Vaibhav Jadhav

Dynamic mRNA and miRNA profiling of CHO‐K1 suspension cell cultures

In spite of the importance of Chinese hamster ovary (CHO) cells for recombinant protein production, very little is known about the molecular and gene regulatory mechanisms that control cellular phenotypes such as enhanced growth under serum‐free conditions or high productivity. Most microarray analyses to this purpose are performed with samples taken during the exponential growth phase. However, the cellular transcriptome is dynamic, changing in response to external and internal stimuli and thus reflecting the current functional capacity of cells as well as their ability to adapt to a changing environment. Therefore, during batch or fed‐batch cultivations it can be expected that the transcription pattern of genes will change and that such changes may give indications on the cellular state in terms of viability, growth, and productivity. In the current study we monitored the change in expression patterns of mRNAs and microRNAs (miRNA) during lag, exponential, and stationary phases in CHO‐K1 suspension cell cultures. In total, over 1400 mRNAs and more than 100 miRNAs were differentially regulated (p<0.05) relative to the batch culture at the starting point. Functional clustering revealed groups of genes with similar expression patterns, which were subjected to functional pathway analysis. In addition, as miRNAs generally act as negative post‐transcriptional regulators of mRNAs, we looked for changes in their expression that were inverse to those of their predicted target mRNAs.

CHO microRNA engineering is growing up : recent successes and future challenges

microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines.

Stable overexpression of miR-17 enhances recombinant protein production of CHO cells

Highlights • Transient overexpression of miR-17 and miR-17–92 cluster enhanced growth rate.• Biological effects of long term and stable overexpression of miRNAs in batch cultures were studied.• Stable miR-17 engineered CHO cells had both improved growth rate and productivity.

A screening method to assess biological effects of microRNA overexpression in Chinese hamster ovary cells.

MicroRNAs (miRNAs) are a novel class of short non‐coding RNAs, which negatively regulate target gene expression at post‐transcriptional level. They mediate an important layer of control in the global regulation of gene networks, controlling a broad range of physiological as well as patho‐physiological pathways including development, cancer, metabolism, proliferation, and stress resistance. So far, more than 365 miRNA genes have been identified in CHO cells. The functional analysis of the physiological effect of such large numbers of miRNAs, however, requires an efficient functional screening method. In the current study, we therefore established and evaluated a protocol to perform miRNA overexpression and to screen their effect on bio‐industrially relevant phenotypes, such as growth, viability and productivity, using a recombinant, Epo‐Fc producing CHO cell line. For protocol optimization, four CHO miRNAs (cgr‐miR‐17, cgr‐miR‐221, cgr‐miR‐21, and cgr‐miR‐210) were cloned into small hairpin vectors including a GFP cassette and transfected. After transfection cells were analyzed for growth and productivity over a 4‐day period. Even from this small set of four miRNAs, the overexpression of miR‐17, one of the members of the oncogenic miR‐17‐92 cluster, gave proof of principle that this method enables the identification of miRNA engineering candidates as its overexpression increased the speed of cell proliferation without negatively impacting specific productivity. The here presented method is applicable for medium‐throughput screening for microRNA, miR‐sponge, siRNA, or mRNA overexpression along with detailed functional characterization using the same experimental set up. As the same procedure can be applied to different production cell lines, the protocol can also be used to test for individual, cell line specific responses to microRNAs. Thus our system represents a general platform to functionally screen candidates for rational cell factory design. Biotechnol. Bioeng. 2012; 109:1376–1385.

Computational identification of microRNA gene loci and precursor microRNA sequences in CHO cell lines

Highlights ► We mapped all known mature CHO miRNAs to two CHO-K1 reference genomes. ► 212 unique genomic miRNA loci and the respective precursor miRNA sequences were identified. ► The genomic loci of 4 polycistronic miRNA cluster were confirmed by PCR. ► The identified sequences were analyzed for SNPs and conservation compared to mouse. ► Sequence data have been prepared for submission to miRBase miRNA sequence repository.

Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time

The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. To investigate the relative contribution of genomic and epigenetic modifications towards phenotype evolution, comprehensive genome and epigenome data are presented for six related CHO cell lines, both in response to perturbations (different culture conditions and media as well as selection of a specific phenotype with increased transient productivity) and in steady state (prolonged time in culture under constant conditions). Clear transitions were observed in DNA‐methylation patterns upon each perturbation, while few changes occurred over time under constant conditions. Only minor DNA‐methylation changes were observed between exponential and stationary growth phase; however, throughout a batch culture the histone modification pattern underwent continuous adaptation. Variation in genome sequence between the six cell lines on the level of SNPs, InDels, and structural variants is high, both upon perturbation and under constant conditions over time. The here presented comprehensive resource may open the door to improved control and manipulation of gene expression during industrial bioprocesses based on epigenetic mechanisms. Biotechnol. Bioeng. 2016;113: 2241–2253.

Endogenous microRNA clusters outperform chimeric sequence clusters in Chinese hamster ovary cells

MicroRNAs (miRNAs) are small non-coding RNAs (∼22 nucleotides) which regulate gene expression by silencing mRNA translation. MiRNAs are transcribed as long primary transcripts, which are enzymatically processed by Drosha/Dgcr8, in the nucleus, and by Dicer in the cytoplasm, into mature miRNAs. The importance of miRNAs for coordinated gene expression is commonly accepted. Consequentially, there is a growing interest in the application of miRNAs to improve phenotypes of mammalian cell factories such as Chinese hamster ovary (CHO) cells. Few studies have reported the targeted over-expression of miRNAs in CHO cells using vector-based systems. These approaches were hampered by limited sequence availability, and required the design of “chimeric” miRNA genes, consisting of the mature CHO miRNA sequence encompassed by murine flanking and loop sequences. Here we show that the substitution of chimeric sequences with CHO-specific sequences for expression of miRNA clusters yields significantly higher expression levels of the mature miRNA in the case of miR–221/222 and miR–15b/16. Our data suggest that the Drosha/Dgcr8-mediated excision from primary transcripts is reduced for chimeric miRNA sequences compared to the endogenous sequence. Overall, this study provides important guidelines for the targeted over-expression of clustered miRNAs in CHO cells. See accompanying commentary by Baik and Lee DOI: 10.1002/biot.201300503

Characterization of a novel cell penetrating peptide derived from human Oct4

BackgroundOct4 is a transcription factor that plays a major role for the preservation of the pluripotent state in embryonic stem cells as well as for efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) or other progenitors. Protein-based reprogramming methods mainly rely on the addition of a fused cell penetrating peptide. This study describes that Oct4 inherently carries a protein transduction domain, which can translocate into human and mouse cells.ResultsA 16 amino acid peptide representing the third helix of the human Oct4 homeodomain, referred to as Oct4 protein transduction domain (Oct4-PTD), can internalize in mammalian cells upon conjugation to a fluorescence moiety thereby acting as a cell penetrating peptide (CPP). The cellular distribution of Oct4-PTD shows diffuse cytosolic and nuclear staining, whereas penetratin is strictly localized to a punctuate pattern in the cytoplasm. By using a Cre/loxP-based reporter system, we show that this peptide also drives translocation of a functionally active Oct4-PTD-Cre-fusion protein. We further provide evidence for translocation of full length Oct4 into human and mouse cell lines without the addition of any kind of cationic fusion tag. Finally, physico-chemical properties of the novel CPP are characterized, showing that in contrast to penetratin a helical structure of Oct4-PTD is only observed if the FITC label is present on the N-terminus of the peptide.ConclusionsOct4 is a key transcription factor in stem cell research and cellular reprogramming. Since it has been shown that recombinant Oct4 fused to a cationic fusion tag can drive generation of iPSCs, our finding might contribute to further development of protein-based methods to generate iPSCs.Moreover, our data support the idea that transcription factors might be part of an alternative paracrine signalling pathway, where the proteins are transferred to neighbouring cells thereby actively changing the behaviour of the recipient cell.

Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting

Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here, we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO‐S, CHO‐K1 8 mM glutamine, and CHO‐K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO‐S and CHO‐K1, with the diversity increasing and new variants appearing, while CHO‐K1 0 mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture.

Analysis of microRNA transcription and post-transcriptional processing by Dicer in the context of CHO cell proliferation

Highlights • The expression of Dicer is correlated to growth rate in different CHO cell lines.• Global perturbation of microRNA levels via DICER knockdown or overexpression directly influences CHO growth behavior.• This provides strong evidence that microRNAs are key growth regulators in CHO cell lines.