Engineered Human Skin Substitutes Undergo Large-Scale Genomic Reprogramming and Normal Skin-Like Maturation after Transplantation to Athymic Mice

Bioengineered skin substitutes can facilitate wound closure in severely burned patients, but deficiencies limit their outcomes compared with native skin autografts. To identify gene programs associated with their in vivo capabilities and limitations, we extended previous gene expression profile analyses to now compare engineered skin after in vivo grafting with both in vitro maturation and normal human skin. Cultured skin substitutes were grafted on full-thickness wounds in athymic mice, and biopsy samples for microarray analyses were collected at multiple in vitro and in vivo time points. Over 10,000 transcripts exhibited large-scale expression pattern differences during in vitro and in vivo maturation. Using hierarchical clustering, 11 different expression profile clusters were partitioned on the basis of differential sample type and temporal stage-specific activation or repression. Analyses show that the wound environment exerts a massive influence on gene expression in skin substitutes. For example, in vivo–healed skin substitutes gained the expression of many native skin-expressed genes, including those associated with epidermal barrier and multiple categories of cell–cell and cell–basement membrane adhesion. In contrast, immunological, trichogenic, and endothelial gene programs were largely lacking. These analyses suggest important areas for guiding further improvement of engineered skin for both increased homology with native skin and enhanced wound healing

High-throughput detection of mutations responsible for childhood hearing loss using resequencing microarrays

Background: Despite current knowledge of mutations in 45 genes that can cause nonsyndromic sensorineural hearing loss (SNHL), no unified clinical test has been developed that can comprehensively detect mutations in multiple genes. We therefore designed Affymetrix resequencing microarrays capable of resequencing 13 genes mutated in SNHL (GJB2, GJB6, CDH23, KCNE1, KCNQ1, MYO7A, OTOF, PDS, MYO6, SLC26A5, TMIE, TMPRSS3, USH1C). We present results from hearing loss arrays developed in two different research facilities and highlight some of the approaches we adopted to enhance the applicability of resequencing arrays in a clinical setting. Results: We leveraged sequence and intensity pattern features responsible for diminished coverage and accuracy and developed a novel algorithm, sPROFILER, which resolved >80% of no-calls from GSEQ and allowed 99.6% (range: 99.2-99.8%) of sequence to be called, while maintaining overall accuracy at >99.8% based upon dideoxy sequencing comparison. Conclusions: Together, these findings provide insight into critical issues for disease-centered resequencing protocols suitable for clinical application and support the use of array-based resequencing technology as a valuable molecular diagnostic tool for pediatric SNHL and other genetic diseases with substantial genetic heterogeneity

Microevolution of Group A Streptococci In Vivo: Capturing Regulatory Networks Engaged in Sociomicrobiology, Niche Adaptation, and Hypervirulence

The onset of infection and the switch from primary to secondary niches are dramatic environmental changes that not only alter bacterial transcriptional programs, but also perturb their sociomicrobiology, often driving minor subpopulations with mutant phenotypes to prevail in specific niches. Having previously reported that M1T1 Streptococcus pyogenes become hypervirulent in mice due to selection of mutants in the covRS regulatory genes, we set out to dissect the impact of these mutations in vitro and in vivo from the impact of other adaptive events. Using a murine subcutaneous chamber model to sample the bacteria prior to selection or expansion of mutants, we compared gene expression dynamics of wild type (WT) and previously isolated animal-passaged (AP) covS mutant bacteria both in vitro and in vivo, and we found extensive transcriptional alterations of pathoadaptive and metabolic gene sets associated with invasion, immune evasion, tissue-dissemination, and metabolic reprogramming. In contrast to the virulence-associated differences between WT and AP bacteria, Phenotype Microarray analysis showed minor in vitro phenotypic differences between the two isogenic variants. Additionally, our results reflect that WT bacteria's rapid host-adaptive transcriptional reprogramming was not sufficient for their survival, and they were outnumbered by hypervirulent covS mutants with SpeB−/Sdahigh phenotype, which survived up to 14 days in mice chambers. Our findings demonstrate the engagement of unique regulatory modules in niche adaptation, implicate a critical role for bacterial genetic heterogeneity that surpasses transcriptional in vivo adaptation, and portray the dynamics underlying the selection of hypervirulent covS mutants over their parental WT cells

BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network

Several homeodomain (HD) proteins are critical for skeletal patterning and respond directly to BMP2 as an early step in bone formation. RUNX2, the earliest transcription factor proven essential for commitment to osteoblastogenesis, is also expressed in response to BMP2. However, there is a gap in our knowledge of the regulatory cascade from BMP2 signaling to the onset of osteogenesis. Here we show that BMP2 induces DLX3, a homeodomain protein that activates Runx2 gene transcription. Small interfering RNA knockdown studies in osteoblasts validate that DLX3 is a potent regulator of Runx2. Furthermore in Runx2 null cells, DLX3 forced expression suffices to induce transcription of Runx2, osteocalcin, and alkaline phosphatase genes, thus defining DLX3 as an osteogenic regulator independent of RUNX2. Our studies further show regulation of the Runx2 gene by several homeodomain proteins: MSX2 and CDP/cut repress whereas DLX3 and DLX5 activate endogenous Runx2 expression and promoter activity in non-osseous cells and osteoblasts. These HD proteins exhibit distinct temporal expression profiles during osteoblast differentiation as well as selective association with Runx2 chromatin that is related to Runx2 transcriptional activity and recruitment of RNA polymerase II. Runx2 promoter mutagenesis shows that multiple HD elements control expression of Runx2 in relation to the stages of osteoblast maturation. Our studies establish mechanisms for commitment to the osteogenic lineage directly through BMP2 induction of HD proteins DLX3 and DLX5 that activate Runx2, thus delineating a transcriptional regulatory pathway mediating osteoblast differentiation. We propose that the three homeodomain proteins MSX2, DLX3, and DLX5 provide a key series of molecular switches that regulate expression of Runx2 throughout bone formation. <br/

Integrated Genomic Analysis of Diverse Induced Pluripotent Stem Cells from the Progenitor Cell Biology Consor tium

The rigorous characterization of distinct induced pluripotent stem cells (iPSC) derived from multiple reprogramming technologies, somatic sources, and donors is required to understand potential sources of variability and downstream potential. To achieve this goal, the Progenitor Cell Biology Consortium performed comprehensive experimental and genomic analyses of 58 iPSC from ten laboratories generated using a variety of reprogramming genes, vectors, and cells. Associated global molecular characterization studies identified functionally informative correlations in gene expression, DNA methylation, and/or copy-number variation among key developmental and oncogenic regulators as a result of donor, sex, line stability, reprogramming technology, and cell of origin. Furthermore, X-chromosome inactivation in PSC produced highly correlated differences in teratoma-lineage staining and regulator expression upon differentiation. All experimental results, and raw, processed, and metadata from these analyses, including powerful tools, are interactively accessible from a new online portal at https://www.synapse.org to serve as a reusable resource for the stem cell community