Tissue-Engineered Disease Modeling of Lymphangioleiomyomatosis Exposes a Therapeutic Vulnerability to HDAC Inhibition.

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring hyperactive mTORC1 signaling. Here, tissue engineering tools are employed to model LAM and identify new therapeutic candidates. Biomimetic hydrogel culture of LAM cells is found to recapitulate the molecular and phenotypic characteristics of human disease more faithfully than culture on plastic. A 3D drug screen is conducted, identifying histone deacetylase (HDAC) inhibitors as anti-invasive agents that are also selectively cytotoxic toward TSC2-/- cells. The anti-invasive effects of HDAC inhibitors are independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Genotype-selective cytotoxicity is seen exclusively in hydrogel culture due to potentiated differential mTORC1 signaling, a feature that is abrogated in cell culture on plastic. Importantly, HDAC inhibitors block invasion and selectively eradicate LAM cells in vivo in zebrafish xenografts. These findings demonstrate that tissue-engineered disease modeling exposes a physiologically relevant therapeutic vulnerability that would be otherwise missed by conventional culture on plastic. This work substantiates HDAC inhibitors as possible therapeutic candidates for the treatment of patients with LAM and requires further study.

Transcriptomically Guided Mesendoderm Induction of Human Pluripotent Stem Cells Using a Systematically Defined Culture Scheme.

Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation.

Metformin Preconditioning of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells Promotes Their Engraftment and Improves Post-Stroke Regeneration and Recovery.

While transplantation of human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSCs) shows therapeutic potential in animal stroke models, major concerns for translating hiPSC therapy to the clinic are efficacy and safety. Therefore, there is a demand to develop an optimal strategy to enhance the engraftment and regenerative capacity of transplanted hiPSC-NSCs to produce fully differentiated neural cells to replace lost brain tissues. Metformin, an FDA-approved drug, is an optimal neuroregenerative agent that not only promotes NSC proliferation but also drives NSCs toward differentiation. In this regard, we hypothesize that preconditioning of hiPSC-NSCs with metformin before transplantation into the stroke-damaged brain will improve engraftment and regenerative capabilities of hiPSC-NSCs, ultimately enhancing functional recovery. In this study, we show that pretreatment of hiPSC-NSCs with metformin enhances the proliferation and differentiation of hiPSC-NSCs in culture. Furthermore, metformin-preconditioned hiPSC-NSCs show increased engraftment 1 week post-transplantation in a rat endothelin-1 focal ischemic stroke model. In addition, metformin-preconditioned cell grafts exhibit increased survival compared to naive cell grafts at 7 weeks post-transplantation. Analysis of the grafts demonstrates that metformin preconditioning enhances the differentiation of hiPSC-NSCs at the expense of their proliferation. As an outcome, rats receiving metformin-preconditioned cells display accelerated gross motor recovery and reduced infarct volume. These studies represent a vital step forward in the optimization of hiPSC-NSC-based transplantation to promote post-stroke recovery.

Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset.

Anaphase onset is an irreversible cell cycle transition that is triggered by the activation of the protease Separase. Separase cleaves the Mcd1 (also known as Scc1) subunit of Cohesin, a complex of proteins that physically links sister chromatids, triggering sister chromatid separation. Separase is regulated by the degradation of the anaphase inhibitor Securin which liberates Separase from inhibitory Securin/Separase complexes. In many organisms, Securin is not essential suggesting that Separase is regulated by additional mechanisms. In this work, we show that in budding yeast Cdk1 activates Separase (Esp1 in yeast) through phosphorylation to trigger anaphase onset. Esp1 activation is opposed by protein phosphatase 2A associated with its regulatory subunit Cdc55 (PP2ACdc55) and the spindle protein Slk19. Premature anaphase spindle elongation occurs when Securin (Pds1 in yeast) is inducibly degraded in cells that also contain phospho-mimetic mutations in ESP1, or deletion of CDC55 or SLK19. This striking phenotype is accompanied by advanced degradation of Mcd1, disruption of pericentric Cohesin organization and chromosome mis-segregation. Our findings suggest that PP2ACdc55 and Slk19 function redundantly with Pds1 to inhibit Esp1 within pericentric chromatin, and both Pds1 degradation and Cdk1-dependent phosphorylation of Esp1 act together to trigger anaphase onset.

Human Pluripotent Stem Cell-Derived TSC2-Haploinsufficient Smooth Muscle Cells Recapitulate Features of Lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a progressive destructive neoplasm of the lung associated with inactivating mutations in the TSC1 or TSC2 tumor suppressor genes. Cell or animal models that accurately reflect the pathology of LAM have been challenging to develop. Here, we generated a robust human cell model of LAM by reprogramming TSC2 mutation-bearing fibroblasts from a patient with both tuberous sclerosis complex (TSC) and LAM (TSC-LAM) into induced pluripotent stem cells (iPSC), followed by selection of cells that resemble those found in LAM tumors by unbiased in vivo differentiation. We established expandable cell lines under smooth muscle cell (SMC) growth conditions that retained a patient-specific genomic TSC2+/- mutation and recapitulated the molecular and functional characteristics of pulmonary LAM cells. These include multiple indicators of hyperactive mTORC1 signaling, presence of specific neural crest and SMC markers, expression of VEGF-D and female sex hormone receptors, reduced autophagy, and metabolic reprogramming. Intriguingly, the LAM-like features of these cells suggest that haploinsufficiency at the TSC2 locus contributes to LAM pathology, and demonstrated that iPSC reprogramming and SMC lineage differentiation of somatic patient cells with germline mutations was a viable approach to generate LAM-like cells. The patient-derived SMC lines we have developed thus represent a novel cellular model of LAM that can advance our understanding of disease pathogenesis and develop therapeutic strategies against LAM. Cancer Res; 77(20); 5491-502. ©2017 AACR.

Tah18 transfers electrons to Dre2 in cytosolic iron-sulfur protein biogenesis.

Cytosolic and nuclear iron-sulfur (Fe-S) proteins play key roles in processes such as ribosome maturation, transcription and DNA repair-replication. For biosynthesis of their Fe-S clusters, a dedicated cytosolic Fe-S protein assembly (CIA) machinery is required. Here, we identify the essential flavoprotein Tah18 as a previously unrecognized CIA component and show by cell biological, biochemical and spectroscopic approaches that the complex of Tah18 and the CIA protein Dre2 is part of an electron transfer chain functioning in an early step of cytosolic Fe-S protein biogenesis. Electrons are transferred from NADPH via the FAD- and FMN-containing Tah18 to the Fe-S clusters of Dre2. This electron transfer chain is required for assembly of target but not scaffold Fe-S proteins, suggesting a need for reduction in the generation of stably inserted Fe-S clusters. The pathway is conserved in eukaryotes, as human Ndor1-Ciapin1 proteins can functionally replace yeast Tah18-Dre2.

Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type.

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.

Survey of allelic expression using EST mining.

Cis-acting allelic variation in gene regulation is a source of phenotypic variation. Consequently, recent studies have experimentally screened human genes in an attempt to initiate a catalog of genes possessing cis-acting variants. In this study, we use human EST data in dbEST as the source of allelic expression data, and the HapMap database to provide expected allele frequencies in human populations. We demonstrate a greater concordance of allele frequencies estimated from human ESTs in dbEST with those derived from the CEPH HapMap sample representing Caucasians from northern and western Europe, than population samples obtained in Asia and Africa. Deviations between allele frequencies observed in EST databases and the ones obtained from the CEPH HapMap samples may result from common heritable cis-acting variants altering the relative allele distribution in RNA. We provide in silico as well as experimental evidence that this strategy does allow significant enrichment of genes harboring common heritable cis-acting polymorphisms in linkage disequilibrium with expressed alleles.

Mutations in the MMAA gene in patients with the cblA disorder of vitamin B12 metabolism.

Mutations in the MMAA gene on human chromosome 4q31.21 result in vitamin B12-responsive methylmalonic aciduria (cblA complementation group) due to deficiency in the synthesis of adenosylcobalamin. Genomic DNA from 37 cblA patients, diagnosed on the basis of cellular adenosylcobalamin synthesis, methylmalonyl-coenzyme A (CoA) mutase function, and complementation analysis, was analyzed for deleterious mutations in the MMAA gene by DNA sequencing of exons and flanking sequences. A total of 18 novel mutations were identified, bringing the total number of mutations identified in 37 cblA patients to 22. A total of 13 mutations result in premature stop codons; three are splice site defects; and six are missense mutations that occur at highly conserved residues. Eight of these mutations were common to two or more individuals. One mutation, c.433C>T (R145X), represents 43% of pathogenic alleles and a common haplotype was identified. Restriction endonuclease or heteroduplex diagnostic tests were designed to confirm mutations. None of the sequence changes identified in cblA patients were found in 100 alleles from unrelated control individuals.

5' flanking variants of resistin are associated with obesity.

Diabetes and obesity have long been known to be related. The recently characterized adipocyte hormone resistin (also called FIZZ3/ADSF) has been implicated as a molecular link between impaired glucose tolerance (IGT) and obesity in mice. A search for sequence variants at the human resistin locus identified nine single-nucleotide polymorphisms (SNPs) but no coding variants. An investigation into the association of these SNPs with diabetes and obesity revealed two 5' flanking variants (g.-537 and g.-420), in strong linkage disequilibrium, that are associated with BMI. In nondiabetic individuals from the Quebec City area and the Saguenay-Lac-St-Jean region of Quebec, the g.-537 mutation (allelic frequency = 0.04) was significantly associated with an increase in BMI (P = 0.03 and P = 0.01, respectively). When the data from these two populations were combined and adjusted for age and sex, both the g.-537 (odds ratio [OR] 2.72, 95% CI 1.28-5.81) and the g.-420 variants (1.58, 1.06-2.35) were associated with an increased risk for a BMI > or =30 kg/m(2). In contrast, in case/control and family-based study populations from Scandinavia, we saw no effect on BMI with either of these promoter variants. No association was seen with diabetes in any of the population samples.