Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.

Hematopoietic Growth Factors Regulate the Entry of Monocytes into the Adult Brain via Chemokine Receptor CCR5.

Monocytes are circulating macrophage precursors generated from bone marrow hematopoietic stem cells. In adults, monocytes continuously replenish cerebral border-associated macrophages under physiological conditions. Monocytes also rapidly infiltrate the brain in pathological settings. The mechanisms of recruiting monocyte-derived macrophages into the brain under pathological conditions have been extensively studied. However, it remains unclear how monocytes enter the brain to renew border-associated macrophages under physiological conditions. Using both in vitro and in vivo approaches, this study reveals that a combination of two hematopoietic growth factors, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF), complementarily and synergistically enhances the adhesion of monocytes to cerebral endothelial cells in a dose-dependent manner. Cysteine-cysteine chemokine receptor 5 (CCR5) in brain endothelial cells, but not the cell adhesion molecules mediating neuroinflammation-related infiltration of monocyte-derived macrophages, modulates SCF+G-CSF-enhanced monocyte-endothelial cell adhesion. Blocking CCR5 or genetically deleting CCR5 reduces monocyte-endothelial cell adhesion induced by SCF+G-CSF. The SCF+G-CSF-enhanced recruitment of bone marrow-derived monocytes/macrophages into the cerebral perivascular space is also reduced in adult CCR5 knockout mice. This study demonstrates the role of SCF and G-CSF in regulating the entry of monocytes into the adult brain to replenish perivascular macrophages.

Effect of Youthful Blood Environment and Its Key Stem Cell Factor on Renal Interstitial Fibrosis in Elderly Mice.

INTRODUCTION: Youthful blood environment was shown to decelerate the aging process of the kidney and to attenuate senile renal fibrosis in a young-old parabiotic animal model; in addition, we identified a stem cell factor (SCF) that is closely linked with the process. This research was to investigate the effect of youthful blood environment on senile renal interstitial fibrosis and the role of SCF. METHODS: We bred SCF receptor c-Kit gene loss-of-function Wps/Wps mice and established a combination mice model that was subjected to unilateral ureteral obstructive (UUO) and parabiotic surgeries. Parabiotic mice were divided into isochronic parabiotic (young-young [Y-IP] and old-old [O-IP]) and heterochronic parabiotic (young-old [HP]) groups. UUO surgery was performed in one of the parabiotic pairs in the IP group (Y-IPuuo and O-IPuuo) and in the elderly mice in the HP group (O-HPuuo). In order to study the role of SCF/c-kit on renal interstitial fibrosis, UUO surgery was performed in wildtype (WT) and Wps/Wps mice. RESULTS: Fourteen days after UUO surgery, the kidney interstitial fibrosis area, kidney function, and the expressions of SCF/c-Kit, pNF-κB, and fibrosis-related proteins in the O-HPuuo group were significantly lower than those in the Ouuo and O-IPuuo groups. Compared with WT UUO mice, the expressions of pNF-κB and fibrosis-related proteins and the kidney function were all significantly decreased in Wps/Wps UUO mice. CONCLUSION: Youthful blood environment downregulated the expressions of SCF/c-Kit in elderly UUO mice and ameliorated UUO-induced kidney fibrosis and function loss.

Differentiation of human primary testicular cells in the presence of SCF using the organoid culture system.

PURPOSE: Development of organoids using human primary testicular cells has remained a challenge due to the complexity of the mammalian testicular cytoarchitecture and culture methods. In this study, we generated testicular organoids derived from human primary testicular cells. Then, we evaluated the effect of stem cell factor (SCF) on cell differentiation and apoptosis in the testicular organoid model. METHODS: The testicular cells were harvested from the three brain-dead donors. Human spermatogonial stem cells (SSCs) were characterized using immunocytochemistry (ICC), RT-PCR and flow cytometry. Testicular organoids were generated from primary testicular cells by hanging drop culture method and were cultured in three groups: control group, experimental group 1 (treated FSH and retinoic acid (RA)), and experimental group 2 (treated FSH, RA and SCF), for five weeks. We assessed the expression of SCP3 (Synaptonemal Complex Protein 3) as a meiotic gene, PRM2 (Protamine 2) as a post-meiotic marker and apoptotic genes of Bax (BCL2-Associated X Protein) and Bcl-2 (B-cell lymphoma 2), respectively by using RT-qPCR. In addition, we identified the expression of PRM2 by immunohistochemistry (IHC). RESULTS: Relative expression of SCP3, PRM2 and Bcl-2 were highest in group 2 after five weeks of culture. In contrast, BAX expression level was lower in experimental group 2 in comparison with other groups. IHC analyses indicated the highest expression of PRM2 as a postmeiotic marker in group 2 in comparison to 2D culture and control groups but not find significant differences between experimental group 1 and experimental group 2 groups. Morphological evaluations revealed that organoids are compact spherical structures and in the peripheral region composed of uncharacterized elongated fibroblast-like cells. CONCLUSION: Our findings revealed that the testicular organoid culture system promote the spermatogonial stem cell (SSC) differentiation, especially in presence of SCF. Developed organoids are capable of recapitulating many important properties of a stem cell niche.

In vivo promotion of primordial follicle activation by stem cell factor treatment in mice with premature ovarian insufficiency and advanced age.

Dormant primordial follicles (PFs) are the most abundant reproductive resource in mammalian ovaries. With advances in the mechanism of study of the regulation of PF activation, PFs have been used to improve fertility in clinical practice. As a central controlling element of follicle activation signaling, the pre-granulosa cell-secreted stem cell factor (SCF; also known as KIT ligand, KITL), which initiates the growth of dormant oocytes, is an ideal natural activator that stimulates follicle activation. However, no systematic study has been conducted to identify the activating effect of SCF in vivo and in vitro. In this study, by combining an in vitro whole ovary culture system and several mouse models, we provide a series of experimental evidence that SCF is an efficient activator for improving PF activation in mouse ovaries. Our in vitro study showed that SCF increased phosphatidylinositol 3-kinase (PI3K) signaling and PF activation ratio in neonatal ovaries. In vivo ovarian non-invasive topical administrations of SCF to the ovaries efficiently improved follicle activation and development, oocyte retrieval ratio and fertility in inducible premature ovarian insufficiency mouse models and aged mice. Our study suggests that SCF is an efficient growth factor that can be applied to improve PF activation.

The SCF/KIT axis in human mast cells: Capicua acts as potent KIT repressor and ERK predominates PI3K.

BACKGROUND: The SCF/KIT axis regulates nearly all aspects of mast cell (MC) biology. A comprehensive view of SCF-triggered phosphorylation dynamics is lacking. The relationship between signaling modules and SCF-supported functions likewise remains ill-defined. METHODS: Mast cells were isolated from human skin; upon stimulation by SCF, global phosphoproteomic changes were analyzed by LC-MS/MS and selectively validated by immunoblotting. MC survival was inspected by YoPro; BrdU incorporation served to monitor proliferation. Gene expression was quantified by RT-qPCR and cytokines by ELISA. Pharmacological inhibitors were supplemented by ERK1 and/or ERK2 knockdown. CIC translocation and degradation were studied in nuclear and cytoplasmic fractions. CIC's impact on KIT signaling and function was assessed following RNA interference. RESULTS: ≈5400 out of ≈10,500 phosphosites experienced regulation by SCF. The MEK/ERK cascade was strongly induced surpassing STAT5 > PI3K/Akt > p38 > JNK. Comparison between MEK/ERK's and PI3K's support of basic programs (apoptosis, proliferation) revealed equipotency between modules. In functional outputs (gene expression, cytokines), ERK was the most influential kinase. OSM and LIF production was identified in skin MCs. Strikingly, SCF triggered massive phosphorylation of a protein not associated with KIT previously: CIC. Phosphorylation was followed by CIC's cytoplasmic appearance and degradation, the latter sensitive to protease but not preoteasome inhibition. Both shuttling and degradation were ERK-dependent. Conversely, CIC-siRNA facilitated KIT signaling, functional outputs, and survival. CONCLUSION: The SCF/KIT axis shows notable strength in MCs, and MEK/ERK as most prominent module. An inhibitory circuit exists between KIT and CIC. CIC stabilization in MCs may turn out as a therapeutic option to interfere with allergic and MC-driven diseases.

IL3 Has a Detrimental Effect on Hematopoietic Stem Cell Self-Renewal in Transplantation Settings.

The ex vivo expansion and maintenance of long-term hematopoietic stem cells (LT-HSC) is crucial for stem cell-based gene therapy. A combination of stem cell factor (SCF), thrombopoietin (TPO), FLT3 ligand (FLT3) and interleukin 3 (IL3) cytokines has been commonly used in clinical settings for the expansion of CD34+ from different sources, prior to transplantation. To assess the effect of IL3 on repopulating capacity of cultured CD34+ cells, we employed the commonly used combination of STF, TPO and FILT3 with or without IL3. Expanded cells were transplanted into NSG mice, followed by secondary transplantation. Overall, this study shows that IL3 leads to lower human cell engraftment and repopulating capacity in NSG mice, suggesting a negative effect of IL3 on HSC self-renewal. We, therefore, recommend omitting IL3 from HSC-based gene therapy protocols.

Granulocyte colony-stimulating factor directly acts on mouse lymphoid-biased but not myeloid-biased hematopoietic stem cells.

Granulocyte colony-stimulating factor (G-CSF) is widely used in clinical settings to mobilize hematopoietic stem cells (HSCs) into the circulation for HSC harvesting and transplantation. However, whether G-CSF directly stimulates HSCs to change their cell cycle state and fate is controversial. HSCs are a heterogeneous population consisting of different types of HSCs, such as myeloid-biased HSCs and lymphoid-biased HSCs. We hypothesized that G-CSF has different effects on different types of HSCs. To verify this, we performed serum-free single-cell culture and competitive repopulation with cultured cells. Single highly purified HSCs and hematopoietic progenitor cells (HPCs) were cultured with stem cell factor (SCF), SCF + G-CSF, SCF + granulocyte/macrophage (GM)-CSF, or SCF + thrombopoietin (TPO) for 7 days. Compared with SCF alone, SCF + G-CSF increased the number of divisions of cells from the lymphoid-biased HSC-enriched population but not that of cells from the My-bi HSC-enriched population. SCF + G-CSF enhanced the level of reconstitution of lymphoid-biased HSCs but not that of myeloid-biased HSCs. Clonal transplantation assay also showed that SCF + G-CSF did not increase the frequency of myeloid-biased HSCs. These data showed that G-CSF directly acted on lymphoid-biased HSCs but not myeloid-biased HSCs. Our study also revised the cytokine network at early stages of hematopoiesis: SCF directly acted on myeloid-biased HSCs; TPO directly acted on myeloid-biased HSCs and lymphoid-biased HSCs; and GM-CSF acted only on HPCs. Early hematopoiesis is controlled differentially and sequentially by a number of cytokines.

Stem cell-secreted factor therapy regenerates the ovarian niche and rescues follicles.

BACKGROUND: Ovarian senescence is a normal age-associated phenomenon, but increasingly younger women are affected by diminished ovarian reserves or premature ovarian insufficiency. There is an urgent need for developing therapies to improve ovarian function in these patients. In this context, previous studies suggest that stem cell-secreted factors could have regenerative properties in the ovaries. OBJECTIVE: This study aimed to test the ability of various human plasma sources, enriched in stem cell-secreted factors, and the mechanisms behind their regenerative properties, to repair ovarian damage and to promote follicular development. STUDY DESIGN: In the first phase, the effects of human plasma enriched in bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization, umbilical cord blood plasma, and their activated forms on ovarian niche, follicle development, and breeding performance were assessed in mouse models of chemotherapy-induced ovarian damage (n=7 per group). In addition, the proteomic profile of each plasma was analyzed to find putative proteins and mechanism involved in their regenerative properties in ovarian tissue. In the second phase, the most effective plasma treatment was validated in human ovarian cortex xenografted in immunodeficient mice (n=4 per group). RESULTS: Infusion of human plasma enriched bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization or of umbilical cord blood plasma-induced varying degrees of microvessel formation and cell proliferation and reduced apoptosis in ovarian tissue to rescue follicular development and fertility in mouse models of ovarian damage. Plasma activation enhanced these effects. Activated granulocyte colony-stimulating factor plasma was the most potent inducing ovarian rescue in both mice and human ovaries, and proteomic analysis indicated that its effects may be mediated by soluble factors related to cell cycle and apoptosis, gene expression, signal transduction, cell communication, response to stress, and DNA repair of double-strand breaks, the most common form of age-induced damage in oocytes. CONCLUSION: Our findings suggested that stem cell-secreted factors present in both granulocyte colony-stimulating factor-mobilized and umbilical cord blood plasma could be an effective treatment for increasing the reproductive outcomes in women with impaired ovarian function owing to several causes. The activated granulocyte colony-stimulating factor plasma, which is already enriched in both stem cell-secreted factors and platelet-enclosed growth factors, seems to be the most promising treatment because of its most potent restorative effects on the ovary together with the autologous source.

Kit activates interleukin-4 receptor and effector signal transducer and activator of transcription 6 independent of its cognate ligand in mouse mast cells.

Signaling by Kit has been extensively studied in hematopoietic cells and is essential for the survival, proliferation and maintenance of hematopoietic stem and progenitor cells. In addition to the activation of intrinsic signaling pathways, Kit has been shown to interact with lineage-restricted type I cytokine receptors and produce cross signals, e.g. erythropoietin receptor, interleukin-7 receptor (IL-7R), IL-3R. Based on the earlier studies, we hypothesize that Kit activate other type I cytokine receptors in a cell-specific manner and execute cell-specific function. To investigate other Kit-activated receptors, we tested Kit and IL-4R cross-receptor activation in murine bone-marrow-derived mast cells, which express both Kit and IL-4R at the surface level. Kit upon activation by Kit ligand (KL), activated IL-4Rα, γC , and signal transducer and activator of transcription 6 independent of its cognate ligand IL-4. Though KL and IL-4 are individually mitogenic, combinations of KL and IL-4 synergistically promoted mast cell proliferation. Furthermore, inhibition of lipid raft formation by methyl-ß-cyclodextrin resulted in loss of synergistic proliferation. Together the data suggest IL-4R as a novel Kit-activated receptor. Such cross-receptor activations are likely to be a universal mechanism of Kit signaling in hematopoiesis.

Inhibition of mast cells: a novel mechanism by which nintedanib may elicit anti-fibrotic effects.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease which presents a grave prognosis for diagnosed patients. Nintedanib (a triple tyrosine kinase inhibitor) and pirfenidone (unclear mechanism of action) are the only approved therapies for IPF, but have limited efficacy. The pathogenic mechanisms of this disease are not fully elucidated; however, a role for mast cells (MCs) has been postulated. OBJECTIVES: The aim of this work was to investigate a role for MCs in IPF and to understand whether nintedanib or pirfenidone could impact MC function. METHODS AND RESULTS: MCs were significantly elevated in human IPF lung and negatively correlated with baseline lung function (FVC). Importantly, MCs were positively associated with the number of fibroblast foci, which has been linked to increased mortality. Furthermore, MCs were increased in the region immediately surrounding the fibroblast foci, and co-culture studies confirmed a role for MC-fibroblast crosstalk in fibrosis. Nintedanib but not pirfenidone inhibited recombinant stem cell factor (SCF)-induced MC survival. Further evaluation of nintedanib determined that it also inhibited human fibroblast-mediated MC survival. This was likely via a direct effect on ckit (SCF receptor) since nintedanib blocked SCF-stimulated ckit phosphorylation, as well as downstream effects on MC proliferation and cytokine release. In addition, nintedanib ablated the increase in lung MCs and impacted high tissue density frequency (HDFm) in a rat bleomycin model of lung fibrosis. CONCLUSION: Nintedanib inhibits MC survival and activation and thus provides a novel additional mechanism by which this drug may exert anti-fibrotic effects in patients with IPF.

Engineering Stem Cell Factor Ligands with Different c-Kit Agonistic Potencies.

Receptor tyrosine kinases (RTKs) are major players in signal transduction, regulating cellular activities in both normal regeneration and malignancy. Thus, many RTKs, c-Kit among them, play key roles in the function of both normal and neoplastic cells, and as such constitute attractive targets for therapeutic intervention. We thus sought to manipulate the self-association of stem cell factor (SCF), the cognate ligand of c-Kit, and hence its suboptimal affinity and activation potency for c-Kit. To this end, we used directed evolution to engineer SCF variants having different c-Kit activation potencies. Our yeast-displayed SCF mutant (SCFM) library screens identified altered dimerization potential and increased affinity for c-Kit by specific SCF-variants. We demonstrated the delicate balance between SCF homo-dimerization, c-Kit binding, and agonistic potencies by structural studies, in vitro binding assays and a functional angiogenesis assay. Importantly, our findings showed that a monomeric SCF variant exhibited superior agonistic potency vs. the wild-type SCF protein and vs. other high-affinity dimeric SCF variants. Our data showed that action of the monomeric ligands in binding to the RTK monomers and inducing receptor dimerization and hence activation was superior to that of the wild-type dimeric ligand, which has a higher affinity to RTK dimers but a lower activation potential. The findings of this study on the binding and c-Kit activation of engineered SCF variants thus provides insights into the structure-function dynamics of ligands and RTKs.

Dose optimisation of PTEN inhibitor, bpV (HOpic), and SCF for the in-vitro activation of sheep primordial follicles.

The in-vitro development of primordial follicles is critical for improving mammalian fertility and wildlife conservation. This study aimed to optimise the effective doses of bpV (HOpic) and stem cell factor (SCF) for the in-vitro activation of sheep primordial follicles. To do this, sheep ovarian cortex was treated with bpV (1.5, 15, and 150 µM) and SCF (50 and 100 ng/ml). Follicular count indicated that 15 µM bpV and 100 ng/ml SCF significantly increased normal primary follicles compared to other groups (p < 0.05). Also, a significant downregulation of P53 and PTEN, as well as the increased expression of PI3K was observed. The in-vitro maturation was more pronounced when the fragmented tissues were co-treated with selected doses of bpV and SCF. In conclusion, the combination of 15 µM bpV and 100 ng/ml SCF was the most effective treatment strategy for the activation and survival of primordial follicles in sheep ovarian fragments.

Rap1 signal modulators control the maintenance of hematopoietic progenitors in bone marrow and adult long-term hematopoiesis.

Adult long-term hematopoiesis depends on sustaining hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM) niches, where their balance of quiescence, self-renewal, and hematopoietic differentiation is tightly regulated. Although various BM stroma cells that produce niche factors have been identified, regulation of the intrinsic responsiveness of HSPC to the niche factors remains elusive. We previously reported that mice deficient for Sipa1, a Rap1 GTPase-activating protein, develop diverse hematopoietic disorders of late onset. Here we showed that transplantation of BM cells expressing membrane-targeted C3G (C3G-F), a Rap1 GTP/GDP exchanger, resulted in the progressive decline of the numbers of HSPC repopulated in BM with time and impaired long-term hematopoiesis of all cell lineages. C3G-F/HSPC were sustained for months in spleen retaining hematopoietic potential, but these cells inefficiently contributed to overall hematopoietic reconstitution. C3G-F/HSPC showed enhanced proliferation and differentiation with accelerated progenitor cell exhaustion in response to stem cell factor (SCF). Using a Ba/F3 cell line, we confirmed that the increased basal Rap1GTP levels with C3G-F expression caused a markedly prolonged activation of c-Kit receptor and downstream signaling through SCF ligation. A minor population of C3G-F/HSPC also showed enhanced proliferation in the presence of thrombopoietin (TPO) compared to Vect/HSPC. Current results suggest an important role of basal Rap1 activation status of HSPC in their maintenance in BM for sustaining long-term adult hematopoiesis.

Large-scale production and directed induction of functional dendritic cells ex vivo from serum-free expanded human hematopoietic stem cells.

BACKGROUND: Dendritic cells (DCs) that are derived from hematopoietic stem cells (HSCs) are the most potent antigen-presenting cells and play a pivotal role in initiating the immune response. Hence, large-scale production and direct induction of functional DCs ex vivo from HSCs are crucial to HSC research and clinical potential, such as vaccines for cancer and immune therapy. METHODS: In a previous study, we developed a serum-free HSC expansion system (SF-HSC medium) to expand large numbers of primitive HSCs ex vivo. Herein, a DC induction and expansion medium (DC medium) was proposed to further generate large numbers of functional DCs from serum-free expanded HSCs, which were developed and optimized by factorial design and the steepest ascent method. RESULTS: The DC medium is composed of effective basal medium (Iscove's modified Dulbecco's medium [IMDM]) and cytokines (2.9 ng/mL stem cell factor [SCF], 2.1 ng/mL Flt-3 ligand, 3.6 ng/mL interleukin [IL]-1ß, 19.3 ng/mL granulocyte-macrophage colony-stimulating factor [GM-CSF] and 20.0 ng/mL tumor necrosis factor-α [TNF-α]). After 10-day culture in DC medium, the maximum fold expansion for accumulated CD1a+CD11c+ DCs was more than 4000-fold, and the induced DCs were characterized and confirmed by analysis of growth kinetics, surface antigen expression, endocytosis ability, mixed lymphocyte reaction, specific cytokine secretion and lipopolysaccharide stimulation. DISCUSSION: In conclusion, the combination of DC medium and SF-HSC medium can efficiently induce and expand a large amount of functional DCs from a small scale of HSCs and might be a promising source of DCs for vaccine and immune therapy in the near future.

Molecular Mechanisms Responsible for Anti-inflammatory and Immunosuppressive Effects of Mesenchymal Stem Cell-Derived Factors.

Mesenchymal stem cells (MSCs) are self-renewable cells capable for multilineage differentiation and immunomodulation. MSCs are able to differentiate into all cell types of mesodermal origin and, due to their plasticity, may generate cells of neuroectodermal or endodermal origin in vitro. In addition to the enormous differentiation potential, MSCs efficiently modulate innate and adaptive immune response and, accordingly, were used in large number of experimental and clinical trials as new therapeutic agents in regenerative medicine. Although MSC-based therapy was efficient in the treatment of many inflammatory and degenerative diseases, unwanted differentiation of engrafted MSCs represents important safety concern. MSC-based beneficial effects are mostly relied on the effects of MSC-derived immunomodulatory, pro-angiogenic, and trophic factors which attenuate detrimental immune response and inflammation, reduce ischemic injuries, and promote tissue repair and regeneration. Accordingly, MSC-conditioned medium (MSC-CM), which contains MSC-derived factors, has the potential to serve as a cell-free, safe therapeutic agent for the treatment of inflammatory diseases. Herein, we summarized current knowledge regarding identification, isolation, ontogeny, and functional characteristics of MSCs and described molecular mechanisms responsible for MSC-CM-mediated anti-inflammatory and immunosuppressive effects in the therapy of inflammatory lung, liver, and kidney diseases and ischemic brain injury.

Establishment and Characterization of a Murine Mucosal Mast Cell Culture Model.

Accumulating evidence suggests that mast cells play critical roles in disruption and maintenance of intestinal homeostasis, although it remains unknown how they affect the local microenvironment. Interleukin-9 (IL-9) was found to play critical roles in intestinal mast cell accumulation induced in various pathological conditions, such as parasite infection and oral allergen-induced anaphylaxis. Newly recruited intestinal mast cells trigger inflammatory responses and damage epithelial integrity through release of a wide variety of mediators including mast cell proteases. We established a novel culture model (IL-9-modified mast cells, MCs/IL-9), in which murine IL-3-dependent bone-marrow-derived cultured mast cells (BMMCs) were further cultured in the presence of stem cell factor and IL-9. In MCs/IL-9, drastic upregulation of Mcpt1 and Mcpt2 was found. Although histamine storage and tryptase activity were significantly downregulated in the presence of SCF and IL-9, this was entirely reversed when mast cells were cocultured with a murine fibroblastic cell line, Swiss 3T3. MCs/IL-9 underwent degranulation upon IgE-mediated antigen stimulation, which was found to less sensitive to lower concentrations of IgE in comparison with BMMCs. This model might be useful for investigation of the spatiotemporal changes of newly recruited intestinal mast cells.

IL-7 and immobilized Kit-ligand stimulate serum- and stromal cell-free cultures of precursor B-cell lines and clones.

Long-term proliferating, DH JH -rearranged mouse precursor B-cell lines have previously been established in serum- and IL-7-containing media from fetal liver, but not from bone marrow. Serum and stromal cells expose these pre-B cells to undefined factors, hampering accurate analyses of ligand-dependent signaling, which controls pre-B cell proliferation, survival, residence and migration. Here, we describe a novel serum-free, stromal cell-free culture system, which allows us to establish and maintain pre-B cells not only from fetal liver, but also from bone marrow with practically identical efficiencies in proliferation, cloning and differentiation. Surprisingly, recombinant kit-ligand, also called stem cell factor, produced as a kit-ligand-Fc fusion protein, suffices to replace stromal cells and serum, provided that it is presented to cultured pre-B cells in an optimal density in plate-bound, insolubilized, potentially crosslinking form. Additional recombinant CXCL12 and fibronectin have a minor influence on the establishment and maintenance of pre-B cell lines and clones from fetal liver, but are necessary to establish such cell lines from bone marrow.

Cytokine therapy-mediated neuroprotection in a Friedreich's ataxia mouse model.

OBJECTIVES: Friedreich's ataxia is a devastating neurological disease currently lacking any proven treatment. We studied the neuroprotective effects of the cytokines, granulocyte-colony stimulating factor (G-CSF) and stem cell factor (SCF) in a humanized murine model of Friedreich's ataxia. METHODS: Mice received monthly subcutaneous infusions of cytokines while also being assessed at monthly time points using an extensive range of behavioral motor performance tests. After 6 months of treatment, neurophysiological evaluation of both sensory and motor nerve conduction was performed. Subsequently, mice were sacrificed for messenger RNA, protein, and histological analysis of the dorsal root ganglia, spinal cord, and cerebellum. RESULTS: Cytokine administration resulted in significant reversal of biochemical, neuropathological, neurophysiological, and behavioural deficits associated with Friedreich's ataxia. Both G-CSF and SCF had pronounced effects on frataxin levels (the primary molecular defect in the pathogenesis of the disease) and a regulators of frataxin expression. Sustained improvements in motor coordination and locomotor activity were observed, even after onset of neurological symptoms. Treatment also restored the duration of sensory nerve compound potentials. Improvements in peripheral nerve conduction positively correlated with cytokine-induced increases in frataxin expression, providing a link between increases in frataxin and neurophysiological function. Abrogation of disease-related pathology was also evident, with reductions in inflammation/gliosis and increased neural stem cell numbers in areas of tissue injury. INTERPRETATION: These experiments show that cytokines already clinically used in other conditions offer the prospect of a novel, rapidly translatable, disease-modifying, and neuroprotective treatment for Friedreich's ataxia. Ann Neurol 2017;81:212-226.

MRGPRX2 is negatively targeted by SCF and IL-4 to diminish pseudo-allergic stimulation of skin mast cells in culture.

MRGPRX2 was recently uncovered as the "missing link" in clinically relevant mast cell (MC) activation explaining previously puzzling phenomena. It is the receptor for various endogenous ligands and exogenous compounds alike, whose binding evokes rapid degranulation much like allergen-mediated exocytosis. While the perceivable outcomes are similar, the two activation routes differ regarding mechanism and regulation. We recently reported that acute SCF administration curbs responses evoked by MRGPRX2 in human skin MCs. Maintenance of MCs in culture requires the presence of MC supportive factors and renders the cells functionally and molecularly unequal to ex vivo counterparts. Here, we asked whether expansion in culture impacts the pseudo-allergic route, and if so, what contribution SCF and IL-4 play in this scenario. We report that the in vitro micromilieu dampens (but does not erase) pseudo-allergic responses and that this is accompanied by strongly reduced MRGPRX2 expression. Withdrawal of SCF or IL-4 individually, but most potently of both collectively, partially reinstates the MRGPRX2 pathway, revealing that SCF and IL-4 make negative adjustments to the pseudo-allergic pathway. Under all conditions, the FcεRI-triggered route showed the inverse pattern of regulation, substantiating that allergic and pseudo-allergic MC activation can obey opposite rules, hinting at possible competition between them.