karyotype analyzed, were characterized by flow cytometry and underwent in vitro adipogenic, chondrogenic, osteogenic, and myogenic differentiation.ResultsHere we show for the first time that hFTs, which are discarded after some gynecological procedures, are a rich additional source of MSCs, which we designated as human tube MSCs (htMSCs).ConclusionHuman tube MSCs can be easily isolated, expanded in vitro, present a mesenchymal profile and are able to differentiate into muscle, fat, cartilage and bone in vitro.

New Source of Muscle-Derived Stem Cells with Potential for Alveolar Bone Reconstruction in Cleft Lip and/or Palate Patients

Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in nonimmunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.

Fat grafts supplemented with adipose-derived stromal cells in the rehabilitation of patients with craniofacial microsomia.

Background: Although first reports of the clinical use of adipose-derived stromal cells suggest that this approach may be feasible and effective for soft-tissue augmentation, there is a lack of randomized, controlled clinical trials in the literature. Thus, this study aimed to investigate whether a faster protocol for isolation of adipose-derived stromal cells and their use in combination with fat tissue improve the long-term retention of the grafts in patients with craniofacial microsomia. Methods: Patients with craniofacial microsomia (n = 14) were grafted either with supplementation of adipose-derived stromal cells (experimental group) or without supplementation of adipose-derived stromal cells (control group). The number of viable cells isolated before and after the supplementation of the grafts was calculated, and these cells were examined for mesenchymal cell surface markers using flow cytometry. Computed tomography was performed to assess both hemifaces preoperatively and at 6 months postoperatively. Results: The average number of viable cells isolated before and after the supplementation of the grafts was 5.6 × 105 and 9.9 × 105 cells/ml of fat tissue (p = 0.015). Flow cytometric analysis revealed that the adipose-derived stromal cells were positive for mesenchymal cell markers (>95 percent for CD73 and CD105). Surviving fat volume at 6 months was 88 percent for the experimental group and 54 percent for the control group (p = 0.003). Conclusion: These results suggest that this strategy for isolation and supplementation of adipose-derived stromal cells is effective, safe, and superior to conventional lipoinjection for facial recontouring in patients with craniofacial microsomia. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.

IRF6 is a risk factor for nonsyndromic cleft lip in the Brazilian population

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a complex disorder with a worldwide incidence estimated at 1:700. Among the putative susceptibility loci, the IRF6 gene and a region at 8q24.21 have been corroborated in different populations. To test the role of IRF6 in NSCL/P predisposition in the Brazilian population, we conducted a structured association study with the SNPs rs642961 and rs590223, respectively, located at 5′ and 3′ of the IRF6 gene and not in strong linkage disequilibrium (LD), in patients from five different Brazilian locations. We also evaluated the effect of these SNPs in IRF6 expression in mesenchymal stem cells (MSC). We observed association between rs642961 and cleft lip only (CLO) (P = 0.009; odds ratio (OR) for AA genotype = 1.83 [95% Confidence interval (CI), 0.64–5.31]; OR for AG genotype = 1.72 [95% CI, 1.03–2.84]). This association seems to be driven by the affected patients from Barbalha, a location which presents the highest heritability estimate (H2 = 0.85), and the A allele at rs642961 is acting through a dominant model. No association was detected for the SNP rs590223. We did not find any correlation between expression levels and genotypes of the two loci, and it is possible that these SNPs have a functional role in some specific period of embryogenesis.

Human Stem Cell Cultures from Cleft Lip/Palate Patients Show Enrichment of Transcripts Involved in Extracellular Matrix Modeling By Comparison to Controls

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.

Novel Mutations in IRF6 in Nonsyndromic Cleft Lip With or Without Cleft Palate: When Should IRF6 Mutational Screening be Done?

Novel Mutations in IRF6 in Nonsyndromic Cleft Lip With or Without Cleft Palate: When Should IRF6 Mutational Screening be Done? Fernanda Sarquis Jehee, Beatriz A. Burin, K atia M. Rocha, Roseli Zechi-Ceide, Daniela F. Bueno, Luciano Brito, Josiane Souza, Gabriela Ferraz Leal, Antonio Richieri-Costa, Nivaldo Alonso, Paulo A. Otto, and Maria Rita Passos-Bueno* Centro de Estudos do Genoma Humano, Departamento de Gen etica e Biologia Evolutiva, Instituto de Biociencias, Universidade de S~ao Paulo, S~ao Paulo, SP, Brazil Departamento de Gen etica Cl inica, Hospital de Rehabilitac~ao de Anomalias Craniofaciais (HRAC), Universidade de S~ao Paulo, Bauru, Brazil Sobrapar, Instituto de Cirurgia Pl astica, Campinas, S~ao Paulo, Brazil CAIF, Centro de Atendimento Integral ao Fissurado L abio Palatal, Curitiba, Paran a, Brazil Centro de Atenc~ao aos Defeitos da Face do Instituto Materno-Infantil Prof. Fernando Figueira (CADEFI), Recife, Brazil Departamento de Gen etica e Biologia Evolutiva, Instituto de Biociencias, USP, S~ao Paulo, Brazil

Susceptibility to DNA damage as a molecular mechanism for non-syndromic cleft lip and palate

Non-syndromic cleft lip/palate (NSCL/P) is a complex, frequent congenital malformation, determined by the interplay between genetic and environmental factors during embryonic development. Previous findings have appointed an aetiological overlap between NSCL/P and cancer, and alterations in similar biological pathways may underpin both conditions. Here, using a combination of transcriptomic profiling and functional approaches, we report that NSCL/P dental pulp stem cells exhibit dysregulation of a co-expressed gene network mainly associated with DNA double-strand break repair and cell cycle control (p = 2.88×10−2–5.02×10−9). This network included important genes for these cellular processes, such as BRCA1, RAD51, and MSH2, which are predicted to be regulated by transcription factor E2F1. Functional assays support these findings, revealing that NSCL/P cells accumulate DNA double-strand breaks upon exposure to H2O2. Furthermore, we show that E2f1, Brca1 and Rad51 are co-expressed in the developing embryonic orofacial primordia, and may act as a molecular hub playing a role in lip and palate morphogenesis. In conclusion, we show for the first time that cellular defences against DNA damage may take part in determining the susceptibility to NSCL/P. These results are in accordance with the hypothesis of aetiological overlap between this malformation and cancer, and suggest a new pathogenic mechanism for the disease.

Genetic contribution for non-syndromic cleft lip with or without cleft palate (NS CL/P) in different regions of Brazil and implications for association studies†

Non‐syndromic cleft lip with or without cleft palate (NS CL/P) is a complex disease in which heritability estimates vary widely depending on the population studied. To evaluate the importance of genetic contribution to NS CL/P in the Brazilian population, we conducted a study with 1,042 families from five different locations (Santarém, Fortaleza, Barbalha, Maceió, and Rio de Janeiro). We also evaluated the role of consanguinity and ethnic background. The proportion of familial cases varied significantly across locations, with the highest values found in Santarém (44%) and the lowest in Maceió (23%). Heritability estimates showed a higher genetic contribution to NS CL/P in Barbalha (85%), followed by Santarém (71%), Rio de Janeiro (70%), Fortaleza (64%), and Maceió (45%). Ancestry was not correlated with the occurrence of NS CL/P or with the variability in heritability. Only in Rio de Janeiro was the coefficient of inbreeding significantly larger in NS CL/P families than in the local population. Recurrence risk for the total sample was approximately 1.5–1.6%, varying according to the location studied (0.6–0.7% in Maceió to 2.2–2.8% in Barbalha). Our findings show that the degree of genetic contribution to NS CL/P varies according to the geographic region studied, and this difference cannot be attributed to consanguinity or ancestry. These findings suggest that Barbalha is a promising region for genetic studies. The data presented here will be useful in interpreting results from molecular analyses and show that care must be taken when pooling samples from different populations for association studies.