Using Fucci System in mBMSC

BMSC generate heterotopic ossicles and are capable of establishing the hematopoietic environment in vivo (Sacchetti et al. 2007). Thus the capacity to establish bone and hematopoietic microenvironment (HME) by the transplantation of M2 cells was evaluated. This experiment was performed by Dr Benedetto Sacchetti (Department of Molecular Medicine, Sapienza University, Rome, Italy). To assess the osteogenic potential, scaffolds of osteoconductive material (hydroxyapatite/tricalcium phosphate particles, HA/TCP) loaded with M2 cells in early (P8-10) or late (P40-44) passages were generated. When transplanted subcutaneously into immunocompromised mice, early- and late-derived M2 cell strains were able to generate complete heterotopic ossicles establishing full bone, adipocytes, vessels and host derived, heterotopic hematopoietic tissue clusters (see Figure 15) proving that the M2 cell line possesses full skeletal stem cell characteristics in vivo.

Figure 16 Phenotype of isolated Fucci-mBMSC

(A) Cell cycle dependent expression of mKO2 (G1/s-phase) and mAG (S-and G2/M-phase) (Sakaue-Sawano et al. 2008). (B) Representative images of a time-lapse movie. At the indicated times cell cycle progression of one representative cell can be seen. White arrow indicate cell.

Cells were isolated as previously described for wild type C57Bl6 mice. To verify successful BMSC isolation multipotency was tested. The isolated Fucci-BMSC were capable of differentiation towards osteogenic, adipogenic, and chondrogenic lineage,

proving multipotency (see Figure 17A). With these Fucci-BMSCs a model system was generated to assess cell cycle regulation in multipotent cells with stem cell characteristics.

Figure 17 Phenotype and multipotency of isolated Fucci-mBMSC

(A) Differentiation of bone marrow stromal cells isolated from a Fucci-transgenic mouse. BMSC were cultured in vitro in adipogenic, osteogenic, or chondrogenic media to assess multilineage differentiation capability. The cells were fixed and stained with Alizarin Red (osteogenesis), Oil Red O (adipogenesis) or Alcian Blue (chondrogenesis) for osteoblast, adipocyte, or chondrocyte

differentiation. For adipogenic and osteogenic differentiation cells cultured in standard growth medium (control) served as a control. For chondrogenic differentiation cells in chondrogenic media without TGF-ß3 served as a control. (B) left: representative microscopic images of cells stained with giantin.

Pictures were inverted for better visualization of Golgi morphology. Right panel: red, yellow and green cell nuclei showing typical Fucci-dependent fluorescence. Blue: giantin with Alexa 633.

Next, the cell cycle state of single cells was analyzed by fluorescence microscopy.

Additionally, Golgi morphology was analyzed and brought in relation to cell cycle phases (see Figure 17B).

Figure 18 Effects of PKD und MEK inhibitor on asynchronous Fucci-BMSC

Fucci-BMSC were incubated with (A) culture medium for 24 h, (B) culture medium supplemented with 10 µM PKD inhibitor CID755673 (C) or culture medium supplemented with 10 µM MEK-inhibitor UO126. The cells were fixed and stained with giantin to visualize the Golgi complex. Percentage of cells with red, green, and colourless nuclei was determined in microscopic images. In each category Golgi morphology was determined. 60-150 cells were analyzed for each condition n=2 for (A) and (B), n=1 for (C).

The influence of PKD and MEK specific inhibitors on cell cycle phase length was investigated. Surprisingly nearly 40% of cell nuclei in untreated control cells did not show detectable fluorescence. However, the remaining 60% of the cell population showed green and red fluorescence indicative of G1 and S/G2/M, phase respectively (see Figure 18A). Interestingly, treatment of Fucci-BMSC with the PKD inhibitor CID755673 for 24 h increased the percentage of cells with green nuclei indicating a prolonged S/G2/M phase. A decreased number of cells in S/G2/M harboured a fragmented Golgi complex compared to control cells in S/G2/M (see Figure 18A & B).

However, this is also the case for cells in G1 phase. Treatment with the MEK inhibitor UO126 did not result in an increased number of cells with green nuclei. Notably, under these conditions, almost all cells in green phase had an intact Golgi complex compared to control cells in S/G2/M phase (see Figure 18C).

One disadvantage of the analysis of asynchronous cells is that inhibition of the different kinases affects the cells in all phases of cell cycle. To analyze the impact of PKD and MEK activity specifically during the S/G2/M phase, the cells had to be synchronized. First, different synchronization strategies were tested in Fucci-BMSC.

Figure 19 Synchronisation of Fucci-mBMSC

(A) Synchronisation of Fucci-BMSCs with the CDK1-Inhibitor RO 3306 (9 µM). (B) Synchronisation and release of Fucci-BMSCs with DNA-polymerase inhibitor aphidicolin (5 µg/ml). Cells were fixed and microscopic images were used to analyze nucleus specific fluorescence. 50 cells were analyzed for each condition.

The incubation of Fucci-BMSCs with the CDK1 inhibitor RO3306 resulted in an increase in cells in green phase (S/G2/M). However, most of the cells died during this treatment. After releasing the cells for 24 h all remaining cells died. Thus, synchronization with a CDK1 inhibitor was not tolerated by Fucci-BMSC (see Figure 19A). Treatment with the DNA polymerase inhibitor aphidicolin also led to an

increase of cells in S/G2/M phase. After the release, part of the cells progressed through S/G2/M phase, which can be seen in the increase in G1 phase cells 24 hours later (see Figure 19B). Similarly to treatment with RO3306, aphidicolin showed toxic effects and cells died during synchronization and also during release. Finally, cells were synchronized by 24 hours of serum starvation resulting in a nearly complete cell cycle arrest in G1. However, after the release all cells died (data not shown). Taken together, a successful synchronization of Fucci-BMSC was unfortunately not possible.

Alternatively, the effect of PKD and MEK inhibitors specifically in S/G2/M phase was investigated by live cell imaging experiments.

Figure 20 Live cell experiments und manual tracking of Fucci-BMSC

Representative live cell frame showing examples of cell cycle phase length verification by manual tracking.

To compare data 4-well chambers were used to image different conditions in one experiment. In a first time lapse movie cells incubated without and with 10 µM UO126 were imaged for 18 hours. A picture was taken every 10 minutes. The single cells were manually tracked throughout the movie (see Figure 20). Figure 21 shows all analyzed cells.

Figure 21 Result of live cell experiment

Graphical results of manual tracking of an 18 hours live cell movie in control and 10 µM UO126 treated Fucci-BMSC. The colours indicate the red (G1) and green (S/G2/M) phase. Black highlighted bars represent cells that died during observation.

It was obvious that a higher percentage of cells died during observation time in the MEK inhibited population. Notably, the average phase length was longer for MEK inhibited cells for both red and green phase. In line with this, there was less cell division in this population compared to control cells (see Figure 22A).

Figure 22 Analysis of live cell imaging with Fucci-BMSC

(A) Population based evaluation of cell cycle phase length and cell divisions per cell number of manual tracking data. (B) Graphical display of entire cell cycles during 28 hours of observation in live cell imaging Fucci-BMSC

Because MEK inhibition caused a delay in both, G1 and S/G2/M phases, it can be suggested that MEK has important functions in the cell cycle in Fucci-BMSC.

Nevertheless, cells did not tolerate the MEK inhibitor very well resulting in enhanced cell death. The effect of PKD inhibition was analyzed in a second 28 hours live cell imaging experiment. An effect of PKD inhibition on G1 phase was not observed. Most importantly, the S/G2/M phase was strongly prolonged pointing to an involvement of PKD in this phase (see Figure 22A & B).

To get more robust primary cells tolerating the inhibitors, Fucci-MEF were isolated and immortalized by Dr Kornelia Ellwanger. Similarly to Fucci-BMSC, the expression of red and green tagged proteins was only visible in some cells. Heterogenous expression was detected in living and fixed cells confirming that a potential loss of the dye by fixation is not likely (see Figure 23). Instead, a heterogenic or too weak expression of the fusion proteins mKO2-Cdt1 and mAG-Geminin seems to be a possible reason. Because only 15-20% of the Fucci-MEF cells were showing detectable amounts of fluorescence (data not shown), further experiments with these cells were not conducted.

Figure 23 Phenotype of Fucci-MEF

(A) Comparison of living and fixed Fucci-MEF. Scale bar represents 200 µm.