Niels Heinz

Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery

The Sleeping Beauty (SB) transposase and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy. The potential cytotoxicity of transposases requires careful assessment, considering that residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and resulting uncontrollable transposition. Comparing retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, we found that conventional SB transposase, SB100X and a newly developed codon-optimized SB100Xo may trigger premitotic arrest and apoptosis. Cell stress induced by continued SB overexpression was self-limiting due to the induction of cell death, which occurred even in the absence of a co-transfected transposable element. The cytotoxic effects of SB transposase were strictly dose dependent and heralded by induction of p53 and c-Jun. Inactivating mutations in SB’s catalytic domain could not abrogate cytotoxicity, suggesting a mechanism independent of DNA cleavage activity. An improved approach of retrovirus particle-mediated mRNA transfer allowed transient and dose-controlled expression of SB100X, supported efficient transposition and prevented cytotoxicity. Transposase-mediated gene transfer can thus be tuned to maintain high efficiency in the absence of overt cell damage.

Activation of Evi1 inhibits cell cycle progression and differentiation of hematopoietic progenitor cells

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.

Serum- and Stromal Cell-Free Hypoxic Generation of Embryonic Stem Cell-Derived Hematopoietic Cells In Vitro, Capable of Multilineage Repopulation of Immunocompetent Mice

Induced pluripotent stem cells (iPSCs) may become a promising source for the generation of patient‐specific hematopoietic stem cells (HSCs) in vitro. A crucial prerequisite will be the availability of reliable protocols for the directed and efficient differentiation toward HSCs. So far, the most robust strategy for generating HSCs from pluripotent cells in vitro has been established in the mouse model involving ectopic expression of the human transcription factor HOXB4. However, most differentiation protocols include coculture on a xenogenic stroma cell line and the use of animal serum. Involvement of any of both would pose a major barrier to the translation of those protocols to human autologous iPSCs intended for clinical use. Therefore, we asked whether long‐term repopulating HSCs can, in principle, be generated from embryonic stem cells without stroma cells or serum. Here, we showed that long‐term multilineage engraftment could be accomplished in immunocompetent mice when HSCs were generated in serum‐free medium without stroma cell support and when hypoxic conditions were used. Under those conditions, HOXB4+ embryonic stem cell‐derived hematopoietic stem and progenitor cells were immunophenotypically similar to definitive bone marrow resident E‐SLAM+ (CD150+CD48−CD45+CD201+) HSCs. Thus, our findings may ease the development of definitive, adult‐type HSCs from pluripotent stem cells, entirely in vitro.

Angptl4 maintains in vivo repopulation capacity of CD34+ human cord blood cells

Methods to expand hematopoietic stem cells (HSCs) ex vivo encompass an attractive approach that would substantially broaden the clinical applicability of HSCs derived from cord blood (CB). Recently, members of the angiopoietin‐like (Angptl) family of growth factors were shown to expand both murine and human HSCs. Specifically, Angptl5 has been implicated in the expansion of human NOD/SCID‐repopulating cells (SRCs) ex vivo. Here, we sought to evaluate the potential of additional Angptls to expand human SRCs from CB. Additionally, the purpose of this study was to evaluate the reproducibility of Angptl‐mediated expansion of SRCs across independent experiments.

TCR-engineered T cells: A model of inducible TCR expression to dissect the interrelationship between two TCRs

TCR gene modified T cells for adoptive therapy simultaneously express the Tg TCR and the endogenous TCR, which might lead to mispaired TCRs with harmful unknown specificity and to a reduced function of TCR‐Tg T cells. We generated dual TCR T cells in two settings in which either TCR was constitutively expressed by a retroviral promoter while the second TCR expression was regulable by a Tet‐on system. Constitutively expressed TCR molecules were reduced on the cell surface depending on the induced TCR expression leading to strongly hampered function. Besides that, using fluorescence resonance energy transfer we detected mispaired TCR dimers and different pairing behaviors of individual TCR chains with a mutual influence on TCR chain expression. The loss of function and mispairing could not be avoided by changing the TCR expression level or by introduction of an additional cysteine bridge. However, in polyclonal T cells, optimized TCR formats (cysteineization, codon optimization) enhanced correct pairing and function. We conclude from our data that (i) the level of mispairing depends on the individual TCRs and is not reduced by increasing the level of one TCR, and (ii) modifications (cysteineization, codon optimization) improve correct pairing but do not completely exclude mispairing (cysteineization).

A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.

Graded or threshold response of the tet-controlled gene expression: all depends on the concentration of the transactivator

BackgroundCurrently, the step-wise integration of tet-dependent transactivator and tet-responsive expression unit is considered to be the most promising tool to achieve stable tet-controlled gene expression in cell populations. However, disadvantages of this strategy for integration into primary cells led us to develop an “All-In-One” vector system, enabling simultaneous integration of both components. The effect on tet-controlled gene expression was analyzed for retroviral “All-In-One” vectors expressing the M2-transactivator either under control of a constitutive or a new type of autoregulated promoter.ResultsDetermination of luciferase activity in transduced cell populations indicated improvement of the dynamic range of gene expression for the autoregulated system. Further differences were observed regarding induction kinetics and dose–response. Most notably, introduction of the autoregulated system resulted in a threshold mode of induction, whereas the constitutive system exhibited pronounced effector-dose dependence.ConclusionTet-regulated gene expression in the applied autoregulated system resembles a threshold mode, whereby full induction of the tet-unit can be achieved at otherwise limiting doxycycline concentrations.

Comparison of Different Cytokine Conditions Reveals Resveratrol as a New Molecule for Ex Vivo Cultivation of Cord Blood‐Derived Hematopoietic Stem Cells

Human cord blood (CB)‐derived hematopoietic stem cells (HSCs) are an interesting source for HSC transplantation. However, the number of collected CB‐HSCs is often too low for one transplantation; therefore, ex vivo expansion of CB‐HSCs is desirable. Current expansion protocols are based on the use of cytokine combinations, including insulin‐like growth factor‐binding protein 2 (IGFBP2) and angiopoietin‐like proteins, or combinations with “small molecules” such as stemregenin‐1. The aim of our project was to compare the potential of different CB‐HSC expansion strategies side‐by‐side by phenotypical analysis in vitro and serial engraftment properties in NOD/SCID/IL2rg−/− (NSG) immunodeficient mice. We further identified resveratrol, a naturally occurring polyphenol, as a new, alternative small molecule combined with cytokines to facilitate serum‐free ex vivo expansion of human CB‐HSCs. The cultivation in resveratrol preserved the CB‐HSC phenotype in vitro most efficiently and was ∼2 times more potent than commonly used cytokine conditions (including stem cell factor, thrombopoietin, Fms‐related tyrosine kinase 3 ligand, interleukin‐6) and the recently established serum‐free culture, including IGFBP2 and angiopoietin‐like 5. Serial transplantation studies further confirmed resveratrol to support robust multilineage engraftment in primary and secondary NSG recipients. Therefore, our work proposes resveratrol as a new small molecule for improved ex vivo culture and modification of human HSCs based on an efficient ex vivo propagation of the HSC fate.

Deoxycytidine-kinase knockdown as a novel myeloprotective strategy in the context of fludarabine, cytarabine or cladribine therapy

Deoxycytidine-kinase knockdown as a novel myeloprotective strategy in the context of fludarabine, cytarabine or cladribine therapy