Pedro J. Real

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.

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

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.

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.

The Notch ligand DLL4 specifically marks human hematoendothelial progenitors and regulates their hematopoietic fate

Notch signaling is essential for definitive hematopoiesis, but its role in human embryonic hematopoiesis is largely unknown. We show that in hESCs the expression of the Notch ligand DLL4 is induced during hematopoietic differentiation. We found that DLL4 is only expressed in a sub-population of bipotent hematoendothelial progenitors (HEPs) and segregates their hematopoietic versus endothelial potential. We demonstrate at the clonal level and through transcriptome analyses that DLL4high HEPs are enriched in endothelial potential, whereas DLL4low/– HEPs are committed to the hematopoietic lineage, albeit both populations still contain bipotent cells. Moreover, DLL4 stimulation enhances hematopoietic differentiation of HEPs and increases the amount of clonogenic hematopoietic progenitors. Confocal microscopy analysis of whole differentiating embryoid bodies revealed that DLL4high HEPs are located close to DLL4low/– HEPs, and at the base of clusters of CD45+ cells, resembling intra-aortic hematopoietic clusters found in mouse embryos. We propose a model for human embryonic hematopoiesis in which DLL4low/– cells within hemogenic endothelium receive Notch-activating signals from DLL4high cells, resulting in an endothelial-to-hematopoietic transition and their differentiation into CD45+ hematopoietic cells.

Maintenance of Human Embryonic Stem Cells in Mesenchymal Stem Cell-Conditioned Media Augments Hematopoietic Specification

The realization of human embryonic stem cells (hESC) as a model for human developmental hematopoiesis and in potential cell replacement strategies relies on an improved understanding of the extrinsic and intrinsic factors regulating hematopoietic-specific hESC differentiation. Human mesenchymal stem cells (hMSCs) are multipotent cells of mesodermal origin that form a part of hematopoietic stem cell niches and have an important role in the regulation of hematopoiesis through production of secreted factors and/or cell-to-cell interactions. We have previously shown that hESCs may be successfully maintained feeder free using hMSC-conditioned media (MSC-CM). Here, we hypothesized that hESCs maintained in MSC-CM may be more prone to differentiation toward hematopoietic lineage than hESCs grown in standard human foreskin fibroblast-conditioned media. We report that specification into hemogenic progenitors and subsequent hematopoietic differentiation and clonogenic progenitor capacity is robustly enhanced in hESC lines maintained in MSC-CM. Interestingly, co-culture of hESCs on hMSCs fully abrogates hematopoietic specification of hESCs, thus suggesting that the improved hematopoietic differentiation is mediated by MSC-secreted factors rather than by MSC-hESC physical interactions. To investigate the molecular mechanism involved in this process, we analyzed global (LINE-1) methylation and genome-wide promoter DNA methylation. hESCs grown in MSC-CM showed a decrease of 17% in global DNA methylation and a promoter DNA methylation signature consisting of 45 genes commonly hypomethylated and 102 genes frequently hypermethylated. Our data indicate that maintenance of hESCs in MSC-CM robustly augments hematopoietic specification and that the process seems mediated by MSC-secreted factors conferring a DNA methylation signature to undifferentiated hESCs which may influence further predisposition toward hematopoietic specification.

FLT3 activation cooperates with MLL-AF4 fusion protein to abrogate the hematopoietic specification of human ESCs

Mixed-lineage leukemia (MLL)-AF4 fusion arises prenatally in high-risk infant acute pro-B-lymphoblastic leukemia (pro-B-ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hematoendothelial specification but was insufficient for transformation, suggesting that additional oncogenic insults seem required for MLL-AF4-mediated transformation. MLL-AF4+ pro-B-ALL expresses enormous levels of FLT3, occasionally because of activating mutations, thus representing a candidate cooperating event in MLL-AF4+ pro-B-ALL. Here, we explored the developmental impact of FLT3 activation alone, or together with MLL-AF4, in the hematopoietic fate of hESCs. FLT3 activation does not affect specification of hemogenic precursors but significantly enhances the formation of CD45(+) blood cells, and CD45(+)CD34(+) blood progenitors with clonogenic potential. However, overexpression of FLT3 mutations or wild-type FLT3 (FLT3-WT) completely abrogates hematopoietic differentiation from MLL-AF4-expressing hESCs, indicating that FLT3 activation cooperates with MLL-AF4 to inhibit human embryonic hematopoiesis. Cell cycle/apoptosis analyses suggest that FLT3 activation directly affects hESC specification rather than proliferation or survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling of hESC-derived CD45(+) cells supports the FLT3-mediated inhibition of hematopoiesis in MLL-AF4-expressing hESCs, which is associated with large transcriptional changes and downregulation of genes involved in hematopoietic system development and function. Importantly, FLT3 activation does not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells, suggesting the need of alternative (epi)-genetic cooperating hits.

Large-scale transcriptional profiling and functional assays reveal important roles for Rho-GTPase signalling and SCL during haematopoietic differentiation of human embryonic stem cells

Understanding the transcriptional cues that direct differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells to defined and functional cell types is essential for future clinical applications. In this study, we have compared transcriptional profiles of haematopoietic progenitors derived from hESCs at various developmental stages of a feeder- and serum-free differentiation method and show that the largest transcriptional changes occur during the first 4 days of differentiation. Data mining on the basis of molecular function revealed Rho-GTPase signalling as a key regulator of differentiation. Inhibition of this pathway resulted in a significant reduction in the numbers of emerging haematopoietic progenitors throughout the differentiation window, thereby uncovering a previously unappreciated role for Rho-GTPase signalling during human haematopoietic development. Our analysis indicated that SCL was the 11th most upregulated transcript during the first 4 days of the hESC differentiation process. Overexpression of SCL in hESCs promoted differentiation to meso-endodermal lineages, the emergence of haematopoietic and erythro-megakaryocytic progenitors and accelerated erythroid differentiation. Importantly, intrasplenic transplantation of SCL-overexpressing hESC-derived haematopoietic cells enhanced recovery from induced acute anaemia without significant cell engraftment, suggesting a paracrine-mediated effect.