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Hematopoietic stem cells (HSC) represent a rare cell population of particular interest for biomedical research and regenerative medicine. Various marker combinations enable the isolation of HSCs bu...

Retroviral insertional mutagenesis (RIM) is both a relevant risk in gene therapy and a powerful tool for identifying genes that enhance the competitiveness of repopulating hematopoietic stem and pr...

Summary Mesenchymal stromal cells (MSCs) are used in many clinical applications. However, ex vivo expansion is required to reach clinically relevant cell numbers, which might lead to selection of clones with different characteristics. To follow clonal selection, we transduced MSC progenitors in umbilical cord pieces (UCPs) with vectors encoding fluorescent proteins and genetic barcodes. After marked MSC cultures grew out from UCPs, we investigated the influence of cytokines on MSC functionality. Specific cytokine conditions selected for clones from common progenitors. MSC secretome analyses revealed differences dependent on the culture conditions used. Clones expanded in human serum containing culture medium secreted a plethora of growth factors. When expanded in the same medium containing TGF-β, MSCs secreted negligible amounts of cytokines but at the same time led to an increased human chimerism after hematopoietic stem cell transplantation into immunodeficient mice. Our results suggest a major influence of cytokine additives on MSC functionality., Graphical Abstract, Highlights • Cytokine supplementation selects for common MSC culture-initiating cells • TGFB1 markedly decreases the cytokine production of MSCs • Medium of TGF-β-selected MSC supports HSC engraftment, Rothe and colleagues transduced mesenchymal stromal cell initiating cells (MSC-I) in the umbilical cord pieces with lentiviral barcode vectors and observed a cytokine-dependent selection of MSC clones with common MSC-I. TGFB1 drastically decreased the cytokine production of selected MSCs, whereas the conditioned medium from these cultures enhanced the engraftment of human hematopoietic stem cells.

Regulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The vector design and drug-dependent selection approach identified new retroviral insertion (RVI) sites that potentially collaborate with Meis1/Hoxa9 and define H9M-ciMP fate. For most cell lines, myelomonocytic conditions supported reversible H9M-ciMP differentiation into neutrophils and macrophages with DOX-dependent modulation of Hoxa9/Meis1 and CD11b/Gr-1 expression. Here, up-regulation of Meis1/Hoxa9 promoted reconstitution of exponential expansion of immature H9M-ciMPs after DOX reapplication. Stem cell maintaining conditions supported selective H9M-ciMP exponential growth. H9M-ciMPs that had Ninj2 RVI and were cultured under myelomonocytic or stem cell maintaining conditions revealed the development of DOX-dependent acute myeloid leukaemia in a murine transplantation model. Transcriptional dysregulation of Ninj2 and distal genes surrounding RVI (Rad52, Kdm5a) was detected. All studied H9M-ciMPs demonstrated adaptation to T-lymphoid microenvironmental conditions while maintaining immature myelomonocytic features. Thus, the established system is relevant to leukaemia and stem cell biology.

Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro. Here we demonstrate that genotoxic vectors induce a unique gene expression signature linked to stemness and oncogenesis in transduced murine hematopoietic stem and progenitor cells. Based on this finding, we developed the surrogate assay for genotoxicity assessment (SAGA). SAGA classifies integrating retroviral vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential, SAGA achieved an accuracy of 90.9%. SAGA is more robust and sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemic severe adverse events in clinical trials. Our work provides a fast and robust tool for preclinical risk assessment of gene therapy vectors, potentially paving the way for safer gene therapy trials. ispartof: Molecular Therapy vol:29 issue:12 pages:3383-3397 ispartof: location:United States status: published

Retroviral insertional mutagenesis (RIM) is both a relevant risk in gene therapy and a powerful tool for identifying genes that enhance the competitiveness of repopulating hematopoietic stem and progenitor cells (HSPCs). However, focusing only on the gene closest to the retroviral vector insertion site (RVIS) may underestimate the effects of RIM, as dysregulation of distal and/or multiple genes by a single insertion event was reported in several studies. As a proof of concept, we examined the common insertion site (CIS)

Summary Induced pluripotent stem cells (iPSCs) offer a promising platform to model early embryonic developmental processes, to create disease models that can be evaluated by drug screens as well as proof-of-concept experiments for regenerative medicine. However, generation of iPSC-derived hemato-endothelial and hematopoietic progenitor cells for these applications is challenging due to variable and limited cell numbers, which necessitates enormous up-scaling or development of demanding protocols. Here, we unravel the function of key transcriptional regulators SCL, LMO2, GATA2, and ETV2 (SLGE) on early hemato-endothelial specification and establish a fully inducible and stepwise hemato-endothelial forward programming system based on SLGE-regulated overexpression. Regulated induction of SLGE in stable SLGE-iPSC lines drives very efficient generation of large numbers of hemato-endothelial progenitor cells (CD144+/CD73–), which produce hematopoietic progenitor cells (CD45+/CD34+/CD38–/CD45RA−/CD90+/CD49f+) through a gradual process of endothelial-to-hematopoietic transition (EHT)., Highlights • Inducible and robust hemato-endothelial forward programming of human iPSCs • Efficient, scalable generation of hemato-endothelial progenitor cells • Production of HPCs with HSC-like immunophenotype and multi-lineage potential • Whole transcriptome screen for potential regulators of definitive hematopoiesis, Lange, Schambach, and colleagues established an inducible hemato-endothelial forward programming protocol, utilizing the combinatorial and timely regulated overexpression of hematopoietic master regulators SCL, LMO2, GATA2, and ETV2 (SLGE). Regulated induction of this combination in SLGE-iPSCs generated large numbers of hemato-endothelial progenitor cells (CD144+/CD73–) capable of producing hematopoietic progenitor cells (CD45+/CD34+) with multi-lineage potential. Furthermore, transcriptome analysis identified potential regulators of definitive hematopoiesis.

Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings. Piezo2 is the main mechanotransducer found in LTMRs. Recent evidence shows that Piezo1, the other mechanotransducer considered absent in dorsal root ganglia (DRG), is expressed at low level in somatosensory neurons. We established a differentiation protocol to generate, from iPSC-derived neuronal precursor cells, human LTMR recapitulating bulbous sensory nerve endings and heterogeneous expression of Piezo1 and Piezo2. The derived neurons express LTMR-specific genes, convert mechanical stimuli into electrical signals and have specialized axon termini that morphologically resemble bulbous nerve endings. Piezo2 is concentrated within these enlarged axon termini. Some derived neurons express low level Piezo1, and a subset co-express both channels. Thus, we generated a unique, iPSCs-derived human model that can be used to investigate the physiology of bulbous sensory nerve endings, and the role of Piezo1 and 2 during mechanosensation., This work was supported by N-RENNT of the Ministry of Science and Culture of Lower Saxony to A.V.B., by a Marie Curie Career Integration Grant to A.V.B. (FP7-PEOPLE-2013-CIG, project number 631792, acronym INMA), by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2155 “RESIST” – Project ID 39087428, by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - SFB-900 – 158989968 to R.F. (B1) and A.V.B. (B9), and by a Spanish Government project to A.G. (PID2019-108194RB-I00).

Site specific recombinases are frequently used as gene switches in transgenic animals where recombination is induced by drug treatment or by tissue specific recombinase expression. Alternatively, lentiviral gene transfer can be utilized for the genetic modification of a wide variety of cell types, albeit systems for tight control of transcriptional activity are scarce. Here, we combined lentiviral gene transfer and the development of a tightly drug-controlled FLP recombinase for the construction of “All-in-One” inducible gene expression systems. Tight control of FLP activity was achieved through N-terminal fusion with a FKBP12-derived conditional destruction domain and a C-terminal estrogen receptor binding domain making recombination dependent on the presence of Shield–1 and 4-hydroxytamoxifen. Exploiting the capacity of FLP to mediate excision and inversion, “All-in-One” lentiviral gene switch vector systems were generated where drug-induced recombination resulted in abrogation of FLP expression and subsequent overexpression or knockdown of the prototypical tumor suppressor phosphatase and tensin homolog PTEN. “All-in-One” vectors proved their functionality in a variety of hematopoietic cell lines, and primary murine bone marrow cells. Our new vector system thus combines the ease of lentiviral gene transfer and the power of site specific recombinases for analysis of gene function.

Tetracycline-regulated systems with efficient temporal and dose regulation of transgene expression are useful for development of new physiologic/pathophysiologic experimental models and gene therapy approaches. Lentiviral vectors with improved tetracycline-regulated promoters help to overcome the existing limitations such as basal activity in the drug absence, poor inducibility or unstable transgene expression. To compare conventional and improved tetracycline-regulated promoters in lentiviral based vectors in vivo, we investigated doxycycline-regulated gene transfer/expression levels in a long-term murine transplantation model and demonstrated that the lentiviral vector with the improved T11 promoter exhibited more efficient inducibility and higher gene transfer level. The time required to reverse transgene expression after doxycycline removal was increased for animals with higher gene expression levels and vector copy numbers. Examination of peripheral blood leukocytes and splenocytes revealed similar cell lineage distributions for transgene positive and negative cell populations from experimental and control mice, but increased variability in the percentages of myeloid and lymphoid cells was detected in transgene positive bone marrow cells. However, no indication of lineage bias in total bone marrow cells and no signs of hematopoietic disease were observed seven months after transplantation. Our results showed that the T11 tetracycline-regulated promoter enabled improved transgene expression in a murine transplantation model. The established system allows further development of tetracycline-regulated experimental models to investigate normal and malignant hematopoiesis.

The transcription factor Oct4 (Pou5f1) is a critical regulator of pluripotency in embryonic and induced pluripotent stem cells. Therefore, Oct4 expression might identify somatic stem cell populations with inherent multipotent potential or a propensity for facilitated reprogramming. However, analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms. Systematic analysis of a transgenic Oct4-EGFP reporter mouse identified testis and skin as two principle sources of Oct4+ cells in postnatal mice. While the prevalence of GFP+ cells in testis rapidly declined with age, the skin-resident GFP+ population expanded in a cyclical fashion. These cells were identified as epidermal stem cells dwelling in the stem cell niche of the hair follicle, which endogenously expressed all principle reprogramming factors at low levels. Interestingly, skin wounding or non-traumatic hair removal robustly expanded the GFP+ epidermal cell pool not only locally, but also in uninjured skin areas, demonstrating the existence of a systemic response. Thus, the epithelial stem cell niche of the hair follicle harbors an expandable pool of Oct4+ stem cells, which might be useful for therapeutic cell transfer or facilitated reprogramming. Electronic supplementary material The online version of this article (doi: 10.1186/2045-9769-3-9) contains supplementary material, which is available to authorized users.

Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells and provide the opportunity for time- and dose-controlled reversible transgene expression in hematopoietic stem cells in vitro and in vivo. However, the background activity of tetracycline-regulated promoters (tetPs) in the absence of induction or vector integration in the vicinity of proto-oncogenes can diminish the advantages of the system. Here we investigated the effect of lentiviral transduction rate on tetP background activity, vector copy number (VCN), and clonal variability as a consequence of vector integration. We found an exponential relationship between VCN and gene transfer/expression level, accompanied by a linear relationship between VCN and tetP background activity. Long-term murine transplantation studies demonstrated stable and reversible transgene expression in serial recipients. Although analysis of associated clonal composition revealed development of clonal dominance in the presence and absence of induction, no indications of disease presented during the observation period. The majority of tetracycline-regulated vector integration sites were identified in intron/exons of metabolic/housekeeping and signaling genes or in noncoding/repeat regions of the genome. Furthermore, we demonstrated that the nature of the selected transgene might affect tetP background activity and inducibility in vivo. Limiting tetP-regulated gene transfer may avoid generation of clones with high VCN and enhanced tetP background activity. Our data help to establish physiologic and pathophysiologic systems to study dose-dependent mechanisms triggered by different levels of transgene expression in the context of basic HSC biology and cellular transformation models.

The transcription factor Evi1 has an outstanding role in the formation and transformation of hematopoietic cells. Its activation by chromosomal rearrangement induces a myelodysplastic syndrome with progression to acute myeloid leukemia of poor prognosis. Similarly, retroviral insertion-mediated upregulation confers a competitive advantage to transplanted hematopoietic cells, triggering clonal dominance or even leukemia. To study the molecular and functional response of primary murine hematopoietic progenitor cells to the activation of Evi1, we established an inducible lentiviral expression system. EVI1 had a biphasic effect with initial growth inhibition and retarded myeloid differentiation linked to enhanced survival of myeloblasts in long-term cultures. Gene expression microarray analysis revealed that within 24 h EVI1 upregulated 'stemness' genes characteristic for long-term hematopoietic stem cells (Aldh1a1, Abca1, Cdkn1b, Cdkn1c, Epcam, among others) but downregulated genes involved in DNA replication (Cyclins and their kinases, among others) and DNA repair (including Brca1, Brca2, Rad51). Cell cycle analysis demonstrated EVI1's anti-proliferative effect to be strictly dose-dependent with accumulation of cells in G0/G1, but preservation of a small fraction of long-term proliferating cells. Although confined to cultured cells, our study contributes to new hypotheses addressing the mechanisms and molecular targets involved in preleukemic clonal dominance or leukemic transformation by Evi1.

Previous gene therapy trials for X-linked chronic granulomatous disease (X-CGD) lacked long-term engraftment of corrected hematopoietic stem and progenitor cells (HSPCs). Chronic inflammation and high levels of interleukin-1 beta (IL1B) might have caused aberrant cell cycling in X-CGD HSPCs with a concurrent loss of their long-term repopulating potential. Thus, we performed a targeted CRISPR-Cas9-based sgRNA screen to identify candidate genes that counteract the decreased repopulating capacity of HSPCs during gene therapy. The candidates were validated in a competitive transplantation assay and tested in a disease context using IL1B-challenged or X-CGD HSPCs. The sgRNA screen identified Mapk14 (p38) as a potential target to increase HSPC engraftment. Knockout of p38 prior to transplantation was sufficient to induce a selective advantage. Inhibition of p38 increased expression of the HSC homing factor CXCR4 and reduced apoptosis and proliferation in HSPCs. For potential clinical translation, treatment of IL1B-challenged or X-CGD HSPCs with a p38 inhibitor led to a 1.5-fold increase of donor cell engraftment. In summary, our findings demonstrate that p38 may serve as a potential druggable target to restore engraftment of HSPCs in the context of X-CGD gene therapy.