Chromosomal Number Aberrations and Transformation in Adult Mouse Retinal Stem Cells In Vitro

Purpose. The potential of stem cells (SCs) as a source for cell-based therapy on a wide range of degenerative diseases and damaged tissues such as retinal degeneration has been recognized. Generation of a high number of retinal stem cells (RSCs) in vitro would thus be beneficial for transplantation in the retina. However, as cells in prolonged cultivation may be unstable and thus have a risk of transformation, it is important to assess the stability of these cells. Methods. Chromosomal aberrations were analyzed in mouse RSC lines isolated from adult and from postnatal day (PN)1 mouse retinas. Moreover, selected cell lines were tested for anchorage-dependent proliferation, and SCs were transplanted into immunocompromised mice to assess the possibility of transformation. Results. Marked aneuploidy occurred in all adult cell lines, albeit to different degrees, and neonatal RSCs were the most stable and displayed a normal karyotype until at least passage 9. Of interest, the level of aneuploidy of adult RSCs did not necessarily correlate with cell transformation. Only the adult RSC lines passaged for longer periods and with a higher dilution ratio underwent transformation. Furthermore, we identified several cell cycle proteins that might support the continuous proliferation and transformation of the cells. Conclusions. Adult RSCs rapidly accumulated severe chromosomal aberrations during cultivation, which led to cell transformation in some cell lines. The culture condition plays an important role in supporting the selection and growth of transformed cells.

Multidisciplinary Analysis of the Metabolic Shift to Lactate Consumption in CHO Cell Culture

Francesca Zagari

Mammalian cells represent the most widely used host system for the industrial production of recombinant therapeutic proteins. In order to increase productivity, chemically defined media are often used and optimized to provide the cells with the necessary nutrients. However, a deregulated cell metabolism is often observed in these culture conditions. In particular, carbon sources are fast and inefficiently consumed with a consequent accumulation of byproducts, mainly lactate and ammonia. Lactate, in particular, causes a decrease of the medium pH which can be detrimental for cells growth and productivity. Its metabolic profile is normally characterized by a first production phase, which is associated to the cells exponential growth. Afterwards, when the cells enter into the stationary phase, a shift to net lactate consumption can occur under optimal culture conditions. However, such a metabolic shift is not easily controlled, since the mechanisms modulating lactate production in cell culture are still under investigation. This work aims to understand which factors could influence the lactate metabolic shift in CHO cell culture, focusing, in particular, on the mitochondrial role. To this purpose, cell lines with opposite lactate profiles were compared. The initial lactate production phase was common to all the fast growing cultures and was concomitant to a rapid glutamine consumption. After glutamine depletion, two different scenario occurred. In one case, the cells started to consume lactate until complete depletion. Alternatively, lactate continued to be accumulated, causing a decrease of media pH. The mitochondrial oxidative capacity was hypothesized as a possible cause of the different lactate profile. Indeed, mitochondrial membrane potential and oxygen consumption measurements highlighted a correlation between a reduced oxidative metabolism and a state of high lactate production. This correlation was confirmed by evaluating other cell lines or media compositions which resulted in the same divergence of lactate metabolism. Afterwards, the expression of selected genes was analysed in correlation with the observed lactate profile. Among 22 genes, two were identified as significantly downregulated (absolute fold change ≥2) in conditions of high lactate accumulation; namely, the mitochondrial aspartate-glutamate carrier (aralar1) and the translocase of the inner mitochondrial membrane 8 (timm8a). Aralar1, in particular, is an important component of the malate-aspartate shuttle (MAS). This system promotes the recycling of the cytosolic NADH pool, which is necessary for maintaining the glycolytic flux. Alternatively, NADH can be oxidised through pyruvate conversion into lactate, catalysed by the lactate dehydrogenase enzyme. Therefore, an inefficient MAS activity can engender an increased lactate accumulation. Finally, stable cell lines, which overexpressed aralar1 or timm8a, were generated. Clones derived from a lactate-producing cell line showed an improvement of lactate metabolism. In particular, they switched more easily to lactate consumption compared to the untransfected control, while maintaining a similar glucose consumption rate. On the other hand, no impact of the transgene expression was observed in the clones derived from the lactate-consuming cell line, since the switch to lactate consumption was maintained and no other effect on cell growth or glucose metabolism was detected. The data obtained indicate that lactate consumption was most likely promoted by a better NADH oxidation through the malate-aspartate shuttle, which resulted in a more efficient link between glycolysis and the mitochondrial oxidative metabolism. In conclusion, the results presented in this thesis indicate that the mitochondrial oxidative capacity plays a central role in the control of lactate production. Media composition and intrinsic cell characteristics have both an impact on mitochondrial activity. Moreover, the expression of specific genes can also be influenced by the culture media. In particular, aralar1 and timm8a have been identified as promising targets for the generation of a host cell line with an improved lactate metabolism.

EPFL2012

Quantitative analysis of SOX2 and OCT4 expression in pluripotency and differentiation

Daniel Strebinger

Embryonic stem (ES) cells have the capacity to give rise to all cell types of the adult organism and can be used as a model to study early cell fate decisions as they closely recapitulate in vivo events. The M and G1 phases have been suggested to be the key time windows for pluripotent cells to engage toward differentiation and SOX2 and OCT4 have been shown to orchestrate the dissolution of pluripotency and induction of cell fate commitment. Furthermore, it is known that the levels of some pluripotency transcription factors fluctuate over time, leading to the notion that ES cells experience dynamic heterogeneity, where for example low levels of NANOG can be found in cells prone to differentiation, whereas cells with high NANOG expression are in a robust pluripotent state. However, it remains unclear if and how (i) the action of transcription factors in specific cell cycle phases and (ii) protein level variations of factors that show mild differences between single cells impact cell fate decisions. This is in part due to the lack of quantitative single-cell meth-ods allowing dissecting the origin and functional consequences of gene expression heterogeneity. In this work, we established a method based on internal tagging of endogenous proteins with an excisable reporter to perform absolute molecule quantification in single living cells. This enabled us to investigate fluctuations of endogenous protein expression levels, the heterogeneity of degradation rates between individual cells and determine absolute amounts of proteins synthesized over the cell cycle. Additionally, inducible excision of the tag enabled us to estimate endogenous mRNA half-lives. Next, by using live-cell microscopy of fusion proteins, we show that SOX2 and OCT4 stay bound to mitotic chromosomes through their DNA binding domains. We then characterized the function of SOX2 at the M-G1 transition, showing that its absence specifically in this phase impairs pluripotency maintenance and abolishes its ability to induce neuroectodermal commitment. This suggests, that transcription factor activity at the M-G1 transition is essential for cell fate maintenance and differentiation. Lastly, we show that mild endogenous fluctuations in the levels of key transcription factors in ES cells bias cell fate decisions. We found that high OCT4 levels at the onset of differentiation increase the probability of single cells to commit to both neuroectoderm and mesendoderm. Further, we showed that high SOX2 levels result in a higher number of cells acquiring a neuroectodermal cell fate. This not only shows that the differentiation potential of embryonic stem cells is highly sensitive to small variations in intracellular concentrations of pluripotency transcription factors but further suggests that endogenous fluctuations of these are a major source of heterogeneity in cell fate decisions. In conclusion, the presented work identifies that the SOX2 activity in the M-G1 transition is important for cell fate maintenance and differentiation. Furthermore, the heterogeneity of protein levels governs cell fate decisions, suggesting the existence of different short-lived cell states in populations of presumed homogenous cells. We think that the minor differences in the levels of transcription factors and the cell cycle specific sensitivity of cells to transcription factor activity could be important mechanisms guiding maintenance and differentiation potential in various stem cell types.

EPFL2018

In vivo pulse labeling of isochronic cohorts of cells in the central nervous system using FlashTag

Subashika Govindan

The tracing of neuronal cell lineages is critical to our understanding of cellular diversity in the CNS. This protocol describes a fluorescence birth-dating technique to label, track and isolate isochronic cohorts of newborn cells in the CNS in vivo in mouse embryos. Injection of carboxyfluorescein esters (CFSEs) into the cerebral ventricle allows pulse labeling of mitotic (M phase) ventricular zone (VZ) progenitors and their progeny across the CNS, a procedure we termed FlashTag. Specificity for M-phase apical progenitors is a result of the somata of these cells transiently contacting the ventricular wall during this cell-cycle phase, exposing them to CFSE injected into the cerebrospinal fluid. Using this approach, the developmental trajectory of progenitors and their daughter neurons can be tracked. Labeled cells can be imaged ex vivo or in fixed tissue, targeted for electrophysiological experiments or isolated using FACS for cell culture or (single-cell) RNA sequencing. Multiple embryos can be labeled within 30 min. The dye is retained for several weeks, allowing labeled cells to be identified postnatally. This protocol describes the labeling procedure using in utero injection, the isolation of live cells using FACS and the processing of labeled tissue for immunohistochemistry.