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STUDY HYPOTHESIS Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? STUDY FINDING We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture,often accompanied by erosion of XCI-specific methylation,and a frequent loss of random XCI in the cultures. WHAT IS KNOWN ALREADY Variable XCI patterns have been reported in female hPSC,not only between different hPSC lines,but also between sub-passages of the same cell line,however the reasons for this variability remain unknown. Moreover,while non-random XCI-linked DNA methylation patterns have been previously reported,their origin and extent have not been investigated. STUDY DESIGN,SAMPLES/MATERIALS,METHODS We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines,during long-term culture and after differentiation,by gene expression analysis,histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence,XIST expression by real-time PCR,and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR,and more in depth by massive parallel bisulphite sequencing. MAIN RESULTS AND THE ROLE OF CHANCE All hPSC lines showed XCI,but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation,it did not result in X chromosome reactivation. Moreover,lines without strong erosion of methylation frequently displayed non-random DNA methylation,which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture take-over of chromosome abnormalities and was independent of the parental origin of the X chromosome. Therefore,we suggest that a culture advantage conferred by alleles on the X chromosome or by XCI-related mechanisms may be at the basis of this phenomenon. Finally,differentiated populations inherited the aberrant XCI patterns from the undifferentiated cells they were derived from. LIMITATIONS,REASONS FOR CAUTION All hPSC lines in this study were cultured in highly similar conditions. Our results may therefore be specific for these conditions and alternative culture conditions might lead to different findings. Our findings are only a first step towards elucidating the molecular events leading to the phenomena we observed. WIDER IMPLICATIONS OF THE FINDINGS Our results highlight the significant extent of aberrant XCI in female hPSC. The fact that these aberrations are inherited by the differentiated progeny may have a significant impact on downstream research and clinical uses of hPSC. In order to achieve the full potential of hPSC,more insight into the XCI status and its stability in hPSC and its effect on the properties of the differentiated progeny is needed. LARGE SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS Our research is supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen,grant 1502512N),Generalitat de Catalunya (2014SGR-005214) and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel,on name of K.S. L.V.H. is funded by EMBO (ALTF 701-2013). The authors declare no potential conflict of interest. View Publication

Fetuses with homozygous alpha-thalassemia usually die at the third trimester of pregnancy or soon after birth. Hence,the disease could potentially be a target for fetal gene therapy. We have previously established a mouse model of alpha-thalassemia. These mice mimic the human alpha-thalassemic conditions and can be used as preclinical models for fetal gene therapy. We tested a lentiviral vector containing the HS 2,3,and 4 of the beta-LCR,a central polypurine tract element,and the beta-globin gene promoter directing either the EGFP or the human alpha-globin gene. We showed that the GFP expression was erythroid-specific and detected in BFU-E colonies and the erythroid progenies of CFU-GEMM. For in utero gene delivery,we did yolk sac vessel injection at midgestation of mouse embryos. The recipient mice were analyzed after birth for human alpha-globin gene expression. In the newborn,human alpha-globin gene expression was detected in the liver,spleen,and peripheral blood. The human alpha-globin gene expression was at the peak at 3-4 months,when it reached 20% in some recipients. However,the expression declined at 7 months. Colony-forming assays in these mice showed low abundance of the transduced human alpha-globin gene in their BFU-E and CFU-GEMM and the lack of its transcript. Thus,lentiviral vectors can be an effective vehicle for delivering the human alpha-globin gene into erythroid cells in utero,but,in the mouse model,delivery at late midgestation could not transduce hematopoietic stem cells adequately to sustain gene expression. View Publication

Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders that involve defects that preclude the normal function of osteoclasts,which differentiate from hematopoietic precursors. In half of human cases,ARO is the result of mutations in the TCIRG1 gene,which codes for a subunit of the vacuolar proton pump that plays a fundamental role in the acidification of the cell-bone interface. Functional mutations of this pump severely impair the resorption of bone mineral. Although postnatal hematopoietic stem cell transplantation can partially rescue the hematological phenotype of ARO,other stigmata of the disease,such as secondary neurological and growth defects,are not reversed. For this reason,ARO is a paradigm for genetic diseases that would benefit from effective prenatal treatment. Using the oc/oc mutant mouse,a murine model whose osteopetrotic phenotype closely recapitulates human TCIRG1-dependent ARO,we report that in utero transplantation of adult bone marrow hematopoietic stem cells can correct the ARO phenotype in a limited number of mice. Here we report that in utero injection of allogeneic fetal liver cells,which include hematopoietic stem cells,into oc/oc mouse fetuses at 13.5 days post coitum produces a high level of engraftment,and the oc/oc phenotype is completely rescued in a high percentage of these mice. Therefore,oc/oc pathology appears to be particularly sensitive to this form of early treatment of the ARO genetic disorder. View Publication

Liver becomes the predominant site of hematopoiesis by 11.5 dpc (days after coitus) in the mouse and 15 gestational weeks in humans and stays so until the end of gestation. The reason the liver is the major hematopoietic site during fetal life is not clear. In this work,we tried to define which of the fetal liver microenvironmental cell populations would be associated with the development of hematopoiesis and found that a population of cells with mixed endodermal and mesodermal features corresponded to hematopoietic-supportive fetal liver stroma. Stromal cells generated from primary cultures or stromal lines from mouse or human fetal liver in the hematopoietic florid phase expressed both mesenchymal markers (vimentin,osteopontin,collagen I,alpha smooth muscle actin,thrombospondin-1,EDa fibronectin,calponin,Stro-1 antigens,myocyte-enhancer factor 2C) and epithelial (alpha-fetoprotein,cytokeratins 8 and 18,albumin,E-cadherin,hepatocyte nuclear factor 3 alpha) markers. Such a cell population fits with the description of cells in epithelial-to-mesenchymal transition (EMT),often observed during development,including that of the liver. The hematopoietic supportive capacity of EMT cells was lost after hepatocytic maturation,induced by oncostatin M in the cell line AFT024. EMT cells were observed in the fetal liver microenvironment during the hematopoietic phase but not in nonhematopoietic liver by the end of gestation and in the adult. EMT cells represent a novel stromal cell type that may be generated from hepatic endodermal or mesenchymal stem cells or even from circulating hematopoietic stem cells (HSCs) seeding the liver rudiment. View Publication

Here,we show that as human embryonic stem cells (ESCs) exit the pluripotent state,NANOG can play a key role in determining lineage outcome. It has previously been reported that BMPs induce differentiation of human ESCs into extraembryonic lineages. Here,we find that FGF2,acting through the MEK-ERK pathway,switches BMP4-induced human ESC differentiation outcome to mesendoderm,characterized by the uniform expression of T (brachyury) and other primitive streak markers. We also find that MEK-ERK signaling prolongs NANOG expression during BMP-induced differentiation,that forced NANOG expression results in FGF-independent BMP4 induction of mesendoderm,and that knockdown of NANOG greatly reduces T induction. Together,our results demonstrate that FGF2 signaling switches the outcome of BMP4-induced differentiation of human ESCs by maintaining NANOG levels through the MEK-ERK pathway. View Publication

Dysregulation of the receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) plays a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase,TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates RSK2 at Y529,which consequently regulates RSK2 activation. Here we identified Y707 as an additional tyrosine in RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation,through a putative disruption of the autoinhibitory alphaL-helix on the C terminus of RSK2,unlike Y529 phosphorylation,which facilitates ERK binding. Moreover,we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2 and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707,as well as the subsequent RSK2 activation. Furthermore,in a murine bone marrow transplant assay,genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild-type cells,suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation. Our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases. View Publication

In this study,we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells,in serum-free medium,supplemented only with fibroblast growth factor (FGF)-1,FGF-2,or FGF-1 +2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assays,high levels of stem cell activity were detectable at 1,3,and 5 weeks after initiation of culture. FGFs as single growth factors failed to support cultures of highly purified Lin(-)Sca-1(+)c-Kit(+)(LSK) cells. However,cocultures of purified CD45.1 LSK cells with whole BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant,showing that HSC activity originated from LSK cells. Subsequently,we tested the reconstituting potential of cells cultured in FGF-1 + 2 with the addition of early acting stimulatory molecules,stem cell factor +interleukin-11 + Flt3 ligand. The addition of these growth factors resulted in a strong mitogenic response,inducing rapid differentiation and thereby completely overriding FGF-dependent stem cell conservation. Importantly,although HSC activity is typically rapidly lost after short-term culture in vitro,our current protocol allows us to sustain stem cell repopulation potential for periods up to 5 weeks. View Publication

Pluripotent or multipotent cell-based therapeutics are vital for skeletal reconstruction in non-healing critical-sized defects since the local endogenous progenitor cells are not often adequate to restore tissue continuity or function. However,currently available cell-based regenerative strategies are hindered by numerous obstacles including inadequate cell availability,painful and invasive cell-harvesting procedures,and tumorigenesis. Previously,we established a novel platform technology for inducing a quiescent stem cell-like stage using only a single extracellular proteoglycan,fibromodulin (FMOD),circumventing gene transduction. In this study,we further purified and significantly increased the reprogramming rate of the yield multipotent FMOD reprogrammed (FReP) cells. We also exposed the 'molecular blueprint' of FReP cell osteogenic differentiation by gene profiling. Radiographic analysis showed that implantation of FReP cells into a critical-sized SCID mouse calvarial defect,contributed to the robust osteogenic capability of FReP cells in a challenging clinically relevant traumatic scenario in vivo. The persistence,engraftment,and osteogenesis of transplanted FReP cells without tumorigenesis in vivo were confirmed by histological and immunohistochemical staining. Taken together,we have provided an extended potency,safety,and molecular profile of FReP cell-based bone regeneration. Therefore,FReP cells present a high potential for cellular and gene therapy products for bone regeneration. View Publication

Despite many years of intensive effort,there is surprisingly little consensus on the most suitable markers with which to locate and isolate stem cells from adult tissues. By comparison,the study of cancer stem cells is still in its infancy; so,unsurprisingly,there is great uncertainty as to the identity of these cells. Stem cell markers can be broadly categorized into molecular determinants of self-renewal,clonogenicity,multipotentiality,adherence to the niche,and longevity. This review assesses the utility of recognizing cancer stem cells by virtue of high expression of aldehyde dehydrogenases (ALDHs),probably significant determinants of cell survival through their ability to detoxify many potentially cytotoxic molecules,and contributing to drug resistance. Antibodies are available against the ALDH enzyme family,but the vast majority of studies have used cell sorting techniques to enrich for cells expressing these enzymes. Live cells expressing high ALDH activity are usually identified by the ALDEFLUOR kit and sorted by fluorescence activated cell sorting (FACS). For many human tumours,but notably breast cancer,cell selection based upon ALDH activity appears to be a useful marker for enriching for cells with tumour-initiating activity (presumed cancer stem cells) in immunodeficient mice,and indeed the frequency of so-called ALDH(bri) cells in many tumours can be an independent prognostic indicator. View Publication

The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors,particularly radial glia (RG),are rare and are defined by a combination of intracellular markers,position and morphology. To circumvent these problems,we developed Fixed and Recovered Intact Single-cell RNA (FRISCR),a method for profiling the transcriptomes of individual fixed,stained and sorted cells. Using FRISCR,we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB,and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles,an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers. View Publication

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells,whereas mural cells are believed to derive from mesoderm,neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation,whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system. View Publication