Enabling stem cell based therapies: Adaptable and scalable manufacturing of human pluripotent stem cells

Enabling stem cell-base therapies requires innovative solutions to close the gaps which exist between research and commercialization. Allogeneic cell therapy indications that target large patient populations will necessitate the use of flexible cell production platforms to meet required cell quantities. Here we will show how moving away from conventional 2D culture platforms and developing a truly scalable, controlled bioreactor platforms for cell expansion enables meeting cell quantity demand for clinical applications while allowing comparability between the various scales. Likewise, it enhances process automation and allows integration of online monitoring systems. These bioreactor platforms are flexible cell production platforms, applicable to various cell types. Utilizing many common components, such as bioreactor controllers and centralized up-stream and down-stream hardware, while being able to quickly and easily change components such as vessels, media and microcarriers. The capability of effectively culturing adherent stem cells, namely pluripotent stem cells, will be presented. Cells are expanded in suspension, in a controlled bioreactor, obtaining high fold expansion without compromising cell quality, and the capacity to be further differentiated. This achieved through avoiding 2D cell culture steps, reduces footprint, labor and cost, while enhancing process control and cell product quality

Characterization of Oxidative Guanine Damage and Repair in Mammalian Telomeres

8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) are among the most common oxidative DNA lesions and are substrates for 8-oxoguanine DNA glycosylase (OGG1)–initiated DNA base excision repair (BER). Mammalian telomeres consist of triple guanine repeats and are subject to oxidative guanine damage. Here, we investigated the impact of oxidative guanine damage and its repair by OGG1 on telomere integrity in mice. The mouse cells were analyzed for telomere integrity by telomere quantitative fluorescence in situ hybridization (telomere–FISH), by chromosome orientation–FISH (CO–FISH), and by indirect immunofluorescence in combination with telomere–FISH and for oxidative base lesions by Fpg-incision/Southern blot assay. In comparison to the wild type, telomere lengthening was observed in Ogg1 null (Ogg1−/−) mouse tissues and primary embryonic fibroblasts (MEFs) cultivated in hypoxia condition (3% oxygen), whereas telomere shortening was detected in Ogg1−/− mouse hematopoietic cells and primary MEFs cultivated in normoxia condition (20% oxygen) or in the presence of an oxidant. In addition, telomere length abnormalities were accompanied by altered telomere sister chromatid exchanges, increased telomere single- and double-strand breaks, and preferential telomere lagging- or G-strand losses in Ogg1−/− mouse cells. Oxidative guanine lesions were increased in telomeres in Ogg1−/− mice with aging and primary MEFs cultivated in 20% oxygen. Furthermore, oxidative guanine lesions persisted at high level in Ogg1−/− MEFs after acute exposure to hydrogen peroxide, while they rapidly returned to basal level in wild-type MEFs. These findings indicate that oxidative guanine damage can arise in telomeres where it affects length homeostasis, recombination, DNA replication, and DNA breakage repair. Our studies demonstrate that BER pathway is required in repairing oxidative guanine damage in telomeres and maintaining telomere integrity in mammals

Translational accuracy: Ribosomal protein -protein interactions and stop codon recognition by variant-code release factors

Maintenance of accuracy during translation is important for optimal fitness of the cell. The protein synthetic machinery of the cell, including the ribosome, ensures fidelity during decoding. Structural components of the ribosome play an important role in maintaining fidelity. Decoding is also affected by trans-acting factors such as tRNAs, elongation factors and peptide release factors. The concentration of substrates probing the A site also influences the rate of decoding. Slow decoding at the A site not only increases misreading but also promotes the occurrence of alternate recoding events like frameshifting. To understand how different factors affect translational accuracy, I have studied the roles of ribosomal protein-protein interactions near the decoding center and that of variant code release factors in decoding. The binding of a cognate tRNA at the A site induces a closed conformation of the ribosomal small subunit. The closure of the small subunit was suggested to disrupt the interface between two accuracy modulating proteins rpS4 and rpS5. Mutations in either protein confer an error-prone or ram phenotype. Mutations in these proteins have been proposed to promote disruption of the interface, facilitating domain closure even in the absence of the cognate tRNA leading to error-prone phenotype. I used a yeast two hybrid system to look at the effects of the ram mutations on the interactions between rpS4 and rpS5. My results confirm the predicted interactions between rpS4 and rpS5. But the fact that some of the error-prone mutations do not affect the protein-protein interaction at the interface contradicts the proposed model. I have also studied the efficiency of stop codon recognition by variant-code ciliate chimeric release factors. Stop codon recognition at a shifty stop frameshift site influences frameshifting efficiency. Using a yeast genetic system, I showed that the chimeric yeast release factors with domain 1 replaced by that of Euplotes and Tetrahymena, impaired recognition of all three stop codons. My results support our hypothesis that poor recognition of UAA and UAG stop codons at the shifty stop frameshift site AAA-UAA/UAG-A promotes frequent frameshifting in Euplotes spp

Translational accuracy: Ribosomal protein-protein interactions and stop codon recognition by variant-code release factors

Maintenance of accuracy during translation is important for optimal fitness of the cell. The protein synthetic machinery of the cell, including the ribosome, ensures fidelity during decoding. Structural components of the ribosome play an important role in maintaining fidelity. Decoding is also affected by trans- acting factors such as tRNAs, elongation factors and peptide release factors. The concentration of substrates probing the A site also influences the rate of decoding. Slow decoding at the A site not only increases misreading but also promotes the occurrence of alternate recoding events like frameshifting. To understand how different factors affect translational accuracy, I have studied the roles of ribosomal protein- protein interactions near the decoding center and that of variant code release factors in decoding. The binding of a cognate tRNA at the A site induces a closed conformation of the ribosomal small subunit. The closure of the small subunit was suggested to disrupt the interface between two accuracy modulating proteins rpS4 and rpS5. Mutations in either protein confer an error- prone or ram phenotype. Mutations in these proteins have been proposed to promote disruption of the interface, facilitating domain closure even in the absence of the cognate tRNA leading to error- prone phenotype. I used a yeast two hybrid system to look at the effects of the ram mutations on the interactions between rpS4 and rpS5. My results confirm the predicted interactions between rpS4 and rpS5. But the fact that some of the error- prone mutations do not affect the protein- protein interaction at the interface contradicts the proposed model. I have also studied the efficiency of stop codon recognition by variant- code ciliate chimeric release factors. Stop codon recognition at a shifty stop frameshift site influences frameshifting efficiency. Using a yeast genetic system, I showed that the chimeric yeast release factors with domain 1 replaced by that of Euplotes and Tetrahymena, impaired recognition of all three stop codons. My results support our hypothesis that poor recognition of UAA and UAG stop codons at the shifty stop frameshift site AAA-UAA/ UAG- A promotes frequent frameshifting in Euplotes spp

Translational accuracy: Ribosomal protein-protein interactions and stop codon recognition by variant-code release factors

Maintenance of accuracy during translation is important for optimal fitness of the cell. The protein synthetic machinery of the cell, including the ribosome, ensures fidelity during decoding. Structural components of the ribosome play an important role in maintaining fidelity. Decoding is also affected by trans- acting factors such as tRNAs, elongation factors and peptide release factors. The concentration of substrates probing the A site also influences the rate of decoding. Slow decoding at the A site not only increases misreading but also promotes the occurrence of alternate recoding events like frameshifting. To understand how different factors affect translational accuracy, I have studied the roles of ribosomal protein- protein interactions near the decoding center and that of variant code release factors in decoding. The binding of a cognate tRNA at the A site induces a closed conformation of the ribosomal small subunit. The closure of the small subunit was suggested to disrupt the interface between two accuracy modulating proteins rpS4 and rpS5. Mutations in either protein confer an error- prone or ram phenotype. Mutations in these proteins have been proposed to promote disruption of the interface, facilitating domain closure even in the absence of the cognate tRNA leading to error- prone phenotype. I used a yeast two hybrid system to look at the effects of the ram mutations on the interactions between rpS4 and rpS5. My results confirm the predicted interactions between rpS4 and rpS5. But the fact that some of the error- prone mutations do not affect the protein- protein interaction at the interface contradicts the proposed model. I have also studied the efficiency of stop codon recognition by variant- code ciliate chimeric release factors. Stop codon recognition at a shifty stop frameshift site influences frameshifting efficiency. Using a yeast genetic system, I showed that the chimeric yeast release factors with domain 1 replaced by that of Euplotes and Tetrahymena, impaired recognition of all three stop codons. My results support our hypothesis that poor recognition of UAA and UAG stop codons at the shifty stop frameshift site AAA-UAA/ UAG- A promotes frequent frameshifting in Euplotes spp

Accuracy modulating mutations of the ribosomal protein S4-S5 interface do not necessarily destabilize the rps4-rps5 protein–protein interaction

During the process of translation, an aminoacyl tRNA is selected in the A site of the decoding center of the small subunit based on the correct codon–anticodon base pairing. Though selection is usually accurate, mutations in the ribosomal RNA and proteins and the presence of some antibiotics like streptomycin alter translational accuracy. Recent crystallographic structures of the ribosome suggest that cognate tRNAs induce a “closed conformation” of the small subunit that stabilizes the codon–anticodon interactions at the A site. During formation of the closed conformation, the protein interface between rpS4 and rpS5 is broken while new contacts form with rpS12. Mutations in rpS12 confer streptomycin resistance or dependence and show a hyperaccurate phenotype. Mutations reversing streptomycin dependence affect rpS4 and rpS5. The canonical rpS4 and rpS5 streptomycin independent mutations increase translational errors and were called ribosomal ambiguity mutations (ram). The mutations in these proteins are proposed to affect formation of the closed complex by breaking the rpS4-rpS5 interface, which reduces the cost of domain closure and thus increases translational errors. We used a yeast two-hybrid system to study the interactions between the small subunit ribosomal proteins rpS4 and rpS5 and to test the effect of ram mutations on the stability of the interface. We found no correlation between ram phenotype and disruption of the interface

Connection between stop codon reassignment and frequent use of shifty stop frameshifting

Ciliated protozoa of the genus Euplotes have undergone genetic code reassignment, redefining the termination codon UGA to encode cysteine. In addition, Euplotes spp. genes very frequently employ shifty stop frameshifting. Both of these phenomena involve noncanonical events at a termination codon, suggesting they might have a common cause. We recently demonstrated that Euplotes octocarinatus peptide release factor eRF1 ignores UGA termination codons while continuing to recognize UAA and UAG. Here we show that both the Tetrahymena thermophila and E. octocarinatus eRF1 factors allow efficient frameshifting at all three termination codons, suggesting that UGA redefinition also impaired UAA/UAG recognition. Mutations of the Euplotes factor restoring a phylogenetically conserved motif in eRF1 (TASNIKS) reduced programmed frameshifting at all three termination codons. Mutation of another conserved residue, Cys124, strongly reduces frameshifting at UGA while actually increasing frameshifting at UAA/UAG. We will discuss these results in light of recent biochemical characterization of these mutations

Telomere length and DNA damage foci in <i>Nth1^+/+Tert^−/−</i> and <i>Nth1^−/−Tert^−/−</i> mouse cells.

<p>(A) Q-FISH analysis of bone marrow cells derived from <i>Nth1^+/+ Tert^−/−</i> and <i>Nth1^−/−Tert^−/−</i> mice (n = 8). Representative quantitative measurement of telomere signal intensity is shown in jitter plot displaying complete distribution of telomeres with diverse signal intensity (left panel) and in a combined histogram displaying relative frequency of telomeres plotted against telomere signal intensity (right panel). Bars (in green) denote mean telomere signal intensity. (B) Representative metaphase spreads from indicated genotypes, showing enlarged chromosome ends with or without telomere signals (Normal and SFE, respectively). Arrows depict SFEs. (C) Quantification of SFEs in bone marrow cells with indicated genotypes (8 mice). At least 50 metaphases/sample are counted. Values depict mean values ± SD from each sample. P-values are calculated using a Student's <i>t</i>-test. * represents P = 0.01. (D–E) Percentage of <i>Nth1^+/+ Tert^−/−</i> and <i>Nth1^−/−Tert^−/−</i> cells with various numbers of γ-H2AX foci in the genome and telomeres by IF and IF-telomere FISH analysis, respectively. At least 100 cells/sample are counted. Error bars indicate standard deviation.</p

DNA damage foci in wild-type and <i>Nth1^−/−</i> primary MEFs.

<p>53BP1 foci at genome or telomeres are detected by IF and IF-telomere FISH, respectively. (A) Percentage of wild-type and <i>Nth1^−/−</i> cells with various numbers of 53BP1 foci in the genome. (B) Percentage of wild-type and <i>Nth1^−/−</i> cells with greater than or equal to three 53BP1 foci that colocalize with telomere DNA. APH: cells treated with 0.2 uM aphidicolin for 16 hours. (C) A representative <i>Nth1^−/−</i> cell showing telomeric DNA (red) and 53BP1 foci (green). N = 4 mice, at least 100 cells/sample are counted. Error bars indicate standard deviation. P-values are calculated using a Student's <i>t</i>-test and adjusted using Benjamini-Hochberg False Discovery Rate -controlling method. P-values<0.05 are statistically significant using the above method.</p

A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae

The process of protein synthesis must be sufficiently rapid and sufficiently accurate to support continued cellular growth. Failure in speed or accuracy can have dire consequences, including disease in humans. Most estimates of the accuracy come from studies of bacterial systems, principally Escherichia coli, and have involved incomplete analysis of possible errors. We recently used a highly quantitative system to measure the frequency of all types of misreading errors by a single tRNA in E. coli. That study found a wide variation in error frequencies among codons; a major factor causing that variation is competition between the correct (cognate) and incorrect (near-cognate) aminoacyl-tRNAs for the mutant codon. Here we extend that analysis to measure the frequency of missense errors by two tRNAs in a eukaryote, the yeast Saccharomyces cerevisiae. The data show that in yeast errors vary by codon from a low of 4 × 10−5 to a high of 6.9 × 10−4 per codon and that error frequency is in general about threefold lower than in E. coli, which may suggest that yeast has additional mechanisms that reduce missense errors. Error rate again is strongly influenced by tRNA competition. Surprisingly, missense errors involving wobble position mispairing were much less frequent in S. cerevisiae than in E. coli. Furthermore, the error-inducing aminoglycoside antibiotic, paromomycin, which stimulates errors on all error-prone codons in E. coli, has a more codon-specific effect in yeast