Increasing The Genetic Admixture of Available Lines of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) may significantly improve drug development pipeline, serving as an in vitro system for the identification of novel leads, and for testing drug toxicity. Furthermore, these cells may be used to address the issue of differential drug response, a phenomenon greatly influenced by genetic factors. This application depends on the availability of hPSC lines from populations with diverse ancestries. So far, it has been reported that most lines of hPSCs derived worldwide are of European or East Asian ancestries. We have established 23 lines of hPSCs from Brazilian individuals, and we report the analysis of their genomic ancestry. We show that embryo-derived PSCs are mostly of European descent, while induced PSCs derived from participants of a national-wide Brazilian cohort study present high levels of admixed European, African and Native American genomic ancestry. Additionally, we use high density SNP data and estimate local ancestries, particularly those of CYP genes loci. Such information will be of key importance when interpreting variation among cell lines with respect to cellular phenotypes of interest. The availability of genetically admixed lines of hPSCs will be of relevance when setting up future in vitro studies of drug response.

The segregation of different submicroscopic imbalances underlying the clinical variability associated with a familial karyotypically balanced translocation.

BACKGROUND: About 7 % of karyotypically balanced chromosomal rearrangements (BCRs) are associated with congenital anomalies due to gene or regulatory element disruption, and cryptic imbalances on rearranged chromosomes. Rare familial BCRs segregating with clinical features are a powerful source for the identifying of causative genes due to the presence of several affected carriers. CASE PRESENTATION: We report on a karyotypically balanced translocation t(2;22)(p13;q12.2) associated with variable learning disabilities, and craniofacial and hand dysmorphisms, detected in six individuals in a three-generation family. Combined a-CGH, FISH and mate-pair sequencing revealed a ten-break complex rearrangement, also involving chromosome 5. As the consequence of the segregation of the derivative chromosomes der(2), der(5) and der(22), different imbalances were present in affected and clinically normal family members, thus contributing to the clinical variability. A 6.64 Mb duplication of a 5q23.2-23.3 segment was the imbalance common to all affected individuals. Although LMNB1, implicated in adult-onset autosomal dominant leukodystrophy (ADLD) when overexpressed, was among the 18 duplicated genes, none of the adult carriers manifested ADLD, and LMNB1 overexpression was not detected in the two tested individuals, after qRT-PCR. The ectopic location of the extra copy of the LMBN1 gene on chromosome 22 might have negatively impacted its expression. In addition, two individuals presenting with more severe learning disabilities carried a 1.42 Mb 2p14 microdeletion, with three genes (CEP68, RAB1A and ACTR2),which are candidates for the intellectual impairment observed in the previously described 2p14p15 microdeletion syndrome, mapping to the minimal overlapping deleted segment. A 5p15.1 deletion, encompassing 1.47 Mb, also detected in the family, did not segregate with the clinical phenotype. CONCLUSION: The disclosing of the complexity of an apparently simple two-break familial rearrangement illustrates the importance of reconstructing the precise structure of derivative chromosomes for establishing genotype-phenotype correlations.

Presumptive germ cells derived from mouse pluripotent somatic cell hybrids.

The fusion of embryonic stem (ES) cells with differentiated somatic cells is an approach that reverses a somatic cell nucleus to a state of pluripotency. The resulting ES-somatic cell hybrids (ES-SCH) retain most of the properties of ES cells: differentiate into multiple cell types and have the ability to produce embryoid bodies (EB) and chimeras. However, it is still unknown whether ES-SCH will be able to complete the differentiation into germ cells (GC) in vitro similar to ES cells. Here, we show that near diploid ES-SCH, obtained by the fusion of mouse ES and spleen cells, were able to differentiate in vitro into presumptive GC. Differentiation of ES-SCH was induced through EB formation and by the addition of retinoic acid. Presumptive GC obtained reacted positively with anti-EMA, Vasa, Fragilis and Dazl antibodies and expressed GC-specific genes, such as Vasa, Stella, Dazl, Piwil 2, Tex14, Bmp8b, Tdrd1 and Rnf17. Fluorescent in situ hybridization analysis indicates chromosome reduction in the GC-like cells. Expression of meiotic and postmeiotic GC-specific genes such as Haprin, Acrosin, Scyp1, Scyp3 and Stra-8 were also detected. Transmission electron microscopy confirmed ES-SCH differentiation into presumptive GC. The presence of several autosomes and the X chromosome originated from the "somatic" partner did not prevent ES-SCH differentiation towards presumptive GC. Overall our study suggests an interesting in vitro model, which allows the study GC differentiation in reprogrammed somatic cells.

Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

BACKGROUND: The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. METHODS: Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. RESULTS AND DISCUSSION: We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age. CONCLUSION: Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.

Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs Local or systemic?

Background: The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. Methods: Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. Results and Discussion: We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes.

In vitro differentiation of male mouse embryonic stem cells into both presumptive sperm cells and oocytes.

Pioneer work in male mouse embryonic stem (ES) cells differentiation into germ cells (GC) showed generations of male or female gametes in separate experiments, using genetically manipulated or preselected ES cells. In an attempt to produce both types of gametes from male mouse ES cells without any genetic manipulation or preselection, we induce the differentiation by retinoic acid (RA) within nonadherent embryoid bodies (EB). It seems that gamete-like cell formation occurs in the correct manner based on the expression of early and late GC-specific genes such as Oct-4, Mvh, Stella, Dazl, Piwil 2, Pdrd 1, Rex 14, Rnf 17, Bmp8b, Acrosin, Stra-8, Haprin, LH-R, Gdf9, Zp3, Zp2, Sycp1, and Sycp3. Immunofluorescence analysis of morphologically well-formed GC and presumptive gametes showed positive labeling for SSEA1, Oct-4, EMA-1, FE-J1, Dazl, Fragilis, Mvh, Acrosin, and acetylated alpha-tubulin. Conventional cytogenetic and FISH analysis indicated a chromosome reduction in ES-derived GC. Our data suggest that ES cells with XY chromosomes can produce under the same experimental conditions both types of presumptive gametes, and this production depends on their positional and temporal information within the EB context.