Identification of Spectral Modifications Occurring during Reprogramming of Somatic Cells

Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However, research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly, this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose

An Intron-Retaining Splice Variant of Human Cyclin A2, Expressed in Adult Differentiated Tissues, Induces a G1/S Cell Cycle Arrest In Vitro

BACKGROUND: Human cyclin A2 is a key regulator of S phase progression and entry into mitosis. Alternative splice variants of the G1 and mitotic cyclins have been shown to interfere with full-length cyclin functions to modulate cell cycle progression and are therefore likely to play a role in differentiation or oncogenesis. The alternative splicing of human cyclin A2 has not yet been studied. METHODOLOGY/PRINCIPAL FINDINGS: Sequence-specific primers were designed to amplify various exon-intron regions of cyclin A2 mRNA in cell lines and human tissues. Intron retaining PCR products were cloned and sequenced and then overexpressed in HeLa cells. The subcellular localization of the splice variants was studied using confocal and time-lapse microscopy, and their impact on the cell cycle by flow cytometry, immunoblotting and histone H1 kinase activity. We found a splice variant of cyclin A2 mRNA called A2V6 that partly retains Intron 6. The gene expression pattern of A2V6 mRNA in human tissues was noticeably different from that of wild-type cyclin A2 (A2WT) mRNA. It was lower in proliferating fetal tissues and stronger in some differentiated adult tissues, especially, heart. In transfected HeLa cells, A2V6 localized exclusively in the cytoplasm whereas A2WT accumulated in the nucleus. We show that A2V6 induced a clear G1/S cell cycle arrest associated with a p21 and p27 upregulation and an inhibition of retinoblastoma protein phosphorylation. Like A2WT, A2V6 bound CDK2, but the A2V6/CDK2 complex did not phosphorylate histone H1. CONCLUSION/SIGNIFICANCE: This study has revealed that some highly differentiated human tissues express an intron-retaining cyclin A2 mRNA that induced a G1/S block in vitro. Contrary to full-length cyclin A2, which regulates cell proliferation, the A2V6 splice variant might play a role in regulating nondividing cell states such as terminal differentiation or senescence

A2V6-GFP binds CDK2 without inducing H1 kinase activity.

<p>(<b>A</b>) Anti-GFP immunoblot (IB) from cell lysates immunoprecipitated (IP) with anti-CDK2 antibody. The A2V6-GFP fusion protein (≈75 kDa) interacts with CDK2 like the A2WT-GFP fusion protein (≈85 kDa). IgG: non-specific antibody control. (<b>B</b>) Autoradiography of ³²P-labeled histone H1 performed after anti-GFP immunoprecipitation in lysates from A2WT- and A2V6-GFP transfected cells. IgG: non-specific antibody control. Input: non-immunoprecipitated lysate. No phosphorylated histone H1 was detected in A2V6-GFP transfected cells, indicating that the A2V6/CDK2 complexes, contrary to the A2WT/CDK2 ones, are not functional. The phosphorylated histone H1 in the input lanes corresponds to the global kinase activity of cycling asynchronous cells.</p

Distribution of the A2V6 splice variant of cyclin A2 in human tissues.

<p>(<b>A</b>) Exon-Intron organization of the Human cyclin A2 gene. Black boxes: exons. Grey boxes: 5′ and 3′ UTR. D-box: destruction box. MRAIL: CDK2 binding motif belonging to the cyclin box. Cyclin A2: gene. A2WT: mature cyclin A2 messenger resulting from alternate RNA splicing and amplified using the X1S-E8B primer pair. A2V6: cyclin A2 splice variant mRNA retaining a portion of Intron 6 (empty box) amplified using the X1S-A2I6 primer pair. The indicated sequence comprises the last nine nucleotides of Exon 6 (uppercase letters) and the retained Intron 6 sequence (lowercase letters). (<b>B</b>) Gene-expression analysis of cyclin A2 using PCR-Southern blotting in human adult and fetal tissue samples from the indicated organs. A2WT: wild-type cyclin A2 mRNA (2.5 kb). The 3.3 kb PCR product was determined to be A2WT plus Intron-1 by sequencing. A2V6: cyclin A2 splice variant (1.3 kb). GAPDH: Glyceraldehyde-3-phosphate dehydrogenase (loading control).</p

Cytoplasmic localization of A2V6-GFP during the cell cycle.

<p>HeLa cells were transfected with wild-type cyclin A2-GFP (A2WT-GFP) or Intron 6-retaining cyclin A2-GFP (A2V6-GFP) vectors. (<b>A</b>) Immunofluorescence images of transfected HeLa cells using anti-GFP (green) and anti-cyclin A2 (red) antibodies. Nuclei (blue) were counterstained with Hoechst dye 33258. The A2V6-GFP protein is located in the cytoplasm of transfected cells. No endogenous cyclin A2 is detected in cells expressing the A2V6 protein. (<b>B</b>) Time-lapse imaging of A2WT- and A2V6-GFP during a cell cycle. Upper panel: Phase contrast microscopy. Lower panel: Fluorescence microscopy. Arrow: parent cell. Labels 1 and 2: daughter cells. Wild-type cyclin A2-GFP is accumulated in G2 nuclei (−1.3 h), degraded in mitosis and reappears mostly in the nuclei in S phase, like endogenous cyclin A2. In contrast, A2V6-GFP remains in the cytoplasm throughout the cell cycle. Scale bar: 20 µm.</p

Internal Radiotherapy of Liver Cancer with Rat Hepatocarcinoma-Intestine-Pancreas Gene as a Liver Tumor-Specific Promoter

International audienceThe hepatocarcinoma-intestine-pancreas (HIP) gene, also called pancreatitis-associated protein-1 (PAP1) or Reg IIIα, is activated in most human hepatocellular carcinomas (HCCs) but not in normal liver, which suggests that HIP regulatory sequence could be used as efficient liver tumor-specific promoters to express a therapeutic polynucleotide in liver cancer. The sodium iodide symporter (NIS), which has recognized therapeutic and reporter gene properties, is appropriate to evaluate the transcriptional strength and specificity of the HIP promoter in HCC. For this purpose, we constructed a recombinant rat HIP–NIS adenoviral vector (AdrHIP-NIS), and evaluated its performance as a mediator of selective radioiodide uptake in tumor hepatocytes. Western blot, immunofluorescence, and iodide uptake assays were performed in AdrHIP-NIS-infected primary hepatocytes and transformed hepatic and nonhepatic cells. Nuclear imaging, tissue counting and immunohistochemistry were performed in normal and HCC-bearing Wistar rats infected with AdrHIP-NIS intratumorally or via the hepatic artery. In AdrHIP-NIS-infected transformed hepatic cells, functional NIS was strongly expressed, as in cells infected with a cytomegalovirus-NIS vector. No NIS expression was found in AdrHIP-NIS-infected normal hepatocytes or transformed nonhepatic cells. In rats bearing multinodular HCC, AdrHIP-NIS triggered functional NIS expression that was preferential in tumor hepatocytes. Administration of 18 mCi of 131I resulted in the destruction of AdrHIP-NIS-injected nodules. This study has identified the rHIP regulatory sequence as a potent liver tumor-specific promoter for the transfer of therapeutic genes, and AdrHIP-NIS-mediated 131I therapy as a valuable option for the treatment of multinodular HCC.</p

Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines

International audienceHematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process

Generation of Multipotent Early Lymphoid Progenitors from Human Embryonic Stem Cells

International audienceDuring human embryonic stem cell (ESC) hematopoietic differentiation, the description of the initial steps of lymphopoiesis remains elusive. Using a two-step culture procedure, we identified two original populations of ESC-derived hematopoietic progenitor cells (HPCs) with CD34+CD45RA+CD7- and CD34+CD45RA+CD7+ phenotypes. Bulk cultures and limiting dilution assays, culture with MS5 cells in the presence of Notch ligand Delta-like-1 (DL-1), and ex vivo colonization tests using fetal thymic organ cultures showed that although CD34+CD45RA+CD7- HPCs could generate cells of the three lymphoid lineages, their potential was skewed toward the B cell lineages. In contrast, CD34+CD45RA+CD7+ HPCs predominantly exhibited a T/natural killer (NK) cell differentiation potential. Furthermore these cells could differentiate equivalently into cells of the granulo-macrophagic lineage and dendritic cells and lacked erythroid potential. Expression profiling of 18 markers by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that CD34+CD45RA+CD7- and CD34+CD45RA+CD7+ HPCs express genes of the lymphoid specification and that CD34+CD45RA+CD7- cells express B-cell-Associated genes, while CD34+CD45RA+CD7+ HPCs display a T-cell molecular profile. Altogether, these findings indicate that CD34+CD45RA+CD7- and CD34+CD45RA+CD7+ HPCs correspond to candidate multipotent early lymphoid progenitors polarized toward either the B or T/NK lineage, respectively. This work should improve our understanding of the early steps of lymphopoiesis from pluripotent stem cells and pave the way for the production of lymphocytes for cell-based immunotherapy and lymphoid development studies. © Copyright 2014, Mary Ann Liebert, Inc. 2014

Human engraftment of NOG mice transplanted with ES or iPS cells.

<p>Human engraftment of NOG mice transplanted with ES or iPS cells.</p

Expression of hematopoietic transcription factors for ES and iPS cell lines.

<p>Gene expression level of <i>RUNX1</i>, <i>HOXB4</i>, <i>TAL1</i>, <i>PU</i>.<i>1</i>, <i>GATA1</i>, <i>GATA2</i>, <i>GATA3</i>, <i>MPO</i> and <i>IKAROS</i> was evaluated by Q-RT-PCR (A) at pluripotent stage and (B) after differentiation in EB at day 16 and human BM as a positive control of expression hematopoietic genes. Gene expression was normalized relative to the endogenous RNA control human <i>HPRT</i>.</p