X-Linked Clonality Analysis of Human Embryonic Stem Cells ( hESC ) Before and after induction of Hematopoietic Differentiation
Authors: Mitjavila-Garcia Maria Teresa , Yates Frank , Bonnet Marie-Laure , Rougeulle Claire , Makhlouf Melanie , Feraud Olivier , Magniez Aurelie , Bennaceur-Griscelli Annelise , Turhan Ali G 
Published: April 2020

Abstract
Human ES cell lines represent an invaluable tool of research for the study of normal and pathological development. In this study we asked whether the human ES cell lines currently used for research purposes are derived from single outgrowths using X-linked clonality analysis. Amongst the cell lines tested, the female H9 cell line was informative at the Humara (human androgen receptor) locus by the presence of two CAG repeats allowing distinction of paternal (Xp) and maternal (Xm) X chromosomes. Clonal analysis was performed upon Hpa II digestion of DNA purified from total H9 cells (passages 27–61), single H9 cell clones generated from single cell cloning, single embryoid bodies (EB) (day 4, day 14) as well as from single hematopoietic progenitors. The same analysis was performed after induction of hematopoietic differentiation in liquid cultures in the presence of hematopoietic permissive conditions (co-culture in the presence of OP-9 cell line). Clonality was evaluated by calculating the Relative Corrected Index (RCI) which is the ratio of the intensity of peaks generated by Hpa II-digested and undigested DNA. In several experiments, all passages of H9 cell lines examined ( p27, p28, p31, p61) were found to exhibit a monoclonal pattern with disappearance of the same allele A upon Hpa II digestion (RCI &gt; 10). 13 clonal cultures of H9 obtained by single cell cloning exhibited also the same clonal pattern with digestion of the same allele and RCI values between 6–14. Similarly, DNA purified from EB’s at different stages was found to be clonal. In DNA obtained from 75 individual hematopoietic progenitors (CFU-GM, CFU-G), clonal analysis revealed the same clonal pattern in each, with disappearance of the same allele A. However, clonal analysis performed in 10 hematopoietic colonies, revealed unexpectedly, an equal digestion of Humara alleles with RCI &lt; 2 (polyclonal pattern). Finally, in two experiments, hematopoietic cells recovered from liquid cultures exhibited an absolute clonal pattern (Total disappearance of the same allele A). Interestingly, quantification of X-Inactivation Specific Transcript (XIST) RNA in H9 and day14 EB’s by RT-PCR did not show any evidence of XIST expression both before and after differentiation. Altogether, our data suggest that XCI has already been initiated in undifferentiated H9 cells and this inactivation is maintained, at least in the Humara locus in the absence of XIST. The monoclonal pattern of the cell line can be due to the fact that H9 ES cell line might have arisen from a limited number of stem cells having undergone XCI at a very early stage, with maintenance of the methylation status at this locus during mesodermal and hematopoietic commitment. Conversely, hematopoietic differentiation might occur from several mesodermal progenitors having inactivated the same X chromosome in H9 at the Humara locus. Despite the fact that hematopoietic progenitors and differentiated hematopoietic cells originating from H9 exhibited also a clonal pattern, a partial reactivation of the inactive X chromosome during hematopoietic differentiation could occur, and may explain the “polyclonal” pattern obtained in 10 CFU-GM analyzed. Overall, X-linked clonality testing could be an important issue before considering the potential clinical applications, in all female hES cell lines.