Proinflammatory phenotype of iPS cell-derived JAK2 V617F megakaryocytes induces fibrosis in 3D in vitro bone marrow niche.

The myeloproliferative disease polycythemia vera (PV) driven by the JAK2 V617F mutation can transform into myelofibrosis (post-PV-MF). It remains an open question how JAK2 V617F in hematopoietic stem cells induces MF. Megakaryocytes are major players in murine PV models but are difficult to study in the human setting. We generated induced pluripotent stem cells (iPSCs) from JAK2 V617F PV patients and differentiated them into megakaryocytes. In differentiation assays, JAK2 V617F iPSCs recapitulated the pathognomonic skewed megakaryocytic and erythroid differentiation. JAK2 V617F iPSCs had a TPO-independent and increased propensity to differentiate into megakaryocytes. RNA sequencing of JAK2 V617F iPSC-derived megakaryocytes reflected a proinflammatory, profibrotic phenotype and decreased ribosome biogenesis. In three-dimensional (3D) coculture, JAK2 V617F megakaryocytes induced a profibrotic phenotype through direct cell contact, which was reversed by the JAK2 inhibitor ruxolitinib. The 3D coculture system opens the perspective for further disease modeling and drug discovery.

Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms.

The role of hematopoietic Hedgehog signaling in myeloproliferative neoplasms (MPNs) remains incompletely understood despite data suggesting that Hedgehog (Hh) pathway inhibitors have therapeutic activity in patients. We aim to systematically interrogate the role of canonical vs. non-canonical Hh signaling in MPNs. We show that Gli1 protein levels in patient peripheral blood mononuclear cells (PBMCs) mark fibrotic progression and that, in murine MPN models, absence of hematopoietic Gli1, but not Gli2 or Smo, significantly reduces MPN phenotype and fibrosis, indicating that GLI1 in the MPN clone can be activated in a non-canonical fashion. Additionally, we establish that hematopoietic Gli1 has a significant effect on stromal cells, mediated through a druggable MIF-CD74 axis. These data highlight the complex interplay between alterations in the MPN clone and activation of stromal cells and indicate that Gli1 represents a promising therapeutic target in MPNs, particularly that Hh signaling is dispensable for normal hematopoiesis.

KIT D816V Mast Cells Derived from Induced Pluripotent Stem Cells Recapitulate Systemic Mastocytosis Transcriptional Profile.

Mast cells (MCs) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MCs occur in low abundance, which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells in the body and established a novel and robust protocol for human iPS cell differentiation toward MCs. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MCs that recapitulate SM disease features: increased number of MCs, abnormal maturation kinetics and activated phenotype, CD25 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MCs are a reliable, inexhaustible, and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics.

A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation.

Transcription factors play a determining role in lineage commitment and cell differentiation. Interferon regulatory factor 8 (IRF8) is a lineage determining transcription factor in hematopoiesis and master regulator of dendritic cells (DC), an important immune cell for immunity and tolerance. IRF8 is prominently upregulated in DC development by autoactivation and controls both DC differentiation and function. However, it is unclear how Irf8 autoactivation is controlled and eventually limited. Here, we identified a novel long non-coding RNA transcribed from the +32 kb enhancer downstream of Irf8 transcription start site and expressed specifically in mouse plasmacytoid DC (pDC), referred to as lncIrf8. The lncIrf8 locus interacts with the lrf8 promoter and shows differential epigenetic signatures in pDC versus classical DC type 1 (cDC1). Interestingly, a sequence element of the lncIrf8 promoter, but not lncIrf8 itself, is crucial for mouse pDC and cDC1 differentiation, and this sequence element confers feedback inhibition of Irf8 expression. Taken together, in DC development Irf8 autoactivation is first initiated by flanking enhancers and then second controlled by feedback inhibition through the lncIrf8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of Irf8 that orchestrates its own expression and thereby controls DC differentiation.

CRISPR/Cas9-engineered human ES cells harboring heterozygous and homozygous c-KIT knockout.

The receptor tyrosine kinase c-KIT (CD117) has a key role in hematopoiesis and is a marker for endothelial and cardiac progenitor cells. In vivo, deficiency of c-KIT is lethal and therefore using CRISPR/Cas9 editing we generated heterozygous and homozygous c-KIT knockout human embryonic stem cell (ES cell) lines. The c-KIT knockout left ES cell pluripotency unaffected as shown by immunofluorescence and trilineage differentiation potential. Heterozygous and homozygous c-KIT knockouts showed complete loss of exon 17, resulting in ablation of c-KIT protein from the cell surface. c-KIT knockout ES cells provide a valuable tool for further investigating c-KIT biology.

CRISPR/Cas9 editing in conditionally immortalized HoxB8 cells for studying gene regulation in mouse dendritic cells.

HoxB8 multipotent progenitors (MPP) are obtained by expression of the estrogen receptor hormone binding domain (ERHBD) HoxB8 fusion gene in mouse BM cells. HoxB8 MPP generate (i) the full complement of DC subsets (cDC1, cDC2, and pDC) in vitro and in vivo and (ii) allow CRISPR/Cas9-mediated gene editing, for example, generating homozygous deletions in cis-acting DNA elements at high precision, and (iii) efficient gene repression by dCas9-KRAB for studying gene regulation in DC differentiation.

Human DC3 Antigen Presenting Dendritic Cells From Induced Pluripotent Stem Cells.

Dendritic cells (DC) are professional antigen-presenting cells that develop from hematopoietic stem cells. Different DC subsets exist based on ontogeny, location and function, including the recently identified proinflammatory DC3 subset. DC3 have the prominent activity to polarize CD8+ T cells into CD8+ CD103+ tissue resident T cells. Here we describe human DC3 differentiated from induced pluripotent stem cells (iPS cells). iPS cell-derived DC3 have the gene expression and surface marker make-up of blood DC3 and polarize CD8+ T cells into CD8+ CD103+ tissue-resident memory T cells in vitro. To test the impact of malignant JAK2 V617F mutation on DC3, we differentiated patient-specific iPS cells with JAK2 V617Fhet and JAK2 V617Fhom mutations into JAK2 V617Fhet and JAK2 V617Fhom DC3. The JAK2 V617F mutation enhanced DC3 production and caused a bias toward erythrocytes and megakaryocytes. The patient-specific iPS cell-derived DC3 are expected to allow studying DC3 in human diseases and developing novel therapeutics.

Agrobacterium tumefaciens Small Lipoprotein Atu8019 Is Involved in Selective Outer Membrane Vesicle (OMV) Docking to Bacterial Cells.

Outer membrane vesicles (OMVs), released from Gram-negative bacteria, have been attributed to intra- and interspecies communication and pathogenicity in diverse bacteria. OMVs carry various components including genetic material, toxins, signaling molecules, or proteins. Although the molecular mechanism(s) of cargo delivery is not fully understood, recent studies showed that transfer of the OMV content to surrounding cells is mediated by selective interactions. Here, we show that the phytopathogen Agrobacterium tumefaciens, the causative agent of crown gall disease, releases OMVs, which attach to the cell surface of various Gram-negative bacteria. The OMVs contain the conserved small lipoprotein Atu8019. An atu8019-deletion mutant produced wildtype-like amounts of OMVs with a subtle but reproducible reduction in cell-attachment. Otherwise, loss of atu8019 did not alter growth, susceptibility against cations or antibiotics, attachment to plant cells, virulence, motility, or biofilm formation. In contrast, overproduction of Atu8019 in A. tumefaciens triggered cell aggregation and biofilm formation. Localization studies revealed that Atu8019 is surface exposed in Agrobacterium cells and in OMVs supporting a role in cell adhesion. Purified Atu8019 protein reconstituted into liposomes interacted with model membranes and with the surface of several Gram-negative bacteria. Collectively, our data suggest that the small lipoprotein Atu8019 is involved in OMV docking to specific bacteria.

A SHH-independent regulation of Gli3 is a significant determinant of anteroposterior patterning of the limb bud.

The family of GLI proteins (GLI1-3) comprises the intracellular mediators of the hedgehog pathway, which regulates a myriad of developmental processes, one of which is limb development. Whereas GLI1 and GLI2 seem to be dispensable during limb development, GLI3 is especially crucial since all GLI3-associated human congenital diseases comprise limb malformations. Furthermore, Gli3(-/-) mouse embryos exhibit pronounced polydactyly in conjunction with a loss of digit identities. Here we examined how the quantity of GLI3 contributes to its function by using different Gli3 mutants in order to vary overall GLI3 levels. In addition, we made use of the Gli3(Delta699) allele, which encodes a C-terminally truncated version of GLI3, thus mimicking the processed GLI3 isoform (GLI3R). The Gli3(Delta699) mutant made it feasible to analyze isoform-specific contributions of GLI3 within the context of anteroposterior patterning of the limb bud. We revealed a so far unappreciated variation in the quantitative demand for GLI3 within different phases and aspects of distal limb formation. In addition, our analyses provide evidence that unprocessed full-length GLI3 is dispensable for anteroposterior patterning of the limb bud. Instead, digit identities are most likely defined by GLI3 repressor activity alone. Furthermore, we present evidence that the anteroposterior grading of GLI3 activity by the action of SHH is supported by a prototype patterning, which regulates Gli3 independently from SHH.

Thalidomide induces limb anomalies by PTEN stabilization, Akt suppression, and stimulation of caspase-dependent cell death.

Thalidomide, a drug used for the treatment of multiple myeloma and inflammatory diseases, is also a teratogen that causes birth defects, such as limb truncations and microphthalmia, in humans. Thalidomide-induced limb truncations result from increased cell death during embryonic limb development and consequential disturbance of limb outgrowth. Here we demonstrate in primary human embryonic cells and in the chicken embryo that thalidomide-induced signaling through bone morphogenetic proteins (Bmps) protects active PTEN from proteasomal degradation, resulting in suppression of Akt signaling. As a consequence, caspase-dependent cell death is stimulated by the intrinsic and Fas death receptor apoptotic pathway. Most importantly, thalidomide-induced limb deformities and microphthalmia in chicken embryos could be rescued by a pharmacological PTEN inhibitor as well as by insulin, a stimulant of Akt signaling. We therefore conclude that perturbation of PTEN/Akt signaling and stimulation of caspase activity is central to the teratogenic effects of thalidomide.

Homozygous Ft embryos are affected in floor plate maintenance and ventral neural tube patterning.

Sonic hedgehog (Shh), produced by the notochord and floor plate cells of the neural tube, plays a critical role in organizing dorsal-ventral patterning in the developing neural tube. We have investigated neural tube development in mouse embryos homozygous for the Fused toes (Ft) mutation, a deletion composed of genes of the Iroquois B (IrxB) cluster and of Fts, Ftm, and Fto. In Ft mutants starting from embryonic day 10.5, the floor plate appeared to degenerate and the notochord failed to undergo ventral displacement from the spinal cord. Consistent with the loss of Shh signalling from the floor plate, V3 neuron generation was reduced in Ft/Ft embryos and the domain of motor neuron generation expanded ventrally at the expense of V2 neurons. These data support the idea that Ft genes play an important role in dorsal-ventral patterning of the neural tube acting to define the extent of motor neuron generation; moreover, the data reveal a previously unanticipated function for Ft genes in the maintenance of the floor plate.