Clara Bueno

Human Induced Pluripotent Stem Cells Develop Teratoma More Efficiently and Faster Than Human Embryonic Stem Cells Regardless the Site of Injection

Human embryonic stem cell (hESC) and reprogrammed/induced pluripotent stem cell (iPSC) research is becoming the “flavor of the month” for downstream applications such as drug screening, disease modeling, and future regenerative medicine and cell therapies [1–4]. Pluripotency (the ability to give rise to any cell type of the three germ layers: mesoderm, ectoderm, and endoderm) is the defining feature of hESCs and iPSCs [5]. In vivo teratoma formation in immune-compromised mice is the “gold-standard” assay to define bona fide pluripotent stem cells capable of generating tumoral disorganized structures containing tissues representing the three germ layers [5,6]. Despite the importance of teratoma assay as an extended screen for the pluripotency of hESCs and iPSCs and as in vivo assay to explore molecular and cellular mechanisms underlying the biology of human teratomas and their transition to teratocarcinomas, there are no standard procedures for performing this assay [5–7]. Different studies on hESCs have correlated the site of implantation with the efficiency of teratoma formation and histology tissue composition [6,8]. However, limited data are available regarding the teratoma development latency. More importantly, no study so far has compared side-by-side the efficiency, latency, and histological tumor composition of hESCs- and iPSCs-derived teratomas. In addition, a new generation of immunodeficient mice has been developed: the NOD/SCID IL2Rγ−/− mouse. This strain carries a IL2Rγ-chain deficiency that blocks signaling through multiple cytokine receptors leading to many innate immune defects [9,10]. The non obese diabetic/severe combined immune-deficient (NOD/SCID) IL2Rγ−/− strain facilitates engraftment and tumor formation and does not develop thymic lymphoma, ensuring a longer lifespan of inoculated mice. Here, we followed the improved teratoma protocol previously developed by Prokhorova et al. [6,11–13] to transplant side-by-side as few as 1 × 106 of either fully characterized undifferentiated hESCs or iPSCs in 6- to 8-week-old non obese diabetic/severe combined immune-deficient (NOD/SCID) IL2Rγ−/− mice [11,13–15]. The following hESC lines were used: H9, H1, AND1, AND2, AND3, HS181, and ECAT. The following iPSC lines were used: MSHU-001, iAND4, CB-CD34+ iPSC1, and CB-CD34+ iPSC2. These lines have been fully characterized and deposited according to Spanish Legislation at The Spanish Stem Cell Bank (http://www.isciii.es/htdocs/terapia/terapia_lineas.jsp) [16]. Briefly, cells were resuspended in phosphate buffered saline (PBS) supplemented with 30% matrigel (Becton Dickinson, San Jose, CA, http://www.bd.com) [6] and transplanted subcutaneously (200 μl volume) or by intratesticular injection (60 μl volume). Figure ​Figure1A1A depicts the experimental strategy used. We then analyzed efficiency, latency, and histological tumor composition. In hESCs, the rate of teratoma formation was 81% subcutaneously versus 94% intratesticularly (n = 30 mice; Fig. ​Fig.1B).1B). However, the intratesticular injection, despite showing higher efficiency of teratoma formation, displayed a slightly longer latency (66 vs. 59 days; p-value > 0.05). There were no site-specific differences in the teratoma composition at the histological level (Fig. ​(Fig.1C).1C). Interestingly, when iPSCs were transplanted the rate of teratoma formation was 100% (n = 16 mice), regardless the type of injection. More importantly, iPSCs seem more aggressive in vivo as the latency was shortened 52% (from 59 days to 31 days) upon subcutaneous injection and 26% (from 66 days to 49 days) upon intratesticular injection. As with hESCs, no differences in teratoma composition were observed either. Figure 1 Human iPSCs form teratomas faster and with higher efficiency than hESCs regardless the site of injection. (A): Cartoon summarizing the experimental design. (B): Table summarizing the efficiency, latency, and histological analysis of the teratomas developed ... To the best of our knowledge, this is the first study comparing side-by-side the efficiency, latency, and teratoma composition between hESCs and iPSCs. We found clear differences in the efficiency and latency but not in the teratoma histological composition. Further experiments are still demanded to gain insights into the higher aggressiveness in vivo of iPSCs as compared with hESCs. Ploidy, analyzed by conventional G-banding karyotype, could not explained these differences because all but two pluripotent stem cell lines were euploid: the aneuploid lines were one hESC (AND1) and one iPSC (iAND4). It is worth emphasizing, however, that karyotype analysis is not a high-resolution technique detecting fine genomic aberrations, with a euploid karyotype not being therefore indicative of an overall cellular genomic stability. Whether or not specific tiny genomic insults (detectable by high-resolution methods such as comparative genomic hybridazation (CGH)-arrays and single-nucleotide polymorphism analysis) or epigenetic differences may explain the higher aggressiveness in vivo of iPSCs still needs to be elucidated. We envision that these data may be useful not only for stem cells scientists addressing pluripotency issues and studying mechanisms underlying specific germ-layer/tissue differentiation but also for cancer researchers developing in vivo models for germ cell tumors.

Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties?

BackgroundThe use of human embryonic stem cells (hESCs) in research is increasing and hESCs hold the promise for many biological, clinical and toxicological studies. Human ESCs are expected to be chromosomally stable since karyotypic changes represent a pitfall for potential future applications. Recently, several studies have analysed the genomic stability of several hESC lines maintained after prolonged in vitro culture but controversial data has been reported. Here, we prompted to compare the chromosomal stability of three hESC lines maintained in the same laboratory using identical culture conditions and passaging methods.ResultsMolecular cytogenetic analyses performed in three different hESC lines maintained in parallel in identical culture conditions revealed significant differences among them in regard to their chromosomal integrity. In feeders, the HS181, SHEF-1 and SHEF-3 hESC lines were chromosomally stable up to 185 passages using either mechanical or enzymatic dissection methods. Despite the three hESC lines were maintained under identical conditions, each hESC line behaved differently upon being transferred to a feeder-free culture system. The two younger hESC lines, HS181 (71 passages) and SHEF-3 (51 passages) became chromosomally unstable shortly after being cultured in feeder-free conditions. The HS181 line gained a chromosome 12 by passage 17 and a marker by passage 21, characterized as a gain of chromosome 20 by SKY. Importantly, the mosaicism for trisomy 12 gradually increased up to 89% by passage 30, suggesting that this karyotypic abnormality provides a selective advantage. Similarly, the SHEF-3 line also acquired a trisomy of chromosome 14 as early as passage 10. However, this karyotypic aberration did not confer selective advantage to the genetically abnormal cells within the bulk culture and the level of mosaicism for the trisomy 14 remained overtime between 15%–36%. Strikingly, however, a much older hESC line, SHEF-1, which was maintained for 185 passages in feeders did not undergo any numerical or structural chromosomal change after 30 passages in feeder-free culture and over 215 passages in total.ConclusionThese results support the concept that feeder-free conditions may partially contribute to hESC chromosomal changes but also confirm the hypothesis that regardless of the culture conditions, culture duration or splitting methods, some hESC lines are inherently more prone than others to karyotypic instability.

Bone marrow mesenchymal stem cells from infants with MLL-AF4+ acute leukemia harbor and express the MLL-AF4 fusion gene

MLL-AF4 fusion is a hallmark genetic abnormality in infant B-acute lymphoblastic leukemia (B-ALL) known to arise in utero. The cellular origin of leukemic fusion genes during human development is difficult to ascertain. The bone marrow (BM) microenvironment plays an important role in the pathogenesis of several hematological malignances. BM mesenchymal stem cells (BM-MSC) from 38 children diagnosed with cytogenetically different acute leukemias were screened for leukemic fusion genes. Fusion genes were absent in BM-MSCs of childhood leukemias carrying TEL-AML1, BCR-ABL, AML1-ETO, MLL-AF9, MLL-AF10, MLL-ENL or hyperdiploidy. However, MLL-AF4 was detected and expressed in BM-MSCs from all cases of MLL-AF4+ B-ALL. Unlike leukemic blasts, MLL-AF4+ BM-MSCs did not display monoclonal Ig gene rearrangements. Endogenous or ectopic expression of MLL-AF4 exerted no effect on MSC culture homeostasis. These findings suggest that MSCs may be in part tumor-related, highlighting an unrecognized role of the BM milieu on the pathogenesis of MLL-AF4+ B-ALL. MLL-AF4 itself is not sufficient for MSC transformation and the expression of MLL-AF4 in MSCs is compatible with a mesenchymal phenotype, suggesting a differential impact in the hematopoietic system and mesenchyme. The absence of monoclonal rearrangements in MLL-AF4+ BM-MSCs precludes the possibility of cellular plasticity or de-differentiation of B-ALL blasts and suggests that MLL-AF4 might arise in a population of prehematopoietic precursors.

Enrichment of Human ESC‐Derived Multipotent Mesenchymal Stem Cells with Immunosuppressive and Anti‐Inflammatory Properties Capable to Protect Against Experimental Inflammatory Bowel Disease

Human ESCs provide access to the earliest stages of human development and may serve as an unlimited source of functional cells for future cell therapies. The optimization of methods directing the differentiation of human embryonic stem cells (hESCs) into tissue‐specific precursors becomes crucial. We report an efficient enrichment of mesenchymal stem cells (MSCs) from hESCs through specific inhibition of SMAD‐2/3 signaling. Human ESC‐derived MSCs (hESC‐MSCs) emerged as a population of fibroblastoid cells expressing a MSC phenotype: CD73+ CD90+ CD105+ CD44+ CD166+ CD45− CD34− CD14− CD19− human leucocyte antigen‐DR (HLA‐DR)−. After 28 days of SMAD‐2/3 inhibition, hESC cultures were enriched (>42%) in multipotent MSCs. CD73+CD90+ hESC‐MSCs were fluorescence activated cell sorting (FACS)‐isolated and long‐term cultures were established and maintained for many passages displaying a faster growth than somatic tissue‐derived MSCs while maintaining MSC morphology and phenotype. They displayed osteogenic, adipogenic, and chondrocytic differentiation potential and exhibited potent immunosuppressive and anti‐inflammatory properties in vitro and in vivo, where hESC‐MSCs were capable of protecting against an experimental model of inflammatory bowel disease. Interestingly, the efficient enrichment of hESCs into MSCs through inhibition of SMAD‐2/3 signaling was not reproducible with distinct induced pluripotent stem cell lines. Our findings provide mechanistic insights into the differentiation of hESCs into immunosuppressive and anti‐inflammatory multipotent MSCs with potential future clinical applications. STEM CELLS 2011;29:251–262

A promoter DNA demethylation landscape of human hematopoietic differentiation

Global mechanisms defining the gene expression programs specific for hematopoiesis are still not fully understood. Here, we show that promoter DNA demethylation is associated with the activation of hematopoietic-specific genes. Using genome-wide promoter methylation arrays, we identified 694 hematopoietic-specific genes repressed by promoter DNA methylation in human embryonic stem cells and whose loss of methylation in hematopoietic can be associated with gene expression. The association between promoter methylation and gene expression was studied for many hematopoietic-specific genes including CD45, CD34, CD28, CD19, the T cell receptor (TCR), the MHC class II gene HLA-DR, perforin 1 and the phosphoinositide 3-kinase (PI3K) and results indicated that DNA demethylation was not always sufficient for gene activation. Promoter demethylation occurred either early during embryonic development or later on during hematopoietic differentiation. Analysis of the genome-wide promoter methylation status of induced pluripotent stem cells (iPSCs) generated from somatic CD34+ HSPCs and differentiated derivatives from CD34+ HSPCs confirmed the role of DNA methylation in regulating the expression of genes of the hemato-immune system, and indicated that promoter methylation of these genes may be associated to stemness. Together, these data suggest that promoter DNA demethylation might play a role in the tissue/cell-specific genome-wide gene regulation within the hematopoietic compartment.

The TEL-AML1 leukemia fusion gene dysregulates the TGF-β pathway in early B lineage progenitor cells

Chromosome translocation to generate the TEL-AML1 (also known as ETV6-RUNX1) chimeric fusion gene is a frequent and early or initiating event in childhood acute lymphoblastic leukemia (ALL). Our starting hypothesis was that the TEL-AML1 protein generates and maintains preleukemic clones and that conversion to overt disease requires secondary genetic changes, possibly in the context of abnormal immune responses. Here, we show that a murine B cell progenitor cell line expressing inducible TEL-AML1 proliferates at a slower rate than parent cells but is more resistant to further inhibition of proliferation by TGF-beta. This facilitates the competitive expansion of TEL-AML1-expressing cells in the presence of TGF-beta. Further analysis indicated that TEL-AML1 binds to a principal TGF-beta signaling target, Smad3, and compromises its ability to activate target promoters. In mice expressing a TEL-AML1 transgene, early, pre-pro-B cells were increased in number and also showed reduced sensitivity to TGF-beta-mediated inhibition of proliferation. Moreover, expression of TEL-AML1 in human cord blood progenitor cells led to the expansion of a candidate preleukemic stem cell population that had an early B lineage phenotype (CD34+CD38-CD19+) and a marked growth advantage in the presence of TGF-beta. Collectively, these data suggest a plausible mechanism by which dysregulated immune responses to infection might promote the malignant evolution of TEL-AML1-expressing preleukemic clones.

Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGF-β and IGF-II

A paracrine regulation was recently proposed in human embryonic stem cells (hESCs) grown in mouse embryonic fibroblast (MEF)-conditioned media (MEF-CM), where hESCs spontaneously differentiate into autologous fibroblast-like cells to maintain culture homeostasis by producing TGF-β and insulin-like growth factor-II (IGF-II) in response to basic fibroblast growth factor (bFGF). Although the importance of TGF-β family members in the maintenance of pluripotency of hESCs is widely established, very little is known about the role of IGF-II. In order to ease hESC culture conditions and to reduce xenogenic components, we sought (i) to determine whether hESCs can be maintained stable and pluripotent using CM from human foreskin fibroblasts (HFFs) and human mesenchymal stem cells (hMSCs) rather than MEF-CM, and (ii) to analyze whether the cooperation of bFGF with TGF-β and IGF-II to maintain hESCs in MEF-CM may be extrapolated to hESCs maintained in allogeneic mesenchymal stem cell (MSC)-CM and HFF-CM. We found that MSCs and HFFs express all FGF receptors (FGFR1-4) and specifically produce TGF-β in response to bFGF. However, HFFs but not MSCs secrete IGF-II. Despite the absence of IGF-II in MSC-CM, hESC pluripotency and culture homeostasis were successfully maintained in MSC-CM for over 37 passages. Human ESCs derived on MSCs and hESCs maintained in MSC-CM retained hESC morphology, euploidy, expression of surface markers and transcription factors linked to pluripotency and displayed in vitro and in vivo multilineage developmental potential, suggesting that IGF-II may be dispensable for hESC pluripotency. In fact, IGF-II blocking had no effect on the homeostasis of hESC cultures maintained either on HFF-CM or on MSC-CM. These data indicate that hESCs are successfully maintained feeder-free with IGF-II-lacking MSC-CM, and that the previously proposed paracrine mechanism by which bFGF cooperates with TGF-β and IGF-II in the maintenance of hESCs in MEF-CM may not be fully extrapolated to hESCs maintained in CM from human MSCs.

Cancer Genes Hypermethylated in Human Embryonic Stem Cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.

Human embryonic stem cells

The derivation of human embryonic stem cells (hESCs), whose in vitro differentiation might be directed toward different cell types, has raised the hope for cell replacement therapies. Despite the emerging reports to differentiate hESCs into specific lineages and then to distinct mature cell subsets, there are still several issues that need to be resolved before transplantation of these cells can be realized. In this context, immune rejection by the host immune system has been considered to be one of the greatest hurdles for cellular transplantation. However, recent data support the concept that hESCs and/or their differentiated derivatives possess immune-privileged properties, suggesting that cells derived from hESC may provide a potential tool for induction of immunetolerance. Currently, our understanding of the tolerogenic potential of hESCs is limited to assessment by in vitro assays or xenogenic transplantation approaches in vivo. Human ESCs express low levels of major histocompatability complex (MHC)-I antigens and lack expression of MHC-II antigens and costimulatory molecules, and are not recognized by natural killer cells and inhibit T-cell induced-stimulation by third-party antigen-presenting cells. Upon injection into immunocompetent mice, hESCs are unable to induce an immune response as demonstrated by their inability to induce an inflammatory response. Based on these initial observations, further studies in hESCs immunobiology are warranted and may reveal unique mechanisms that account for the immunological properties of hESCs. Here, we explore the prospect of using hESCs and their derivatives for immunomodulation and tolerance induction.

NF-κB activation impairs somatic cell reprogramming in ageing.

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor–Guillermo progeria syndrome and Hutchinson–Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.