Differentiation-inducing Signals Research Articles

Kidney organoids: steps towards better organization and function.

Kidney organoids - 3D representations of kidneys made either from pluripotent or tissue stem cells - have been available for well over a decade. Their application could confer notable benefits over longstanding in vivo approaches with the potential for clinically aligned human cells and reduced ethical burdens. They been used, at a proof-of-concept level, in development in disease modeling (including with patient-derived stem cells), and in screening drugs for efficacy/toxicity. They differ from real kidneys: they represent only foetal-stage tissue, in their simplest forms they lack organ-scale anatomical organization, they lack a properly arranged vascular system, and include non-renal cells. Cell specificity may be improved by better techniques for differentiation and/or sorting. Sequential assembly techniques that mimic the sequence of natural development, and localized sources of differentiation-inducing signals, improve organ-scale anatomy. Organotypic vascularization remains a challenge: capillaries are easy, but the large vessels that should serve them are absent from organoids and, even in cultured real kidneys, these large vessels do not survive without blood flow. Transplantation of organoids into hosts results in their being vascularized (though probably not organotypically) and in some renal function. It will be important to transplant more advanced organoids, with a urine exit, in the near future to assess function more stringently. Transplantation of human foetal kidneys, followed by nephrectomy of host kidneys, keeps rats alive for many weeks, raising hope that, if organoids can be produced even to the limited size and complexity of foetal kidneys, they may one day be useful in renal replacement.

CXCR3 regulates stem and proliferative CD8+ T cells during chronic infection by promoting interactions with DCs in splenic bridging channels.

SUMMARYProduction of effector CD8+ T cells during persistent infection requires a stable pool of stem-like cells that can give rise to effector cells via a proliferative intermediate population. In infection models marked by T cell exhaustion, this process can be transiently induced by checkpoint blockade but occurs spontaneously in mice chronically infected with the protozoan intracellular parasite Toxoplasma gondii. We observe distinct locations for parasite-specific T cell subsets, implying a link between differentiation and anatomical niches in the spleen. Loss of the chemokine receptor CXCR3 on T cells does not prevent white pulp-to-red pulp migration but reduces interactions with CXCR3 ligand-producing dendritic cells (DCs) and impairs memory-to-intermediate transition, leading to a buildup of memory T cells in the red pulp. Thus, CXCR3 increases T cell exposure to differentiation-inducing signals during red pulp migration, providing a dynamic mechanism for modulating effector differentiation in response to environmental signals.

DYRK1A Negatively Regulates CDK5-SOX2 Pathway and Self-Renewal of Glioblastoma Stem Cells.

Glioblastoma display vast cellular heterogeneity, with glioblastoma stem cells (GSCs) at the apex. The critical role of GSCs in tumour growth and resistance to therapy highlights the need to delineate mechanisms that control stemness and differentiation potential of GSC. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) regulates neural progenitor cell differentiation, but its role in cancer stem cell differentiation is largely unknown. Herein, we demonstrate that DYRK1A kinase is crucial for the differentiation commitment of glioblastoma stem cells. DYRK1A inhibition insulates the self-renewing population of GSCs from potent differentiation-inducing signals. Mechanistically, we show that DYRK1A promotes differentiation and limits stemness acquisition via deactivation of CDK5, an unconventional kinase recently described as an oncogene. DYRK1A-dependent inactivation of CDK5 results in decreased expression of the stemness gene SOX2 and promotes the commitment of GSC to differentiate. Our investigations of the novel DYRK1A-CDK5-SOX2 pathway provide further insights into the mechanisms underlying glioblastoma stem cell maintenance.

Cell Cycle Heterogeneity Can Generate Robust Cell Type Proportioning

SummaryCell-cell heterogeneity can facilitate lineage choice during embryonic development because it primes cells to respond to differentiation cues. However, remarkably little is known about the origin of heterogeneity or whether intrinsic and extrinsic variation can be controlled to generate reproducible cell type proportioning seen in vivo. Here, we use experimentation and modeling in D. discoideum to demonstrate that population-level cell cycle heterogeneity can be optimized to generate robust cell fate proportioning. First, cell cycle position is quantitatively linked to responsiveness to differentiation-inducing signals. Second, intrinsic variation in cell cycle length ensures cells are randomly distributed throughout the cell cycle at the onset of multicellular development. Finally, extrinsic perturbation of optimal cell cycle heterogeneity is buffered by compensatory changes in global signal responsiveness. These studies thus illustrate key regulatory principles underlying cell-cell heterogeneity optimization and the generation of robust and reproducible fate choice in development.

OCT4 Acts as an Integrator of Pluripotency and Signal-Induced Differentiation

Cell type specification relies on the capacity of undifferentiated cells to properly respond to specific differentiation-inducing signals. Using genomic approaches along with loss- and gain-of-function genetic models, we identified OCT4-dependent mechanisms that provide embryonic stem cells with the means to customize their response to external cues. OCT4 binds a large set of low-accessible genomic regions. At these sites, OCT4 is required for proper enhancer and gene activation by recruiting co-regulators and RAR:RXR or β-catenin, suggesting an unexpected collaboration between the lineage-determining transcription factor and these differentiation-initiating, signal-dependent transcription factors. As a proof of concept, we demonstrate that overexpression of OCT4 in a kidney cell line is sufficient for signal-dependent activation of otherwise unresponsive genes in these cells. Our results uncover OCT4 as an integral and necessary component of signal-regulated transcriptional processes required for tissue-specific responses.

CCND1-CDK4-mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo.

Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1-CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1-CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1-CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis.

Lineage conversion induced by pluripotency factors involves transient passage through an iPSC stage.

Brief expression of pluripotency-associated factors such as Oct4, Klf4, Sox2 and c-Myc (OKSM), in combination with differentiation-inducing signals, was reported to trigger transdifferentiation of fibroblasts into other cell types. Here we show that OKSM expression in mouse fibroblasts gives rise to both induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) under conditions previously shown to induce only iNSCs. Fibroblast-derived iNSC colonies silenced retroviral transgenes and reactivated silenced X chromosomes, both hallmarks of pluripotent stem cells. Moreover, lineage tracing with an Oct4-CreER labeling system demonstrated that virtually all iNSC colonies originated from cells transiently expressing Oct4, whereas ablation of Oct4+ cells prevented iNSC formation. Lastly, an alternative transdifferentiation cocktail that lacks Oct4 and was reportedly unable to support induced pluripotency yielded iPSCs and iNSCs carrying the Oct4-CreER-derived lineage label. Together, these data suggest that iNSC generation from fibroblasts using OKSM and other pluripotency-related reprogramming factors requires passage through a transient iPSC state.

Hierarchical differentiation competence in response to retinoic acid ensures stem cell maintenance during mouse spermatogenesis.

Stem cells ensure tissue homeostasis through the production of differentiating and self-renewing progeny. In some tissues, this is achieved by the function of a definitive stem cell niche. However, the mechanisms that operate in mouse spermatogenesis are unknown because undifferentiated spermatogonia (Aundiff) are motile and intermingle with differentiating cells in an ‘open’ niche environment of seminiferous tubules. Aundiff include glial cell line-derived neurotrophic factor receptor α1 (GFRα1)+ and neurogenin 3 (NGN3)+ subpopulations, both of which retain the ability to self-renew. However, whereas GFRα1+ cells comprise the homeostatic stem cell pool, NGN3+ cells show a higher probability to differentiate into KIT+ spermatogonia by as yet unknown mechanisms. In the present study, by combining fate analysis of pulse-labeled cells and a model of vitamin A deficiency, we demonstrate that retinoic acid (RA), which may periodically increase in concentration in the tubules during the seminiferous epithelial cycle, induced only NGN3+ cells to differentiate. Comparison of gene expression revealed that retinoic acid receptor γ (Rarg) was predominantly expressed in NGN3+ cells, but not in GFRα1+ cells, whereas the expression levels of many other RA response-related genes were similar in the two populations. Ectopic expression of RARγ was sufficient to induce GFRα1+ cells to directly differentiate to KIT+ cells without transiting the NGN3+ state. Therefore, RARγ plays key roles in the differentiation competence of NGN3+ cells. We propose a novel mechanism of stem cell fate selection in an open niche environment whereby undifferentiated cells show heterogeneous competence to differentiate in response to ubiquitously distributed differentiation-inducing signals.

Transgenic human ES and iPS reporter cell lines for identification and selection of pluripotent stem cells in vitro

Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency, and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3, H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers, such as OCT4 (POU5F1), TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors.

Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions

Cardiovascular progenitor cells (CVPCs) derived from human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold great promise for the study of cardiovascular development and cell-based therapy of heart diseases, but their applications are challenged by the difficulties in their efficient generation and stable maintenance. This study aims to develop chemically defined systems for robust generation and stable propagation of hPSC-derived CVPCs by modulating the key early developmental pathways involved in human cardiovascular specification and CVPC self-renewal. Herein we report that a combination of bone morphogenetic protein 4 (BMP4), glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021 and ascorbic acid is sufficient to rapidly convert monolayer-cultured hPSCs, including hESCs and hiPSCs, into homogeneous CVPCs in a chemically defined medium under feeder- and serum-free culture conditions. These CVPCs stably self-renewed under feeder- and serum-free conditions and expanded over 10(7)-fold when the differentiation-inducing signals from BMP, GSK3 and Activin/Nodal pathways were simultaneously eliminated. Furthermore, these CVPCs exhibited expected genome-wide molecular features of CVPCs, retained potentials to generate major cardiovascular lineages including cardiomyocytes, smooth muscle cells and endothelial cells in vitro, and were non-tumorigenic in vivo. Altogether, the established systems reported here permit efficient generation and stable maintenance of hPSC-derived CVPCs, which represent a powerful tool to study early embryonic cardiovascular development and provide a potentially safe source of cells for myocardial regenerative medicine.

Hepatic differentiation of human embryonic stem cells on growth factor-containing surfaces

Embryonic stem cells (ESCs) hold considerable promise in tissue engineering and regenerative medicine as a source of tissue-specific cells. Hepatocytes derived from ESCs will be useful for therapies, bioartificial liver assistance devices and drug discovery. In traditional stem cell cultivation/differentiation experiments, growth factors (GFs) are added in soluble form in order to provide signals for tissue-specific differentiation. In contrast, we investigated differentiation of hESCs cultured on top of GFs. In these experiments, glass substrates were imprinted with a mixture of ECM and GF molecules to form 500 µm diameter spots. hESCs were cultured onto these GF-containing ECM spots for up to 12 days to induce differentiation towards the hepatic lineage. The dynamics of differentiation were examined by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and immunocytochemistry. Stem cells cultured on GF-containing surfaces stained positive for the endoderm markers SOX17 and FOXA2, as well as early liver markers such as α-fetoprotein and albumin. qRT-PCR confirmed that pluripotency, endoderm and liver gene expression of hESCs cultured on GF-containing surfaces was consistent with hepatic differentiation. In comparison, hESCs cultured on ECM spots without GFs showed less pronounced loss of pluripotency and lower levels of liver gene expression. In summary, our study demonstrates that hESCs receive differentiation-inducing signals from GF-containing surfaces and may be pushed along the hepatic lineage when cultured on such surfaces. Compared to traditional approaches, the advantages of GF immobilization include reduction in the cost of experiments, stronger and longer stimulation and the possibility of screening GF-stem cell interactions in a multiplexed manner.

Cell type-specific filamin complex regulation by a novel class of HECT ubiquitin ligase is required for normal cell motility and patterning

Differential cell motility, which plays a key role in many developmental processes, is perhaps most evident in examples of pattern formation in which the different cell types arise intermingled before sorting out into discrete tissues. This is thought to require heterogeneities in responsiveness to differentiation-inducing signals that result in the activation of cell type-specific genes and 'salt and pepper' patterning. How differential gene expression results in cell sorting is poorly defined. Here we describe a novel gene (hfnA) that provides the first mechanistic link between cell signalling, differential gene expression and cell type-specific sorting in Dictyostelium. HfnA defines a novel group of evolutionarily conserved HECT ubiquitin ligases with an N-terminal filamin domain (HFNs). HfnA expression is induced by the stalk differentiation-inducing factor DIF-1 and is restricted to a subset of prestalk cells (pstO). hfnA(-) pstO cells differentiate but their sorting out is delayed. Genetic interactions suggest that this is due to misregulation of filamin complex activity. Overexpression of filamin complex members phenocopies the hfnA(-) pstO cell sorting defect, whereas disruption of filamin complex function in a wild-type background results in pstO cells sorting more strongly. Filamin disruption in an hfnA(-) background rescues pstO cell localisation. hfnA(-) cells exhibit altered slug phototaxis phenotypes consistent with filamin complex hyperactivity. We propose that HfnA regulates filamin complex activity and cell type-specific motility through the breakdown of filamin complexes. These findings provide a novel mechanism for filamin regulation and demonstrate that filamin is a crucial mechanistic link between responses to differentiation signals and cell movement in patterning based on 'salt and pepper' differentiation and sorting out.

Cell type-specific filamin complex regulation by a novel class of HECT ubiquitin ligase is required for normal cell motility and patterning.

Differential cell motility, which plays a key role in many developmental processes, is perhaps most evident in examples of pattern formation in which the different cell types arise intermingled before sorting out into discrete tissues. This is thought to require heterogeneities in responsiveness to differentiation-inducing signals that result in the activation of cell type-specific genes and 'salt and pepper' patterning. How differential gene expression results in cell sorting is poorly defined. Here we describe a novel gene (hfnA) that provides the first mechanistic link between cell signalling, differential gene expression and cell type-specific sorting in Dictyostelium. HfnA defines a novel group of evolutionarily conserved HECT ubiquitin ligases with an N-terminal filamin domain (HFNs). HfnA expression is induced by the stalk differentiation-inducing factor DIF-1 and is restricted to a subset of prestalk cells (pstO). hfnA(-) pstO cells differentiate but their sorting out is delayed. Genetic interactions suggest that this is due to misregulation of filamin complex activity. Overexpression of filamin complex members phenocopies the hfnA(-) pstO cell sorting defect, whereas disruption of filamin complex function in a wild-type background results in pstO cells sorting more strongly. Filamin disruption in an hfnA(-) background rescues pstO cell localisation. hfnA(-) cells exhibit altered slug phototaxis phenotypes consistent with filamin complex hyperactivity. We propose that HfnA regulates filamin complex activity and cell type-specific motility through the breakdown of filamin complexes. These findings provide a novel mechanism for filamin regulation and demonstrate that filamin is a crucial mechanistic link between responses to differentiation signals and cell movement in patterning based on 'salt and pepper' differentiation and sorting out.

Immortalisation of Normal Human Urothelial Cells Compromises Differentiation Capacity

BackgroundThe development of urothelial malignancy is not solely a consequence of loss of proliferation constraints but also involves loss of cellular differentiation, defined histopathologically as grade. Although tumour grade is an independent prognostic marker for urothelial carcinoma (UC), the molecular events underpinning the loss of urothelial differentiation are poorly understood. ObjectiveTo examine the effect of gene alterations implicated in UC development on the ability of human urothelial cells to undergo molecular differentiation and form a functional urothelial barrier. Design, setting, and participantsLaboratory study. InterventionNormal human urothelial (NHU) cell cultures were transduced with recombinant retroviruses to produce stable sublines overexpressing wild-type or oncogenic mutated fibroblast growth factor receptor 3 or human telomerase reverse transcriptase (hTERT). Previously generated NHU sublines carrying dominant-negative CDK4 and p53 mutant genes or immortalised with the human papillomavirus 16 E6 oncoprotein were included. MeasurementsThe activity of introduced transgenes was demonstrated by comparing phenotypes of transgene-expressing and isogenic control NHU cells. Modified and control sublines were compared for changes in generational potential (life span) and capacity to respond to differentiation-inducing signals by transcript expression of uroplakins 2 and 3. The ability to form a barrier epithelium was assessed by measuring the transepithelial electrical resistance. Results and limitationsBy contrast to tumour suppressor loss of function or oncogene overactivation, hTERT overexpression alone led to life span extension and immortalisation. The hTERT immortalised cells carried no gross genomic alterations but became progressively insensitive to differentiation signals and lost the ability to form an epithelial barrier. Further characterisation of hTERT cells revealed a downregulation of p16 cyclin-dependent kinase inhibitor expression and loss of responsiveness to peroxisome proliferator-activated receptor γ, providing mechanistic explanations for the subjugation of senescence constraints and the abrogation of differentiation capability, respectively. Although immortalised urothelial cell lines without karyotypic aberrations may be generated, such cell lines are compromised in terms of differentiation and functional capacity. ConclusionsOverexpression of hTERT promotes development of an immortalised differentiation-insensitive urothelial cell phenotype. Although such cells offer a useful insight into the grade/stage paradigm of UC, they have limited value for investigating normal urothelial cell/tissue biology and physiology.

Negative regulation of UCP2 by TGFβ signaling characterizes low and intermediate-grade primary breast cancer

The histological manifestation of growth-regulating and differentiation-inducing signals in cancer cells is considered as a key component for clinical outcome prediction and commonly defined as tumor differentiation grade. However, the molecular and functional framework underlying this clinical parameter remains poorly understood. Our correlative data display a significant association (P>0.001) between mitochondrial uncoupling protein 2 (UCP2) and tumor grade in primary breast cancer (n=234). Through mechanistic analyses, we show a synergistic link between UCP2 and established cellular pathways in conferring grade-associated functional phenotypes. Here, the application of well to moderately differentiated primary tumor cell lines has enabled direct observation of SMAD recruitment to the UCP2 promoter underlying repression of gene transcription. In contrast, poorly differentiated tumor cells, known to be TGFβ resistant, displayed aberrant UCP2 regulation, and consequently, gene overexpression, which reduced mitochondrial calcium and facilitated the maintenance of mitochondrial membrane potential, thereby significantly decreasing oxidative stress and inhibiting cell death. Conversely, UCP2 silencing in such cells rapidly led to the induction of apoptosis and cell differentiation, concurrent with reduced cell survival and proliferation, confirming gene-specific effects. Demonstration of a biologically driven role for UCP2 dysregulation in promoting multiple characteristics of tumor aggressiveness strongly endorses assessment of gene expression at clinical presentation to augment therapeutic decision-making and improve patient outcome through personalized targeting approaches.

Heterogeneity of Embryonic and Adult Stem Cells

New studies suggest that stem cells of embryonic, neural, and hematopoietic origin are heterogeneous, with cells moving between two or more metastable states. These cell states show a bias in their differentiation potential and correlate with specific patterns of transcription factor expression and chromatin modifications.

Alkaline Phosphatase-Positive Colony Formation Is a Sensitive, Specific, and Quantitative Indicator of Undifferentiated Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific, robust, and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP(+)) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows. (a) Most colonies obtained under these conditions consist of >or=30 AP(+) cells that coexpress OCT4, NANOG, SSEA3, SSEA4, TRA-1-60, and TRA-1-81. (b) Most such colonies are derived from SSEA3(+) cells. (c) Primary colonies contain cells that produce secondary colonies of the same composition, including cells that initiate multilineage differentiation in embryoid bodies (EBs). (d) Colony formation is independent of plating density or the colony-forming cell (CFC) content of the test population over a wide range of cell concentrations. (e) CFC frequencies decrease when differentiation is induced by exposure either to retinoic acid or to conditions that stimulate EB formation. Interestingly, this loss of AP(+) clonogenic potential also occurs more rapidly than the loss of SSEA3 or OCT4 expression. The CFC assay thus provides a simple, reliable, broadly applicable, and highly specific functional assay for quantifying undifferentiated hESCs with self-renewal potential. Its use under standardized assay conditions should enhance future elucidation of the mechanisms that regulate hESC propagation and their early differentiation.

Drosophila Pgc protein inhibits P-TEFb recruitment to chromatin in primordial germ cells

Germ cells are the only cells that transmit genetic information to the next generation, and they therefore must be prevented from differentiating inappropriately into somatic cells. A common mechanism by which germline progenitors are protected from differentiation-inducing signals is a transient and global repression of RNA polymerase II (RNAPII)-dependent transcription. In both Drosophila and Caenorhabditis elegans embryos, the repression of messenger RNA transcription during germ cell specification correlates with an absence of phosphorylation of Ser 2 residues in the carboxy-terminal domain of RNAPII (hereafter called CTD), a critical modification for transcriptional elongation. Here we show that, in Drosophila embryos, a small protein encoded by polar granule component (pgc) is essential for repressing CTD Ser 2 phosphorylation in newly formed pole cells, the germline progenitors. Ectopic Pgc expression in somatic cells is sufficient to repress CTD Ser 2 phosphorylation. Furthermore, Pgc interacts, physically and genetically, with positive transcription elongation factor b (P-TEFb), the CTD Ser 2 kinase complex, and prevents its recruitment to transcription sites. These results indicate that Pgc is a cell-type-specific P-TEFb inhibitor that has a fundamental role in Drosophila germ cell specification. In C. elegans embryos, PIE-1 protein segregates to germline blastomeres, and is thought to repress mRNA transcription through interaction with P-TEFb. Thus, inhibition of P-TEFb is probably a common mechanism during germ cell specification in the disparate organisms C. elegans and Drosophila.

Infection with Anaplasma phagocytophilum Inhibits Proliferation and Differentiation of Myeloid Progenitors: New Insight into Infection-Related Pancytopenia.

Human granulocytic anaplasmosis (HGA) is a potentially fatal tick-borne disease caused by the obligate intracellular bacterium Anaplasma phagocytophilum, which has a tropism for neutrophils. Clinical symptoms of HGA include fever, headache, myalgia, neutropenia and thrombocytopenia. Our group and others have identified several mechanisms utilized by A. phagocytophilum to establish a safe environment within the normally hostile mature neutrophil to facilitate its survival and propagation, including inhibition of the phagocyte respiratory burst, inhibition of apoptosis, and induction of cytokine secretion to recruit fresh neutrophils. However, the occurrence of thrombocytopenia as the most prominent hematologic abnormality in infected individuals suggests that neutrophils are not the only reservoir of infection. We postulated that A. phagocytophilum infects bone marrow progenitors and inhibits their proliferation and maturation to cause more global marrow suppression and induce pancytopenia. To test this hypothesis, we infected several hematopoietic cell models and determined whether infection inhibits the proliferation and differentiation of myeloid progenitors at several distinct stages of development. Using three distinct in vitro myeloid models, we have shows that A. phagocytophilum can efficiently infect promyelocytes and late myeloblasts, and that infection inhibits their responses to both proliferative and differentiation-inducing signals. The first model is the EPRO cell line, which shows dramatic decreases in colony formation upon GM-CSF-induced growth when infected with A. phagocytophilum. For our second model we used a myeloblast cell line generated by transducing bone marrow progenitors with an empty pBabepuro vector with subsequent selection in puromycin and growth in stem cell factor (SCF). These cells rapidly undergo neutrophil differentiation upon induction with G-CSF in the absence of SCF. Infection with A. phagocytophilum causes a pronounced decrease in G-CSF induced differentiation, and inhibits the initial stages of proliferation prior to differentiation. Using a third cell line model, we show that A. phagocytophilum can also infect the EML multipotent progenitor cell line. Furthermore, we demonstrate that A. phagocytophilum infection causes decreased proliferation in response to SCF. Parallel studies in primary progenitor cells are ongoing. Together these data suggest that A. phagocytophilum can infect progenitors at multiple stages of blood cell development, and that infection inhibits both the proliferation of myeloid progenitors and their ability to differentiate towards mature neutrophils. Whether infection also inhibits thrombopoiesis is currently under investigation. Our studies provide a new paradigm of susceptibility to A. phagocytophilum, where both peripheral blood neutrophils and bone marrow cells become infected upon exposure to the bacterium. These data also suggest that the pancytopenia observed in A. phagocytophilum-infected patients may be due to inhibitory effects on the growth and development of bone marrow progenitors.

New prestalk and prespore inducing signals in Dictyostelium

The differentiation-inducing signals (DIFs) currently known in Dictyostelium appear unable to account for the full diversity of cell types produced in development. To search for new signals, we analyzed the differentiation in monolayers of cells expressing prestalk (ecmAO, ecmA, ecmO, ecmB and cAR2) and prespore (psA) markers. Expression of each marker drops off as the cell density is reduced, suggesting that cell interaction is required. Expression of each marker is inhibited by cerulenin, an inhibitor of polyketide synthesis, and can be restored by conditioned medium. However, the known stalk-inducing polyketide, DIF-1, could not replace conditioned medium and induce the ecmA or cAR2 prestalk markers, suggesting that they require different polyketide inducers. Polyketide production by fungi is stimulated by cadmium ions, which also dramatically stimulates differentiation in Dictyostelium cell cultures and the accumulation of medium factors. Factors produced with cadmium present were extracted from conditioned medium and fractionated by HPLC. A new factor inducing prespore cell differentiation, called PSI-2, and two inducing stalk cell differentiation (DIFs 6 and 7) were resolved. All are distinct from currently identified factors. DIF-6, but not DIF-7 or PSI-2, appears to have an essential carbonyl group. Thus Dictyostelium may use extensive polyketide signaling in its development.