Using Pluripotent Stem Cells to Understand Normal and Leukemic Hematopoietic Development.

Several decades have passed since the generation of the first embryonic stem cell (ESC) lines both in mice and in humans. Since then, stem cell biologists have tried to understand their potential biological and clinical uses for their implementation in regenerative medicine. The hematopoietic field was a pioneer in establishing the potential use for the development of blood cell products and clinical applications; however, early expectations have been truncated by the difficulty in generating bonafide hematopoietic stem cells (HSCs). Despite some progress in understanding the origin of HSCs during embryonic development, the reproduction of this process in vitro is still not possible, but the knowledge acquired in the embryo is slowly being implemented for mouse and human pluripotent stem cells (PSCs). In contrast, ESC-derived hematopoietic cells may recapitulate some leukemic transformation processes when exposed to oncogenic drivers. This would be especially useful to model prenatal leukemia development or other leukemia-predisposing syndromes, which are difficult to study. In this review, we will review the state of the art of the use of PSCs as a model for hematopoietic and leukemia development.

Generation of heterozygous SAMD9 CRISPR/Cas9-edited iPSC line (ESi086-A-3), carrying p.I1567M mutation.

Germline SAMD9 mutations are one of the most common alterations that predispose to pediatric myelodysplastic syndrome (MDS), a clonal disorder characterized by ineffective hematopoiesis, increasing the risk of developing acute myeloid leukemia (AML). Up to date, a disease model to study the role of SAMD9 mutation in MDS is still lacking. Here, we have generated a human induced pluripotent stem cell (hiPSC) line carrying SAMD9mut (p.I1567M), taking advantage of CRISPR/Cas9 system. As a result, the genetic engineered hiPSC line represent a new in vitro disease model to understand the impact of SAMD9 mutation at molecular and cellular level during hematopoiesis.

GATA2 deficiency and MDS/AML: Experimental strategies for disease modelling and future therapeutic prospects.

The importance of predisposition to leukaemia in clinical practice is being increasingly recognized. This is emphasized by the establishment of a novel WHO disease category in 2016 called "myeloid neoplasms with germline predisposition". A major syndrome within this group is GATA2 deficiency, a heterogeneous immunodeficiency syndrome with a very high lifetime risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). GATA2 deficiency has been identified as the most common hereditary cause of MDS in adolescents with monosomy 7. Allogenic haematopoietic stem cell transplantation is the only curative option; however, chances of survival decrease with progression of immunodeficiency and MDS evolution. Penetrance and expressivity within families carrying GATA2 mutations is often variable, suggesting that co-operating extrinsic events are required to trigger the disease. Predictive tools are lacking, and intrafamilial heterogeneity is poorly understood; hence there is a clear unmet medical need. On behalf of the ERAPerMed GATA2 HuMo consortium, in this review we describe the genetic, clinical, and biological aspects of familial GATA2-related MDS, highlighting the importance of developing robust disease preclinical models to improve early detection and clinical decision-making of GATA2 carriers.

Data quality and FAIR principles applied to marine litter data in Europe.

High quality and integrated information able to show marine litter distribution at a global scale is a crucial goal to tackle the environmental problem. One of the important gaps is the definition of a global monitoring protocol and reporting. Large data infrastructures can provide a sustainable framework to host harmonized and standard litter data that can be used and re-used for any purpose, including assessment. EMODnet Chemistry has collected marine litter data since 2016 and has adopted different strategies for the management of the diverse litter data types, exploiting the advantages deriving from the application of the FAIR principles in marine litter data stewardship. The quality of the released data sets is improved allowing a better consistency within data values collected in different contexts (several countries, different techniques, …).

Generation of two heterozygous GATA2 CRISPR/Cas9-edited iPSC lines, R398W and R396Q, for modeling GATA2 deficiency.

Germline heterozygous GATA2 mutations underlie a complex disorder characterized by bone marrow failure, immunodeficiency and high risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our understanding about GATA2 deficiency is limited due to the lack of relevant disease models. Here we generated high quality human induced pluripotent stem cell (iPSC) lines carrying two of the most recurrent germline GATA2 mutations (R389W and R396Q) associated with MDS, using CRISPR/Cas9. These hiPSCs represent an in vitro model to study the molecular and cellular mechanisms underlying GATA2 deficiency, when differentiated into blood progenitors.

Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer.

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas.

Retrieval of germinal zone neural stem cells from the cerebrospinal fluid of premature infants with intraventricular hemorrhage.

Intraventricular hemorrhage is a common cause of morbidity and mortality in premature infants. The rupture of the germinal zone into the ventricles entails loss of neural stem cells and disturbs the normal cytoarchitecture of the region, compromising late neurogliogenesis. Here we demonstrate that neural stem cells can be easily and robustly isolated from the hemorrhagic cerebrospinal fluid obtained during therapeutic neuroendoscopic lavage in preterm infants with severe intraventricular hemorrhage. Our analyses demonstrate that these neural stem cells, although similar to human fetal cell lines, display distinctive hallmarks related to their regional and developmental origin in the germinal zone of the ventral forebrain, the ganglionic eminences that give rise to interneurons and oligodendrocytes. These cells can be expanded, cryopreserved, and differentiated in vitro and in vivo in the brain of nude mice and show no sign of tumoral transformation 6 months after transplantation. This novel class of neural stem cells poses no ethical concerns, as the fluid is usually discarded, and could be useful for the development of an autologous therapy for preterm infants, aiming to restore late neurogliogenesis and attenuate neurocognitive deficits. Furthermore, these cells represent a valuable tool for the study of the final stages of human brain development and germinal zone biology.

GATA2 Promotes Hematopoietic Development and Represses Cardiac Differentiation of Human Mesoderm.

In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates.

Generation of two transgene-free human iPSC lines from CD133+ cord blood cells.

We have generated two human induced pluripotent stem cell (iPSC) lines from CD133+ cells isolated from umbilical cord blood (CB) of a female child using non-integrative Sendai virus. Here we describe the complete characterization of these iPSC lines: PRYDi-CB5 and PRYDi-CB40.

Genetic Rescue of Mitochondrial and Skeletal Muscle Impairment in an Induced Pluripotent Stem Cells Model of Coenzyme Q10 Deficiency.

Coenzyme Q10 (CoQ10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ10 ], CoQ10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4ed -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4ed -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687-1703.

Induced Pluripotency and Gene Editing in Fanconi Anemia.

Induced pluripotent stem cells (iPSCs) represent an invaluable tool in a chromosomal instability syndrome such as Fanconi anemia (FA), as they can allow to study of the molecular defects underlying this disease. Many other applications, such as its use as a platform to test different methods or compounds, could also be of interest. But the greatest impact of iPSCs may be in bone marrow failure diseases, as iPSCs could represent an unlimited source of autologous cells to apply in advanced treatments such as gene therapy. At the same time, genome editing constitutes the next generation of technology to further develop a safer, personalized, targeted gene therapy. Despite the promising advantages that these two technologies would present in a disease such as FA, the specific characteristics of the disease make both of these processes especially challenging. Efficient and safer FA-hiPSC (human induced pluripotent stem cell) generation methods, robust and reliable differentiation protocols for iPSCs, as well as really efficient delivery methods to perform targeted gene correction should be developed.

Proinflammatory signals are insufficient to drive definitive hematopoietic specification of human HSCs in vitro.

Recent studies in zebrafish and mice have revealed that proinflammatory signaling is a positive regulator of definitive hematopoietic development. Whether proinflammatory signaling also regulates human hematopoietic specification remains unknown. Here, we explored the impact of the proinflammatory cytokines tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), and interleukin-1ß (IL1ß) on in vitro hematopoietic differentiation using human pluripotent stem cells. Gene expression analysis and enzyme-linked immunosorbent assay revealed the absence of a proinflammatory signature during hematopoietic development of human pluripotent stem cells. Functionally, the emergence of hemogenic endothelial progenitors (CD31+CD34+CD45- or CD34+CD43-CD73-) and hematopoietic cells (CD43+CD45+) was not affected by treatment with increasing doses of TNFα, IFNγ, and IL1ß irrespective of the developmental window or the differentiation protocol used (embryoid body or OP9 co-culture based). Similarly, knockdown of endogenous NF-kB signaling had no impact on hematopoietic differentiation of human pluripotent stem cells. This study serves as a demonstration that TNFα, IFNγ, and IL1ß signals do not improve hematopoietic differentiation of human pluripotent stem cells using current protocols and suggests that proinflammatory signaling is insufficient to drive definitive hematopoietic specification of human hematopoietic stem cells in vitro.

Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency.

Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming "boosters" also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

Fast and Efficient Neural Conversion of Human Hematopoietic Cells.

A major challenge in regenerative medicine is the generation of functionally effective target cells to replace or repair damaged tissues. The finding that most somatic cells can be directly converted into cells of another lineage by the expression of specific transcription factors has paved the way to novel applications. Induced neurons (iNs) represent an alternative source of neurons for disease modeling, drug screening, and potentially, for cell replacement therapy. This unit describes methods for the efficient conversion of blood cells into iNs, including protocols to isolate cord blood CD133+ cells, infect them with Sendai virus vectors that express SOX2 and c-MYC, and differentiate the infected cells (PB-MNCs) into mature neurons. A method to reprogram peripheral blood mononuclear cells into iNs is also described. Support protocols describe how to culture rat astrocytes and characterize the electrophysiology of iNs. © 2016 by John Wiley & Sons, Inc.

Fast and efficient neural conversion of human hematopoietic cells.

Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency.

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.

Analysis of protein-coding mutations in hiPSCs and their possible role during somatic cell reprogramming.

Recent studies indicate that human-induced pluripotent stem cells contain genomic structural variations and point mutations in coding regions. However, these studies have focused on fibroblast-derived human induced pluripotent stem cells, and it is currently unknown whether the use of alternative somatic cell sources with varying reprogramming efficiencies would result in different levels of genetic alterations. Here we characterize the genomic integrity of eight human induced pluripotent stem cell lines derived from five different non-fibroblast somatic cell types. We show that protein-coding mutations are a general feature of the human induced pluripotent stem cell state and are independent of somatic cell source. Furthermore, we analyse a total of 17 point mutations found in human induced pluripotent stem cells and demonstrate that they do not generally facilitate the acquisition of pluripotency and thus are not likely to provide a selective advantage for reprogramming.