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Gene expression for mouse feeder cells (inactivated MEFs)

Gene expression for mouse feeder cells (inactivated MEFs)


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I'm looking for a paper with gene expression data for mouse feeder cells, inactivated by gamma radiation or mitomycin C. Ideally I'd like RNA-seq data but I'll use microarray data if that's all there is.

I've spent quite a while looking (in GEO, Google Scholar, PubMed, etc) with no luck. Any leads would be appreciated.


Different murine-derived feeder cells alter the definitive endoderm differentiation of human induced pluripotent stem cells

The crosstalk between cells is important for differentiation of cells. Murine-derived feeder cells, SNL76/7 feeder cells (SNLs) or mouse primary embryonic fibroblast feeder cells (MEFs) are widely used for culturing undifferentiated human induced pluripotent stem cells (hiPSCs). It is still unclear whether different culture conditions affect the induction efficiency of definitive endoderm (DE) differentiation from hiPSCs. Here we show that the efficiency of DE differentiation from hiPSCs cultured on MEFs was higher than that of hiPSCs cultured on SNLs. The qPCR, immunofluorescent and flow cytometry analyses revealed that the expression levels of mRNA and/or proteins of the DE marker genes, SOX17, FOXA2 and CXCR4, in DE cells differentiated from hiPSCs cultured on MEFs were significantly higher than those cultured on SNLs. Comprehensive RNA sequencing and molecular network analyses showed the alteration of the gene expression and the signal transduction of hiPSCs cultured on SNLs and MEFs. Interestingly, the expression of non-coding hXIST exon 4 was up-regulated in hiPSCs cultured on MEFs, in comparison to that in hiPSCs cultured on SNLs. By qPCR analysis, the mRNA expression of undifferentiated stem cell markers KLF4, KLF5, OCT3/4, SOX2, NANOG, UTF1, and GRB7 were lower, while that of hXIST exon 4, LEFTY1, and LEFTY2 was higher in hiPSCs cultured on MEFs than in those cultured on SNLs. Taken together, our finding indicated that differences in murine-feeder cells used for maintenance of the undifferentiated state alter the expression of pluripotency-related genes in hiPSCs by the signaling pathways and affect DE differentiation from hiPSCs, suggesting that the feeder cells can potentiate hiPSCs for DE differentiation.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Experimental procedure for DE differentiation…

Fig 1. Experimental procedure for DE differentiation from SNL- and MEFP1-201B7 and -253G1 cells.

Fig 2. Experimental procedure for DE differentiation…

Fig 2. Experimental procedure for DE differentiation from MEFP2- or MEFP1-SNL-201B7 cells.

Fig 3. Expression analysis of SOX17 and…

Fig 3. Expression analysis of SOX17 and FOXA2 mRNA in DE differentiated SNL-201B7 and -253G1…

Fig 4. Immunofluorescent staining of SOX17 and…

Fig 4. Immunofluorescent staining of SOX17 and FOXA2 proteins in DE differentiated SNL-201B7 and -253G1…

Fig 5. Flow cytometric analysis of CXCR4…

Fig 5. Flow cytometric analysis of CXCR4 protein expression in DE differentiated SNL-201B7 and -253G1…

Fig 6. Expression analysis of SOX17 and…

Fig 6. Expression analysis of SOX17 and FOXA2 mRNAs in DE differentiated MEFP2- and MEFP1-SNL-201B7…

Fig 7. Immunofluorescent staining of SOX17 and…

Fig 7. Immunofluorescent staining of SOX17 and FOXA2 proteins in DE differentiated MEFP2- and MEFP1-SNL-201B7…

Fig 8. Flow cytometric analysis of CXCR4…

Fig 8. Flow cytometric analysis of CXCR4 expression in DE differentiated MEFP2- and MEFP1-SNL-201B7 cells.

Fig 9. Undifferentiated states of SNL- and…

Fig 9. Undifferentiated states of SNL- and MEFP1-201B7 cells.

(A and B) Quantification of mouse…

Fig 10. Molecular network analysis of SNL-…

Fig 10. Molecular network analysis of SNL- and MEFP1-201B7 cells by KeyMolnet program using the…

Fig 11. Mapping of RNA-sequencing of SNL-201B7…

Fig 11. Mapping of RNA-sequencing of SNL-201B7 and MEFP1-201B7 on the human genome, hg38.

Fig 12. Approximately 32-nucleotide conserved RNA fragment…

Fig 12. Approximately 32-nucleotide conserved RNA fragment in hXIST exon4 highly expressed in MEFP1-201B7.

Fig 13. Changes in gene-expression of hXIST…

Fig 13. Changes in gene-expression of hXIST exons and of the undifferentiated stem cell marker…


Abstract

Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.

Citation: Huang B, Ning S, Zhuang L, Jiang C, Cui Y, Fan G, et al. (2015) Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells. PLoS ONE 10(6): e0130332. https://doi.org/10.1371/journal.pone.0130332

Academic Editor: Austin John Cooney, Baylor College of Medicine, UNITED STATES

Received: April 17, 2014 Accepted: May 18, 2015 Published: June 19, 2015

Copyright: © 2015 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: Data have been uploaded to Figshare database and are accessible through: (http://dx.doi.org/10.6084/m9.figshare.1431806), (http://dx.doi.org/10.6084/m9.figshare.1431818), and (http://dx.doi.org/10.6084/m9.figshare.14318187). For any queries related to the protocol for preparing eiMEF, contact Dr Lianju Qin: [email protected]

Funding: This work was supported by the grants from the State Major Research Program of China (2012CB944902), National Natural Science Foundation of China (81370764) Jiangsu projects (BL2012009, FXK201221 and the PAPD project) and China Scholarship Council (201307060009).

Competing interests: The authors have declared that no competing interests exist.


Results

Dynamics of alternative splicing in C/EBPα-enhanced B cell reprogramming occur independently from gene expression changes

To study the dynamics of changes in alternative splicing (AS) during cell reprogramming, primary mouse pre-B cells (hereafter referred to as “B cells”) were reprogrammed as previously described [18,19,20]. Briefly, B cells were isolated from bone marrow of reprogrammable mice, containing a doxycycline-inducible OSKM cassette and an OCT4-GFP reporter. These cells were infected with a retroviral construct containing an inducible version of C/EBPα fused to the estrogen receptor ligand-binding domain (ER). Infected cells were selected and re-plated on a feeder layer of inactivated MEFs. This was followed by a 18 h-long pulse of β-estradiol, triggering the translocation of C/EBPα-ER to the nucleus and poising the B cells for efficient and homogeneous reprogramming [18, 19]. After washout of β-estradiol, reprogramming was induced by growing the cells for 8 days in reprogramming medium containing doxycycline (see “Methods” and [20]). RNA was isolated every other day from duplicates and subjected to paired-end sequencing (RNA-seq), resulting in high coverage (more than 100 million reads per sample) (Fig. 1A). As positive controls, mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells were also sequenced.

Dynamics of alternative splicing and gene expression changes during B cell reprogramming. A Schematic representation of C/EBPα-mediated B cell reprogramming time points and related controls analyzed by RNA-seq. Bα cells: B cells treated for 18 h with β-estradiol to activate C/EBPα. B Stacked bar plot representing cumulative number of events differentially spliced between B cells and subsequent reprogramming stages (x-axis), as well as controls (iPS and ES cells). The y-axis represents the number of differentially spliced events compared to B cells. The upper part corresponds to events with positive ∆PSI values compared to B cells (> 10%), the lower part to events with negative ∆PSI values compared to B cells (< − 10%). Red/orange areas: alternative 3′/5′ splice sites (Alt3/Alt5) respectively grey areas: retained introns blue-green areas: cassette exons of increasing complexity (see “Methods”). See also Additional file 1: Figure S1A. C Clusters of cassette exons displaying related profiles of inclusion level changes during B cell reprogramming. Six clusters (out of a total of 12 identified, see Additional file 1: Figure S1B-C) are shown and classified into 4 categories, corresponding to the timing of the main shift observed (left). The y-axis represents scaled percent spliced in (PSI) values. The color of each line corresponds to the membership score of each exon relative to the general trend of the cluster. The size (number of events) of each cluster is indicated (n). D RT-PCR validation of selected cassette exon changes inferred from RNA-seq analyses. AS events assigned to different clusters were analyzed by semi-quantitative RT-PCR and quantified by capillary electrophoresis. Each panel includes a gel representation of the inclusion (top) and skipping (bottom) amplification products of one of the replicates and the corresponding quantification of the duplicates (PSI = molarity of inclusion product / molarity of inclusion + skipping products). Light grey columns: PSI values quantified by RT-PCR dark grey columns: PSI values quantified by RNA-seq analysis using vast-tools software (n = 2). E Validation of Grhl1 exon 6 and Dnmt3b exon 10 inclusion level changes, previously associated with reprogramming and pluripotency, performed as in panel D. Crosses indicate time points for which PSI values were calculated with low coverage (less than 10 actual reads). F Heatmap displaying correlations between B cell reprogramming stages according to gene expression (blue, top heatmap) and AS (red, bottom heatmap). Color scales represent Pearson correlation coefficient values calculated on the cpm values of the 25% most variably expressed genes or upon the PSI values of the 25% most variable cassette exons. G Gene expression patterns of genes containing the exons belonging to each of the AS clusters in panel C. Genes with expression changes correlating with the cluster centroid or its negative (membership > 0.3) are highlighted in blue and green, respectively, while the grey portion represents the (majority of) genes which follow gene expression profiles that do not match the changes in inclusion patterns of their exon(s). Percentage of concordant/contrasting patterns are displayed for each cluster. See also Additional file 1: Figure S1D. H Stacked bar plot representing the percentage of cassette exons in each of the AS clusters in panel C classified according to the following categories: disrupting the open reading frame (ORF) upon exclusion or inclusion, preserving the transcript ORF, mapping in non-coding RNAs, 3′ UTRs or 5′ UTRs or uncertain. The first column represents all exons differentially spliced between at least one pair of conditions, while the following ones represent the exons belonging to the each AS cluster (indicated below the bar). Fractions < 5% are not indicated. Where indicated, statistical significance was calculated by Fisher’s exact test on the proportion of exons in the cluster compared to the general distribution of all AS exons (*, **, *** = p value < 0.05, 0.01, 0.001 respectively)

AS analysis was performed using vast-tools [21], an event-based software that quantifies percent spliced in (PSI) values of annotated AS events and constitutive exons in all samples. These analyses revealed more than 14,000 AS changes during the entire reprogramming time course (for any possible pair of conditions: minimum absolute ∆PSI of 10 between PSI averages and minimum difference of 5 between any individual replicates across conditions) and a gradual increase in the number of differentially spliced events when comparing B cells to progressive stages of somatic cell reprogramming (Fig. 1B and Additional file 1: Figure S1A). Different classes of AS events were detected, with similar relative proportions at the various time points: 31–47% cassette exons, 5–11% alternative 3′ splice sites and 6–11% alternative 5′ splice sites, and 33–57% retained introns (Fig. 1B).

To classify the dynamics of AS changes during reprogramming, we selected cassette exon (CEx) events differentially spliced in at least one comparison (4556 exons) and performed a fuzzy c-means clustering analysis on their scaled PSI values (Mfuzz [22]). This analysis revealed diverse kinetics of exon inclusion occurring during B cell reprogramming (Fig. 1C, Additional file 1: Figure S1B-C and Additional file 2: Table S1). Six major clusters of AS dynamic profiles were detected: (1) exons that become included already after the C/EBPα pulse (2) and (3) exons that are regulated either towards increased inclusion or skipping early after OSKM induction (day 2) (4) and (5) exons that display changes in inclusion at middle stages of reprogramming (day 4 onwards) (6) a cluster of exons only included at the latest steps of reprogramming (day 8 onwards) (Fig. 1C). Each of these clusters consisted of 300–500 exons. The inclusion levels of examples of exons belonging to different cluster types were validated by semi-quantitative RT-PCR (Fig. 1D). For reference, changes in the PSI values of Grhl1 exon 6 and Dnmt3B exon 10, previously described to be associated with reprogramming and pluripotency [17, 23, 24] were also quantified and found to follow similar inclusion patterns in B cell reprogramming, compared to the ones previously described in other systems (Fig. 1E).

We next sought to compare general AS and gene expression dynamics during reprogramming. Gene expression was analyzed using the edgeR package [25] and the level of similarity between each pair of conditions was estimated using a Pearson correlation coefficient on the cpm (counts per million) values of the most variable genes (3rd quartile coefficient of variation, n = 2961). In addition, Pearson correlation coefficient was calculated based on the PSI values from the most variable cassette exons (3rd quartile coefficient of variation, n = 1140). Both analyses showed pronounced switches between days 4 and 6 post-OSKM induction (Fig. 1F). Overall, however, most clusters displayed matching profiles in gene expression and AS of any included exon in less than 10% of the genes, reaching a maximum of 20% in a subset of AS clusters (Fig. 1G and Additional file 1: Figures S1D-E). These results argue, as observed before in a variety of other systems (e.g., [26]), that global programs of regulation of gene expression and AS are uncoupled from each other.

Interestingly, a larger proportion of exons included (or skipped) at early stages of reprogramming are predicted to disrupt the open reading frame (ORF) of the transcripts upon exon skipping (or inclusion, respectively), compared to middle/late exons and to the general distribution of mapped alternative exons (Fig. 1H, classification as described in [21]). This suggests a higher impact of AS-mediated on/off regulation of the corresponding protein expression via nonsense-mediated decay (NMD), and a switch to expression of full-length proteins during early steps of reprogramming. Middle clusters, instead, contain more exons predicted to preserve the coding potential of their transcripts, implying modulation of the functions of their encoded protein isoforms rather than on/off switches (Fig. 1H). Consistent with these concepts, while PSI values of cassette exons tend to increase throughout reprogramming (Fig. 1B, blue bars), intron retention—generally leading to NMD—tends to decrease in the course of reprogramming (Fig. 1B, grey bars).

AS changes at intermediate reprogramming stages show commonalities with MEF reprogramming

As a first step to identify key AS events and potential regulators important for reprogramming, we compared our transcriptome analysis of B cell reprogramming with that of MEF reprogramming [17]. The two datasets differ in the experimental design and time frame (compare Figs. 1A and 2A). Specifically, the MEF system required sorting of cells undergoing reprogramming using the SSEA1 surface marker, while this was not necessary for B cell reprogramming due to its efficiency. To facilitate the comparison between the two transcriptome datasets, vast-tools analysis was applied to the MEF dataset [17], which yielded 843 cassette exons differentially spliced (|∆PSI| ≥ 10, range ≥ 5) between any pair of samples of the MEF reprogramming dataset. Despite differences in the experimental setup, 79% of these exons (669 out of 843) were also found to be differentially spliced in B cell reprogramming (Additional file 1: Figure S2A and Additional file 3: Table S2). A similar level of overlap was also observed at the level of other AS events (72% of AS events in general, Additional file 1: Figure S2A). The overlap between differentially spliced exons in the two systems was higher for middle and late AS clusters than for early clusters of B cell reprogramming (Fig. 2B), as might be expected from the convergence on a common program of AS related to pluripotency.

B cell and MEF reprogramming systems share a program of AS changes. A Schematic representation of MEF reprogramming time points analyzed by RNA-seq in [17]. B Percentage of exons in each B cell reprogramming AS cluster that are also detected as differentially spliced in the MEF reprogramming dataset of [17]. The number of events in each cluster is indicated at the bottom of the corresponding bar. The magenta dashed line indicates the average percentage for all 12 clusters. C PCA analysis of the 25% most variably expressed genes, segregated using k-means into 4 clusters: differentiated cells, early and late reprogramming and pluripotent cells, highlighted by colors and boxes. Circles: B cell reprogramming time points. Squares: MEF reprogramming time points. D Heatmap representing scaled PSI values (average between replicates) of exons differentially spliced in at least one time point in both B cell (left) and MEF reprogramming (right), with the corresponding hierarchical clustering. E Heatmap representing the expression of RNA-binding proteins known to be involved in pluripotency, somatic cell reprogramming, and/or development. Scaled cpm values (average between replicates) of both datasets are shown, with the corresponding hierarchical clustering. The bar plot (right) represents (when available) the fold change expression between ES cells and differentiated mouse tissues calculated in [7] and the corresponding ranking (color of the bar).

To further compare the two datasets, we performed a Principal Component Analysis (PCA) on the gene expression of the most variable genes in both datasets (3rd quartile coefficient of variation, n = 2679) separating the stages into four distinct groups by k-means clustering (Fig. 2C). These groups clearly distinguish between starting cells, early and late stages of reprogramming and pluripotent cells. The PCA allowed us to outline a “reprogramming pseudotime” which was subsequently used to select AS exons and regulators for functional characterization. It also further highlighted the transition between days 4 and 6 in B cell reprogramming, juxtaposing them with days 7/10 in MEF reprogramming. A heatmap displaying the scaled PSI values of the 669 common differentially spliced exons of the two datasets at the different steps of the reprogramming process shows substantial similarities in exon inclusion (Fig. 2D).

In contrast to the similarities in AS profiles between the two reprogramming systems, we observed that expression of RNA-binding proteins (RBPs) previously associated with pluripotency, somatic cell reprogramming, and/or development [7, 9, 14, 17] and presumably mechanistically linked to different aspects of post-transcriptional regulation, differed significantly between the two datasets (Fig. 2E). Despite these more divergent profiles, hierarchical clustering captured three known functional groups, with cluster (a) containing factors with higher expression in iPS/ES samples (fold change > 0 and high rank score according to 7, right panel) and known to promote pluripotency/reprogramming, such as U2af1 or Srsf2/3 [9, 14]. In contrast, cluster (b) contains factors with higher expression in the starting somatic cells, which includes known repressors of reprogramming such as Mbnl1/2, Celf2, and Zcchc24 [7, 11, 17]. Finally, cluster (c) contains factors with more variegated expression patterns at early and intermediate reprogramming steps (and mildly repressed in iPS/ES cells), including Esrp1/2 [16, 17] (Fig. 2E).

Taken together, our analyses revealed widespread AS changes during B cell reprogramming, which significantly overlapped with those of MEF reprogramming, especially at intermediate phases of the process.

Predicted regulators of alternative splicing during somatic cell reprogramming

To infer potential regulators of exons differentially spliced during B cell reprogramming, we extracted gene expression profiles of 507 RBPs (as annotated in the Uniprot database), which were detectably expressed (cpm ≥ 5 in at least 33% of samples) and featured a minimum of variation across the B cell reprogramming dataset (coefficient of variation ≥ 0.2). Using the membership function of the Mfuzz package, we correlated (positively or negatively) the scaled gene expression profile of each RBP to each AS cluster centroid. This allowed us to derive a list of potential regulators whose changes in levels of expression correlate (or anti-correlate) with the profiles of AS changes in each cluster (membership > 0.3) (Fig. 3, Additional file 1: Figure S3 and Additional file 4: Table S3). In line with previous work [7, 11, 14], our analysis identified known AS regulators involved in the induction or repression of pluripotency/developmental decisions, such as Mbnl1/2, Celf2 (both potential negative regulators of pulse/late clusters 1/6), and U2af1 (potential positive regulator of middle cluster 4) (Fig. 3). Importantly, additional RBPs and splicing factors without previously known functions in reprogramming emerged as possible regulators.

Inferred regulators of AS changes during B cell reprogramming. A Gene expression profiles of RNA-binding proteins (RBPs) correlating with the centroid of each AS cluster (positive regulators). Average scaled cpm values are represented by each line and the number of putative positive or negative regulators of each cluster is indicated (1). Expression profiles of Cspf3, Hnrnpul1 and Tia1 are highlighted. See also Additional file 1: Figure S3A. B Gene expression profiles of RBPs correlating with the negative of the centroid of each AS cluster (negative regulators). Displays as in panel A. See also Additional file 1: Figure S3B

To further define such regulators, we focused on RBPs that change their expression after the C/EBPα pulse or at early stages of reprogramming for functional validation during the induction of pluripotency, as we speculated that these could drive the inclusion/skipping of both early and intermediate AS exons during the reprogramming of C/EBPα-poised B cells. We selected CPSF3 as a putative positive regulator of very early events because its profile of expression increases during B cell and MEF reprogramming in parallel with centroid of AS changes in cluster 1 (Fig. 3A). While CPSF3 was originally described as part of cleavage / polyadenylation complexes [27], more recent work implicated components of this complex on alternative splicing regulation, including numerous internal exons not linked to the selection of alternative polyadenylation sites [28, 29]. Following a similar logic, we chose two putative negative regulators of cluster 1, namely TIA1, a well-described AS regulator with roles in cell proliferation and development (see below), and hnRNP UL1, a member of the heterogeneous ribonucleoprotein (hnRNP) family whose involvement in splicing regulation is largely unexplored (Fig. 3B). Due to experimental difficulties of genetic manipulation of the B cell reprogramming system (see “Discussion”), we modulated their expression at early stages of MEF reprogramming [30] and examined the consequences on the dynamics of pluripotency induction and on relevant AS alterations.

Knockdowns of CPSF3 or hnRNP UL1 repress MEF reprogramming

The Cleavage and Polyadenylation Specificity Factor (CPSF) complex is primarily involved in mRNA polyadenylation, but a number of its components, including CPSF3, has been shown to also play a role in splicing [28, 29, 31,32,33]. Cpsf3 expression increased early during reprogramming of both B cells and MEFs (Figs. 3A and 4A). The expression of Hnrnpul1, an hnRNP whose function in RNA metabolism is poorly understood, decreases in B cells after the C/EBPα pulse and then stabilizes at levels similar to those observed throughout MEF reprogramming (Figs. 3B and 4B).

Knockdowns of CPSF3 or hnRNP UL1 impair MEF reprogramming. A Gene expression profiles of Cpsf3 in B cell reprogramming and MEF reprogramming (cpm values, blue and magenta lines, respectively). The x-axis represents “reprogramming pseudotime” in both datasets, calculated through the PCA analysis of Figure 2B. B Gene expression levels of Hnrnpul1 in B cell and MEF reprogramming (cpm values, blue and magenta lines respectively), as in panel A. C Relative expression levels of Cpsf3 mRNA quantified by RT-qPCR in non-infected cells (NI), cells transduced with a scrambled control shRNA (shSCR) or one of two shRNAs specific for Cpsf3. The y-axis represents the relative expression (2^(−∆Ct) value) of Cpsf3 after normalization over Gapdh. D Relative expression levels of Hnrnpul1 mRNA quantified by RT-qPCR as in panel C. E Relative expression levels of Pou5f1 and Nanog quantified by RT-qPCR as in panels C and D. See also Additional file 1: Figure S4B. BE Average of biological triplicates and SD values are shown. Statistical significance was calculated by t-test on ∆Ct values compared to the NI control (*, **, *** = p value < 0.05, 0.01, 0.001 respectively, corrected for multiple testing using Holm-Sidak method). F Reduction of early reprogramming intermediates at day 6 post-OSKM induction upon knockdowns of Cpsf3 and Hnrnpul1. Fold change was calculated from the percentage of SSEA1+EPCAM1− cells (of the total of alive cells) in every condition compared to the shSCR control using flow cytometry analysis. G Reduction of late reprogramming intermediates at days 10 and 12 post-OSKM induction upon knockdown of Cpsf3 or Hnrnpul1. Fold change was calculated from the percentage of SSEA1+EPCAM1+ cells (of the total of alive cells) in every condition compared to the shSCR control using flow cytometry analysis. See Additional file 1: Figure S4C for examples of gates. H Number of colonies stained with alkaline phosphatase (AP) at day 14 post-OSKM induction upon knockdown of Cpsf3 or Hnrnpul1. On the bottom, images of representative wells are shown for every condition. F,G,H Average of biological triplicates and SD values are shown. Statistical significance was calculated by t-test comparing each condition to the shSCR control (*, **, *** = p value < 0.05, 0.01, 0.001 respectively, corrected for multiple testing with Holm-Sidak method)

To test their effects on MEF reprogramming, two different short hairpin RNAs (shRNAs) for each factor were cloned into lentiviral vectors containing a GFP reporter. The protocol used is summarized in Additional file 1: Figure S4A. Briefly, early passage MEFs isolated from reprogrammable mice were transduced with constructs bearing the shRNAs and the cells treated with doxycycline to activate OSKM (day 0). GFP + cells were FACS-sorted 48 h post-infection and seeded on inactivated MEFs serving as feeder layers, to proceed with reprogramming for up to 14 days. Cells were harvested every other day and samples analyzed by RT-qPCR for the expression of the shRNAs target and of pluripotency markers, and by flow cytometry to quantify the proportion of cells expressing the early pluripotency cell surface marker SSEA1 (appearing around days 5–6) and the later pluripotency marker EPCAM1 (appearing around day 8, [34]). At day 14, 2 days after removing doxycycline, cultures were stained for alkaline phosphatase (AP) activity to identify iPS colonies and to assess the efficiency of reprogramming.

Cells infected with shRNAs targeting Cpsf3 and Hnrnpul1 were compared with non-infected (NI) cells, as well as with cells transduced with a scrambled control shRNA (shSCR). Knockdown efficiency, quantified by RT-qPCR (Fig. 4C, D), showed a 51% and 58% reduction in Cpsf3 at day 6 post-infection with shCPSF3#1 and #2, respectively, becoming slightly less efficient during reprogramming (Fig. 4C). Similarly, knockdown of Hnrnpul1 resulted in 81% and 76% reduction of mRNA levels at day 6 post-infection with shUL1#1 and #2, respectively (Fig. 4D). The increase in the mRNA levels of endogenous Pou5f1 (encoding OCT4) and Nanog pluripotency markers was delayed in both Cpsf3 and Hnrnpul1 knockdown cells (compare for example values at day 8 in Fig. 4E and Additional file 1: Figure S4B), suggesting slower reprogramming kinetics. Consistent with these observations, knockdown of Cpsf3 and Hnrnpul1 reduced the percentage of cells expressing SSEA1 at day 6 (Fig. 4F) and cells expressing both SSEA1 and EPCAM1 at days 10–12 (Fig. 4G and Additional file 1: Figure S4C). Survival of cells during reprogramming did not seem to be affected in the knockdowns because no significant increase in the proportion of DAPI-stained cells was observed throughout reprogramming (Additional file 1: Figure S4D). Finally, we counted the number of AP positive colonies at day 14 and found that the amount was significantly reduced in cells infected with either the shRNAs targeting Cpsf3 or those targeting Hnrnpul1 (Fig. 4H). We finally tested the effects of overexpression of a T7-tagged version of CPSF3 from the beginning of reprogramming (Additional file 1: Figure S4A and E). A trend towards increased expression of Pou5f1 and Nanog at late times of reprogramming upon T7-Cpsf3 overexpression was observed (Additional file 1: Figure S4F). However, this was not accompanied by enhanced reprogramming efficiency or redistribution of reprogramming intermediates (Additional file 1: Figure S4G-I), likely because of additional rate-limiting steps required to increase the efficiency of this complex process or because of suboptimal timing or levels of overexpression achieved in the experiment.

Taken together, these data show that the knockdown of Cpsf3 or Hnrnpul1 reduces the MEF to iPS reprogramming efficiency, therefore arguing that both RBPs contribute to the induction of pluripotency.

Overexpression of TIA1 represses MEF reprogramming

TIA1 is an RBP and AS regulator [35,36,37]. Tia1 mRNA levels decrease early during B cell reprogramming (Figs. 3B and 5A), compatible with a potential role as a repressor of cell reprogramming in this system. Because depletion of TIA1 induces mouse embryonic lethality and the protein is important for MEF proliferation, cell cycle progression, autophagy, and numerous signaling pathways [38], we decided to test the effects of TIA1 overexpression during MEF reprogramming. For this purpose, primary MEFs were infected at day 0 (concomitantly with the induction of reprogramming), with retroviral constructs containing a T7-tagged Tia1 cDNA and a GFP reporter to allow sorting of the transduced cells (Additional file 1: Figure S4A). Levels of Tia1 were quantified by RT-qPCR (24-fold increase compared to cells transduced with an empty vector, Fig. 5B). Consistent with our prediction from its expression profile in B cell reprogramming, overexpression of Tia1 repressed the induction of endogenous Pou5f1 and Nanog genes (Fig. 5C and Additional file 1: Figure S5A) and led to a reduction of early SSEA1 + EPCAM1 − cells and of late reprogramming intermediates (SSEA1 + EPCAM1 + cells) compared to empty vector and non-infected controls (Fig. 5D, E and Additional file 1: Figure S5B). In addition, it significantly reduced the count of AP + colonies at day 14 post-OSKM induction compared to controls (Fig. 5F), without substantially affecting the viability of reprogramming cells (Additional file 1: Figure S5C). Of note, overexpression of Tia1 delayed the gradual skipping of Lef1 exon 6, a conserved AS event between B cell and MEF reprogramming (Fig. 5G), which might partially explain the observed reduction in reprogramming efficiency.

Overexpression of TIA1 impairs MEF reprogramming. A Gene expression profiles of Tia1 mRNA in B cell and MEF reprogramming (cpm values, blue and magenta lines respectively). The x-axis represents the “reprogramming pseudotime” in both datasets, calculated through the PCA analysis of Fig. 2B. B Expression levels of Tia1 mRNA relative to Gapdh, quantified by RT-qPCR in non-infected cells (NI), cells transduced with an empty vector (Empty) or with T7-Tia1 cDNA. C Expression levels of Pou5f1 and Nanog relative to Gapdh, quantified by RT-qPCR as in panel B. See also Additional file 1: Figure S4E. B,C Average of biological replicates and SD values are shown. Statistical significance was calculated by t-test on ∆Ct values comparing to the Empty control (*, **, *** = p value < 0.05, 0.01, 0.001 respectively, corrected for multiple testing with Holm-Sidak method). D Percentage of SSEA1+EPCAM1− early reprogramming intermediates (day 6 post-OSKM induction) upon Tia1 overexpression determined by flow cytometry. E Percentage of SSEA1+EPCAM1+ late reprogramming intermediates (days 10 and 12 post-OSKM induction) upon Tia1 overexpression. See Additional file 1: Figure S5B for gating strategy. F Number of alkaline phosphatase (AP) positive colonies at day 14 post-OSKM induction upon Tia1 overexpression. Images of representative wells are shown below. D,E,F Average of biological replicates and SD values (n = 4). Statistical significance was calculated by t-test comparing to the Empty control (*, **, *** = p value < 0.05, 0.01, 0.001 respectively, corrected for multiple testing with Holm-Sidak method). G Inclusion of Lef1 exon 6 upon overexpression of T7-Tia1, quantified by semi-quantitative RT-PCR and capillary electrophoresis. Values represent average and SD. Statistical significance calculated by t-test on average ± SD of the area under the curve in each condition yielded a p value of 0.079

Taken together, the observed reduced expression of TIA1 during B cell reprogramming and its impairment of MEF reprogramming when overexpressed suggest that it functions as a general repressor of pluripotency induction.

CPSF3, hnRNP UL1, and TIA1 regulate alternative splicing during reprogramming

To assess the effects of CPSF3, hnRNP UL1, and TIA1 manipulation on AS during reprogramming, RNA-seq analyses were carried out with RNAs isolated from MEFs at 0 and at 12 days post-OSKM induction, comparing the effects of Cpsf3/Hnrnpul1 knockdown (two different shRNAs for each factor) or Tia1 overexpression with those of the corresponding shSCR/empty vector controls (Fig. 6A). Quantification of gene expression of Tia1, Cpsf3, and Hnrnpul1 at the two timepoints is shown in Additional file 1: Figures S6A-B.

Knockdowns of CPSF3 or hnRNP UL1 and overexpression of TIA1 regulate AS during reprogramming. A Schematic representation of the experiments performed to study the effect of TIA1 overexpression / CPSF3 or hnRNP UL1 knockdown by RNA-seq. B TIA1-dependent events detected during reprogramming. The x-axis represents the ∆PSI value between Empty day 12 and day 0. The y-axis represents the ∆PSI value between T7-TIA1 day 12 and day 0 control. TIA1-dependent events (|∆∆PSI(T7-TIA1 − Empty)| ≥ 10) are represented by colored dots (palette representing the |∆∆PSI(T7-TIA1 − Empty)| value, n = 387). TIA1-independent events (|∆∆PSI(T7-TIA1 − Empty)| < 2) are represented by grey dots (n = 558). C CPSF3- and UL1-dependent events detected during reprogramming (left and right, respectively). The x-axis represents the ∆PSI value between shSCR day 12 and day 0 control. The y-axis represents the ∆PSI value between shCPSF3#1 or shUL1#1 day 12 and day 0 control. CPSF3/UL1-dependent events (∆∆PSI(average_shRNAs − shSCR) ≥ 10) are represented by non-grey-colored dots (palette representing the ∆∆PSI(average_shRNAs − shSCR) value). CPSF3/UL1-independent events (∆∆PSI(average_shRNAs − shSCR) < 2) are represented by grey dots. See Additional file 1: Figure S6C for ∆PSI values of the same events in shRNA#2 conditions. D Venn diagram representing the overlap between CPSF3-, UL1-, and TIA1-dependent events (left, the number of events in each category is shown). Barplot representing the percentage of CPSF3-, UL1-, and TIA1-dependent events which are also differentially spliced in B cell reprogramming (right, the percentage of overlap in each category is indicated). E Violin plots representing the distribution of PSI values of TIA1-dependent events in non-infected cells (NI, day 0) and day 12 cells infected with Empty or T7-Tia1 vectors. F Violin plots representing the distribution of PSI values of TIA1-independent events as in panel E. E,F Statistical significance was calculated by Fisher’s exact test comparing number of events with intermediate (25 < PSI < 75) or extreme PSI values (PSI ≥ 75 or ≤ 25) in each condition against Empty control (*, **, *** = p value < 0.05, 0.01, 0.001 respectively). See also Additional file 1: Figure S6D. G Boxplots representing the distribution of the indicated sequence features of TIA1-dependent exons, compared to TIA1-independent events and a random set of exons with intermediate PSI values not changing throughout reprogramming (Control CEx). Statistical significance was calculated using Matt, by paired Mann-Whitney U test comparing each condition to the Control CEx set (*, **, *** = p value < 0.05, 0.01, 0.001 respectively). H RNA map representing the distribution of TIA1 binding motif in TIA1-dependent exons and flanking introns, compared to TIA1-independent and Control CEx. Thicker segments indicate regions in which enrichment of TIA1 motif is significantly different compared to Control CEx. I Gene Ontology (GO) terms enriched in genes containing TIA1-dependent events, compared to a background of all genes containing mapped AS events in the dataset. GO enrichment was calculated using GOrilla and GO terms were summarized for visualization using REViGO. The x-axis and the size of each bubble represent the −log10(p value) of each GO term. J Violin plots representing the distribution of PSI values of CPSF3-dependent events in non-infected cells (NI, day 0) and day 12 cells infected with shSCR or two shRNAs specific for CPSF3 (shC#1 and shC#2) or UL1 (shU#1 and shU#2). K Violin plots representing the distribution of PSI values of CPSF3-independent events as in panel J. J,K Statistical significance was calculated by Fisher’s exact test comparing number of events with intermediate (25 < PSI < 75) or extreme PSI values (PSI ≥ 75 or ≤ 25) in each condition against shSCR control (*, **, *** = p value < 0.05, 0.01, 0.001 respectively). See also Additional file 1: Figure S6E. L Boxplots representing the distribution of sequence features of CPSF3- and UL1-dependent exons, compared to the corresponding CPSF3- and UL1-independent events and Control CEx. Statistical significance was calculated using Matt, by paired Mann-Whitney U test comparing each condition to the Control CEx set (*, **, *** = p value < 0.05, 0.01, 0.001 respectively). M GO terms enriched in genes containing CPSF3- and UL1-dependent events, compared to a background of all genes containing mapped AS events in the dataset, performed as in panel I

To determine the impact of these perturbations on AS, we calculated differentially spliced events between day 0 and day 12 in each condition as described before (|∆PSI(day12 − day0)| ≥ 10, range ≥ 5). TIA1-dependent events were defined as those changing during reprogramming only in the control or the overexpression condition, with |∆∆PSI(TIA1 − Empty)| ≥ 10 (colored dots in Fig. 6B). Similarly, CPSF3-dependent and UL1-dependent events were defined as those with |∆∆PSI(average shRNAs − shSCR)| ≥ 10 (only events selected by both specific shRNAs were considered, 54% and 60% overlap respectively, colored dots in Fig. 6C and Additional file 1: Figure S6C). For each RBP, control events, changing during reprogramming but not affected by TIA1, CPSF3, or hnRNP UL1 manipulation, were classified as those differentially spliced between days 0 and 12, either in control or knockdown/overexpression conditions (|∆PSI(day12 − day0)| ≥ 10, range ≥ 5), that display minimal differences between the ∆PSI values in the two conditions (e.g., |∆∆PSI(TIA1 − Empty)| < 2) (grey dots in Fig. 6B, C and Additional file 1: Figure S6C). All sets are summarized in Additional file 5: Table S4.

We thus identified 387 TIA1-dependent events and 558 TIA1-independent events. Similarly, we identified 357 CPSF3-dependent and 298 UL1-dependent events (as well as 429 CPSF3-independent and 662 UL1-independent events). Interestingly, CPSF3-dependent and UL1-dependent events showed a significant overlap (e.g., 70% of UL1-dependent events were also CPSF3-dependent events, of which 98% changed in the same direction) whereas little overlap was observed with TIA1-dependent events (Fig. 6D, left panel). As we found no strong evidence for cross-regulation between CPSF3 and hnRNP UL1 factors (either in AS or in gene expression, Additional file 1: Figure S6B), these data suggest that CPSF3 and hnRNP UL1 contribute to a common program of AS changes relevant for cell reprogramming. Moreover, TIA1-, CPSF3-, and UL1-dependent events detected in MEFs showed high overlap with events that were also differentially spliced during B cell reprogramming, suggesting that these factors regulate AS events that generally contribute to the induction of pluripotency (Fig. 6D, right panel).

Focusing on cassette exon events, TIA1-dependent exons (but not TIA1-independent exons) displayed significantly higher PSI values at day 12 compared to day 0 of reprogramming, as well as a relative increase in exons displaying intermediate PSI values (Fig. 6E, F and Additional file 1: Figure S6E). These effects are altered by TIA1 overexpression (Fig. 6E), suggesting that in this system, the typical role of TIA1 is to repress exon inclusion.

Analysis of sequence features associated with TIA1-regulated exons performed using Matt [39] revealed weaker 5′ splice sites, shorter median length of the flanking downstream introns and a larger difference in GC content between the alternative exons and their flanking upstream introns (Fig. 6G), suggestive of a strong dependence of these exons on the process of exon definition [40]. Notably, an enrichment in putative TIA1 binding motifs was detected about 100 nucleotides upstream of the distal 3′ splice site (Fig. 6H), suggesting that binding of TIA1 to this region might repress inclusion of the alternative exon (or enhance pairing between the distal splice sites), leading to exon skipping.

Gene Ontology (GO) enrichment analysis (GOrilla [41]) on the set of genes containing TIA1-dependent AS events compared to a background list of all genes containing mapped AS events (n = 11132) showed an enrichment in functions related to peptide/hormone secretion, T-helper cell functions, and embryonic development (Fig. 6I and Additional file 6: Table S5).

CPSF3- and UL1-dependent exons showed similar inclusion patterns: an increased proportion of intermediate PSIs was observed at day 12 in CPSF3- and UL1-dependent but not in CPSF3- and UL1-independent events (Fig. 6J, K and Additional file 1: Figure S6E). CPSF3- and UL1-dependent exons also showed higher PSI values at day 12 compared to day 0, and an enrichment in exons displaying intermediate PSI values at the end of the reprogramming process (Fig. 6J, K and Additional file 1: Figure S6E). These effects were attenuated, specifically for CPSF3- and UL1-dependent exons, upon knockdown of either of these factors (Fig. 6J, K and Additional file 1: Figure S6E), supporting the notion that their regulatory programs overlap. Both CPSF3- and UL1-dependent exons displayed weaker 3′ and 5′ splice sites (Fig. 6L). GO analyses revealed that CPSF3- and UL1-dependent events belong to genes enriched in functional categories related to the regulation of cell morphogenesis, cell-substrate adhesion, and cytoskeleton organization (Fig. 6M and Additional file 6: Table S5). Changes in inclusion levels of selected CPSF3- and UL1-dependent events were validated by semi-quantitative RT-PCR (Additional file 1: Figure S6F).

As CPSF3 was originally described as a cleavage / polyadenylation factor, we asked whether the CPSF3-regulated exons could be the result of changes in alternative polyadenylation (APA) sites. Not unexpectedly, quantification of poly-A usage performed with QAPA [42] revealed hundreds of genes with APA events affected by Cpsf3 knockdown during reprogramming (277 genes) and, interestingly, also many genes with APA events affected by Hnrnpul1 knockdown (237 genes) (|∆∆PAU(shRNA–shSCR) ≥ 10). However, the overlap between the genes / exons whose AS or APA were affected by Cpsf3 or Hnrnpul1 knockdown was very limited (Additional file 1: Figures S6G-H), suggesting that the mechanisms by which CPSF3 and hnRNP UL1 regulate alternative splicing and 3′ end processing are distinct and independent.

Taken together, these results suggest that the AS regulators TIA1, CPSF3, and hnRNP UL1 function in cell reprogramming through their activities on genes relevant for cell fate decisions. Moreover, CPSF3 and hnRNP UL1 act through a highly overlapping program of splicing changes, while TIA1 affects a different set of AS events. Distinct programs of AS and APA were associated with CPSF3 and hnRNP UL1.


Commentary

Background Information

ESCs are pluripotent cells that are isolated from the inner cell mass of the blastocyst-stage embryo and can be cultured indefinitely in vitro (Evans and Kaufman, 1981 Martin, 1981 Thomson et al., 1998 ). Their ability to differentiate into multiple cell types (Evans and Kaufman, 1981 Nagy et al., 1993 Thomson et al., 1998 Reubinoff et al., 2000 ) makes them a suitable substrate for studies involving drug discovery (McNeish, 2004 ), human development, and cell therapies (Menendez et al., 2005 ).

hESCs are often cultured as dense colonies which are mechanically cut into small pieces and transferred from one organ culture dish to another (Thomson et al., 1998 Reubinoff et al., 2000 ). This method is preferred for long-term maintenance of hESCs as it reduces the level and frequency of stress associated with passaging and reduces the incidence of karyotypic abnormalities. Collagenase Type IV is commonly used for the large-scale passaging of hESCs (Amit et al., 2000 ) however, because this enzyme usually yields cell clumps, precise cell numbers can only be estimated making this method unsuitable for situations where precise cell numbers or single cells are required. Replacing Collagenase Type IV with either trypsin or TrypLE Select enables hESCs to be passaged as a single-cell suspension. All enzymatic passaging methods eventually select for cells which are adapted to such methods. Over time, such adaptations may include the acquisition of chromosomal aberrations (Draper et al., 2004 ) that provide a selective advantage to cells grown under these conditions. For this reason it is recommended that enzymatic passaging only be used transiently for the generation of large cell numbers required for experiments rather than as a method for routine long-term hESC maintenance. Uses for enzymatically passaged cells include electroporation of ∼1 × 10 7 cells, typical spin EB (embryoid bodies) method for differentiation of 3 × 10 5 hESCs per 96-well plate with 3000 cells per well. Most experiments would use 10 to 30 plates.

Critical Parameters and Troubleshooting

hESCs are cultured using two different techniques, each with different requirements. The two techniques are listed below.

Maintenance culture in organ culture dishes: the feeder density influences the thickness of the hESC colony. If the feeder density is too high then the colonies will be very thick and will tend to tear when being cut for passaging. If the feeder density is sparse, the colonies will be thin and the cut pieces will fray when being manually dislodged from the dish during passaging.

Bulk culture: The time it takes to achieve confluence from one passage to the next is influenced by the number and size of colonies present in the initiating culture. Although the hESCs are eventually passaged as a single-cell suspension they reform colonies on the dish as the cells proliferate. The longer these colonies are left to regrow between passages, the harder it is to dissociate them, which in turn leads to higher levels of cell death once passaged. Passaging the cells twice a week regardless of the number of colonies per flask prevents the colonies from becoming too large and difficult to dissociate.

Anticipated Results

This protocol generates large numbers of karyotypically normal hESCs, suitable for numerous applications such as electroporation and differentiation. After 5 bulk (enzymatic) passages, expect 4 × 150-cm 2 flasks each containing ∼8 × 10 6 hESCs (∼3.2 × 10 7 total).

Time Considerations

hESC colonies from one organ culture dish containing 10 colonies should be able to be distributed among six new dishes. It should take 2 weeks to go from 1 dish to 18. During the third week the colonies on the 18 dishes are mechanically transferred to a single 75-cm 2 flask (passage 1) which is subsequently passaged 3 days later (passage 2) using either trypsin or TrypLE select. By passage 4 (week 4), there should be sufficient cells to generate two confluent 150-cm 2 flasks. At this stage, cells which are to be used for experiments are passaged onto flasks seeded with MEFS at a reduced density (passage 5). Alternatively, cells can be enzymatically passaged 20 to 25 times without the appearance of chromosomal abnormalities. Under such circumstances, excess cells generated at each passage can be fed into other applications, kept for future experiments, or discarded.

Acknowledgement

We thank Elizabeth Ng for her valuable contribution to the development of the maintenance culture protocol described in this unit.


The culture and induction of ES-like colonies from SSCs

Kanatsu-Shinohara et al. cultured SSCs in such a way that these cells propagated themselves, while retaining their capacity to repopulate a recipient mouse testis upon transplantation(Kanatsu-Shinohara et al.,2004). A special medium was used, designed to culture hematopoietic stem cells, to which several growth factors, including GDNF,were added. In this culture system, a feeder layer is first formed that is composed of the contaminating somatic cells of the neonatal testis. Then,after 2 weeks and two passages, mitomycin-treated mouse embryonic fibroblasts(MEFs) are used as a feeder layer. During the first weeks of culture, the only colonies that formed consisted of SSCs, but, within 4-7 weeks, colonies formed that morphologically resembled ES cell colonies. Further work indicated that these colonies were indeed composed of multipotent ES-like cells. In order to maintain the multipotent character of these ES-like cells, they subsequently had to be cultured under standard ES cell culture conditions in medium containing 15% fetal calf serum and LIF. Under these conditions, the cultured SSCs could not be propagated because of the lack of GDNF. ES-like colonies could only be obtained when the starting population of SSCs was derived from neonatal mice when it was derived from older mice, ES-like colonies did not appear. However, cultures of SSCs derived from adult p53(Trp53)-null mice did produce ES-like colonies. P53 is involved in the cellular response to DNA damage and a lack of P53 increases the chances of teratoma development. Possibly, P53-deficient SSCs are more capable of undergoing the transition into ES-like cells.

An essentially similar protocol was followed by Seandel et al.(Seandel et al., 2007), except that this group used inactivated testicular stromal cells consisting of a mixture of CD34 + peritubular cells,α-smooth-muscle-actin-positive peritubular cells and cells positive for the Sertoli cell marker vimentin, as a feeder layer because they had less success using MEFs. By this method, ES-like colonies only appeared after more than 3 months in culture, more slowly than reported by Kanatsu-Shinohara et al. (Kanatsu-Shinohara et al.,2004). A substantially different approach was taken by Guan et al.(Guan et al., 2006). Their starting material was derived from 4- to 6-week-old mice and they did not use the stem cell medium described by Kanatsu-Shinohara et al.(Kanatsu-Shinohara et al.,2004) but simply Dulbecco's Modified Eagle's Medium (DMEM) with serum and added GDNF, in which testicular cells were initially cultured for 4 to 7 days. These cells were then sorted for the expression of STRA8 and subsequently cultured in DMEM under various conditions, but without adding GDNF. Colonies of ES-like cells formed when LIF was added to the medium and/or when the cells were cultured on a feeder layer of MEFs. The ES-like cells were further expanded by culture on MEFs and added LIF.

Hu et al. (Hu et al., 2007)cultured germ cells of prepubertal mice under conditions that favor osteoblast differentiation and reported the emergence of cells that had characteristics of osteoblasts after several weeks in culture. In this system, there was no period of culture with added GDNF. Finally, Boulanger et al.(Boulanger et al., 2007)employed no culture step at all. This group transplanted cells isolated from adult mouse seminiferous tubules, together with mammary cells, into mammary fat pads to obtain the differentiation of SSCs into mammary epithelial cells.

Taken together, it does not seem that a very specific approach is required to obtain the transformation of SSCs into ES-like cells (see Table 1). This transformation can occur on different feeder layers and even without a feeder layer, provided that LIF is added to the culture medium. Furthermore, the culture medium also does not seem to play a decisive role in the transformation of SSCs into ES-like cells, as the groups of Kanatsu-Shinohara et al.(Kanatsu-Shinohara et al.,2004) and Seandel et al.(Seandel et al., 2007) used a specific stem cell medium, whereas Guan et al.(Guan et al., 2006) used DMEM. All three groups did add GDNF to the culture, either continuously(Kanatsu-Shinohara et al.,2004 Seandel et al.,2007) or only at the start(Guan et al., 2006). However,to obtain the transformation of SSCs into cells of another lineage, it might not be necessary for them to become ES-like cells first. Putting the SSCs in an osteoblast-inductive environment in culture(Hu et al., 2007) or transplanting them into a mammary gland-inductive environment in vivo(Boulanger et al., 2007) might be enough for these cells to change their lineage. This rather suggests that SSCs are restricted to the spermatogenic lineage owing to the seminiferous tubular environment in which they reside. Once outside of this environment,they can switch to another lineage depending on the particular niche in which they are placed.

An overview of the different protocols for deriving ES-like cells from cultured mouse spermatogonial stem cells (SSCs) and the differentiation potential of the ES-like cells obtained

Starting material . Initial culture protocol . Second culture . Differentiation potential . References .
Germ cells from neonatal mouse testes Culture in stem cell medium + GDNF on MEFs for 4-7 months Standard ES cell medium Teratoma, EBs and chimaeras form with differentiation/contribution to endodermal, mesodermal and ectodermal derivatives. Using ES cell differentiation protocols, differentiation into hematopoietic cells, neurons,glial cells, vascular cells and myocytes was achieved (Kanatsu-Shinohara et al., 2004 Baba et al., 2007)
Germ cells of 4- to 6-week-old mice, purified for STRA8 expression DMEM + serum + GDNF, 4-7 days ES cell-like colonies when cultured + LIF and/or on MEFs Teratomas and EBs. Both form endodermal, mesodermal and ectodermal derivatives. Cardiomyocytes formed from EBs (Guan et al., 2006 Guan et al., 2007)
Germ cells of 3-week to 8-month-old mice Culture in stem cell medium + GDNF on inactivated testicular somatic cells for at least 3 months On MEFs in ES cell medium Teratoma and EBs. Both form endodermal, mesodermal and ectodermal derivatives. Chimaera formation upon transplantation into blastocyst (Seandel et al., 2007)
Germ cells from 6-to 8-day-old mouse testes * IMDM + serum for 3 days Culture under osteoblast-promoting conditions (+ DMSO and FGF2) Osteoblast formation (Hu et al., 2007)
Cell suspension from adult seminiferous tubules * Not applicable Not applicable Mammary epithelial cell differentiation upon inoculation together with mammary cells in mammary fat pad in vivo (Boulanger et al., 2007)
Starting material . Initial culture protocol . Second culture . Differentiation potential . References .
Germ cells from neonatal mouse testes Culture in stem cell medium + GDNF on MEFs for 4-7 months Standard ES cell medium Teratoma, EBs and chimaeras form with differentiation/contribution to endodermal, mesodermal and ectodermal derivatives. Using ES cell differentiation protocols, differentiation into hematopoietic cells, neurons,glial cells, vascular cells and myocytes was achieved (Kanatsu-Shinohara et al., 2004 Baba et al., 2007)
Germ cells of 4- to 6-week-old mice, purified for STRA8 expression DMEM + serum + GDNF, 4-7 days ES cell-like colonies when cultured + LIF and/or on MEFs Teratomas and EBs. Both form endodermal, mesodermal and ectodermal derivatives. Cardiomyocytes formed from EBs (Guan et al., 2006 Guan et al., 2007)
Germ cells of 3-week to 8-month-old mice Culture in stem cell medium + GDNF on inactivated testicular somatic cells for at least 3 months On MEFs in ES cell medium Teratoma and EBs. Both form endodermal, mesodermal and ectodermal derivatives. Chimaera formation upon transplantation into blastocyst (Seandel et al., 2007)
Germ cells from 6-to 8-day-old mouse testes * IMDM + serum for 3 days Culture under osteoblast-promoting conditions (+ DMSO and FGF2) Osteoblast formation (Hu et al., 2007)
Cell suspension from adult seminiferous tubules * Not applicable Not applicable Mammary epithelial cell differentiation upon inoculation together with mammary cells in mammary fat pad in vivo (Boulanger et al., 2007)

DMEM, Dulbecco's Modified Eagle's Medium DMSO, dimethylsulfoxide EB,embryoid body ES cell, embryonic stem cell FGF2, fibroblast growth factor 2GDNF, glial cell line derived neurotrophic factor IMDM, Iscove's Modified Dulbecco's Medium LIF, leukemia inhibitory factor MEFs, mouse embryonic fibroblasts STRA8, stimulated by retinoic acid gene 8.

Two studies in which direct transdifferentiation of SSCs into other cell lineages was reported.


Cryopreservation and quality control of mouse embryonic feeder cells ☆

Stem cell research is a highly promising and rapidly progressing field inside regenerative medicine. Embryonic stem cells (ESCs), reprogrammed “induced pluripotent” cells (iPS), or lately protein induced pluripotent cells (piPS) share one inevitable factor: mouse embryonic feeder cells (MEFs), which are commonly used for ESC long term culture procedures and colony regeneration. These MEFs originate from different mouse strains, are inactivated by different methods and are differently cryopreserved. Incomprehensibly, there are to date no established quality control parameters for MEFs to insure consistency of ESC experiments and culture. Hence, in this work, we developed a bench-top quality control for embryonic feeder cells.

According to our findings, MEFs should be inactivated by irradiation (30 Gy) and cryopreserved with optimal 10% DMSO at 1 K/min freezing velocity. Thawed cells should be free of mycoplasma and should have above 85 ± 13.1% viability. Values for the metabolic activity should be above 150 ± 10.5% and for the combined gene expression of selected marker genes above 225 ± 43.8% compared to non-irradiated, cryopreserved controls. Cells matching these criteria can be utilized for at least 12 days for ESC culture without detaching from the culture dish or disruption of the cell layer.

Highlights

► In ESC culture quality of MEFs should be regularly checked. ► Optimal cryopreservation of 30 Gy γ-irradiated MEF is 1 K/min plus 10% DMSO. ► Thawed cells should have 85% viability and be free of mycoplasma. ► Cells should have 150% metabolic activity and 225% selected marker gene expression.


Materials and Methods

Cell Culture

The GS cells were established from the transgenic mouse line B6-TgR(ROSA26)26Sor (The Jackson Laboratory) bred into a DBA/2 background [ 6]. For primary culture of gonocytes, we used 0-day-old pups in ICR background (Japan SLC). Gonocytes were enriched by gelatin selection and cultures initiated as described previously [ 6]. For primary culture of spermatogonia, we used 8-day-old pups in DBA/2 background (Japan SLC). Spermatogonia were collected by using a magnetic bead selection technique with biotinylated rat anti-mouse CD9 antibody (KMC8 BD Bioscience) and streptavidin-conjugated Dynabeads (Invitrogen) as previously described [ 20]. After selection, 2–3 × 10 5 cells were plated in six-well plates coated with laminin (20 μg/ml BD Biosciences).

Medium A (original GS cell medium) was made by supplementing StemPro-34 serum-free medium (Invitrogen) with 20 ng/ml of mouse EGF, 10 ng/ml of human FGF2, and 15 ng/ml of rat GDNF (all from PeproTech EC) as described previously [ 6]. This medium contained 1% FBS (Thermo Fisher Scientific). Medium B was modified from medium A by replacing serum and bovine serum albumin (BSA ImmunO, fraction V MP Biomedicals) with 3 mg/ml of lipid-rich BSA (Albumax II Invitrogen), 1 mg/ml of fetuin (Sigma), 1:100 Lipid Mixture 1 (Sigma), and 1:1000 Lipoprotein-Cholesterol Concentrate (MP Biomedicals). Cells were cultured on laminin-coated dishes at 37°C under of 5% CO2 in air. The cells were passaged by incubation with 0.25% trypsin for 5 min. Trypsin reaction was stopped by adding two volumes of medium B. In some experiments, we also used sphingosine kinase inhibitor 2 (SKI II 10 μM Cayman Chemical) at the time of cell plating. When indicated, heat-inactivated FBS or charcoal-treated FBS (Thermo Fisher Scientific) was added to the medium as a control. Sera from sheep, horse, goat, rabbit, and pig (all from Invitrogen) were used after heat inactivation for 30 min at 55°C. The cells were cryopreserved after adding 10% dimethyl sulfoxide (DMSO Sigma).

For laminin adhesion assays, 2 × 10 5 cells/well were plated in six-well plates coated with laminin and incubated overnight at 37°C. Adherent cells were recovered by trypsin digestion after washing the plates twice with PBS as previously described [ 21].

Flow Cytometry

The primary antibodies used were mouse anti-FUT4 (MC-480 Chemicon), rat anti-mouse EPCAM (G8.8), rat anti-human ITGA6 (CD49f) (GoH3), biotinylated hamster anti-rat ITGB1 (CD29) (Ha2/5), biotinylated rat anti-mouse CD9 (KMC8), and rat anti-mouse KIT (CD117) (2B8) (all from BD Biosciences). Allophycocyanin (APC)-conjugated goat anti-rat immunoglobulin G (Cedarlane Laboratories) and APC-conjugated streptavidin (BD Biosciences) were used to detect the primary antibodies. Antibodies were used at 5 μg/ml.

Transplantation Procedure

For germ cell transplantation, cultured cells were microinjected into the seminiferous tubules of WBB6F1-W/W v (W) mice via an efferent duct (Japan SLC). Approximately 80–90% of the tubules were filled in each recipient testis. To avoid rejection of donor cells, the recipient mice were treated with anti-CD4 antibody (GK1.5) to induce tolerance to the donor cells [ 22]. The Institutional Animal Care and Use Committee of Kyoto University approved all of the animal experiments.

Analysis of Recipient Testes

The donor cell colonization levels were determined by staining the recipient testes for the LacZ gene product, β-galactosidase with X-gal (Wako Pure Chemical Industries), as previously described [ 23]. Tubules that stained blue were counted under a stereomicroscope. Colonies were defined as germ cell clusters longer than 0.1 mm that occupied the entire circumference of the tubule. For histological analyses, the samples were embedded in paraffin blocks and processed for sectioning. All sections were 12 μm in thickness and were stained with hematoxylin and eosin.

Analysis of Gene Expression

Total RNA was isolated using TRIzol reagent (Invitrogen), and first-strand cDNA was produced by Superscript II (RNase H − reverse transcriptase Invitrogen). RT-PCR was performed using the specific primers listed in Supplemental Table S1 (available online at www.biolreprod.org). To quantify mRNA expression by real-time PCR, we used a StepOnePlus real-time PCR system and Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's recommendations. The transcript levels were normalized to those of Hprt. The PCR conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The experiments were performed on six independent samples, and each PCR reaction was run in triplicate.

Combined Bisulfite Restriction Analysis

Genomic DNA was treated with sodium bisulfite, which deaminates unmethylated cytosines to uracils but does not affect 5-methylated cytosines. Using this template, we amplified the indicated differentially methylated regions (DMRs) by PCR with the specific primers listed in Table S1. The PCR products were digested with the indicated restriction enzymes, which had recognition sequences containing CpG in the original unconverted DNA. The intensity of the digested bands was assessed using Image Gauge software (Fuji Photo Film).

Karyotype Analysis

Cultured cells were harvested, treated with 75 mM KCl for 15 min, and fixed with methanol/acetic acid (3:1). Metaphase spreads were prepared using standard procedures, and the slides were stained with Hoechst 33258 (Sigma).

Microinsemination

Spermatozoa were collected by mechanically dissociating the seminiferous tubule segments of fresh or frozen recipient testes [ 24]. These cells were microinjected into C57BL/6 × DBA/2 (BDF1) oocytes using a piezo-driven micropipetter (PrimeTech) [ 25]. Embryos at the 2-cell stage after 24 h in culture were transferred to the uteri of ICR recipient females.

Statistical Analyses

Results are presented as the mean ± SEM. Data were analyzed by Student t-test.


Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are conventionally maintained on mouse embryonic fibroblast (MEF) feeder layers. However, downstream applications, such as directed differentiation protocols, are primarily optimized for feeder-free cultures. Therefore, hPSCs must often be adapted to feeder-free conditions. Here we propose a novel feeder-free adaptation protocol using StemFlex medium, which can be directly applied to thawed hPSC lines.

The direct feeder-free adaptation protocol using StemFlex culture medium on Geltrex coating led to robust hPSC cultures in approximately 2 weeks. This approach was tested with three human embryonic stem cell (hESC) lines. All lines were confirmed to be pluripotent, expressing POU5F1, SOX2, and NANOG. No chromosomal imbalances were induced by the feeder-free adaptation.

StemFlex medium enabled the efficient adaptation of hPSCs to feeder-free conditions directly after thawing. This protocol is easy to implement in laboratories that perform feeder-free cultures, allowing more convenient adaptation and more robust expansion of cryopreserved hPSCs, even in cases when sample quality is low or unknown.

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Feeder Cell Type Affects the Growth of In Vitro Cultured Bovine Trophoblast Cells.

There are two distinct cell populations in the blastocysts of mammals, the trophoblast and the inner cell mass. During embryogenesis, trophoblast cells are believed to be the first to differentiate [1]. The trophoblastic cells form most of the placenta, whereas the inner cell mass grows to give the embryo and its related membranes [2]. So, although the implantation and placental formation step is dependent on the differentiation of the trophoblast, this process involves unknown participating factors and remains weakly understood. Recently, a number of specific molecules has been linked as markers to trophoblastic cells, during bovine periimplantation processes, such as interferon tau (IFNT) [3, 4], early trophoblastic marker CDX2 [5], and cytokeratin-8 (KRT8) in in vitro produced bovine embryos [6].

Studying development and functions of trophoblastic cells is critical therefore, in vitro models for culturing primary trophoblast cells are essential. However, the finite lifespan of primary trophoblast cells limits their long-term culture and use in investigations. In an attempt to prolong trophoblast cells lifespan in goat, telomerase immortalized trophoblast cells (hTERT-transfected cells) showed more telomerase activity and proliferated persistently for at least 50 passages without any signs of senescence [7]. But, many researchers are skeptic about using transfected cell lines in their research because their regeneration is costly and complex, and little is known about how transfection could inadvertently change the outcomes of their research. Consequently, maintenance of trophoblastic cell lines using feeder cells comes as a more convenient and feasible approach. In this regard, recently, some trophoblastic cell lines were established, on mouse embryonic fibroblasts as feeders, in several species as porcine [8], murine [9], and bovine [10,11]. Henceforward, improving the culture system is of particular interest and advantage, to facilitate the in vitro investigation of the trophoblastic cells. In the present study, and for the first time, porcine granulosa cells were used as alternative feeder cell for culturing primary trophoblastic cells which were isolated from in vitro produced bovine blastocysts.

2.1. Oocyte Collection and In Vitro Maturation (IVM). Cow ovaries were obtained from a nearby abattoir, washed with saline and kept in it at 35[degrees] C, and promptly sent (within 2 hrs) to the lab. These cows' management was as previously described [12]. Ovarian follicles (diameter = 2-8mm) were aspirated with an 18-gauge needle connected to 10 mL disposable syringe to obtain cumulus-oocyte complexes (COCs). The COCs were selected, if they had evenly granulated cytoplasm and enclosed by three or more layers of compact cumulus cells and washed 3x in HEPES-buffered TCM199 (Invitrogen, USA) supplemented with 10% FBS, 2mM NaHCO3 (Sigma, USA), and 1% penicillin-streptomycin (v/v, Sigma, USA). Followed by IVM, COCs were cultivated in 4-well plates (30-40 oocytes per well Falcon, BD, UK) in 450 [micro]L TCM-199 with 10% FBS, 0.005 AU/mL FSH (Antrin, Tokyo, Japan), and 1 [micro]g/mL 17[beta]-estradiol (Sigma, USA) at 39[degrees] C in a humidified atmosphere of 5% C[O.sub.2] for 24 hrs.

2.2. In Vitro Fertilization (IVF) and In Vitro Culture (IVC) of Embryos. This protocol followed our previously published protocol [11]. Motile spermatozoa were obtained by purification using Percoll gradient method as previously described [13]. Shortly, semen straws were thawed and spermatozoa were collected by centrifugation on a (45-90%) Percoll discontinuous gradient at 1500 rpm for 15 minutes. The 45% Percoll solution was prepared using 90% Percoll and TALP medium (1: 1 ratio). The obtained sperm-pellet was washed (2x) with TALP medium then centrifuged at 1500 rpm for 5 minutes. Then, active and motile sperms (1-2 x 106 sperm/mL) were used for inseminating the IVM oocytes (day 0) for 18 hrs in 30 [micro]L IVF/TALP medium blanketed with mineral oil at 39[degrees]C in 5% C[O.sub.2] humidified atmosphere. Plausible zygotes were stripped and cultivated in a twostep defined culture medium as previously described [14,15] and overlaid with mineral oil (Sigma, USA). Fertilization was repeated six times (n = 150, 25 oocytes each). We obtained forty-nine blastocysts (32.6%) among them thirty-six blastocysts were hatched (24%) on day 10 of IVC that were used for trophoblast culture.

2.3. Preparation of Feeder Cells. Two different feeders were used: the conventional method using mouse embryonic fibroblasts (MEFs) as we described before [11] and new feeder porcine granulosa cells (PGCs). PGCs were obtained through aspiration of follicular fluid of 4-6 mm porcine ovarian follicles with an 18-gauge needle. The follicular fluid was centrifuged at 1500 rpm for 2 minutes and then washed three times using PBS and then with culture medium (DMEM and FBS 10%). The two cell types were mitotically inactivated using mitomycin-C (Sigma-Aldrich Corp.) followed by culturing (well density = 5 x [10.sup.4] cell/mL) on 4-well plates coated with 0.1% (w/v) gelatin. The medium comprised of DMEM-199 and 10% (v/v) FBS, with addition of nonessential amino acids (NEAA), [beta]-mercaptoethanol, and nucleosides as previously described [10].

2.4. Measurement of Steroid Hormones in the Culture Medium. Steroids (estrogen and progesterone) were measured in the culture medium using commercial kits, following producer's directions. Culture media were aspirated from five different replicates and were centrifuged at 1500 rpm for 5 min. The supernatant was divided into aliquots and preserved at -20[degrees]C until analyzed. Progesterone (P4) levels were measured by radioimmunoassay (RIA) using commercial progesterone kit (Coat-a-Count, Siemens, USA). The kit contains rabbit antiP4 antibody, and the minimum detection limit is 0.02 ng/mL. The intra- and interassay coefficient of variation (CV%) ranged from 2.7 to 8.8 and from 3.9 to 9.7, respectively. Also, estradiol (E2) levels were determined by electrochemiluminescence immune-assay with commercial kit (Estradiol II kit, Roche, USA). The kit contains rabbit anti-E2 antibody and the kit minimum detection limit is 5.0 pg/mL. The intra- and interassay coefficient of variation (CV%) ranged from 2.3 to 6.2 and from 6.2 to 13.0, respectively. Each sample from each replicate was measured three times and the data were recorded as mean [+ or -] SEM.

2.5. Isolation and Culture of Bovine Trophoblastic Cells. Thirty-six IVF hatched blastocysts on days 10-11 of IVC were randomly distributed into two 4-well plates (Nunc, Thermo Scientific, Denmark), grouped into MEF and PGCs groups, eighteen embryos each and three embryos per each replicate. The plates were precoated with 0.1% gelatin and followed by culturing of mitotically inactivated feeder layers of either mouse embryonic fibroblasts (MEF) or porcine granulosa cells (PGCs). The blastocysts were primary cultivated in 1 mL DMEM/F12 medium [composed of DMEM/F12 provided with 10% FBS, 0.1 mM [beta]-mercaptoethanol, 1% NEAA (Invitrogen), 2mM GlutaMax, and 1% penicillin/streptomycin (Invitrogen)], with medium changes with fresh medium every 3-4 days. Subculturing of the trophoblastic cells was by mechanical detachment and chopping of the colonies into relatively equal small pieces and recultivating those pieces on new feeder-plates. The small chops were centrifuged at 1500 rpm for 2 min and pelleted in 1.5 mL rounded bottom centrifuge tubes. Then, resuspension of the pellet was done with DMEM/F12, followed by plating onto new feeder-plates (split ratio = 1: 4-1: 6). Incubations were done at 39[degrees]C in 5% C[O.sub.2] humidified atmosphere.

2.6. RT-PCR. Trophoblast colonies (n = 5, three replicates) of the 1st and 10th passages were mechanically isolated and washed three times with PBS. Total RNA was extracted from trophoblast colonies using an RNeasy total extraction kit (Qiagen, USA) following the manufacturer directions, and as we described previously [11]. Reverse transcription reactions were done in 20 [micro]l reactions at 50[degrees]C for 50 minutes using random-hexamers and SuperScript[TM] III Reverse-Transcriptase (Invitrogen). One [micro]g cDNA was subjected to RT-PCR using a Maxime PCR-PreMix kit (i-starTaq, Intron, Republic of Korea). Primers and their sequences, annealing temperatures and expected sizes of products are listed in Table 1. The amplification cycle was done with initial denaturation at 95[degrees]C for 5 minutes, followed by cycles of denaturation at 95[degrees]C for 30 seconds, annealing for 30 seconds, extension at 72[degrees]C for 45 seconds and final extension at 72[degrees]C for 5 minutes. PCR products (10 [micro]L) were ran on 1% agarose gel (Intron) stained with RedSafe[TM] (Intron). Densitometry scanning with ImageJ v1.45 software (NIH, USA) was done to quantify the intensity of RT-PCR signals and specific target's values were normalized using the internal control (GAPDH) to calculate relative expression units. All controls in RT-PCR, reactions without cDNA template and those without reverse transcription, gave no amplification reaction.

2.7. Statistical Analysis. Data were analyzed with one-way ANOVA, Tukey's test was done to conclude significant differences between the different experimental groups using GraphPad (Version 4.0). Data were considered statistically significant when P value was <0.05.

In this study, the primary culture of trophoblasts was done by culturing of hatching/hatched blastocysts on feeder cells, which were either mouse embryonic fibroblasts (MEFs) or, for the first time, porcine granulosa cells (PGCs).

When the feeders were cultured alone, PGCs showed higher proliferation with approximately 24 hrs doubling time comparing to MEFs (P < 0.05). In addition, PGCs were easier to recover from monolayer cultures and offered a fair amount of sex steroids, 17[beta]-estradiol (E2, 31.21 [+ or -] 3.1 ng/mL) and progesterone (P4, 6.36 [+ or -] 0.4 ng/mL). Steroids production by cultured porcine granulosa cells has been previously reported [16].

After coculture with mitotically inactivated MEFs or PGCs, blastocysts were seen attached and from it outgrowths were observed then it was kept in culture for 10 days till it reached approximately 1 cm diameter. Following that, secondary and succeeding subcultures underwent mechanical detachment and cutting/chopping of these cell-growths and subculturing of the relatively similar sized small pieces/chops on fresh feeder-plates every 7 days (after reaching about 0.5 cm diameter). In all subcultures, trophoblasts were morphologically visible as large cuboidal cells under phasecontrast microscope (Figure 1).

The isolated trophoblastic cells showed faster growth pattern in primary culture on PGCs, in one-week colony diameter measured 7.0 [+ or -] 0.3 mm, compared to those cultured on MEFs colonies grew to 5.7 [+ or -] 0.4 mm (P < 0.05 Figures 2(a) and 2(b)), whereas, on subsequent cultures (2nd-10th) the difference in colony morphology, although it was visually larger when trophoblasts were cultured on PGCs compared to MEFs, did not reach statistical significance (P > 0.05Figures 2(c), 2(d), 2(e), and 2(f)).

In contrast to the colony size, the isolated cells showed high expression of trophoblast gene-markers throughout the study these were IFNT, KRT8, and CDX2 (Figure 3). Of importance, the expression levels of IFNT, KRT8, and CDX2 mRNA were higher when trophoblastic cells were cultured on PGCs compared to MEFs, particularly at the 10th passage (P [less than or equal to] 0.05 Figure 3). The culture was stopped at passage 10 that is, the culture duration was approximately 70 days after the primary culture.

Isolated trophoblastic cells on both feeders were morphologically similar, which visually is a good sign that culturing the cells on PGCs resulted in a normal morphology similar to using MEFS. What is more, the trophoblast cells isolated on PGCs showed superior quality, indicated by higher expression of IFNT, KRT8, and CDX2 and long-term culture for up to 70 days. Interferon tau is produced early in preimplantation [3, 4] and its expression indicates normal trophoblast function. So, in case of PGCs as feeders, higher expression of trophoblast IFNT fairly indicates an improved trophoblast function, while the expression of the homeobox protein CDX2 is considered as a healthy sign of preimplantation trophectoderm cells [5,15] and again it was highly expressed in cells cultured on PGCs, favoring PGCs as feeders over MEFs. Moreover, KRT8 expression as a marker for trophectoderm is also consistent with other studies on in vitro produced bovine embryos [6,17].

In addition, PGCs provided the cultured trophoblastic cells with a considerable amount of E2 and P4. Previous work revealed that estrogens and progesterone receptors were expressed in trophoblast and caruncular epithelium and suggested that E2 and P4 are important factors controlling caruncular growth, differentiation, and function [18]. Interestingly, it was found recently that genes encoding steroid hormones were also expressed by bovine trophoblastic cells [19, 20]. Moreover, steroidogenic proteins were expressed in bovine placental tissue during the first half of gestation [21]. Hence, we assume that the constant supplementation of sex steroids and other yet to be determined factors, during trophoblasts coculture with PGCs, is better and superior to using MEFs as feeders. Nevertheless, steroids synthesis and functions during early implantation remain understudied and more research is needed to understand their roles(s) throughout the peri-implantation period.

In this report, we described the isolation of trophoblastic cell line from bovine IVF embryos for the first time using granulosa cells as feeders. The isolated trophoblastic cells had normal shape and superior trophoblast quality, indicated by increased expression of trophoblast markers IFNT, KRT8, and CDX2 compared to cells isolated on MEFs. Using this model, we anticipate more studies aiming at elucidating the fetal-maternal crosstalk at implantation. Also, it will be a valuable model for studies interested in explaining effects of diseases, infections, or toxicity on the placental formation and early development and gestation.

All procedures and handling described in this study (of mice or animal tissue) followed directions and protocols approved by the Animal Care and Ethics Committee of the King Saud University.

Islam M. Saadeldin and Ahmed Abdelfattah-Hassan are cofirst authors. All data generated or analyzed during this study are included in this article.

The authors declare that they have no conflicts of interest.

All authors shared in the design and implementation of this study. Also, all authors read and approved the final manuscript. Islam M. Saadeldin and Ahmed Abdelfattah Hassan are equally contributing authors to the work.

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through the Research Group Project (no. RG-1438-018).

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Islam M. Saadeldin, (1,2) Ahmed Abdelfattah-Hassan, (3) and Ayman Abdel-Aziz Swelum (1,4)

(1) Department of Animal Production, College of Food and Agricultural Sciences, KingSaud University, Riyadh 11451, Saudi Arabia

(2) Department of Physiology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt

(3) Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt

(4) Department of Theriogenology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt

Correspondence should be addressed to Islam M. Saadeldin [email protected]

Received 23 March 2017 Accepted 4 May 2017 Published 24 May 2017

Academic Editor: Enrique Gomez

Caption: Figure 1: Representative phase-contrast light micrograph showing the typical cuboidal morphology of cultured bovine trophoblastic cells (10th passage) on mouse embryonic fibroblasts, MEFs (a), and on porcine granulosa cells, PGCs (b) [200x].

Caption: Figure 2: Phase-contrast light micrographs showing culture of bovine trophoblast on mouse embryonic fibroblast (MEFs) and on porcine granulosa cells (PGCs). The cells were subcultured until the 10th passage [100x].

Caption: Figure 3: (a) RT-PCR analysis using primers specific for interferon tau (IFNT), keratin-8 (KRT8), and homeobox protein (CDX2) expression in trophectoderm colonies. In all analyses, reactions without cDNA template or reverse transcriptions resulted in negative amplification. ((b), (c), and (d)) Densitometric relative expression values of IFNT, KRT8, and CDX2, respectively (normalized to those of the internal control GAPDH) using ImageJ v1.45 software (NIH, USA). Trophectoderm cells were grown on mouse embryonic fibroblasts, primary culture (F1) and 10th passage (F10), and on porcine granulosa cells, primary culture (G1) and 10th passage (G10) [* p value [less than or equal to] 0.05].


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