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Highlights of the 3rd Meeting of the French Society for...
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Highlights of the 3rd Meeting of the French Society for Stem Cell Research (FSSCR)

A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Stem Cells".

Deadline for manuscript submissions: closed (29 February 2020) | Viewed by 8822

Special Issue Editors

Dr. Pierre Savatier

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Guest Editor
INSERM U1208, Stem Cell and Brain Research Institute, 18 avenue Doyen Lepine, 69500 Bron, France
Interests: pluripotent stem cells; embryo chimeras; cycle regulation
Dr. Cécile Martinat

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Guest Editor
INSERM UEVE UMR 861, I-STEM, Association Française des Myopathies, 28 Rue Henri Desbruères, 91100 Corbeil Essonnes, France
Interests: human pluripotent stem cells; differentiation; disease modeling; drug screening; cell therapy
Dr. Thierry Jaffredo

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Guest Editor
Institut de Biologie Paris Seine, CNRS UMR7622, INSERM U1156, Sorbonne Université, 9 Quai St Bernard, CEDEX 05, 75252 Paris, France
Interests: development; stem cells; hematopoietic stem cells; niche cells; stem cell niche interactions; high troughput approaches

Special Issue Information

Dear Colleagues,

The 3rd annual meeting of the French Society for Stem Cell Research (FSSCR) will be held in Lyon on November 18 and 19, 2019. It will be organized around four sessions: “Gastruloids and Blastoids”, “Organoids”, “Stem Cells at the Single Cell Level”, and “Epigenetics and Aging”. All participants wishing to submit an Abstract at any of these four sessions, whether for a poster or a short presentation, have the opportunity to also submit an article for publication in this Special Issue of Cells. This article can be a review or an original manuscript.

Dr. Thierry Jaffredo
Dr. Pierre Savatier
Dr. Cécile Martinat
Guest Editors

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22 pages, 6983 KiB  
Article
FAK Deficiency in Bone Marrow Stromal Cells Alters Their Homeostasis and Drives Abnormal Proliferation and Differentiation of Haematopoietic Stem Cells
by Yuenv Wu, Lydia Campos, Elisabeth Daguenet, Zhiguo He, Tiphanie Picot, Emmanuelle Tavernier-Tardy, Gilbert Soglu, Denis Guyotat and Carmen-Mariana Aanei
Cells 2020, 9(3), 646; https://doi.org/10.3390/cells9030646 - 6 Mar 2020
Cited by 5 | Viewed by 3184
Abstract
Emerging evidence indicates that in myelodysplastic syndromes (MDS), the bone marrow (BM) microenvironment may also contribute to the ineffective, malignant haematopoiesis in addition to the intrinsic abnormalities of haematopoietic stem precursor cells (HSPCs). The BM microenvironment influences malignant haematopoiesis through indirect mechanisms, but [...] Read more.
Emerging evidence indicates that in myelodysplastic syndromes (MDS), the bone marrow (BM) microenvironment may also contribute to the ineffective, malignant haematopoiesis in addition to the intrinsic abnormalities of haematopoietic stem precursor cells (HSPCs). The BM microenvironment influences malignant haematopoiesis through indirect mechanisms, but the processes by which the BM microenvironment directly contributes to MDS initiation and progression have not yet been elucidated. Our previous data showed that BM-derived stromal cells (BMSCs) from MDS patients have an abnormal expression of focal adhesion kinase (FAK). In this study, we characterise the morpho-phenotypic features and the functional alterations of BMSCs from MDS patients and in FAK knock-downed HS-5 cells. The decreased expression of FAK or its phosphorylated form in BMSCs from low-risk (LR) MDS directly correlates with BMSCs’ functional deficiency and is associated with a reduced level of haemoglobin. The downregulation of FAK in HS-5 cells alters their morphology, proliferation, and differentiation capabilities and impairs the expression of several adhesion molecules. In addition, we examine the CD34+ healthy donor (HD)-derived HSPCs’ properties when co-cultured with FAK-deficient BMSCs. Both abnormal proliferation and the impaired erythroid differentiation capacity of HD-HSPCs were observed. Together, these results demonstrate that stromal adhesion mechanisms mediated by FAK are crucial for regulating HSPCs’ homeostasis. Full article
(This article belongs to the Special Issue Highlights of the 3rd Meeting of the French Society for Stem Cell Research (FSSCR))
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Figure 1

Figure 1
<p>Intrinsic abnormalities related to focal adhesion kinase (FAK) deficiency in BMSCs from MDS patients correlate with the reduced clonogenic potential of HSPCs and with a degree of anaemia. (<b>A</b>,<b>B</b>) Evaluation of CFU-F and <b>C</b>, proliferative capacities (measured by MTT Cell Proliferation Assay) in BMSCs derived from MDS patients compared with healthy donors as controls (HC). (<b>D</b>) Quantification of oil red (adipogenic differentiation) and alizarin red (osteogenic differentiation) staining at day 14 in MSC derived from HC, LR-MDS (low-risk) and HR-MDS (high-risk) patients. (<b>E</b>) Morphological evaluation of MDS-derived MSCs compared to HC MSCs. (<b>F</b>) Phenotypic differences in BMSCs selected from LR-MDS patients compared to HC. (<b>G</b>) Significant correlation between PTK2 expression in BMSCs and the haemoglobin level in an MDS setting. (<b>H</b>) Evaluation of the clonogenic capacity of HSPCs selected from MDS patients compared to HC. I, SDF-1 mRNA expression in BMSCs isolated from LR-MDS and HR-MDS patients compared to HC. HC, HD controls; LR-MDS, low-risk MDS; HR-MDS, high-risk MDS. <span class="html-italic">p</span> &lt; 0.05(*); <span class="html-italic">p</span> &lt; 0.01(**); <span class="html-italic">p</span> &lt; 0.0001(****).</p> Full article ">Figure 2
<p>The pharmacological inhibition of Y397-FAK autophosphorylation and short hairpin RNA (shRNA)-mediated knockdown of FAK in HS-5 cells lead to morphological, phenotypic, and functional abnormalities in BMSCs (<b>A</b>) Western blot, detection of protein level of FAK and pFAK upon treatment with increasing doses of VS-4718; (<b>B</b>,<b>C</b>) Relative concentration of FAK and pFAK in HS-5 cells after 24 h, 48 h, and 72 h exposure to increasing doses of VS-4718. (<b>D</b>) Viability evaluation of HS-5 cells after exposure to 2 μM VS-4718. (<b>E</b>) Representative image of HS-5 cells’ morphology after 72 h exposure to 2 µM VS-4718. Giemsa staining; 200× magnification. Morphological alterations are depicted with red arrows. (<b>F</b>) FSC and SSC determination by flow cytometry in HS-5 cells after treatment with 2 µM VS-4718. (<b>G</b>) HS-5 cell proliferation assay using carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) tracing (untreated HS-5 cells, black column, n = 3; HS-5 cells exposed to 2 µM VS-4718, grey column, n = 3). (<b>H</b>) qRT-PCR measurement of mRNA levels of haematopoiesis-supporting genes after pharmacological inhibition of FAK phosphorylation in HS-5 cells (n<sub>HS-5+VS-4718</sub> = 3; n<sub>HS-5</sub> = 3). (<b>I</b>,<b>J</b>) Western blot, detection of protein level of FAK in HS-5 cells after FAK silencing by shRNA (HS-5, non-infected HS-5 cells, n = 3; shRNA, control shRNA, n = 3; shFAK KD, specific shRNA, n = 3). (<b>K</b>) Representative microscopic images at 40×, 100× magnification from HS-5 cultures with (right) and without FAK shRNA knockdown (left), Giemsa staining. (<b>L</b>) FSC and SSC evaluation by flow cytometry of HS-5 cells after FAK silencing (Control, control shRNA, n = 3; shFAK, specific shRNA, n = 3). (<b>M</b>) Cell growth curves of HS-5 cells after shRNA FAK KD (n = 3) compared to control shRNA (n = 3). (<b>N</b>) qRT-PCR measurement of mRNA levels of haematopoiesis-supporting genes in HS-5 cells after FAK silencing by shRNA (n<sub>shFAK KD HS-5</sub> = 3, n<sub>shRNA control</sub> = 3). <span class="html-italic">p</span> &lt; 0.05(*); <span class="html-italic">p</span> &lt; 0.01(**); <span class="html-italic">p</span> &lt; 0.001(***); <span class="html-italic">p</span> &lt; 0.0001(****).</p> Full article ">Figure 3
<p>ShRNA-mediated FAK knockdown impairs HS-5 homeostasis by controlling the phosphorylation of several proteins from PTEN-Akt-p21 and ERK-p38 MAPK signalling pathways (<b>A</b>,<b>B</b>); Western blot analysis of key signalling pathways. (<b>C</b>) Doubling time assay of control shRNA and FAK shRNA in HS-5 cells. Initial inoculum cell concentration was 10<sup>5</sup> cells/cm². (<b>D</b>) Cell cycle analysis with Hoechst 33342 and Pyronin Y (n<sub>control shRNA</sub> = 3, n<sub>shFAK</sub> = 3). (<b>E</b>) Scratch-wound assay of HS-5 cells after FAK shRNA compared to control shRNA (n<sub>control shRNA</sub> = 3, n<sub>shFAK</sub> = 3). (<b>F</b>) Re-expression of WT FAK in shRNA FAK cells and Western blot analysis of signalling proteins. <span class="html-italic">p</span> &lt; 0.01(**); <span class="html-italic">p</span> &lt; 0.001(***).</p> Full article ">Figure 4
<p>FAK deficiency in HS-5 cells promotes the proliferation of CD34+ HD-HSPCs cells in short-term co-cultures (<b>A</b>) Reliable discrimination between the HS-5 cells (grey dots) and CFSE-labelled CD34+ HD-HSPCs (red, blue, and pink dots). Evaluation of HSPCs’ compartment using immature stem cells markers and differentiation antigens. Three main populations are observed: The immature HSPCs CD133+ CD34+low CD38+var CD117+low CD33- CD19- (red dots) and more mature HSPCs CD133- CD34- can be divided by CD38 into two separate subpopulations: CD117+ CD38+ CD33- CD19- (blue dots) and CD38- CD117- CD33+ CD19- (pink dots). (<b>B</b>) Absolute cell count of HD-HSPCs recovered after five days of co-cultures between CD34+ HD-HSCs and HS-5 cells (shFAK cells or control shRNA; n<sub>Control shRNA</sub> = 3, n<sub>shFAK</sub> = 3). <span class="html-italic">p</span> &lt; 0.01(**); <span class="html-italic">p</span> &lt; 0.001(***).</p> Full article ">Figure 5
<p>FAK-deficient HS-5 cells negatively regulate the differentiation capacity of CD34+ HD-HSPCs towards erythroid lineage and promote the differentiation of other myeloid progenitors. (<b>A</b>) Flow cytometry evaluation of the differentiation capacity of CD34+ HD-HSCs after direct contact with FAK shRNA HS-5 cells during five days versus co-cultures with control shRNA. (<b>B</b>) Megakaryocyte/erythroid progenitors (MEPs) CD38- HLADR- CD33- (yellow dots), erythroid precursors (EPs) CD36+ CD33- HLADR- (pale pink dots), and other myeloid precursors (MyPs) CD33+ can be divided by the expression of CD36 in the monocyte-committed precursors CD38+ CD36+ CD33+ HLADR+ (MPs, pale blue dots) and non-monocytic precursors CD38+low/- CD36- CD33+low/- HLADR+ (red dots). The absolute cell count of EPs (n<sub>control shRNA</sub> = 3, n<sub>shFAK</sub> = 3; <a href="#cells-09-00646-f005" class="html-fig">Figure 5</a>B1) and of other MyPs (n<sub>control shRNA</sub> = 3, n<sub>shFAK</sub> = 3; <a href="#cells-09-00646-f005" class="html-fig">Figure 5</a>B2) recovered from CD34+ HD-HSCs from after five days of direct co-cultures with FAK shRNA HS-5 cells compared to control shRNA. (<b>C</b>) BFU-E colonies recovered from non-adherent HSPCs after five days of co-cultures with FAK shRNA HS-5 cells compared to control shRNA (n<sub>control shRNA</sub> = 3, n<sub>shFAK</sub> = 3). <span class="html-italic">p</span> &lt; 0.05(*); <span class="html-italic">p</span> &lt; 0.01(**).</p> Full article ">Figure 6
<p>Reduced immature haematopoietic stem cells’ recovery and impairment of erythroid differentiation are observed after co-culture between CD34+ HD-HSCs with FAK shRNA HS-5 cells compared to control shRNA cells. (<b>A</b>) Three populations can be identified inside the CSFE+ HSPC population recovered after two weeks of co-culture with FAK shRNA HS-5 or control shRNA: Immature HSCs CD45+low CD133+ HLADR-/+low CD117+low CD71+low CD33/CD36- cells (red dots), erythroid precursors’ EPs CD45-/+low CD133- HLADR-/+low CD117-/+low CD38+ CD33/36- CD71+low (pale pink dots), and other myeloid precursors’ MyPs CD45+low CD38+ HLADR+low CD117-/+low CD33/CD36+ CD71- (blue dots). (<b>B</b>) Absolute cell count evaluation of the immature CD133+ HSCs, Eps, and MyPs after two weeks of direct contact with FAK shRNA HS-5 cells compared to control shRNA and FAK shRNA cells after WT FAK re-expression (n<sub>shFAK KD</sub> = 5, n<sub>shRNA</sub> = 5, n<sub>shFAK restored</sub> = 3). (<b>C</b>) Burst-forming unit-erythroid evaluation of non-adherent HSPCs recovered from long-term direct co-culture with FAK shRNAHS-5 cells compared to control shRNA. (<b>D</b>) Evaluation of the proliferation capacity and erythroid differentiation ability of HSPCs recovered from indirect long-term co-culture between CD34+ HD-HSCs and FAK shRNA HS-5 cells compared to control shRNA and FAK shRNA cells after WT FAK re-expression (n<sub>shRNA Control</sub> = 5, n<sub>shFAK KD</sub> = 5, n<sub>shFAK restored</sub> = 3). <span class="html-italic">p</span> &lt; 0.05(*); <span class="html-italic">p</span> &lt; 0.01(**).</p> Full article ">Figure 7
<p>Upon FAK downregulation, the adhesion molecules profile is remodelled in HSPCs and stromal cells when co-cultured in direct contact. (<b>A</b>) HSPCs are discriminated from HS-5 cells based on their expression for CSFE (grey dots, non-haematopoietic HS-5 cells; dark green dots, CD133+ HSCs after direct contact with control shRNA cells; light green dots, more mature haematopoietic precursors after direct contact with control shRNA cells <a href="#cells-09-00646-f007" class="html-fig">Figure 7</a>A left; dark red dots, CD133+ HSCs after direct contact with FAK shRNA HS-5 cells; orange dots, more mature haematopoietic precursors after direct contact with FAK shRNA HS-5 cells <a href="#cells-09-00646-f007" class="html-fig">Figure 7</a>A middle). Discrimination of HSPC sub-populations based on the expression of CD133, CD34, and HLA-DR markers (<a href="#cells-09-00646-f007" class="html-fig">Figure 7</a>A right). (<b>B</b>,<b>C</b>) MFI values of LFA-1 (CD11a) and CD44 on more mature haematopoietic progenitor cells (grey, non-haematopoietic HS-5 cells; light green, HSPCs after direct contact with control shRNA cells; orange, HSPCs after direct contact with FAK shRNA HS-5 cells). Box plots represent the expression level of LFA-1(CD11a) and CD44 (n<sub>shRNA Control</sub> = 3, n<sub>HSPC+shRNA Control cells</sub> = 3, n<sub>HSPCs+shFAK KD cells</sub> = 3). Data were analysed in a merged file composed of three fcs files from HSPCs + shRNA control HS-5 cell co-cultures and three fcs files from HSPCs + shRNA FAK KD HS-5 cell co-cultures. The upper and lower hinges of the box indicate the 75<sup>th</sup> and 25<sup>th</sup> percentiles of the data set. The median line within the box represents the median value of the intensity of expression. The whiskers indicate the minimum and maximum data values. (<b>D</b>) MFI values of several adhesion molecules evaluated on FAK shRNA HS-5 cells by flow cytometry. <span class="html-italic">p</span> &lt; 0.05(*); <span class="html-italic">p</span> &lt; 0.01(**); <span class="html-italic">p</span> &lt; 0.001(***).</p> Full article ">
24 pages, 6830 KiB  
Article
NOTO Transcription Factor Directs Human Induced Pluripotent Stem Cell-Derived Mesendoderm Progenitors to a Notochordal Fate
by Pauline Colombier, Boris Halgand, Claire Chédeville, Caroline Chariau, Valentin François-Campion, Stéphanie Kilens, Nicolas Vedrenne, Johann Clouet, Laurent David, Jérôme Guicheux and Anne Camus
Cells 2020, 9(2), 509; https://doi.org/10.3390/cells9020509 - 24 Feb 2020
Cited by 22 | Viewed by 5092
Abstract
The founder cells of the Nucleus pulposus, the centre of the intervertebral disc, originate in the embryonic notochord. After birth, mature notochordal cells (NC) are identified as key regulators of disc homeostasis. Better understanding of their biology has great potential in delaying the [...] Read more.
The founder cells of the Nucleus pulposus, the centre of the intervertebral disc, originate in the embryonic notochord. After birth, mature notochordal cells (NC) are identified as key regulators of disc homeostasis. Better understanding of their biology has great potential in delaying the onset of disc degeneration or as a regenerative-cell source for disc repair. Using human pluripotent stem cells, we developed a two-step method to generate a stable NC-like population with a distinct molecular signature. Time-course analysis of lineage-specific markers shows that WNT pathway activation and transfection of the notochord-related transcription factor NOTO are sufficient to induce high levels of mesendoderm progenitors and favour their commitment toward the notochordal lineage instead of paraxial and lateral mesodermal or endodermal lineages. This study results in the identification of NOTO-regulated genes including some that are found expressed in human healthy disc tissue and highlights NOTO function in coordinating the gene network to human notochord differentiation. Full article
(This article belongs to the Special Issue Highlights of the 3rd Meeting of the French Society for Stem Cell Research (FSSCR))
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Figure 1

Figure 1
<p>Schematic workflow of hiPSCs differentiation. The differentiation was initiated by single cell seeding at 35.000 cells/cm<sup>2</sup> (TryplE digestion) on matrigel-coated plates in mTser1 medium supplemented with rock inhibitor for 24 h. From day 0 to day 2, hiPSCs were cultivated in N2B27 in increasing doses of CHIR99021 and Activin A for hiPSC-derived mesendoderm progenitor cell (MEPC) specification. At Day 2, MEPC were dissociated with TryplE and transfected with Lipofectamin RNAimax (5:1) in a single cell suspension with 1500 ng of <span class="html-italic">T</span>, <span class="html-italic">FOXA2</span> or <span class="html-italic">NOTO</span> mRNA for 24 h for MEPC differentiation. Monolayer transfections were then performed on day 3 and day 4. Cells were maintained in N2B27 with 3 or 6 µM CHIR99021 with or without 50 ng/mL FGF2 from day 2 to day 5. For the stabilization phase, transfected cells were maintained in N2B27 supplemented with 3 µM CHIR99021 with or without 50 ng/mL FGF2 and 100 ng/mL SHH from day 5 to day 7. Top panel: representative brightfield images of differentiating hiPSCs upon optimal culture condition for notochordal lineage from day 0 to day 7, including undifferentiated control cells at day 2 (cells without treatment). (*) indicates optimal culture condition for notochordal differentiation at day 7.</p> Full article ">Figure 2
<p>WNT signalling pathway induces hiPSCs differentiation towards mesendoderm progenitors. (<b>A</b>) Modulation of WNT signalling by CHIR; (<b>B</b>) Relative expression of pluripotent markers (<span class="html-italic">SOX2</span>, <span class="html-italic">NANOG</span> and <span class="html-italic">POU5F1</span>), WNT and NODAL target genes (<span class="html-italic">LEF1</span>, <span class="html-italic">LEFTY1</span> and <span class="html-italic">NODAL</span>), primitive streak (<span class="html-italic">T</span>, <span class="html-italic">MIXL1</span> and <span class="html-italic">EOMES</span>) and mesendoderm markers (<span class="html-italic">FOXA2</span>, <span class="html-italic">GSC</span> and <span class="html-italic">CER1</span>), (n = 2 independent experiments, mean values); (<b>C</b>) Brightfield acquisition of differentiating hiPSCs upon CHIR treatment; (<b>D</b>) Immunostainings of T+/FOXA2+ positive cells (cell counting at day 2, n = 2 independent experiments, mean percentage ± SEM). Insets are showing nuclei staining with Hoechst. Scale bars: 100 µm. APS = anterior primitive streak; PPS = posterior primitive streak.</p> Full article ">Figure 3
<p>WNT and NODAL signalling pathways induce hiPSCs differentiation towards mesendoderm progenitors. (<b>A</b>) Modulation of WNT and NODAL signalling by CHIR and ActA; (<b>B</b>) Relative expression of pluripotent markers (<span class="html-italic">SOX2</span>, <span class="html-italic">NANOG</span> and <span class="html-italic">POU5F1)</span>, WNT and NODAL target genes (<span class="html-italic">LEF1</span>, <span class="html-italic">LEFTY1</span> and <span class="html-italic">NODAL</span>), primitive streak (<span class="html-italic">T</span>, <span class="html-italic">MIXL1</span> and <span class="html-italic">EOMES</span>) and mesendoderm markers (<span class="html-italic">FOXA2</span>, <span class="html-italic">GSC</span> and <span class="html-italic">CER1</span>), (n = 3, mean values); (<b>C</b>) Brightfield acquisition of differentiating hiPCSs upon CHIR and ActA treatment; (<b>D</b>) Immunostainings of T+/FOXA2+ positive cells (cell counting at day 2, n = 2 independent experiments, mean values ± SEM). Insets are showing nuclei staining with Hoechst. Scale bars: 100 µm. APS = anterior primitive streak; PPS = posterior primitive streak.</p> Full article ">Figure 4
<p>Generation and differentiation of mesendoderm progenitors upon WNT and NODAL signalling activation. (<b>A</b>) Effects of sustained WNT signalling activation on the differentiation of MEPC; (<b>B</b>) Brightfield acquisition of MEPC and immunostainings of T+/FOXA2+ positive cells (cell counting at day 2, n = 2 independent experiments, mean percentage ± SEM). Insets are showing nuclei staining with Hoechst; (<b>C</b>) Relative expression of axial mesoderm (<span class="html-italic">T</span>, <span class="html-italic">FOXA2</span>, <span class="html-italic">SHH</span>, <span class="html-italic">FOXJ1</span>, <span class="html-italic">NOGGIN</span> and <span class="html-italic">NOTO</span>), endoderm (<span class="html-italic">GSC, CER1</span> and <span class="html-italic">SOX17</span>) and mesoderm (<span class="html-italic">MIXL1</span>, <span class="html-italic">TBX6</span> and <span class="html-italic">FOXF1</span>) markers expression (n = 4, mean values). ND = Non-Detected Ct value. Mean and standard error of mean (SEM) values relative to experiments in panel C are shown in <a href="#app2-cells-09-00509" class="html-app">Appendix A</a>. Statistical analysis (2 way Anova test) relative to experiments in panel C to determine significant differences between conditions at day 2 and day 5 is shown in <a href="#app3-cells-09-00509" class="html-app">Appendix B</a>. Scale bars: 100 µm.</p> Full article ">Figure 5
<p>Generation and differentiation of mesendoderm progenitors upon WNT signalling activation following <span class="html-italic">T</span>, <span class="html-italic">FOXA2</span> or <span class="html-italic">NOTO</span> mRNA transfections. (<b>A</b>) Differentiation of MEPC following <span class="html-italic">T, FOXA2</span> or <span class="html-italic">NOTO</span> mRNA transfections; (<b>B</b>) Relative expression of axial mesoderm (<span class="html-italic">T</span>, <span class="html-italic">FOXA2 NOTO</span>, <span class="html-italic">SHH</span>, <span class="html-italic">NOGGIN</span> and <span class="html-italic">FOXJ1</span>), endoderm (<span class="html-italic">GSC</span>, <span class="html-italic">CER1</span> and <span class="html-italic">SOX17</span>) and mesoderm (<span class="html-italic">MIXL1</span>, <span class="html-italic">TBX6</span> and <span class="html-italic">FOXF1</span>) markers, (n = 3 independent experiments, mean values ± SEM). * indicates endogenous expression analysed by 3′UTR amplification of <span class="html-italic">T</span>, <span class="html-italic">FOXA2</span> and <span class="html-italic">NOTO transcripts</span>; (<b>C</b>,<b>D</b>) Immunostainings and quantifications of T+/FOXA2+, T+/SOX9+ and FOXA2+/SOX17+ cells (n = 2; quantification n = 2 technical replicates, mean values). Insets in C are showing nuclei staining with Hoechst. Scale bars: 100 µm.</p> Full article ">Figure 6
<p>The FGF signalling pathway does not enhance notochordal differentiation. (<b>A</b>) Assessment of MEPC differentiation into NLC following FGF2 supplementation; (<b>B</b>) Relative expression of axial mesoderm (<span class="html-italic">T</span>, <span class="html-italic">FOXA2</span>, <span class="html-italic">NOTO</span>, <span class="html-italic">FOXJ1</span>, <span class="html-italic">NOGGIN</span> and <span class="html-italic">SHH</span>), endoderm (<span class="html-italic">GSC</span>, <span class="html-italic">CER1</span> and <span class="html-italic">SOX17</span>) and mesoderm (<span class="html-italic">MIXL1</span>, <span class="html-italic">TBX6</span> and <span class="html-italic">FOXF1</span>) markers in differentiating MEPC (RT-qPCR, n = 2 independent experiments, mean values ± SEM). * endogenous expression analysed by 3′UTR amplification of <span class="html-italic">NOTO transcript</span>; (<b>C</b>) Immunostaining of T+/FOXA2+ positive cells in differentiating MEPC (n = 2). Insets are showing nuclei stained with Hoechst. Scale bar: 50 μm.</p> Full article ">Figure 7
<p><span class="html-italic">NOTO</span> mRNA transfection and WNT signalling activity are sufficient to induce a stable NLC population. (<b>A</b>) Assessment of NLC stabilization by FGF and SHH signalling activities; (<b>B</b>) Relative expression of axial mesoderm (<span class="html-italic">T</span>, <span class="html-italic">FOXA2</span>, <span class="html-italic">NOTO</span>, <span class="html-italic">SHH</span>, <span class="html-italic">FOXJ1</span>, <span class="html-italic">NOGGIN</span> and <span class="html-italic">SHH</span>), endoderm (<span class="html-italic">GSC</span>, <span class="html-italic">CER1</span>, <span class="html-italic">SOX17</span>) and mesoderm (<span class="html-italic">MIXL1</span>, <span class="html-italic">TBX6</span> and <span class="html-italic">FOXF1</span>) markers (n = 2 independent experiments, mean ± SEM). * Endogenous expression analysed by 3′UTR amplification of <span class="html-italic">NOTO transcript</span>; (<b>C</b>) Immunostainings of T+/FOXA2+ positive cells. Insets are showing nuclei staining with Hoechst. Scale bars: 50 µm; (<b>D</b>) Heatmap representation of gene expression profiles during control and <span class="html-italic">NOTO</span>-transfected cell differentiation (n = 2, mean values).</p> Full article ">Figure 8
<p><span class="html-italic">NOTO</span> mRNA transfection induced a distinct molecular signature. (<b>A</b>) Differentiation of MEPC following <span class="html-italic">NOTO</span> or <span class="html-italic">FOXA2</span> mRNA transfections; (<b>B</b>) Expression levels of genes used as markers of mesoderm, ectoderm and endoderm across our samples (left) or Tsankov et al. samples (right); (<b>C</b>) RNAseq expression profile of differentially expressed genes during the course of differentiation. Differentially expressed genes were distributed in 5 clusters based on their kinetic of expression; (<b>D</b>) Expression levels of immediate <span class="html-italic">NOTO</span> response genes during the course of <span class="html-italic">NOTO</span>- and <span class="html-italic">FOXA2</span>-driven MEPC differentiation (this study) and in hESC-derived mesoderm, ectoderm and endoderm [<a href="#B59-cells-09-00509" class="html-bibr">59</a>]; (<b>E</b>) Expression levels of delayed <span class="html-italic">NOTO</span> response genes during the course of <span class="html-italic">NOTO</span>- and <span class="html-italic">FOXA2</span>-driven MEPC differentiation (this study) and in hESC-derived mesoderm, ectoderm and endoderm [<a href="#B59-cells-09-00509" class="html-bibr">59</a>]; (<b>F</b>) Top 15 Biological Processes associated with the up-regulated genes in <span class="html-italic">NOTO</span>-transfected condition compared to <span class="html-italic">FOXA2</span>-transfected condition. Cluster details for mesendoderm genes, NOTO inhibited genes and FOXA2 response genes are presented in <a href="#app1-cells-09-00509" class="html-app">Figure S2</a>.</p> Full article ">Figure 9
<p>Visual summary of the main findings of the study. Human iPSCs were generated from dermal fibroblasts using 4 reprogramming factors (Oct4, Klf4, Sox2 and c-Myc = OKSM). Activation at day 2: WNT/β-catenin pathway activity (intermediate concentration of CHIR = Mid WNT) induced an increase in <span class="html-italic">NODAL</span> and <span class="html-italic">LEFTY1</span> gene expression. In this condition, high levels of bipotent mesendoderm progenitors (T+/FOXA2+ cells) were generated. Supplementation with Activin A resulted in hiPSCs commitment toward endoderm lineage (T-/FOXA2+ progenitor cells). High WNT pathway activation (high concentration of CHIR = High WNT) resulted in hiPSCs commitment toward mesoderm lineage (T+/FOXA2- progenitor cells). Differentiation from day 3: Mesendoderm progenitors transfected with synthetic mRNAs encoding human NOTO transcription factor and sustained with Mid WNT signalling activation generated axial mesoderm progenitors (T+/FOXA2+ cells). Stabilization up to day 7: <span class="html-italic">NOTO</span> transfection and Mid WNT signalling activation increased both SHH and FGF signalling pathway activities in axial mesoderm progenitors, which further differentiated into stable notochord-like cell population (NLC) at day 7 (T+/FOXA2+ cells and T+/SOX9+ cells). Human iPSC-derived NLC expressed embryonic notochord-related markers. Blue arrows indicate optimal culture condition for notochordal differentiation. Sets of gene markers relative to lineages or specific cell-types are indicated.</p> Full article ">
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