Verónica Ramos-Mejía

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.

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.

iPSC lines that do not silence the expression of the ectopic reprogramming factors may display enhanced propensity to genomic instability

Here, we provide data suggesting that the absence of silencing of the ectopic reprogramming factors used to reprogram somatic cells to induced pluripotent stem cells (iPSCs) may predispose iPSCs to genomic instability. We encourage stem cell scientists to undertake an extensive characterization and standardization of much larger cohorts of iPSC lines in order to set up rigorous criteria to define safe and stable bona fide iPSCs.

iPSCs from cancer cells: challenges and opportunities

Reprogramming and oncogenic transformation are stepwise processes that share many similarities, and induced pluripotent stem cells (iPSCs) generated from cancer cells could illuminate molecular mechanisms underlying the pathogenesis of human cancer. Deciphering the barriers underlying the reprogramming process of primary cancer cells could reveal information on the links between pluripotency and oncogenic transformation that would be instrumental for therapy development.

Nodal/Activin Signaling Predicts Human Pluripotent Stem Cell Lines Prone to Differentiate Toward the Hematopoietic Lineage

Lineage-specific differentiation potential varies among different human pluripotent stem cell (hPSC) lines, becoming therefore highly desirable to prospectively know which hPSC lines exhibit the highest differentiation potential for a certain lineage. We have compared the hematopoietic potential of 14 human embryonic stem cell (hESC)/induced pluripotent stem cell (iPSC) lines. The emergence of hemogenic progenitors, primitive and mature blood cells, and colony-forming unit (CFU) potential was analyzed at different time points. Significant differences in the propensity to differentiate toward blood were observed among hPSCs: some hPSCs exhibited good blood differentiation potential, whereas others barely displayed blood-differentiation capacity. Correlation studies revealed that the CFU potential robustly correlates with hemogenic progenitors and primitive but not mature blood cells. Developmental progression of mesoendodermal and hematopoietic transcription factors expression revealed no correlation with either hematopoietic initiation or maturation efficiency. Microarray studies showed distinct gene expression profile between hPSCs with good versus poor hematopoietic potential. Although neuroectoderm-associated genes were downregulated in hPSCs prone to hematopoietic differentiation many members of the Nodal/Activin signaling were upregulated, suggesting that this signaling predicts those hPSC lines with good blood-differentiation potential. The association between Nodal/Activin signaling and the hematopoietic differentiation potential was confirmed using loss- and gain-of-function functional assays. Our data reinforce the value of prospective comparative studies aimed at determining the lineage-specific differentiation potential among different hPSCs and indicate that Nodal/Activin signaling seems to predict those hPSC lines prone to hematopoietic specification.

Residual Expression of the Reprogramming Factors Prevents Differentiation of iPSC Generated from Human Fibroblasts and Cord Blood CD34+ Progenitors

Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation.

SCL/TAL1 Regulates Hematopoietic Specification From Human Embryonic Stem Cells

Determining the molecular regulators/pathways responsible for the specification of human embryonic stem cells (hESCs) into hematopoietic precursors has far-reaching implications for potential cell therapies and disease modeling. Mouse models lacking SCL/TAL1 (stem cell leukemia/T-cell acute lymphocytic leukemia 1) do not survive beyond early embryogenesis because of complete absence of hematopoiesis, indicating that SCL is a master early hematopoietic regulator. SCL is commonly found rearranged in human leukemias. However, there is barely information on the role of SCL on human embryonic hematopoietic development. Differentiation and sorting assays show that endogenous SCL expression parallels hematopoietic specification of hESCs and that SCL is specifically expressed in hematoendothelial progenitors (CD45(-)CD31(+)CD34(+)) and, to a lesser extent, on CD45(+) hematopoietic cells. Enforced expression of SCL in hESCs accelerates the emergence of hematoendothelial progenitors and robustly promotes subsequent differentiation into primitive (CD34(+)CD45(+)) and total (CD45(+)) blood cells with higher clonogenic potential. Short-hairpin RNA-based silencing of endogenous SCL abrogates hematopoietic specification of hESCs, confirming the early hematopoiesis-promoting effect of SCL. Unfortunately, SCL expression on its own is not sufficient to confer in vivo engraftment to hESC-derived hematopoietic cells, suggesting that additional yet undefined master regulators are required to orchestrate the stepwise hematopoietic developmental process leading to the generation of definitive in vivo functional hematopoiesis from hESCs.

Concise Review: Generation of Neurons From Somatic Cells of Healthy Individuals and Neurological Patients Through Induced Pluripotency or Direct Conversion

Access to healthy or diseased human neural tissue is a daunting task and represents a barrier for advancing our understanding about the cellular, genetic, and molecular mechanisms underlying neurogenesis and neurodegeneration. Reprogramming of somatic cells to pluripotency by transient expression of transcription factors was achieved a few years ago. Induced pluripotent stem cells (iPSC) from both healthy individuals and patients suffering from debilitating, life‐threatening neurological diseases have been differentiated into several specific neuronal subtypes. An alternative emerging approach is the direct conversion of somatic cells (i.e., fibroblasts, blood cells, or glial cells) into neuron‐like cells. However, to what extent neuronal direct conversion of diseased somatic cells can be achieved remains an open question. Optimization of current expansion and differentiation approaches is highly demanded to increase the differentiation efficiency of specific phenotypes of functional neurons from iPSCs or through somatic cell direct conversion. The realization of the full potential of iPSCs relies on the ability to precisely modify specific genome sequences. Genome editing technologies including zinc finger nucleases, transcription activator‐like effector nucleases, and clustered regularly interspaced short palindromic repeat/CAS9 RNA‐guided nucleases have progressed very fast over the last years. The combination of genome‐editing strategies and patient‐specific iPSC biology will offer a unique platform for in vitro generation of diseased and corrected neural derivatives for personalized therapies, disease modeling and drug screening. Stem Cells 2014;32:2811–2817

A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification.

The MLL-AF4 fusion gene is a hallmark genomic aberration in high-risk acute lymphoblastic leukemia in infants. Although it is well established that MLL-AF4 arises prenatally during human development, its effects on hematopoietic development in utero remain unexplored. We have created a human-specific cellular system to study early hemato-endothelial development in MLL-AF4-expressing human embryonic stem cells (hESCs). Functional studies, clonal analysis and gene expression profiling reveal that expression of MLL-AF4 in hESCs has a phenotypic, functional and gene expression impact. MLL-AF4 acts as a global transcriptional activator and a positive regulator of homeobox gene expression in hESCs. Functionally, MLL-AF4 enhances the specification of hemogenic precursors from hESCs but strongly impairs further hematopoietic commitment in favor of an endothelial cell fate. MLL-AF4 hESCs are transcriptionally primed to differentiate towards hemogenic precursors prone to endothelial maturation, as reflected by the marked upregulation of master genes associated to vascular-endothelial functions and early hematopoiesis. Furthermore, we report that MLL-AF4 expression is not sufficient to transform hESC-derived hematopoietic cells. This work illustrates how hESCs may provide unique insights into human development and further our understanding of how leukemic fusion genes, known to arise prenatally, regulate human embryonic hematopoietic specification.

Phenotypic analyses of mouse embryos with ubiquitous expression of Oct4: Effects on mid–hindbrain patterning and gene expression

Oct4 is a transcription factor that has been associated with pluripotency and fate determination in the initial cell lineages of mammals. On the other hand, Pou2, the ortholog of Oct4 in zebrafish, serves additional later functions during brain development acting as a differentiation switch. In mice, Oct4 is expressed throughout the neural plate of embryos until embryonic day (E) 8.0. In this study, we produced transgenic mouse embryos that ubiquitously express Oct4 and analyzed the consequences during development. We show that, at E8.0, a higher dosage of Oct4 in the neuroectoderm is sufficient to transiently alter mid–hindbrain patterning and produced a strong up‐regulation of Pax2, indicating that Oct4 can regulate this gene in vivo. After E9.5, ectopic Oct4 in this region produced cell death and affected the development of the forebrain, suggesting that, at these later stages, Oct4 down‐regulation is necessary for normal development to proceed. The phenotype of the transgenic embryos was also accompanied with an increase of Fgf8 expression in several of its endogenous domains, suggesting the possibility that Oct4 can participate in the regulation of expression of this ligand. Our observations support the hypothesis that Oct4, like zebrafish Pou2, has a conserved function during early brain patterning in mouse. Developmental Dynamics 232:180–190, 2005.