text:
Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators

The majority of mammalian somatic cells maintain a diploid genome. However, some mammalian cell types undergo multiple rounds of genome replication (endoreplication) as part of normal development and differentiation. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). During the normal mitotic cell cycle, geminin and Cdt1 are involved in ‘licensing’ of replication origins, which ensures that replication occurs only once in a cell cycle. Their protein accumulation is directly regulated by two E3 ubiquitin ligase activities, APCCdh1 and SCFSkp2, which oscillate reciprocally during the cell cycle. Although proteolysis-mediated, oscillatory accumulation of proteins has been documented in endoreplicating Drosophila cells, it is not known whether the ubiquitin oscillators that control normal cell cycle transitions also function during mammalian endoreplication. In this study, we used transgenic mice expressing Fucci fluorescent cell-cycle probes that report the activity of APCCdh1 and SCFSkp2. By performing long-term, high temporal-resolution Fucci imaging, we were able to visualize reciprocal activation of APCCdh1 and SCFSkp2 in differentiating TGCs and MKCs grown in our custom-designed culture wells. We found that TGCs and MKCs both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs varies with the polyploidy level. We also obtained three-dimensional reconstructions of highly polyploid TGCs in whole, fixed mouse placentas. Thus, the Fucci technique is able to reveal the spatiotemporal regulation of the endoreplicative cell cycle during differentiation.

and Cross, 2010; Davoli and de Lange, 2011). In lower eukaryotes, endoreplication is developmentally programmed and is a major phenomenon during embryogenesis and differentiation. For example, salivary gland and follicle cells in Drosophila and leaf epidermal cells in plants have polyploid nuclei (Schaeffer et al., 2004;Lammens et al., 2008;Narbonne-Reveau et al., 2008). In these cell types, anaphase-promoting complex/cyclosome (APC/C) and an activator (Cdh1, Fzr or ccs52a) are required to switch from the mitotic to the endoreplicative state. The cyclic activity of APC/C Cdh1/Fzr/ccs52a is also essential to sustain endoreplication.
Endoreplication is a relatively minor event in mammals and has been intensively studied in two cell types. Trophoblast giant cells (TGCs) and megakaryocytes (MKCs) undergo endoreduplication (bypassed mitosis) and endomitosis (abortive mitosis), respectively. TGCs are polyploid cells in the placenta that are essential for embryonic implantation in the uterus and for post-implantation placental development. TGCs avoid mitosis entirely, and this form of endoreplication is called endoreduplication. TGCs endoreduplicate as they differentiate from trophoblast stem cells (TSCs) in the trophectoderm layer of the blastocyst-stage embryo (Zybina and Zybina, 1996;Rossant and Cross, 2001;Martindill and Riley, 2008;Ullah et al., 2008;Ullah et al., 2009;Hu and Cross, 2010;Davoli and de Lange, 2011;Chen et al., 2012). Loss of fibroblast growth factor 4 (Fgf4) signaling or activation of the transcription factors Hand1 and Tead4 is necessary for TSCs to switch from the mitotic to the endoreduplicative cell cycle (Rossant and Cross, 2001;Martindill and Riley, 2008;Nishioka et al., 2008;Ullah et al., 2008;Chen et al., 2012). However, little is known about the molecular mechanisms responsible for progression of endoreduplication in TSCs.
MKCs in the bone marrow also show polyploidy. Hematopoietic stem cells (HSCs) differentiate first into megakaryoblasts (MKBs) and then into mature MKCs through endomitosis in the presence of the cytokine thrombopoietin (TPO; also known as THPO). Endomitosis is a form of endoreplication in which the cell undergoes early mitotic events such as chromosome condensation. Molecular candidates for the endomitotic switch include cyclin B1 and cyclin E (Carow et al., 2001;Bermejo et al., 2002;Eliades et al., 2010). In addition, live-imaging experiments were performed using the histone2B (H2B)-GFP or YFP-tubulin marker to monitor how mitosis aborted (Geddis et al., 2007;Papadantonakis et al., 2008).
Polyploidization or endoreplication is not only developmentally programmed, but is also an early step in the generation of aneuploid cancers. For instance, persistent DNA damage signals elicited by dysfunctional telomeres causes mitosis to be bypassed in p53deficient cells (Davoli et al., 2010). Oscillations in ubiquitylation by the SCF Skp2 and APC Cdh1 complexes (Ang and Harper, 2004;Vodermaier, 2004;Nakayama and Nakayama, 2006) were observed during endoreplication in these cells using fluorescent ubiquitylation-based cell cycle indicator (Fucci) live-cell imaging.
This technology harnesses the cell cycle-dependent proteolysis of two ubiquitin oscillators, human CDT1 and geminin, which are the direct substrates of SCF Skp2 and APC Cdh1 complexes, respectively (Sakaue-Sawano et al., 2008). The Fucci probe consists of two chimeric proteins: mKO2 (monomeric Kusabira-Orange 2) fused to the ubiquitylation domain of CDT1 [FucciG1=mKO2-hCdt1(30/120)], which labels G1 phase nuclei red, and mAG (monomeric Azami-Green) fused to the ubiquitylation domain of geminin [FucciS/G2/M=mAG-hGem(1/110)], which labels S/G2/M phase nuclei green. We also observed that DNA damage-induced endoreplication occurs when normal murine mammary gland (NMuMG) cells were treated with etoposide, an inhibitor of DNA topoisomerase II . The nuclei of NMuMG cells stably expressing Fucci probes changed from green to red without cell division and failed to exhibit any features consistent with mitosis, including nuclear envelope breakdown (NEB). Thus, Fucci probes can report reciprocal E3 ligase activities in DNA damage-induced endoreplication.
Regulation of ubiquitin oscillators by SCF Skp2 and APC Cdh1 complexes is crucial for normal development. Cdh1 −/− embryos die at embryonic day (E) 9.5-10.5 owing to defects in endoreduplication of TSCs (García-Higuera et al., 2008;Li et al., 2008). Conversely, geminin −/− embryos fail to form an inner cell mass and exhibit premature endoreduplication at the eight-cell stage; as a result, all cells become committed to the trophoblast cell lineage (Gonzalez et al., 2006;Hara et al., 2006). In addition, early embryogenesis was examined using mice deficient in Emi1, which is known to be an APC/C inhibitor (Reimann et al., 2001). Although Emi1 −/− embryos form polyploid TGCs, the inner cell mass does not develop properly, presumably because of the high degree of polyploidy and the abnormal cell-cycle progression (Lee et al., 2006). By contrast, Skp2 appears to be less crucial than Cdh1. Skp2 −/− embryos are viable, but cells in knockout mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, resulting in reduced growth and increased apoptosis (Nakayama et al., 2000). However, these studies do not provide direct evidence that SCF Skp2 and APC Cdh1 activities oscillate reciprocally during the endoreduplication cycle.
Here, we used Fucci technology to study endoreplication in differentiating mouse TGCs and MKCs. To do this, we screened transgenic mouse lines expressing Fucci probes and identified mice that mark differentiating trophoblast cells and hematopoietic cells. We also designed special culture wells to track the cell cycle progression of cells isolated from these mice as they differentiated into TGCs or MKCs. Finally, we used the Scale technique, which renders fixed biological samples optically transparent, but preserves fluorescent signals in the clarified structures (Hama et al., 2011). By this technique, we were able to perform three-dimensional (3D) reconstruction of polyploid cells in whole placentas of transgenic pregnant mice. Using this combination of novel techniques, we show that TGCs and MKCs both skip cytokinesis, but using different mechanisms. In addition, we demonstrate that the transition from the SCF Skp2 -to APC Cdh1 -active state in differentiating MKCs varies with the polyploidy level.
For observation of MKC differentiation, these two lines were not suitable, as they did not show Fucci signals in the hematopoietic organs. We therefore performed extensive flow cytometry analysis using cell surface markers (Noda et al., 2008) to identify transgenic mouse lines expressing Fucci in the hematopoietic system. We found that FucciS/G2/M-#474 and FucciG1-#610 exhibited optimal signals in cells expressing a marker for cycling HSCs that have high repopulating activity (Table 1). We were therefore able to isolate HSCs from the bone marrow of the Fucci transgenic mice (#474/#610) and to observe cell cycle dynamics as they differentiated into MKCs.

Validation of Fucci performance during endoreplication
We first performed validation experiments using TGCs and MKCs isolated from #504/#596 and #474/#610 mice, respectively (supplementary material Fig. S1A-D), to demonstrate that the Fucci system can be used to study cell cycle regulation during endoreplication. Immediately after administration of 5-ethynyl-2′deoxyuridine (EdU) to a pregnant mouse (#504/#596) for 20 minutes, E10.5 placental cells were isolated and subjected to the Click-iT reaction for labeling with Alexa 647 (supplementary material Fig. S1A). Bone marrow (BM) cells were isolated from a mouse (#474/#610), cultured in the presence of TPO for 7 days, treated with EdU for 20 minutes, and then subjected to the Click-iT reaction for labeling with Alexa 647 (supplementary material Fig. S1C). Prior to flow cytometric analysis, both samples were  Fig. S1B,D). Thus, all of the cells that transgenically exhibited the two-color fluorescence signals of Fucci probes produced fluorescence signals for the analysis of DNA synthesis (Alexa 647) and DNA content (DAPI). We performed multi-color flow cytometric analysis using four laser lines (supplementary material Fig. S2) to characterize polyploid populations in the samples. As cells in the cerebral cortex of an adult mouse are non-dividing diploid cells resting in G0/G1, they were used as the reference G0/G1 population (supplementary material Fig. S1E,F). Substantial DNA synthesis was detected in the placental and BM cell samples. Furthermore, prominent polyploidy associated with DNA synthesis was identified in both samples. By contrast, neither DNA synthesis nor polyploidization was found in the brain cell sample. Next, we obtained four fractions according to the EdU and DAPI signals for placental ( Fig. 1A) and TPO-treated BM ( Fig. 1B) cell samples each, and characterized their cell cycle phases (Fig. 1C,D, respectively). Fractions #1 and #5 produced an mKO2 signal and were supposed to represent the G1 population, whereas fractions #2 and #6 should represent the S population. Remarkably, by contrast, fractions #3, #4, #7 and #8 were composed of cells with ≥4 C. As illustrated in Fig. 1E, fractions #3 and #7 were EdU(-) and thus should consist of cells in the endoG1 and endoG2 populations (Hu and Cross, 2010) as well as the G2 population, whereas fractions #4 and #8 were EdU(+) and should represent the endoS population (Hu and Cross, 2010). These cell population data demonstrated that polyploid cells (>4 C) prepared from the Fucci transgenic mice had green or red nuclei according to cell cycle progression, which indeed involved DNA synthesis.
We also performed similar validation at the single-cell level. Another placenta from an EdU-administered pregnant mouse (#504/#596) was optically cleared using ScaleA2 reagent (Hama et al., 2011) and then subjected to the Click-iT reaction and DAPI staining (Fig. 2). Multi-color imaging using a Zeiss LSM780 system with two-photon excitation and confocal configurations (supplementary material Fig. S2) allowed us to identify gigantic TGCs that carried EdU(-) red nuclei (endoG1), EdU(+) green nuclei (endoS) and EdU(-) green nuclei (endoG2).

Culture well for tracking all individual cells
During extended time-lapse imaging, target cells often migrate away from the field of view. To overcome this problem, we developed a culture well (FulTrac) with a poly(dimethylsiloxane) (PDMS) block stamped onto a glass coverslip (supplementary material Fig. S3). This allows us to fully track or trace individual cells as they progress through the cell cycle and differentiate. As the surface for cell growth is <0.33 mm in diameter, imaged cells remain inside the field of view of a 20× objective. Moreover, the FulTrac well has a stadium shape to maximize the numerical aperture (0.55) of the condenser for transmitted light. This enables high spatial resolution for differential interference contrast (DIC) imaging everywhere in the well, whereas conventional cylindrical wells give rise to shading in the periphery. For example, using an LCV100 microscope equipped with a FulTrac well, we were able to time-lapse image a morula-stage mouse embryo as it developed, all the way until it hatched from the zona pellucida (supplementary material Movie 1). Of particular importance is the design flexibility of FulTrac wells resulting from the ease of PDMS fabrication. For high-throughput analysis, we designed a PDMS block with four wells, performance of which can be maximized by a motorized microscope stage.

etoposide-treated NMuMG cells (supplementary material
To gain a comprehensive 3D perspective of the endoreduplication cycle in TGCs, we examined Fucci signals in optically cleared placentas of #504/#596 embryos. Placentas with intact maternal decidua tissues at various gestation stages were treated with ScaleU2 (Hama et al., 2011) for >2 months. We imaged a whole E10.5 sample from the embryonic side (Fig. 4A). We used a confocal microscopy system (FV1000) equipped with a 40× silicone-oil objective to acquire high-resolution images of Fucci signals residing deep inside the sample and verified their nuclear localization by DAPI staining (Fig. 4B). Next, using a macro-confocal microscopy system (AZ-C1) equipped with a 2× objective, we obtained a 2D projection image of the entire placenta (Fig. 4C). Then we substituted a 4× objective for higher magnification views in central (Fig. 4D-H) and peripheral ( Fig. 4I-M) regions of the placenta. Highly polyploid TGCs with giant nuclei (>100 μm in diameter) were located peripherally at a depth of 0.6-1.0 mm, in the junctional zone ( Fig. 4J-L). At this stage (E10.5), the green (endoS/G2) and red (endoG1) giant nuclei were observed in equal proportion. As their distributions appeared to be random, it is likely that no intercellular or spatial regulation governs progression of the endoreduplication cycle. By contrast, the central region was abundant with green and red nuclei of normal size (~10 μm in diameter), which might belong to diploid spongiotrophoblast (SpT) cells or less differentiated TGCs. The lack of highly polyploid TGCs in the central region was confirmed by 3D reconstruction experiments using two other E10.5 samples. These results suggest a centripetal pattern of differentiation of TGCs in the placenta. Highly polyploid TGCs with nuclear size >150,000 μm 3 were automatically identified using commercial software (Volocity) in the peripheral regions of transparent placentas prepared at E8.5, E9.5, E10.5, E11.5, E12.5 and E13.5 (supplementary material Fig. S5). Their number increased drastically between E9.5 and E10.5, and then the ratio of the green (endoS/G2) to red (endoG1) nuclei decreased. By E13.5, >99% of the gigantic cells had red (endoG1) nuclei. Thus, endoreduplicative cycling appears to be almost complete by E13.5 and produces terminally differentiated TGCs.

Visualizing endomitosis of MKBs and MKCs
To monitor endomitosis in HSCs as they differentiate into MKCs, we used two transgenic lines that mark hematopoietic cell nuclei  with green (FucciS/G2/M-#474) and red (FucciG1-#610) fluorescence (Table 1; Fig. 5). CD34 -/c-Kit + /Sca-1 + /Lincells (HSCs) were isolated from the bone marrow of adult #474/#610 mice and were used for further studies (supplementary material Fig. S6A). First, we analyzed the fluorescence intensities of mKO2 (red) and mAG (green) of these HSC, multipotent progenitor (MPP), mature B and immature B cell fractions by flow cytometry (supplementary material Fig. S6B). In each fraction, we identified two cell populations with high and low intensities of mKO2 signals for CDT1 accumulation, which we hypothesize correspond to quiescent G0 and cycling G1 cells, respectively. Because the HSCs exhibited a high intensity of mKO2 (red) fluorescence (supplementary material Fig. S6B, HSC), the #474/#610-derived HSC fraction was assumed to be enriched with quiescent G0 cells. We transferred the HSCs into the FulTrac well with a medium containing TPO (Fig. 5A). Nearly all of the cell nuclei were red (G0) in the beginning, but turned green (S/G2/M) after ~2 days, as revealed by the time-lapse imaging (supplementary material Fig. S7 and Movie 3). This slow proliferation switch in HSCs is corroborated by previous bromodeoxyuridine (BrdU) incorporation analyses (Passegué et al., 2005). After re-entering the cell cycle, some cells switch to an endomitotic cell cycle. At DIV (days in vitro) 8, giant cells with green (endoS/G2) or red (endoG1) nuclei appeared (Fig. 5B, HSC, DIV 8), which we assumed to be highly polyploid megakaryoblasts (supplementary material Fig. S8). These cell nuclei alternated between green and red multiple times as they grew and differentiated. The cells finally became presumptive MKCs with red nuclei.
We also examined nuclear structures during endomitosis in MKCs (Fig. 5C). The green-to-red conversion (the endoG2/endoG1 transition) was accompanied by breakdown of the nuclear envelope, consistent with the fact that polyploidy in MKCs is achieved via endomitosis. Interestingly, upon the disappearance of the green fluorescence, cells rapidly adhered to the coverslip and then gradually returned to spheres. This transient morphological change was easily quantified by measuring the cell diameter (Fig. 5D). We were also able to determine the C values of individual differentiating cells and observed that nuclei spent a longer time in the green (endoS/G2) or red (endoG1) state as polyploidy increased (supplementary material Fig. S9 and Movie 4). During the green-tored conversion, in particular, the latency time for the emergence of the red signal (endoG1 entry) was longer in highly polyploid cells (≥16 C) (supplementary material Fig. S10).

Comparison of endoreplication in TGCs and MKCs
Although APC Cdh1 is activated at the metaphase/anaphase transition in the mitotic cell cycle, it also functions at the endoG2/endoG1 transition in TGC endoreduplication and MKC endomitosis. However, cellular features consistent with M phase were observed, including NEB, during the endoG2/endoG1 transition of MKCs but not TGCs (Fig. 6A). We detailed the temporal profiles of the transition in multiple cells and attempted to superimpose them ( Fig. 6B-D). In TGCs, the rate at which the green fluorescence disappeared and the red fluorescence appeared was constant, irrespective of polyploidy level (Fig. 6B). By contrast, the polyploidy of MKCs determined the speed of green-to-red conversion (endoG2/endoG1 transition). In low polyploidy MKCs (<16 C), the emergence and subsequent extinction of red fluorescence (endoG1 entry and exit) was variable but rapid (Fig. 6C), whereas the appearance of red fluorescence (endoG1 entry) in high polyploidy MKCs (≥16 C) was consistently slow ( Fig. 6D; supplementary material Fig. S10). To summarize, greento-red conversions of Fucci signals during mitotic cycling, endoreduplication and endomitosis are illustrated with respect to the normal cell cycle phases, from G2 to G1 to M (Fig. 6).

DISCUSSION
Visualizing the cell cycle behavior of individual cells within complex tissues has in the past been extremely difficult. For instance, it has not been clear whether the ubiquitin oscillators that control normal mitotic cell cycle transitions (Ang and Harper, 2004;Vodermaier, 2004;Nakayama and Nakayama, 2006) function during endoreplication. Previous work showed that the protein level of the cyclin-dependent kinase inhibitor p57 (also known as Cdkn1c), which is targeted by SCF Skp2 for degradation, oscillates in endoreduplicating mammalian cells (TGCs) in culture (Hattori et al., 2000;Ullah et al., 2008). Because the promoter activity of p57 is constant in TGCs, it was surmised that the enzymatic activity of SCF Skp2 must oscillate. In endoreplicating Drosophila cells, oscillation in APC Fzr/Cdh1 activity was verified by measuring the protein level of its substrate, Origin recognition complex 1 (Orc1) (Asano and Wharton, 1999;Narbonne-Reveau et al., 2008). Although these findings support the function of  ubiquitin oscillators in endoreplicative cycling, most of the data are snapshots correlating substrate expression levels and S phase markers, such as BrdU incorporation. Importantly, in addition, none of these studies examined both APC Cdh1 and SCF Skp2 activities at the same time. Thus, little was known about the precise timing of the transitions between the APC Cdh1 -and SCF Skp2 -active states during endoreplicative cycles. A recent computational study simulated the oscillatory dynamics of the APC Fzr/Cdh1 and CRL4 CDT2 ubiquitin ligases in endoreplicating Drosophila cells (Zielke et al., 2011).
In the present study, we overcame previous limitations by performing live imaging of TGCs and MKCs using Fucci probes. This allowed us to monitor the anti-phase oscillating activities of the two E3 ligases, with high contrast and high temporal resolution. In particular, our ex vivo imaging using custom FulTrac wells permitted us to observe individual cells over a long time period. This work lays the foundation for future studies of endoreduplication and endomitosis in a multicellular context; for example, in living intact placenta and bone marrow. This is important because the endoreplicative cell cycle is regulated not only by intracellular signals but also by extracellular signals, although a recent integrative model for the cyclin/Cdk network in mammalian endoreplicative cells suggests that it functions as a self-sustained oscillator (Gérard and Goldbeter, 2009).
The temporal profiles of the green-to-red conversion of Fucci signals (Fig. 6) might yield clues as to what activates APC Cdh1 during endoG2 phase or abortive mitosis. It is thus interesting to investigate temporal patterns of the activation of APC Cdh1 regulators, such as Emi1. Furthermore, the reciprocal activation of SCF Skp2 and APC Cdh1 is modified during differentiation into MKCs. The relatively slow conversion seen in high-polyploidy (≥16 C) MKCs suggests the existence of a state in which APC Cdh1 is fully activated whereas SCF Skp2 is not yet fully inactivated. Although the APC Cdh1 complex directly targets the SCF Skp2 complex for degradation and vice versa, there might be other mechanisms that regulate the anti-phase oscillation between the two E3 ligase activities. Notably, similar ploidy-level dependence was observed for the differential downregulation of two guanine nucleotide exchange factors [GEF-H1 (also known as Arhgef2) and ECT2] in endomitosing MKCs (Gao et al., 2012).
The Fucci transgenic mouse lines we have generated will be very useful for a variety of future studies. Crossing Fucci lines to the many mutant lines isolated by forward genetic screens will allow identification of the molecules responsible for the initiation, progression and termination of endoreplication. This information will refine computational models, which attempt to account for mammalian endoreplication. Also, crossing Fucci mice to the numerous transgenic lines expressing fluorescent markers under the control of cell type-or stage-specific promoters will be fruitful. Finally, Fucci transgenic mice will help researchers to re-evaluate polyploidy in cell types other than TGCs and MKCs. For example, the presence of polyploid neurons in vertebrates has been debated for the last half century; tetraploidy has been described in retinal ganglion cells (Morillo et al., 2010), cerebellar Purkinje neurons (Das, 1977) and cortical neurons of Alzheimer's disease patients (Mosch et al., 2007). In addition, an intriguing attempt would be the three-dimensional reconstructions of Fucci-labeled hepatocytes in the developing or regenerating liver, because hepatocytes show unique polyploidization (Duncan et al., 2010).

Cell cycle analysis of endoreplicating cells
Pregnant mice were injected intravenously with EdU solution (Life Technologies) (50 mg/kg body weight). After 20 minutes, placentas at E10.5 were removed, minced, and dissociated with 500 U/ml collagenase I (Worthington) at 37°C for 30 minutes and then with 6.25 U/ml DNase I (Worthington) for 30 minutes. After being washed and passed through a 100-μm nylon mesh, the placental cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes on ice. The fixed cells were treated with azide-conjugated Alexa 647 by the Click-iT reaction (Life Technologies) with slight modification. As copper ion used for the conjugation between EdU and Alexa dyes in the Click-iT reaction tended to quench fluorescent proteins, we attempted to lower the copper ion concentration. We found that one-third of the copper ion concentration recommended by the manufacturer was sufficient for the reaction while effectively avoiding the quenching reaction. Finally, the cell samples were stained with 3 μM DAPI and analyzed using a FACSAria II (BD Biosciences). DAPI was excited by a 355-nm laser line and its emission collected through 450/50BP; mAG was excited by a 488-nm laser line and its emission collected through 530/30BP; mKO2 was excited by a 561-nm laser line and its emission collected through 610/20BP; and Alexa 647 was excited by a 640-nm laser line and its emission collected through 710/50BP. The data were analyzed using FlowJo software (Tree Star).

LCV100 microscopy
Cells were subjected to long-term, time-lapse imaging using a computerassisted fluorescence microscope (Olympus, LCV100) equipped with an objective lens (Olympus, UAPO 40×/340 N.A.=0.90), a 0.5×zoom lens, a halogen lamp, a red light emitting diode (LED) (620 nm), a charge coupled device camera (Olympus, DP30), DIC optical components, and interference filters. The halogen lamp was used with a 470DF35 excitation filter, a 505DRLP dichroic mirror and a 510WB40 emission filter to observe the mAG fluorescence, and a BP520-540HQ excitation filter, a DM545HQ dichroic mirror and a BP555-600HQ emission filter to observe the mKO2 fluorescence. For DIC imaging, the red LED was used with a filter cube containing an analyzer. Image acquisition and analysis were performed using MetaMorph 6.37 and 7.6.0.0 software (Universal Imaging Corporation), respectively.

Time-lapse imaging of trophoblast cell endoreduplication
FucciG1-#596 heterozygous eggs and FucciS/G2/M-#504 homozygous sperm were used for in vitro fertilization. Fertilized eggs were cultured to the two-cell stage and then were frozen as stocks. After thawing, two-cell stage embryos were transplanted into pseudopregnant female mice. After 3 days, blastocysts were collected from the uterus, and were individually cultured in FulTrac wells in KSOM-AA medium for 24 hours in a CO 2 incubator. Then, the medium was changed to RPMI1640 containing 20% fetal calf serum. Trophoectoderm outgrowth from the inner cell mass was time-lapse imaged using an LCV100 microscope.

Estimation of ploidy level
In this study, we determined the ploidy (the C value) for some TGCs and MKCs cultured in FulTrac wells, which enabled full tracking of individual cells over an extended period. We traced TGCs and MKCs back to cells inside the ICM and cells before exposure to TPO, respectively. These original cells had not entered the endoreplicative cell cycle yet and were supposed to be in the normal mitotic cell cycle (2 C ~4 C).


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Engineering

text:
CHO-gmt5, a novel CHO glycosylation mutant for producing afucosylated and asialylated recombinant antibodies

Engineered zinc-finger nucleases (ZFNs) are powerful tools for creating double-stranded-breaks (DSBs) in genomic DNA in a site-specific manner. These DSBs generated by ZFNs can be repaired by homology-directed repair or nonhomologous end joining, in which the latter can be exploited to generate insertion or deletion mutants. Based on published literature, we designed a pair of zinc-finger nucleases and inactivated the GDP-fucose transporter gene (Slc35c1) in a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter gene (Slc35a1). The resulting mutant cell line, CHO-gmt5, lacks functional GDP-fucose transporter and CMP-sialic acid transporter. As a result, these cells can only produce asialylated and afucosylated glycoproteins. It is now widely recognized that removal of the core fucose from the N-glycans attached to Asn297 of human IgG1 significantly enhances its binding to its receptor, FcγRIIIa, and thereby dramatically improves antibody-dependent cellular cytotoxicity (ADCC). Recent reports showed that removal of sialic acid from IgG1 also enhances ADCC. Therefore, CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC. These cells show comparable growth rate to wild type CHO-K1 cells and uncompromised transfection efficiency, which make them desirable for use as a production line.

Recombinant human IgG1 antibodies have been successfully used as therapeutic drugs to target malignant cells in cancer patients. Upon binding to the target molecule expressed on cancer cells, the Fc region of the antibody recruits the effector cells such as natural killer (NK) cells to kill cancer cells by antibody-dependent cellular cytotoxicity (ADCC). Studies have shown that the major interaction sites of the Fc region by the FcγRIII are located in the hinge region and the CH2 domains of the antibody. 1,2 The binding of the Fc and the Fc receptor is known to be dependent on the structures of the N-glycans attached to the conserved glycosylation site at Asn 297 in each of the CH2 domains. 3 It has been demonstrated clearly that removal of the fucose residue from the N-glycan attached to Asn 297 of human IgG1 significantly enhances its binding to FcγRIIIa and thereby dramatically improves ADCC. 4,5 Detailed binding analyses indicated that removal of fucose enhanced binding enthalpy and increased binding constant of IgG1 for FcγRIIIa. 6 The molecular mechanism underlying the enhanced affinity between Fc and FcγRIIIa was further investigated. The structural differences between fucosylated and afucosylated Fc fragments of human IgG1 were compared in X-ray crystallographic and NMR spectroscopic studies. The overall conformations of the fucosylated engineered zinc-finger nucleases (ZFns) are powerful tools for creating double-stranded-breaks (DSBs) in genomic DnA in a site-specific manner. These DSBs generated by ZFns can be repaired by homology-directed repair or nonhomologous end joining, in which the latter can be exploited to generate insertion or deletion mutants. Based on published literature, we designed a pair of zinc-finger nucleases and inactivated the GDp-fucose transporter gene (Slc35c1) in a previously reported cho mutant that has a dysfunctional cMp-sialic acid transporter gene (Slc35a1). The resulting mutant cell line, cho-gmt5, lacks functional GDp-fucose transporter and cMp-sialic acid transporter. As a result, these cells can only produce asialylated and afucosylated glycoproteins. it is now widely recognized that removal of the core fucose from the N-glycans attached to Asn 297 of human igG1 significantly enhances its binding to its receptor, FcγRiiia, and thereby dramatically improves antibody-dependent cellular cytotoxicity (ADcc). Recent reports showed that removal of sialic acid from igG1 also enhances ADcc. Therefore, cho-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADcc. These cells show comparable growth rate to wild type cho-K1 cells and uncompromised transfection efficiency, which make them desirable for use as a production line. and afucosylated Fc fragments are similar except for hydration mode around Tyr 296 . 7 The conformation of Tyr 296 is more flexible for FcγRIIIa in afucosylated Fc than in fucosylated Fc. 7 Tyr 296 has already been indicated in the interaction with FcγRIIIa. 2,8 Enhanced ADCC was not only observed for the afucosylated antibody in in vitro assays, it was also confirmed in vivo in patients or animal models. [9][10][11][12] These data have convincingly shown that removal of fucose from human IgG1 can be a general method for treating cancer patients with antibodies through improved ADCC.
In addition to fucosylation, sialylation of the Fc N-glycan may also affect the cell-killing activity of the antibody. Increased sialylation of the Fc N-glycan was shown to reduce the binding of Fc to FcγRIIIa and consequently, decrease ADCC. 13,14 We have previously reported the isolation of a CHO mutant (CHO-gmt1) that produces asialylated glycoproteins due to the lack of a functional CMP-sialic acid transporter. 15 Using zinc-finger nuclease technology we have inactivated the GDP-fucose transporter in CHO-gmt1. 16 The resulting mutant, CHO-gmt5, is able to produce afucosylated and asialylated recombinant antibodies. The efficacy in ADCC by antibodies produced in CHO-gmt5 cells will be evaluated. ZFNs generated by the combinatorial selection methods may have high DNA-binding affinity and low toxicity. 18 Sangamo Biosciences has used its own proprietary information to create highly specific ZFNs. [19][20][21] However, most laboratories do not have the randomized libraries or the selection expertise to do so. Alternatively, a modular assembly strategy can be used based on publically available information in the literature. 22,23 We also used the modular assembly strategy to design ZFNs to interrupt the GDP-fucose transporter gene in CHO cells. To target a gene with ZFNs, the first step is to identify an ideal target site in the gene of interest. The open reading frame of the cDNA can be analyzed by the web-based ZiFiT program provided by the Zinc Finger Consortium (ZiFiT: software for engineering zinc finger proteins (V3.0)) at: http:// bindr.gdcb.iastate.edu/ZiFiT/. 24 The ZiFiT output will suggest a few potential target sites. The fingers that bind the 5'-GNN-3' sequences are the best studied and strongest DNA-binding fingers. [25][26][27] Two binding sites for ZFNs should be separated by 5-7

CHO-gmt1 Cells
CHO-gmt1 cells (originally called MAR-11) were isolated from CHO-K1 cells treated with Maackia amurensis agglutinin (MAA) which is specific for Neu5Acα2,3Gal structure. The cells have a dysfunctional CMP-sialic acid transporter (CMP-SAT). 15 A point mutation in the CMP-SAT gene in CHO-gmt1 cells results in a premature stop codon. As a result, these cells express a truncated version of CMP-SAT which contains only 100 amino acids, rather than the normal CMP-SAT which consists of 336 amino acids. As a result, glycoproteins produced by this cell line are completely free of sialic acid. These cells have been used to  Fig. 2A). When this population was subsequently stained with FITC and analyzed by FACS, a significant number of the cells were AAL-negative (Fig. 2B). Following two more cycles of this lectin-based cell sorting essentially all the cells in the population became AAL negative cells (data not shown).
In order to confirm that the phenotypic change observed in the flow cytometry was indeed due to the disruption of the GDP-fucose transporter gene, the cells were transfected with a construct that expressed wild type GDP-fucose transporter in a rescue assay. The conversion of the AAL-negative phenotype to AAL-positive following this assay is indicative of a mutated GDP-fucose transporter. The gene locus in the mutants was then amplified by PCR and sequenced. The results further bps which is the optimal distance for the two FokI domains to dimerize and cleave the targeted site. Therefore, an ideal target sequence for two 4-fingered ZFNs should be: 5'-NNC NNC NNC NNC xxx xxx GNN GNN GNN GNN-3'. This sequence ensures each zinc finger binds a 5'-GNN-3' sequence. The 5'-GNG-3' sequences are better binding sites compared with other 5'-GNN-3' sequences. In addition to the 5'-GNN-3' sequences, other sequences have also be successfully used in the literature. These include CTG, TGG, AAG and AAA triplets. It is generally believed that 3-fingered ZFNs should work as well as the 4-fingered ZFNs. In the event that there is no ideal site in the open reading frame, one can try to find two ideal sites that flank an exon in the open reading frame of the targeted gene. ZFNs designed to target two different sites can introduce two concurrent DNA double-strand breaks in the chromosome and create deletions of the genomic segment between the two sites. 28 In this situation, cells will be transfected with two pairs of ZFNs simultaneously in order to generate targeted deletions of genomic segments. A simplified "modular assembly" strategy to design zinc-finger nucleases (ZFNs) based on publically available information is outlined in Figure 1.
The structural scaffold for the ZFNs can be adopted from previous publications. 19,20 To eliminate unwanted homodimerization of FokI cleavage domain, the high-fidelity FokI-KK and FokI-EL variants can be used. 29 The amino acid sequences of the DNA-binding domains in the ZFNs are assembled using an archive of zinc-finger motifs collected from previous publications [25][26][27] and many other related publications which are not listed here.

Using ZFNs to Inactivate GDP-Fucose Transporter Gene in CHO Cells and Fluorescence-Activated Cell Sorting to Rapidly Isolate Mutant Cells
Analysis of the open reading frame of Chinese hamster GDPfucose transporter gene using the ZiFiT program 24 identified one potential target site in the first exon of the GDP-fucose transporter coding region (5'-tAA CCT CTG CCT CAA GTA CGT AGG GGT GGC Ct-3'). As discussed earlier, this sequence allows each zinc finger in the left ZFN and the right ZFN to bind to a 5'-GNN-3' DNA triplet. CHO cells were transiently transfected with plasmids expressing the left and right zinc-finger nucleases as described previously. 16 In our previous report, two days after transfection, single cells were seeded in a 96-well plate for clone isolation. Genomic DNA from each single clone was isolated and the targeted GDP-fucose transporter locus was amplified by two specific PCR primers flanking the targeted site in the gene. The PCR products were sequenced in order to identify the mutation. However, this is a labor intensive and time-consuming process and we identified only one mutant. 16 To increase the selection efficiency, a fucose-specific lectin combined with fluorescenceactivated cell sorting (FACS) strategy was employed. After transfection with the ZFN constructs, the cells were cultured for several generations to allow the daughter cells of successful knockout mutants to express afucosylated glycoproteins on confirmed the genetic mutation at the targeted site. Taken together, the fucose-specific lectin in combination with FACS is a successful method to rapidly isolate mutants.

CHO-gmt5 Cells
The GDP-fucose transporter gene was successfully inactivated by ZFNs in CHO-gmt1 cells. 16 The resultant CHO-gmt5 cells lack functional CMP-sialic acid transporter and GDP-fucose transporter. They can be used to produce afucosylated and asialylated recombinant antibodies such as Rituximab. The ability of fucose-free and sialic acid-free antibodies to enhance ADCC will be assessed. We have also shown that CHO-gmt5 cells have comparable growth rate to wild type CHO-K1 upon adaptation to suspension culture in chemically defined medium, as well as uncompromised transfection efficiency when transfected with GFP and recombinant Rituximab constructs.

Perspectives
This work demonstrated that the "modular assembly" strategy can be successfully utilized by labs without special expertise in zinc finger design. Utilizing the archive of zinc-finger motifs collected from all the zinc finger work published in the literature, researchers will be able to design better zinc fingers.
FACS is a simple method to rapidly isolate mutant cells provided the mutant cell can be identified by a fluorescent tag. In our case, fucosylated proteins on the wild type cell surface can be positively stained by AAL, which consequently excludes the mutant cells. Based on this feature we were able to enrich and isolate the cells with inactivated GDP-fucose transporter gene. FACS can also rapidly enrich and isolate mutant cells in which the dihydrofolate reductase (DHFR) gene is interrupted by ZFNs. Wild type cells that express DHFR bind fluorescent methotrexate. 30 Mutants that are no longer able to bind fluorescent methotrexate can be isolated by FACS. We have successfully used methotrexate to isolate DHFR negative CHO cells using previously reported ZFNs. 21 Thus, the FACS strategy can be applied to the isolation of a variety of mutants, including mutations in the genes that encode cell surface proteins, provided antibodies specific for the proteins are available.
CHO-gmt5 cells lack functional CMP-sialic acid transporter and GDP-fucose transporter. These cells can be used to study structure-function relationships of these two receptors. 16,17 They can also be used to produce afucosylated and asialylated recombinant antibodies. It is of great interest for us to investigate whether these antibodies will have enhanced ADCC against the target cells.

Disclosure of Potential Conflicts of Interest
No conflicts of interest were disclosed.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Psychology

text:
Placenta-derived mesenchymal stem cells improve memory dysfunction in an Aβ1–42-infused mouse model of Alzheimer's disease

Mesenchymal stem cells (MSCs) promote functional recoveries in pathological experimental models of central nervous system (CNS) and are currently being tested in clinical trials for neurological disorders, but preventive mechanisms of placenta-derived MSCs (PD-MSCs) for Alzheimer's disease are poorly understood. Herein, we investigated the inhibitory effect of PD-MSCs on neuronal cell death and memory impairment in Aβ1–42-infused mice. After intracerebroventrical (ICV) infusion of Aβ1–42 for 14 days, the cognitive function was assessed by the Morris water maze test and passive avoidance test. Our results showed that the transplantation of PD-MSCs into Aβ1–42-infused mice significantly improved cognitive impairment, and behavioral changes attenuated the expression of APP, BACE1, and Aβ, as well as the activity of β-secretase and γ-secretase. In addition, the activation of glia cells and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were inhibited by the transplantation of PD-MSCs. Furthermore, we also found that PD-MSCs downregulated the release of inflammatory cytokines as well as prevented neuronal cell death and promoted neuronal cell differentiation from neuronal progenitor cells in Aβ1–42-infused mice. These data indicate that PD-MSC mediates neuroprotection by regulating neuronal death, neurogenesis, glia cell activation in hippocampus, and altering cytokine expression, suggesting a close link between the therapeutic effects of MSCs and the damaged CNS in Alzheimer's disease.

Subject Category: Neuroscience
Mesenchymal stem cell (MSC) has been recently considered to improve disease progression of neurological disorders. 1,2 MSC is a multipotent stem cell with capacity for self-renewal and differentiation with broad tissue distribution. 3 MSC was first identified and isolated from bone marrow (BM) more than 43 years ago. 4 However, within the bone marrow, BM-MSC is a rare cell population, resulting in a low MSC yield when isolated. 5 The progenitor pool may be depleted following extensive proliferation, leading to reducing ability to ensure regeneration after injury. 6,7 For these reasons, novel sources of MSCs are now being investigated for clinical use in diseases. 8,9 It was reported that MSCs derived from placenta (PD-MSCs) showed higher proliferative capacity and safety, 8,10 and also included noninvasive and ethically nonproblematic availability. 11 Thus, PD-MSCs can be obtained in large quantities, and the required numbers of these stem cells can be transplanted as an ideal alternative to adult bone marrow in therapeutic approaches for tissue replacement.
It was reported that BM-MSCs reduced Ab levels in an acutely amyloid b (Ab)-injected mouse model of Alzheimer's disease, 12 and attenuated Ab-induced apoptotic cell death in primary cultured hippocampal neurons by the activation of the cell survival signaling pathway. 13 In amyloid precursor protein (APP) and PS1 double-transgenic mice, BM-MSCs significantly also reduced Ab level associated with defective microglial function and decreased tau hyperphosphorylation and improved cognitive function. 14 Recently, human umbilical cord blood-(hUCB)-derived MSCs significantly also improved learning and memory decline through the modulation of neuroinflammation in an APP and PS1 double-transgenic mice. 15 Interestingly, no studies are available on the various roles played by PD-MSCs in eliciting the therapeutic effects in Alzheimer's diseases. Therefore, it is important to elucidate the novel roles and potential effects mediated by PD-MSCs in Alzheimer's diseases.
Alzheimer's disease as one of the most important neurodegenerative disorders is an irreversible and progressive disorder with observable memory impairment. 16,17 Ab is generated from APP, a membrane-spanning protein to be digested by b-secretase (b-site APP cleaving enzyme 1 (BACE1)). 18,19 It was reported that APP metabolism and Ab generation associated with senile plaques are related to the activation of glia cells. 20 Ab peptide activates glia cells that are found to surround Ab plaques. 21,22 The activation of these cells is associated with many inflammation-associated neurodegenerative diseases including Alzheimer's disease. 23,24 It is also suggested that glia cells could act as a source for Ab because they overexpress BACE1 in response to chronic stress. 25 Thus, the feed-forward vicious cycle by Ab between amyloidogenesis and neuroinflammation culminates in widespread neuronal dysfunction and cell death, leading to progressive Alzheimer's disease associated with extensive Ab pathology.
In the present study, we examined whether the transplantation of PD-MSCs could have beneficial effects in Ab 1-42 -infused mice model. We found that the transplantation of PD-MSCs rescued cognitive impairment, promoted antiamyloidogenic and anti-inflammatory response, and also increased neuronal survival and neurogenesis.

Results
PD-MSC reverses memory impairment in Ab 1-42 -infused mice. In order to identify whether PD-MSCs could affect memory impairment by Ab 1-42 in Alzheimer's diseases, ICR mice were infused with Ab 1-42 (300 pmol per day per mouse) for 14 days and then compared memory deficiency between the control mice and mice transplanted with PD-MSCs (1 Â 10 5 , 5 Â 10 5 , and 1 Â 10 6 cells per mouse) via intravenous injection into mouse tail vein. As shown in Figure 1, we performed behavioral tests for a total of 12 days using the Morris water maze and passive avoidance tests. All mice were trained for three trials per day for 7 days. Escape latency (time, s) and swimming distance (distance, cm) that were traveled to reach a platform in water maze were measured to determine memory impairment effect of PD-MSCs. The Ab 1-42 -infused mice exhibited significantly delayed escape latency (33.2 ± 17.94 s, **Po0.01; Figure 2a) and swimming distance (851.5 ± 123.5 cm, **Po0.01; Figure 2b) compared with the control mice (escape latency: 17 Figures 2a and b).
After the water maze test, a probe test was performed in which the platform was removed to analyze whether the animals used a nonspatial strategy to find the platform. During the probe test, we calculated the time spent in the target quadrant zone during the 60 s test. The memory deficiency in Ab 1-42 -infused mice (15.92 ± 10.32 s, *Po0.05) was significantly improved in PD-MSC mice group (1 Â 10 5 cells: 25.89±7.51%; 5 Â 10 5 cells: 28.58 ±7.63%, # Po0.05; 1 Â 10 6 cells: 30.84 ± 5.27%, # Po0.05; Figure 2c). To determine whether PD-MSC mice improved the contextual memory in Ab 1-42 -infused mice, we next evaluated learning and memory capacities by the passive avoidance test using the step-through method. In the passive avoidance test, there was no significant difference on the learning trial. In contrast, PD-MSC mice ( Figure 2d). Thus, we validated that the beneficial effects of PD-MSCs remained consistent over a prolonged period of time. These results indicate that PD-MSC improves deficits of learning and memory in Ab 1-42 -infused mice.
PD-MSC reveals antiamyloidogenic effects in Ab 1-42 -infused mice. We investigated whether PD-MSCs influenced amyloidogenesis in the Ab 1-42 -infused mice. The number of Ab 1-42 -reactive cells was determined with immunohistochemical analysis and Ab 1-42 expression was significantly lowered by the transplantation of PD-MSCs ( Figure 3a) and the levels of Ab 1-42 were also measured using ELISA (Con: 13.00 ± 8.06; Ab: 33.53 ± 5.44, *Po0.05; Ab þ MSC: 19.79 ± 4.01, # Po0.05; Figure 3e). To clarify how Ab 1-42 deposition had occurred, we analyzed the expression level of APP and BACE1. As shown in Figures 3b-d, there was a significant rise in the level of the proteins in the brain after Ab 1-42 injection, which was inhibited by PD-MSCs. To confirm the speculation, we next attempted to measure b-and g-secretase activity. The activity of b-and g-secretase significantly increased in the brain of Ab 1-42 -infused mice (b-secretase: Ab, 420.5±39.77, *Po0.05; g-secretase: Ab, 245.2 ± 14.15, **Po0.01), whereas the alteration in b-and g-secretase activity was attenuated by the transplantation of PD-MSC (b-secretase: Ab þ MSC, 348.0±7.45, # Po0.05; g-secretase: Ab þ MSC, 141.3±20.71, ## Po0.01; Figures 3f and g). These data suggest that PD-MSC has an antiamyloidogenic effect in Ab 1-42 -infused mice. Figure 1 The scheme of experimental study on an Ab 1-42 -infused mouse model. After Ab infusion for 14 days, the Morris water maze test, probe test, and passive avoidance test were conducted as shown PD-MSC reveals antineuroinflammatory effects in Abb 1-42 -infused mice. It was also reported that activation of glia cells is one of the characteristic feature in the Alzheimer's disease, and they can produce inflammatory cytokines as well as generate Ab when they are activated. Therefore, we compared activation of astrocytes and microglia in the brains between Ab 1-42 -infused mice and Ab 1-42 -infused PD-MSC mice. As shown in Figures 4a and b, GFAP-positive cell numbers (astrocytes) and Iba1-positive cell number (microglia) were much higher in the brains of Ab 1-42 -infused mice as compared with the brains of Ab 1-42 -infused PD-MSC mice. To verify these results, we investigated the expression of GFAP and Iba1 by western blotting. The results revealed that the GFAP and Iba1 levels were significantly increased in Ab 1-42 -infused mice as compared with control mice. However, Ab 1-42 -infused PD-MSC mice were significantly attenuated the expression of GFAP and Iba1 in the Ab 1-42 -infused mice (Figure 4e). We also demonstrated the effect of the transplantation of PD-MSCs on memory formation and Ab deposition was mediated by neuroinflammation. The expression of the inflammatory protein such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the brain of Ab 1-42 -infused mice were significantly increased than the control mice, whereas PD-MSC mice revealed a decrease in the expression of iNOS and COX-2 detected by immunohistochemical analysis (Figures 4c and d) and western blotting ( Figure 4e). To validate the activation of glia cells and inflammatory response, we performed mouse cytokine array using Proteome profiler arrays. We found that the levels of G-CSF, I-309, sICAM-1, IFN-g, IL-1b, IL-7, IL-17, IL-27, IP-10, KC, MIG, CCL5, TIMP-1, and TNF-a by infusion of Ab 1-42 were significantly altered, which is inhibited by PD-MSC (Supplementary Figure 1). Our results indicate that PD-MSC regulates glia cells, inflammatory response, and cytokine release to improve the symptom in Alzheimer's diseases.
PD-MSC prevents neuronal cell death and promotes neuronal differentiation. We explored whether PD-MSC was able to improve memory impairment by preventing Ab 1-42 -induced neuronal cell death. In the CA1, CA3, and dentate gyrus (DG) region of the hippocampus, Ab 1-42 injection significantly increased apoptotic cell death as  (Figure 5f), implying that PD-MSCs did not affect proliferation of adult neural stem cell. As MSC is a multipotent cell that can be differentiated into neural cell lineages, we therefore investigated whether the protective effects of PD-MSCs were mediated by direct differentiation into neuron or glia cells. As shown in Figure 6, PD-MSCs implanted in Ab 1-42 -infused mice was not colocalized with NeuN (mature neuron) (Figure 6a), DCX (newborn neuron; Figure 6b), or GFAP (astrocyte; Figure 6c) positive cells. Taken together, these data suggest that PD-MSC prevents neuronal cell death and induces newly generated neuron regardless of the neural differentiation potential of PD-MSC.

Discussion
In the present study, we originally reported clear evidences in the role of PD-MSCs using an Ab-infused mouse model of Alzheimer's diseases. Ab-infused mouse model has been used to directly investigate the toxicity of Ab that consisted of senile plaques of Alzheimer's disease. 12,13,[26][27][28] Also, we continually investigated amyloidogenesis, neuroinflammation, and memory impairment through the injection of Ab. [29][30][31][32][33] Therefore, to investigate the effect on the Ab toxicity of PD-MSCs, we used the Ab-infused mouse model in the present study. We demonstrated that the transplantation of PD-MSCs in Ab 1-42 -infused mice has antiamyloidogenic and antineuroinflammatory effects to improve neuronal survival and neurogenesis, and prevent memory deficiency. Amyloidogenesis postulates that gradual changes in the metabolism and aggregation of Ab initiate a cascade of neuronal and inflammatory injury that culminates in extensive neuronal dysfunction and cell death in Alzheimer's disease. 34,35 In these experiments, we demonstrated that PD-MSCs reduced the amyloid cascade in the   (a and b) The effect of PD-MSC on reactive astrocytes and activated microglia cells was measured by immunohistochemical analysis. The sections were incubated with antiglial fibrillary acidic protein (GFAP) (a) and anti-ionized calcium binding adaptor molecule 1 (Iba1) (b) antibodies, followed by the biotinylated secondary antibody (n ¼ 8). Immunoperoxidase staining was expressed as brown color. (c and d) The sections were incubated with anti-iNOS (c) and anti-COX-2 (d) antibodies, and then followed by the biotinylated secondary antibody (n ¼ 8). Immunoperoxidase staining was expressed as brown color. (e) The expression of GFAP, Iba1, iNOS, and COX-2 was detected by western blotting using specific antibodies. The signals of GFAP, Iba1, iNOS, and COX2 were normalized using b-actin and the numerical values were expressed as relative fold of control. Significant difference from control mice (*Po0.05 and **Po0.01). Significant difference from Ab 1-42 -infused mice ( # Po0.05). The experiments shown in Figure 3 were repeated in triplicate with similar results activity of g-secretase. Amyloidogenic processing of APP involves sequential cleavages by BACE1 and g-secretase. 35 C-terminal fragment of APP generated by BACE1 cleavage was further processed by g-secretase to produce Ab 40/42 . Our results indicated that the expression of BACE1 and APP, and the activity of g-secretase, were decreased by PD-MSCs in Ab 1-42 -infused mice. Previously, it was reported that BM-MSCs significantly reduced Ab deposition and attenuated Ab-induced memory impairment and apoptosis by inhibiting neuronal cell death. [12][13][14] Adipose tissue-derived MSCs also improved cognitive function in aging mice. 36 Thus, we suggest that PD-MSC improves cognitive dysfunction through BACE1 and APP expression, and g-secretase activities to diminish amyloidogenesis.
APP and BACE1 are upregulated in astrocytes and microglia upon exposure to cytokines influencing APP processing to induce amyloidogenesis, and induced in the proximity of activated astrocytes and microglia. 37,38 We found that the number of astrocytes and microglia were elevated in Ab 1-42 -infused mice that were prevented by PD-MSCs. It was also reported that BACE1 and APP transcriptions are increased by other inflammatory gene expression such as iNOS and COX-2. 39,40 Several studies have indicated that neuroinflammatory reactions could contribute to BACE1 and APP expression and g-secretase activity. These studies also support the idea for neuroinflammation as a direct effect on amyloidogenesis. Especially, hUCB-derived MSCs significantly reduced the levels of Ab, BACE1, and Tau hyperphosphorylation by regulating neuroinflammatory responses including alternatively elevated microglia and decreased proinflammatory cytokines (IL-1b and TNFa). 15 In present study, we also found that PD-MSCs inhibited neuroinflammatory responses through decreased iNOS and COX-2 expression, decreased proinflammatory cytokines including IL-1b and TNFa, and inactivated glia cells. These results suggest that the inhibitory effect of PD-MSCs on the activation of astrocytes and microglia might be significant to modulate neuroinflammatory-mediated amyloidogenesis. Based on previous reports in Alzheimer's diseases patients and animal models, [41][42][43] we speculated whether activated microglia and astrocytes by Ab also caused cytokine releases. As a result of mouse cytokine array, the transplantation of PD-MSCs also inhibited cytokine secretion induced in Ab 1-42 -infused mice. We found that the levels of cytokines including IL-1b, IL-17, TNF-a, and IP-100 were significantly altered by Ab 1-42 that were inhibited by PD-MSCs. IL-1b, IL-17, and TNFa have important roles in affected brain regions in association with plaques and tangles in Alzheimer's disease. 44,45 Ab induces the production and secretion of IL-1b, 46 and astrocytes induce neuronal death by induction of IL-1b and IL-17, together with TNFa. 47 TNFa binds to neuronal Cd120a/b receptors triggering caspase activation through death effector domains. 48 IP-10 is markedly increased in reactive astrocytes in Alzheimer's disease brains, and IP-10positive astrocytes are associated with senile plaques and Ab-induced neurotoxicity. 49,50 Similar to our observation, MSC tends to be migrated and localized to tissues that undergo an inflammatory response, altering the spectrum of local cytokines. 51 MSC is a multipotent cell that can be differentiated into a variety of cell types, including: osteogenic, chondrogenic, adipogenic, and neural cell lineages. 52 However, in the mouse brain, PD-MSC was not differentiated into neural cell. Therefore, our results implied that PD-MSCs might have a role in the memory improvement in Alzheimer's diseases because of the antineuroinflammatory reactionassociated antiamyloidogenesis, but not the multipotency of PD-MSCs.
Inflammation is also frequently associated with hippocampal neurogenesis that causes deficits in cognition. 53 The cognitive deficits associated with Alzheimer's diseases were ameliorated by stimulation of cell proliferation and survival and integration of newborn cells into neuronal network of the SGZ-GCL of the hippocampus. 54 The decrease of hippocampal neurogenesis is associated with behavioral deficits and developed with age, in parallel with the development of Alzheimer's diseases. 55,56 Neural stem cells in the hippocampus are located in the SGZ at the border between the GCL and hilus of the DG. 57 Some of the daughter cells produced by division of those precursor cells differentiate into neurons and develop the prominent apical dendrite that characterizes dentate granule neurons as they move into the GCL. 58 Adultnewborn neurons (DCX-positive cells) project axons to the primary target of dentate granule neurons and the stratum lucidum of CA3 area, and then integrated into the hippocampal circuitry. 59 In Ab 1-42 -infused mice model, a decrease in hippocampal neurogenesis was detected on the SGZ at the border between the GCL and hilus of the DG, whereas PD-MSCs recovered neurogenesis of adult neural progenitor cells. Overall, our data indicated that PD-MSCs could also improve memory function through partially enhanced neurogenesis.
In conclusion, our data not only provide novel evidences of PD-MSCs on inflammation-mediated amyloidogenesis, neurogenesis, and neuroprotection in an Ab 1-42 infused mice model, but also our findings strongly support the therapeutic potential and clinical use of PD-MSCs in Alzheimer's diseases.

Materials and Methods
Animals. Male ICR mice (20-25 g) were purchased from Samtako (Seoul, Korea) and were maintained in accordance with the National Institute of Toxicological Research of the Korea Food and Drug Administration guideline for the humane care and use of laboratory animals. All experimental procedures in the present study were approved by the IACUC of Chungbuk National University (approval number: CBNUA-144-1001-01). Animals were housed in a room that was automatically maintained at 21-251C and relative humidity (45-65%) with a controlled light-dark cycle. All animals had free access to food (Samyang Foods, Seoul, Korea) and water.
Isolation and expansion of human PD-MSCs. Normal human placentas with no evidence of medical, obstetrical, or surgical complications were collected from normal full-term pregnancies (Z37 weeks of gestational age). All donors provided written informed consent before placenta collection. The protocols for sample isolation and their subsequent use for research purposes were approved by the institutional review board (IRB) of the Bundang CHA General Hospital, Korea. PD-MSC was harvested from the amniochorionic membrane of placentas obtained after Cesarean section. We isolated PD-MSCs and characterized them (Supplementary Figure 2) as previously described. 60 Briefly, the chorioamniotic membrane was peeled and separated from the placenta. Subsequently, the amnion and innermost membrane from the chorion and decidua were removed. These tissues were washed several times in phosphate-buffered saline (PBS, Gibco, Grand Island, NY, USA), and then cells of chorionic plate side scraped from the membrane were removed and the remainder membrane was dissected and minced in a Petri dish. The minced tissue was transferred to a 50 ml centrifuge tube and digested for 30 min at 371C shaking incubator containing  Figure 6 The localization of PD-MSCs with NeuN, DCX, and GFAP in Ab 1-42infused mice. (a-c) Immunofluorescence analysis using confocal microscope in Ab 1-42 -infused mice. The sections were fixed and permeabilized. Mature neuron (a), newborn neuron (b), and astrocytes (c) (green, left) were immunostained with anti-NeuN, anti-DCX, and anti-GFAP antibodies, and then followed by Alexa488conjugated secondary antibodies. PD-MSC (red, middle) was immunostained with anti-CD90 antibody, and then followed by Alexa555-conjugated secondary antibodies. The right panels of (a-c) show the merged images of the left and middle panels. The experiments shown in Figure 5 were repeated in triplicate with similar results atmosphere of 5% CO 2 and 3% O 2 . When cells reached 70-80% confluence, adherent cells were dissociated with TrypLE (Gibco), washed, and replated at a concentration of 4 Â 10 3 cells/cm 2 .
Ab 1-42 -infused mouse model. The infusion mouse model was adapted from previous work on the rat infusion model (Nitta et al. 26 ). The anesthetized animals were placed in a sterotaxic instrument and catheters were attached to an osmotic mini-pump (Alzet 1002, ALZA, Mountain View, CA, USA) and brain infusion kit 1 (Alzet kit 3-5 mm, ALZA) that were implanted according to the following coordinates: mouse (unilaterally): À 1.0 mm anterior/posterior, þ 0.5 mm medial/lateral, and À 2.5 mm dorsal/ventral. The pump contents were released over a period of 14 days consisting of 300 pmol aggregated Ab 1-42 (Bachem Chemical, Torrance, CA, USA) dissolved in sterile saline (0.9% NaCl) for each pump.
Water maze test. The learning and memory capacity was assessed using two separate tests (water maze and passive avoidance test). The water maze test is a widely accepted method for testing memory. The examination was performed using the SMART-CS (Panlab, Barcelona, Spain) program and equipment. A circular plastic pool (height 35 cm, diameter 100 cm) was filled with water (containing milk) kept at 22-251C. An escape platform (height 14.5 cm, diameter 4.5 cm) was positioned submerged 0.5-1 cm below the surface of the water. The test was performed three times per day for 7 days. Each trial lasted for 60 s or ended as soon as the mouse reached the submerged platform and was allowed to remain on the platform for 2 s. The mice were allowed to swim until they sought the escape platform. Escape latency, escape distance, swimming speed, and swimming pattern of each mouse were monitored by a camera above the center of the pool connected to a SMART-LD program (Panlab). A quiet environment, consistent lighting, constant water temperature, and fixed spatial frame were maintained throughout the period of the experiment.
Probe test. A probe test to assess memory consolidation was performed 2 days after the 7-day acquisition tests. In this test, the platform was removed from the tank, and the mice were allowed to swim freely. For these tests, the spent time on the target quadrant within 60 s was recorded. The time spent in the target quadrant was taken to indicate the degree of memory consolidation that had taken place after learning. The swimming pattern of each mouse was monitored by a camera above the center of the pool connected to a SMART-LD program as described above.
Passive avoidance performance test. The passive avoidance test is also widely accepted as a simple and rapid method for testing memory. The passive avoidance response was determined using a 'step-through' apparatus (Med Associates, Georgia, VT, USA) that consists of an illuminated and a dark compartment (each 20.3 Â 15.9 Â 21.3 cm) adjoining each other through a small gate with a grid floor of 3.175 mm stainless steel rods set 8 mm apart. At 1 day after the probe test, the training trial was performed. The mouse was placed in the illuminated compartment facing away from the dark compartment. When the mouse moved completely into the dark compartment, it received an electric shock (1 mA, 3 s duration). Then the mouse was returned to its home cage. After 1 day, the mouse was placed in the illuminated compartment and the latency period to enter the dark compartment, defined as 'retention,' was measured. The time when the mouse entered the dark compartment was recorded and described as stepthrough latency. The retention trials were set at a limit of 180 s as cutoff time.
Brain tissue collection and preservation. After behavioral tests, animals were perfused with PBS under inhaled diethyl ether anesthetization. The brains were immediately removed from skull, and the cortex and hippocampus were dissected on ice. All the brain tissues were immediately stored at À 801C until biochemical assays were conducted.
Western blot analysis. Cells and each area of the brain tissue were homogenized and lysed by a 30 min incubation on ice. The lysates were centrifuged at 15 000 Â r.p.m. for 15 min. An equal amount of total protein (20 mg) isolated from brain tissues was resolved on an sodium dodecyl sulfate (SDS) 10 or 12% polyacrylamide gel and then transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA). Blots were blocked for 1 h at room temperature with 5% (w/v) nonfat dried milk in Tris-buffered saline Tween-20 (TBST: 10 mM Tris (pH 8.0) and a 150 mM NaCl solution containing 0.05% Tween-20). After a short wash in TBST, membranes were incubated at 41C with specific antibodies. The blot was then incubated with the HRP-conjugated anti-mouse or anti-rabbit antibodies (1 : 2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactive proteins were detected with the enhanced chemiluminescence (ECL) western blotting detection system.
Immunohistochemistry and immunofluorescence. Mice were anesthetized with ether. While under general anesthesia, the mice received intracardiac perfusion with 50 ml of saline. Brains were fixed in formalin and paraffin-enclosed for examination. Tissue sections, 5 mm thick, were used with immunohistochemistry (also used in immunofluorescence). Paraffin-embedded sections were deparaffinized and rehydrated, washed in distilled water, and then subjected to heat-mediated antigen retrieval treatment. Endogenous peroxidase activity was quenched by incubation in 1% hydrogen peroxide in methanol for 30 min and then cleared in PBS for 5 min. The sections were blocked for 30 min with 3% normal horse/goat serum diluted in PBS. These sections were incubated overnight with appropriate antibodies. After washing in PBS, the sections were incubated in biotinylated goat anti-mouse/rabbit IgG antibody (1 : 1000 dilution, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were subsequently washed and incubated with avidin-conjugated peroxidase complex (ABC kit, 1 : 200 dilution, Vector Laboratories) for 30 min followed by PBS washing. The peroxidase reaction was performed in PBS using 3, 3'-diaminobenzidine tetrahydrochloride (DAB, 0.02%) as the chromogen. Finally, sections were dehydrated in ethanol, cleared in xylene, and mounted with Permount (Fisher Scientific, Waltham, MA, USA), and evaluated on a light microscopy (Olympus, Tokyo, Japan). For immunofluorescence, sections were incubated with an anti-rabbit secondary antibody labeled with Alexa-Fluor 488 (1 : 400 dilution; Invitrogen, Carlsbad, CA, USA) or anti-mouse secondary antibody labeled with Alexa-Fluor 568 (1 : 400 dilution, Invitrogen) for 2 h at room temperature. Final images were acquired using a confocal laser scanning microscope (TCS SP2, Leica Microsystems AG, Werzlar, Germany).  . Lysates of brain tissue were obtained through protein extraction buffer containing protease inhibitor. Ab 1-42 level was determined using each specific enzyme-linked immunosorbent assay (ELISA) Kit (Immuno-Biological Laboratories Co., Ltd, Takasaki-Shi, Gunma, Japan). In brief, 100 ml of sample was added into the precoated plate and was incubated overnight at 41C. After washing each well of the precoated plate with washing buffer, 100 ml of labeled antibody solution was added and the mixture was incubated for 1 h at 41C in the dark. After washing, chromogen was added and the mixture was incubated for 30 min at room temperature in the dark. Finally, the resulting color was assayed at 450 nm using a microplate absorbance reader (SunriseTM, TECAN, Switzerland) after adding stop solution.

Measurement of Ab
TUNEL assay. DNA fragmentation was examined by terminal deoxynucleotidyl transferase-mediated FITC-dUDP nick-end labeling (TUNEL). TUNEL assays were performed using the in situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. Briefly, after fixation of 25-mm cryosections with 4% paraformaldehyde, treatment with 0.1% NaBH4 and 0.1 Triton X-100, the slides were incubated for at least 1 h with a reaction mix containing deoxynucleotidyl transferase and FITC-dUDP (Roche, Reinach, Switzerland). For 4 0 ,6 0 -diamidino-2-phenylindole dihydrochloride (DAPI) staining, slides were incubated for 15 min at room temperature in the dark with mounting medium for fluorescence containing DAPI (Vector Laboratories). The tissues were then observed through a fluorescence microscope (Leica Microsystems AG, Wetzlar, Germany) and the nuclei were visualized by the DAPI staining.
Proteome profiler arrays. The brain tissues were excised and homogenized in PBS with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and Triton X-100 (final concentration 1%). The samples were frozen at À 701C, thawed, and centrifuged at 10 000 Â g for 5 min to remove any cellular debris. Proteins (200 mg) collected from three samples per group were used for mouse cytokine array according to the protocol provided by supplier (Proteome Profiler, ARY006, R&D Systems).
Statistical analysis. The data were analyzed using the GraphPad Prism version 4 program (GraphPad Software, Inc., San Diego, CA, USA). Data are presented as mean ± S.D. Statistical significance was performed on the data