Compact Colony Morphology Research Articles

Rab GDP-dissociation inhibitor gdiA is an essential gene required for cell wall chitin deposition in Aspergillus niger

The cell wall is a distinctive feature of filamentous fungi, providing them with structural integrity and protection from both biotic and abiotic factors. Unlike plant cell walls, fungi rely on structurally strong hydrophobic chitin core for mechanical strength together with alpha- and beta-glucans, galactomannans and glycoproteins. Cell wall stress conditions are known to alter the cell wall through the signaling cascade of the cell wall integrity (CWI) pathway and can result in increased cell wall chitin deposition. A previously isolated set of Aspergillus niger cell wall mutants was screened for increased cell wall chitin deposition. UV-mutant RD15.8#16 was found to contain approximately 60% more cell wall chitin than the wild type. In addition to the chitin phenotype, RD15.8#16 exhibits a compact colony morphology and increased sensitivity towards SDS. RD15.8#16 was subjected to classical genetic approach for identification of the underlying causative mutation, using co-segregation analysis and SNP genotyping. Genome sequencing of RD15.8#16 revealed eight SNPs in open reading frames (ORF) which were individually checked for co-segregation with the associated phenotypes, and showed the potential relevance of two genes located on chromosome IV. In situ re-creation of these ORF-located SNPs in a wild type background, using CRISPR/Cas9 genome editing, showed the importance Rab GTPase dissociation inhibitor A (gdiA) for the phenotypes of RD15.8#16. An alteration in the 5′ donor splice site of gdiA reduced pre-mRNA splicing efficiency, causing aberrant cell wall assembly and increased chitin levels, whereas gene disruption attempts showed that a full gene deletion of gdiA is lethal.

186 Improving Success Rate of Establishment and Maintenance of Porcine Induced Pluripotent Stem Cells by Investigation of Colony Morphology

Induced pluripotent stem cells (iPS cells) are generated by reprogramming of somatic cells using ectopic introduction of 4 transcription factors, including OCT4, SOX2, KLF4, and c-MYC (OSKM). Fibroblast cells are the most commonly used as a primary cell source for iPS cells because they are easy to harvest and culture. However, reprogramming efficiency of porcine fibroblasts is poor (~0.1%). During reprogramming process, mixed populations of primary colonies become the major obstacle in iPS establishment. In this study, we characterised 2 different colony morphologies at colony pick-up (compact and loose iPS morphology). We hypothesised that compact colonies will proceed to long-term culture and pluripotency. The fibroblast cells were isolated from tails of piglets and transfected with retroviral vectors expressing OSKM. The primary colonies were counted on Day 7 after gene transduction. The iPS-like colonies were divided into compact (n = 10) and loose (n = 10) morphology at colony pick-up. The characteristics of iPS-like cell lines were analysed by morphology, alkaline phosphatase staining, G-banding karyotype, immunofluorescence staining (OCT4), pluripotent gene expression (RT-PCR), and embryoid body formation. A total of 1,697 iPS-like colonies (2.34%) were observed. The compact colonies contained with tightly packed cells with distinct border between iPS colony and feeder cells, while colonies with irregular shape and border were classified as loose colonies. These 2 types of iPS-like colonies expressed alkaline phosphatase and OCT4. A total 5 of 10 (50%) compact morphology cell lines could be maintained in the undifferentiated state for more than 50 passages. But only 3 of 10 (30%) loose morphology cell lines demonstrated pluripotent characteristics. G-Banding karyotype analysis revealed normal chromosome number (n = 38). All of the cell lines also expressed endogenous pluripotent genes, including OSKM and NANOG and formed three-dimensional aggregating masses. In this study, we found that the cell lines from compact morphology could be maintained for longer than those of the loose morphology. A high rate of differentiation of loose iPS colony may also indicate that this type of colony has different pluripotency signals or incomplete reprogramming compared with compact colony. In conclusion, selection of compact colony morphology at colony pick-up is simple and reflects long-term propagation of porcine iPS cell lines. This information is important for improving the success rate of establishment and maintenance of porcine iPS cells.

189 SMALL MOLECULE INHIBITORS PD0325901 AND CHIR99021 CAUSE REDUCED EXPRESSION OF PLURIPOTENCY GENES IN PUTATIVE PORCINE INDUCED PLURIPOTENT STEM CELLS

Small molecule inhibitors acting on the MEK and Wnt signalling pathways (PD0325901 and CHIR99021, respectively) have been used successfully for the maintenance of murine and rat pluripotent stem cells in the in vitro culture. The effects of these compounds in other species have not been conclusively determined, and some reports suggest that they may actually cause loss of pluripotency in human embryonic stem cells and decrease of OCT4 expression levels in porcine induced pluripotent stem cells (piPSC). In our experiments, putative piPSC lines derived from transgenic porcine fetal fibroblasts (pFF) and adipose mesenchymal stem cells (pAMSC) that harbor the OCT4-EGFP reporter construct were maintained in piPSC culture medium [DMEM supplemented with 10% Knockout Serum Replacement (Life Technologies, Carlsbad, CA, USA), 10% fetal bovine serum, nonessential amino acids, L-glutamine, penicillin-streptomycin, and 1000 U mL–1 murine leukemia inhibitory factor (LIF)], with or without addition of 1 μM PD0325901 and 3 μM CHIR99021. After five passages in the two experimental culture conditions, three pFF-derived putative piPSC lines were evaluated morphologically and the expression of various pluripotency genes was analysed by real-time quantitative PCR. The results showed decrease of OCT4-EGFP expression and loss of compact colony morphology of the cells cultured in the presence of PD0325901 and CHIR99021. Moreover, the transcriptional expression levels of OCT4, SOX2, NANOG, REX1, UTF1, and TDH were reduced by 12-, 9-, 10-, 3-, 5-, and 20-fold, respectively, compared with the cells cultured without these inhibitors. When putative piPSC derived from pAMSC were cultured in medium supplemented with small molecule inhibitors, the OCT4-EGFP expression was completely lost within a few days. The cells also lost their iPSC-like colony morphology and were further propagated as single, mesenchymal-like cells. The same effects were observed when the cells were cultured with CHIR99021 alone, whereas there were no such changes when we used only PD0325901. This suggests that, similarly to human embryonic stem cells (ESC), the activation of the Wnt pathway in pAMSC-derived iPSC may lead to differentiation in these cells. The effects of the CHIR99021 alone on the pFF-derived piPSC-like cells are still to be determined. In conclusion, the results of our preliminary investigations call into question the effectiveness of PD0325901 and CHIR99021 in the maintenance of piPSC in culture.

Derivation of three new human embryonic stem cell lines

Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines.

The Mll-AF9 Fusion Gene Results in Overexpression of Homeobox Genes and Deregulation of Granulocyte-Monocyte Progenitors.

Our understanding of the biology of MLL fusion gene leukemias is limited by the lack of knowledge of the effects of the different MLL fusion genes on expression of specific homoebox genes and the specific cell compartment(s) that are subsequently deregulated. In this study we investigated whether cellular deregulation was present in committed myeloid precursors and/or the multi-potent hematopoietic stem cells derived from Mll-AF9 knock-in mice. We used the murine knock-in model since it offers the advantage of a single copy of the Mll fusion gene under the control of the endogenous promoter that is present in every hematopoietic stem/progenitor cell. The Mll-AF9 knock-in mice display expansion of the myeloid compartment as early as 6 weeks of age (young adult) and develop myeloid leukemia at approximately 6 months. We purified hematopoietic stem cells (HSCs) and granulocyte-monocyte progenitors (GMPs) from wild type and Mll-AF9 young adult bone marrow. We depleted lineage positive cells using a magnetic separation system and purified the respective populations using fluorescence activated cell sorting with specific panels of antibodies (HSC=Li−/Thy1.1lo/IL-7R−/C-kit+/Sca-1+; GMP=Lin−/IL-7R−/Sca-1+/C-kit+/CD34+/CD16/32hi). We cultured these cells in methylcellulose supplemented with GM-CSF, IL-3, SCF and IL-6, conditions that promote the growth of myeloid colonies. We assessed growth deregulation by increased colony numbers at the end of 7 days of culture and by the predominance of dense, compact colony morphology, the latter comprised of immature myeloid cells. Culture of HSCs from Mll-AF9 and wild type mice yielded an identical number of colonies (1102 and 1315 colonies per 104 cells respectively, average). In contrast, GMPs from Mll-AF9 mice yielded almost four times the number of colonies compared to wild type GMPs (3331 and 920 colonies per 104 cells respectively, average). Additionally, Mll-AF9 GMPs formed a higher number of dense, compact colonies compared to Mll-AF9 HSCs (1314 and 352 colonies per 104 cells respectively, average). Neither HSCs nor GMPs from wild type mice formed dense, compact colonies. These results indicate a greater deregulation of GMPs compared to HSCs in Mll-AF9 mice. MLL fusion gene leukemias are characterized by over-expression of specific homeobox genes, and we have previously shown that Mll-AF9 bone marrow cells display increased expression of 5′ Hox-a genes and of the Hox co-factor Meis1 compared to wild type counterparts. We hypothesized that these genes are over-expressed in Mll-AF9 GMPs compared to wild type GMPs. Real time quantitative RT-PCR showed that expression levels of Hoxa7, Hoxa9 and Meis1 were increased in Mll-AF9 GMPs compared to wild type (2.7 ± 0.8, 11.7 ± 7.8 and 19 ± 11.3 fold respectively, mean ± SEM). Overall, these data support the hypothesis that the Mll-AF9 gene is “instructive” at the molecular level at least in part via specific homeobox gene over-expression, resulting in deregulation and expansion of specific progenitor/stem cells such as the GMP population. This expanded GMP population then becomes a target for secondary mutations and later development of leukemia. Future studies focused on understanding the biology of this compartment in Mll-AF9 mice will help in our understanding of the pathogenesis of leukemia and aid in the development of newer, more effective therapies.

Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates

To study the relation between the number of hyphal tips and protein secretion during growth on a solid substrate, we have constructed two mutant strains of Aspergillus oryzae with increased hyphal branching. We have analysed hydrolytic enzyme activities during growth on wheat kernels (WK) of A. oryzae strains carrying the disrupted allele of the pclA gene encoding a secretion pathway specific (KEX2-like) endo-protease and the disrupted allele of the pg/pi-tp gene encoding a phosphatidylglycerol/phosphatidylinositol transfer protein. The biomass levels produced by the pclA and pg/pi-tp disrupted strains on wheat-based solid media were similar as found for the wild-type strain. However, the pclA disrupted strain showed much more compact colony morphology than the other two strains. Sporulation of the pclA and pg/pi-tp disrupted strains occurred, respectively, 2 days and 1 day later, compared to the wild type during fermentation on ground WK. During surface growth, microscopic analysis revealed that the hyphal growth unit length (L (hgu)) of the pclA and pg/pi-tp disrupted strains was, on average, 50 and 74% of that of the wild-type strain. This implies that in both mutant strains, a higher branching frequency occurs than in the wild-type strain. Compared to the wild-type strain, the pclA and pg/pi-tp disrupted strains produced at least 50% more amylase, at least 100% more glucoamylase and at least 90% more protease activity levels after growth on WK. These results support the hypothesis that branching mutants with an increased branching frequency can improve the solid state fermentation process.

Drosophila as a model host for Pseudomonas aeruginosa infection.

Using the fruit fly Drosophila melanogaster as model host, we have identified mutants of the bacterium Pseudomonas aeruginosa with reduced virulence. Strikingly, all strains strongly impaired in fly killing also lacked twitching motility; most such strains had a mutation in pilGHIJKL chpABCDE, a gene cluster known to be required for twitching motility and potentially encoding a signal transduction system. The pil chp genes appear to control the expression of additional virulence factors, however, since the wild-type fly-killing phenotype of a subset of mutants isolated on the basis of their compact colony morphology indicated that twitching motility itself was not required for full virulence in the fly.

Symbiotic and galactose utilization properties of phage RMP64-resistant mutants affecting three complementation groups inRhizobium meliloti

Random Tn5 insertional mutants were induced inRhizobium meliloti Rmd201, a streptomycin-resistant mutant of AK631 (which is itself a compact colony morphology mutant of the wild-type strain Rm41), and screened for sensitivity to a set of 16 phages. Out of 3000 mutants 240 were found to be phage-resistant. The phage-resistant mutants were separable into six groups on the basis of their sensitivity pattern against test phages. Nodulation tests on alfalfa showed that although all the phage-resistant mutants induced root nodules, 7 mutants out of 12 of a class resistant to phage RMP64 (Sxf-) induced atypical nodules that were ineffective in nitrogen fixation (Fix-). The aberrant nodules were small, white, contained only a few bacteria and no bacteroids, and phenotypically resembled nodules elicited by already knownexoB, exoH, ndvA andndvB mutants ofR. meliloti. Spontaneous mutants selected for resistance to RMP64 also fell into two groups: Fix+ and Fix-. Genetic complementation tests between the Sxf- mutants defined three genessxfA, sxfB andsxfC, of whichsxfA andsxfB comprise an operon. These also demonstrated thatsxfA, sxfB andsxfC must be located on the same replicon. All the Sxf- mutants were Calcofluor-positive, like their parent strains Rmd201 and AK631. Characterization of carbohydrate metabolism of the mutants revealed that while thesxfA (Fix-) andsxfB (Fix+) mutants utilized galactose as sole carbon source,sxfC (Fix-) mutants did not. It has been concluded thatsxf A, sxfB andsxfC are new genetic loci and thatsxfA andsxfC have roles in nodule invasion and development.

Differentiation of Abelson murine leukemia virus-infected promonocytic leukemia cells.

Virus-producing, tumorigenic, promonocytic leukemia cell lines were derived from Abelson murine leukemia virus-infected mice. This study shows that, of these 25 cloned lines, 22 were capable of extensive differentiation. It also shows that granulocyte-macrophage colony-stimulating activity increased the proportion of differentiating cells in 17/22 lines. Cells from agar colonies with a diffuse colony morphology had an increased expression of mature macrophage phenotypic characteristics, and a reduced proliferative capacity in vitro, compared to cells from agar colonies with a compact colony morphology. Cells from diffuse colonies also produced less Abelson virus, and were less tumorigenic in vivo than cells from compact colonies. Together, these results suggest some Abelson virus-producing leukemic cells are not blocked in their capacity to differentiate and are capable of reversing the transformed phenotype.

Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes.

Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured in reduced serum with or without additives (1 microgram/ml insulin, 3 X 10(-7) M linoleic acid, 1 X 10(-8) M H2SeO3) or bovine serum albumin (BSA, 1% wt/vol). With the additives and less than or equal to 0.5% fetal bovine serum (FBS), Ham's F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle's minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology. In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5% FBS; in Ham's F12, 1% FBS + deoxycytidine + BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at the aprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethylnitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels.