Marie Laure Bonnet

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.

Molecular Genetics at the T-Cell Receptor β Locus: Insights into the Regulation of V(D)J Recombination

The V(D)J recombination machinery assembles antigen receptor genes from germline V, D and J segments duringlymphocyte development. In alphabetaT cells, this leads to the production of the T-cell receptor (TCR) alpha and beta chains. Notably, V(D)J recombination at the Tcrb locus is tightly controlled at various levels, including cell-type and stage specificities, intralocus ordering and allelic exclusion. Although many of these controls are partly mediated at the level of genomic accessibility to the V(D)J recombinase, recent studies have uncovered novel mechanisms that are also likely to contribute to the developmental regulation of Tcrb gene rearrangement events. In this chapter, we summarize our current knowledge and highlight unanswered questions regarding the regulation of V(D)J recombination at the Tcrb locus, placing emphasis on mouse transgenesis and gene-targeting approaches.

Partial Reversal of the Methylation Pattern of the X-linked Gene HUMARA during Hematopoietic Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with high efficiency. In this work, we analyzed the methylation status of the X-linked HUMARA (human androgen receptor) gene in hematopoietic cells derived from hESC line H9 before and after induction of hematopoietic differentiation. All passages of H9 and H9-derived hematopoietic cells displayed homogenous methylation pattern with disappearance of the same allele upon HpaII digestion. This pattern persisted in the great majority of different hematopoietic progenitors derived from H9, except in 11 of 86 individually plucked colonies in which an equal digestion of the HUMARA alleles has been found, suggesting that a methylation change occurring at this locus during differentiation. Interestingly, quantification of X inactive-specific transcript (XIST) RNA in undifferentiated H9 cell line and day 14 embryoid bodies (EB) by RT-PCR did not show any evidence of XIST expression either before or after differentiation. Thus, during self-renewal conditions and after induction of commitment towards the formation of EB, the methylation pattern of the HUMARA locus appears locked with the same unmethylated allele. However, hematopoietic differentiation seems to be permissive to the reversal of methylation status of HUMARA in some terminally differentiated progenitors. These data suggest that monitoring methylation of HUMARA gene during induced differentiation could be of use for studying hESC-derived hematopoiesis.