Human Stem Cell Factor Research Articles

Characterization of Human Engraftment and Hemophagocytic Lymphohistiocytosis in NSG-SGM3 Neonate Mice Engrafted with Purified CD34+ Hematopoietic Stem Cells

Immunodeficient mice bearing human immune systems, or "humanized" chimeric mice, are widely used in basic research, along with the preclinical stages of drug development. Nonobese diabetic-severe combined immunodeficiency (NOD-SCID) IL2Rγnull (NSG) mice expressing human stem cell factor, granulocyte-macrophage colony stimulating factor, and interleukin-3 (NSG-SGM3) support robust development of human myeloid cells and T cells but have reduced longevity due to the development of fatal hemophagocytic lymphohistiocytosis (HLH). Here, we describe an optimized protocol for development of human immune chimerism in NSG-SGM3 mice. We demonstrate that efficient human CD45+ reconstitution can be achieved and HLH delayed by engraftment of neonatal NSG-SGM3 with low numbers of human umbilical cord-derived CD34+ hematopoietic stem cells in the absence of preconditioning irradiation.

Docosahexaenoic Acid Alleviates Trimethylamine-N-oxide-mediated Impairment of Neovascularization in Human Endothelial Progenitor Cells

Background: Human endothelial progenitor cells (hEPCs), originating from hemangioblasts in bone marrow (BM), migrate into the blood circulation, differentiate into endothelial cells, and could act as an alternative tool for tissue regeneration. In addition, trimethylamine-N-oxide (TMAO), one of the gut microbiota metabolites, has been identified as an atherosclerosis risk factor. However, the deleterious effects of TMAO on the neovascularization of hEPCs have not been studied yet. Results: Our results demonstrated that TMAO dose-dependently impaired human stem cell factor (SCF)-mediated neovascularization in hEPCs. The action of TMAO was through the inactivation of Akt/endothelial nitric oxide synthase (eNOS), MAPK/ERK signaling pathways, and an upregulation of microRNA (miR)-221. Docosahexaenoic acid (DHA) could effectively inhibit the cellular miR-221 level and induce the phosphorylation level of Akt/eNOS, MAPK/ERK signaling molecules, and neovascularization in hEPCs. DHA enhanced cellular amounts of reduced form glutathione (GSH) through an increased expression of the gamma-glutamylcysteine synthetase (γ-GCS) protein. Conclusions: TMAO could significantly inhibit SCF-mediated neovascularization, in part in association with an upregulation of miR-221 level, inactivation of Akt/eNOS and MAPK/ERK cascades, suppression of γ-GCS protein, and decreased levels of GSH and GSH/GSSG ratio. Furthermore, the DHA could alleviate the detrimental effects of TMAO and induce neovasculogenesis through suppression of miR-221 level, activation of Akt/eNOS and MAPK/ERK signaling cascades, increased expression of γ-GCS protein, and increment of cellular GSH level and GSH/GSSG ratio in hEPCs.

Peanut allergen inhibition prevents anaphylaxis in a humanized mouse model.

Peanut-induced allergy is an immunoglobulin E (IgE)-mediated type I hypersensitivity reaction that manifests symptoms ranging from local edema to life-threatening anaphylaxis. Although there are treatments for symptoms in patients with allergies resulting from allergen exposure, there are few preventive therapies other than strict dietary avoidance or oral immunotherapy, neither of which are successful in all patients. We have previously designed a covalent heterobivalent inhibitor (cHBI) that binds in an allergen-specific manner as a preventive for allergic reactions. Building on previous in vitro testing, here, we developed a humanized mouse model to test cHBI efficacy in vivo. Nonobese diabetic-severe combined immunodeficient γc-deficient mice expressing transgenes for human stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3 developed mature functional human mast cells in multiple tissues and displayed robust anaphylactic reactions when passively sensitized with patient-derived IgE monoclonal antibodies specific for peanut Arachis hypogaea 2 (Ara h 2). The allergic response in humanized mice was IgE dose dependent and was mediated by human mast cells. Using this humanized mouse model, we showed that cHBI prevented allergic reactions for more than 2 weeks when administered before allergen exposure. cHBI also prevented fatal anaphylaxis and attenuated allergic reactions when administered shortly after the onset of symptoms. cHBI impaired mast cell degranulation in vivo in an allergen-specific manner. cHBI rescued the mice from lethal anaphylactic responses during oral Ara h 2 allergen-induced anaphylaxis. Together, these findings suggest that cHBI has the potential to be an effective preventative for peanut-specific allergic responses in patients.

Acceleration of melanocyte senescence by the proinflammatory cytokines IFNγ and TNFα impairs the repigmentation response of vitiligo patients to narrowband ultraviolet B (NBUVB) phototherapy

Vitiligo is a chronic autoimmune disease characterized by the T helper 1 (Th1) cytokine-driven immune destruction of melanocytes (MCs). Although narrowband ultraviolet B (NBUVB) phototherapy has been proven to be an effective therapeutic option, the repigmentation response to that phototherapy varies greatly in different vitiligo patients. Here, we demonstrate that there is an increase of NBUVB-induced cellular senescence in vitiligo MCs exposed to Th1 cytokine interferon γ (IFNγ) and/or tumor necrosis factor α (TNFα) in lesional vitiligo skin from poor responders who had undergone NBUVB phototherapy. Supplementation with exogenous recombinant human stem cell factor (rhSCF) in the culture medium as well as the lentiviral vector–mediated overexpression of cKIT could prevent the MCs from the IFNγ/TNFα-accelerated cellular senescence. Mechanistic studies indicated that the reduced ratio of membrane-bound KIT (mKIT) to the soluble form of KIT (sKIT) is directly related to the cellular senescence of vitiligo MCs following exposure to IFNγ and TNFα. Furthermore, the matrix metalloprotease 9 (MMP9) inhibitor GM6001 attenuates the production of sKIT via the suppression of cKIT ectodomain shedding. Altogether, our study indicates that the presence of Th1 cytokines IFNγ and/or TNFα in the epidermal milieu might impair the repigmentation response of vitiligo patients to NBUVB phototherapy.

Generation and functional evaluation of novel monoclonal antibodies targeting glycosylated human stem cell factor.

Human stem cell factor (hSCF) is an early-acting growth factor that promotes proliferation, differentiation, migration, and survival in several tissues. It plays a crucial role in hematopoiesis, gametogenesis, melanogenesis, intestinal motility, and in development and recovery of nervous and cardiovascular systems. Potential therapeutic applications comprise anemia treatment, mobilization of hematopoietic stem/progenitor cells to peripheral blood, and increasing gene transduction efficiency for gene therapy. Developing new tools to characterize recombinant hSCF in most native-like form as possible is crucial to understand the complexity of its in vivo functions and for improving its biotechnological applications. The soluble domain of hSCF was expressed in HEK293 cells. Highly purified rhSCF showed great molecular mass variability due to the presence of N- and O-linked carbohydrates, and it presented a 2.5-fold increase on proliferative activity compared to bacteria-derived hSCF. Three hybridoma clones producing monoclonal antibodies (mAbs) with high specificity for the glycoprotein were obtained. 1C4 and 2D3 mAbs were able to detect bacteria-derived and glycosylated rhSCF and demonstrated to be excellent candidates to develop a sandwich ELISA assay for rhSCF quantification, with detection limits of 0.18 and 0.07ng/ml, respectively. Interestingly, 1A10 mAb only recognized glycosylated rhSCF, suggesting that sugar moieties might be involved in epitope recognition. 1A10 mAb showed the highest binding affinity, and it constituted the best candidate for immunodetection of the entire set rhSCF glycoforms in western blot assays, and for intracellular cytokine staining. Our work shows that combining glycosylated rhSCF expression with hybridoma technology is a powerful strategy to obtain specific suitable immunochemical assays and thus improve glycoprotein-producing bioprocesses. KEY POINTS: • Soluble glycosylated human SCF exerted improved proliferative activity on UT-7 cells. • Three mAbs with high specificity targeting glycosylated human SCF were obtained. • mAbs applications comprise sandwich ELISA, western blot, and immunofluorescence assays.

Induction of Human T Cell Development In Vitro with OP9-DL4-7FS Cells Expressing Human Cytokines.

For nearly a generation now, OP9-DL1 and OP9-DL4 cells have provided an efficient and reliable cell system to generate T cells from mouse and human hematopoietic stem cells (HSCs) and pluripotent stem cells. OP9-DL1 and OP9-DL4 were originally derived from the OP9 mouse bone marrow stromal cell line, which was transduced to ectopically express Delta-like 1 or 4 proteins, respectively. OP9-DL cells mimic the thymic microenvironment in that when cocultured with mouse or human (h) HSCs, they interact with and activate Notch receptors present on HSCs, required for T cell differentiation. The HSC/OP9-DL cocultures require additional cytokines that are necessary for survival and proliferation of hematopoietic cells. For hHSCs, these factors are interleukin-7 (IL-7), stem cell factor (SCF), and FMS-like tyrosine kinase 3 ligand (FLT3L) that are normally exogenously added to the cocultures. In this chapter, we describe methods for establishing a novel and improved version of OP9-DL4 cells, called OP9-DL4-7FS cells that circumvent the addition of these costly cytokines, by transducing OP9-DL4 cell line to express human IL-7, FLT3L, and SCF (7FS). Herein, we describe the protocol for the generation of OP9-DL4-7FS cells and the conditions for OP9-DL4-7FS/hHSC coculture to support T cell lineage initiation and expansion while comparing it to the now "classic" OP9-DL4 coculture. The use of OP9-DL4-7FS cell system will provide an improved and cost-effective method to the commonly used OP9-DL/HSC coculture for studying both mouse and human T cell development.

Efficacy of SCF drug conjugate targeting c-KIT in gastrointestinal stromal tumor

BackgroundGastrointestinal stromal tumor (GIST) is a rare type of cancer that occurs in the gastrointestinal tract. The majority of GIST cases carry oncogenic forms of KIT, the receptor for stem cell factor (SCF). Small molecule kinase inhibitor imatinib is effective in prolonging the survival of GIST patients by targeting KIT. However, drug resistance often develops during the therapeutic treatment. Here, we produced a SCF-emtansine drug conjugate (SCF-DM1) with favorable drug efficacy towards GIST cells.MethodsRecombinant human SCF (rhSCF) was expressed in E. coli cells and further purified with Ni–NTA Sepharose and Phenyl Sepharose. It was then conjugated with DM1, and the conjugated product SCF-DM1 was evaluated using in vitro cell-based assays and in vivo xenograft mouse model.ResultsSCF-DM1 was effective in inhibiting imatinib-sensitive and -resistant GIST cell lines and primary tumor cells, with IC50 values of < 30 nM. It induced apoptosis and cell cycle arrest in GIST cells. In xenograft mouse model, SCF-DM1 showed favorable efficacy and safety profiles.ConclusionsrhSCF is a convenient and effective vector for drug delivery to KIT positive GIST cells. SCF-DM1 is an effective drug candidate to treat imatinib-sensitive and -resistant GIST.

IFN-γ Inhibitors and Ruxolitinib Therapy for Acquired Severe Aplastic Anemia in an Ex-Vivo Model

Background:Severe aplastic anemia (SAA) is a life-threatening disorder that is associated with multiple etiologies, both inherited and acquired. In acquired SAA, oligoclonal expansion of dysregulated CD8+ cytotoxic T cells, abnormal function of CD4+ T helper cells, along with elevated production of IFN-γ and TNF-α have been associated with the apoptosis of hematopoietic stem and progenitor cells (HSPC) (Young, N Engl J Med, 2018). Currently, the first line treatment for patients who have a suitable HLA matched donor is a hematopoietic progenitor cell transplant (HPCT). When HPCT is not possible, due to lack of a closely matched HLA donor and/or concomitant co-morbidities, then the treatment of choice is immunosuppression with anti-thymocyte globulin, cyclosporine and eltrombopag (ELT)(Georges et al, Blood, 2018). Alvarado et al (Blood, 2019) recently demonstrated that ELT bypasses the inhibitory effect of IFN-γ by alternatively activating TPO signaling. However, ELT cannot overcome other IFN-γ mediated effect through JAK-STAT1 phosphorylation or apoptosis via Fas/FasL. Alternative therapies are in great need for patients with aSAA as treatment response is sub-optimal.Objective:To determine the effects of IFN-γ neutralizing antibodies or Ruxolitinib on HSPCs survival, proliferation and differentiation in an ex vivo culture of human CD34+ cells in the presence of IFN-γ and TNF-α.Design/Methods:Human CD34+ HSPCs were isolated from cord blood, based on CD34 microbeads magnetic selection (Miltenyl Biotec, Germany). The CD34+ cells were cultured in StemSpan Serum-Free Medium II (STEMCELL Technologies) supplemented with 5 ng/mL human stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L) and 5 ng/mL recombinant human TPO. A 1x10 5 CD34+ HSPCs were seeded in a 96 well plate. HSPC alone, with and without IFN-γ (100 ng/mL) and TNF-α (10 ng/mL), were the negative and positive control, respectively. Specific IFN-γ neutralizing antibody, B27 (BD Pharmingen), MD1 (BioLegend) and B133.5 (ImmunoTools) or Ruxolitinib (Jakafi, Incyte) were added to the culture and HSPC were harvested and assayed for their survival at day 7 and 14. Each experimental condition was set up in triplicate. The cells were cultured at 37°C with 5% CO 2. Also, we assessed the multi-lineage differentiation capacity with a selective colony forming units (CFU) assay. Fourteen days after co-culture of each experimental treatment, 500 CD34+ cells were seeded in 6-well plates Smart Dish™ (STEMCELL Technologies). Big burst forming units of erythroid (BFU-E), CFU of granulocyte and megakaryocyte (GM) and granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) were counted and compared between the experimental groups. The signaling pathways were determined using phospho flow-cytometric analysis of pSTAT1, pSTAT3, pSTAT5. All statistically analyzed data is represented as mean ± SD. Differences between groups were analyzed by multiple 2-tailed unpaired Student t tests using Excel. Statistically significant differences were represented as *P <0.05, ** P <0.01 and *** P<0.001.Results:The number of CD34+ cells at day 7 and day 14 of culture in the presence of IFN-γ and TNF-α was significantly lower (21% ± 3 (p<0.0006) and 15% ± 6 (p<0.02), respectively, than that of the control (Fig 1). Importantly, the myelosuppressive effect of IFN-γ and TNF-α was significantly rescued by the addition of Ruxolitinib (at day 14, p<0.05) or IFN-γ neutralizing antibodies (B27 and B133.5, respectively) (day 7 and 14, p<0.01). Improvement in HSPC survival ranged between 1.5-3.3 fold compared to our negative control. Based on the CFU analysis, the CD34+ cells cultured in the presence of B27, B133.5 or Ruxolitinib, were able to produce more CFU at day 7 (p<0.01, p<0.01 and p<0.05 respectively)(Fig 2A). Additionally, Both B27 (p < 0.01) and Ruxolitinb (p ≤ 0.001) were found to produce more CFU-GM on day 14 (Fig 2B). Phospho flow-cytometry demonstrated a significant decrease in STAT1 phosphorylation of CD34+ cells in the presence of B27 and B133.5 (p<0.05, p<0.001, respectively).Conclusions:Our preliminary studies supports the potential benefits of utilizing IFN-γ neutralizing antibodies or Ruxolitinib to improve HSPC survival, proliferation and differentiation in aSAA. Future studies will need to be done to investigate the exact mechanisms of action and the effects of IFN-γ neutralizing antibodies in an animal model of aSAA. [Display omitted] DisclosuresCairo: Amgen: Speakers Bureau; Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Speakers Bureau; Servier: Speakers Bureau; Sobi: Speakers Bureau. OffLabel Disclosure:Ruxolitinib was used to inhibit JAK-STAT signaling pathway in an Ex-Vivo model of Aplastic Anemia. Drug wasn't supplied by drug company.

An Enhanced Zebrafish Xenograft Model to Study the Behavior of Human Hematopoietic Stem Cells and Patient Derived Leukemia In Vivo

Human hematopoietic stem cells (HSCs) are characterized by their unique ability to self-renew and differentiate into multiple cell types. Transplantation of human HSCs into immunocompromised mice is the gold standard for evaluating the in vivo repopulation capacity of these cells and has been adapted to evaluate the leukemic potential of leukemia-initiating cells (LICs) in acute leukemia. In spite of the popularity of this approach, certain cellular subsets do not survive post xenotransplantation as they are dependent on human factors that may be missing in the murine microenvironment. We pioneered human leukemia xenografts in the zebrafish model (Corkery et al., 2011; Bentley et al., 2015) as a parallel strategy, taking advantage of the immuno-permissiveness and transparency of zebrafish larvae to provide unprecedented continuous observation of leukemia evolution. However, as in mice, the addition of human cytokines and growth factors may further improve engraftment and optimize the utility of the zebrafish as a preclinical transplant model for medium to high-throughput therapeutic screening by creating a microenvironment more comparable to that found in patients.Using transgenic technology, we created novel zebrafish lines that express human stem cell factor (SCF/KITLG), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), and stromal cell-derived factor 1 alpha (SDF1α/CXCL12). All of these factors are essential for the survival and expansion of HSCs, progenitors and LICs. CMK, GRWi (Down Syndrome- acute myeloid leukemia (DS-AML)) and Jurkat (T-acute lymphoblastic leukemia) cell lines were injected into the yolk sac of these multi-cytokine zebrafish embryos. These cells engrafted and exhibited increased cell proliferation and physiological migration compared to control casper embryo transplants. Moreover, only in the multi-cytokine fish did these cells home to hematopoietic niches, including the caudal hematopoietic tissue (CHT) (equivalent to the fetal liver) at 7 days-post injection (dpi) and the kidney marrow at 11 dpi.The yolk sac is not the physiological site for hematopoietic cell growth and thus we injected patient-derived AML cells into the circulation of the multi-cytokine fish. We observed similar trends in proliferation and migration as seen with leukemia cell lines. Historically, human HSCs do not survive beyond 24h in zebrafish xenografts (Pruvot et al. 2011). Given our success with patient-derived leukemia samples, we wanted to see if our multi-cytokine fish would provide a more favorable environment for human HSC survival. Indeed, primary human umbilical cord blood-derived HSCs were injected into the circulation of zebrafish larvae and survived past 3 dpi in the multi-cytokine fish but not in casper control larvae.Transgenic zebrafish expressing human cytokines provide an enhanced model for studying human leukemia and stem cell biology in a visually tractable system that more closely recapitulates the human microenvironment. The inherent capacity of the zebrafish for rapid and efficient chemical screening will be exploited in this model for preclinical testing of anti-leukemic therapies and to evaluate HSC enhancing agents in vivo . DisclosuresBerman:AGADA Therapeutics: Research Funding.

Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor.

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b’a’ domain (PDIb’a’) tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

Community-Wide Experimental Evaluation of the PROSS Stability-Design Method

Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science.

SCF/C-Kit Signaling Induces Self-Renewal of Dental Pulp Stem Cells

IntroductionThe maintenance of a stem cell pool is imperative to enable healing processes in the dental pulp tissue throughout life. As such, knowing mechanisms underlying stem cell self-renewal is critical to understand pulp pathophysiology and pulp regeneration. The purpose of this study was to evaluate the impact of stem cell factor (SCF) signaling through its receptor tyrosine kinase (c-Kit) on the self-renewal of human dental pulp stem cells (hDPSCs). MethodsThe hDPSCs were stably transduced with lentiviral vectors expressing shRNA-c-Kit or vector control. The impact of the SCF/c-Kit axis on hDPSC self-renewal was evaluated by using a pulpsphere assay in low attachment conditions and by evaluating the expression of polycomb complex protein Bmi-1 (master regulator of self-renewal) by Western blot and flow cytometry. ResultsThe c-Kit–silenced hDPSCs formed fewer pulpspheres when compared with hDPSCs transduced with control vector (P < .05). Evaluation of pulpsphere morphology revealed the presence of 3 distinct sphere types, ie, holospheres, merospheres, and paraspheres. Although c-Kit silencing decreased the number of holospheres compared with control cells (P < .05), it had no effect on the number of merospheres and paraspheres. Recombinant human stem cell factor (rhSCF) increased the number of holospheres (P < .05) and induced dose-dependent Bmi-1 expression in hDPSCs. As expected, the inductive capacity of rhSCF on Bmi-1 expression and fraction of Bmi-1–positive cells was inhibited when we silenced c-Kit in hDPSCs. ConclusionsThese results unveiled the role of SCF/c-Kit signaling on the self-renewal of hDPSCs and suggested that this pathway enables long-term maintenance of stem cell pools in human dental pulps.

Human granulocyte-colony stimulating factor (G-CSF)/stem cell factor (SCF) fusion proteins: design, characterization and activity.

BackgroundStem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are well-characterized vital hematopoietic growth factors that regulate hematopoiesis. G-CSF and SCF synergistically exhibit a stimulatory effect on hematopoietic progenitors. The combination of G-CSF and SCF has been used for mobilization of peripheral blood progenitor cells in cancer and non-cancerous conditions. To overcome challenges connected with the administration of two cytokines, we developed two fusion proteins composed of human SCF and human G-CSF interspaced by an alpha-helix-forming peptide linker.MethodsThe recombinant proteins SCF-Lα-GCSF and GCSF-Lα-SCF were purified in three steps using an ion-exchange and mixed-mode chromatography. The purity and quantity of the proteins after each stage of purification was assessed using RP-HPLC, SDS-PAGE, and the Bradford assays. Purified proteins were identified using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and the Western blot analyses. The molecular weight was determined by size exclusion HPLC (SE-HPLC). The activity of heterodimers was assessed using cell proliferation assays in vitro. The capacity of recombinant fusion proteins to stimulate the increase of the absolute neutrophil count in rats was determined in vivo. The binding kinetics of the proteins to immobilized G-CSF and SCF receptors was measured using total internal reflection ellipsometry and evaluated by a standard Langmuir kinetics model.ResultsThe novel SCF-Lα-GCSF and GCSF-Lα-SCF proteins were synthesized in Escherichia coli. The purity of the heterodimers reached >90% as determined by RP-HPLC. The identity of the proteins was confirmed using the Western blot and HPLC/ESI-MS assays. An array of multimeric forms, non-covalently associated dimers or trimers were detected in the protein preparations by SE-HPLC. Each protein induced a dose-dependent proliferative response on the cell lines. At equimolar concentration, the heterodimers retain 70–140% of the SCF monomer activity (p ≤ 0.01) in promoting the M-07e cells proliferation. The G-CSF moiety in GCSF-Lα-SCF retained 15% (p ≤ 0.0001) and in SCF-Lα-GCSF retained 34% (p ≤ 0.01) of the monomeric G-CSF activity in stimulating the growth of G-NFS-60 cells. The obtained results were in good agreement with the binding data of each moiety in the fusion proteins to their respective receptors. The increase in the absolute neutrophil count in rats caused by the SCF-Lα-GCSF protein corresponded to the increase induced by a mixture of SCF and G-CSF.

Molecular modeling and co-expression analysis of human stem cell factor as fusion partner to granulocyte colony stimulating factor for improving their bioactivity

Human granulocyte colony stimulating factor (hG-CSF) is an expensive hematopoietic growth factor that is clinically used in human for the treatment of neutropenia in diseases such as AIDS, aplastic anemia, myelodysplastic syndrome and congenital or chemotherapy-induced neutropenia. Here, through a computational biology approach, we show that human stem cell factor (hSCF) could be a better fusion partner than human thyroid peroxidase (hTPO), human erythropoietin (hEPO) and human interleukin-3 (hIL3) for co-expression with hG-CSF. Molecular modeling of hG-CSF-hSCF fusion protein with hG-CSF and hSCF receptors showed that binding of fusion protein with human granulocyte colony stimulating factor receptor (hG-CSFR) did not inhibit its binding to human stem cell factor receptor (hSCFR) and vice versa. To validate the results, coding sequences of hG-CSF and hSCF were cloned and co-expressed as fusion protein and their bioactivity was evaluated on hG-CSF responsive 3T3 cell line. The fused expression vector expressed recombinant hG-CSF-hSCF upon IPTG-induction, as revealed by real-time polymerase chain reaction (RT-PCR), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Bioactivity analysis confirmed that rhG-CSF-hSCF protein had higher bioactivity than hG-CSF. Thus, hSCF could be a good fusion partner for hG-CSF and its co-expression as hG-CSF-hSCF may offer an alternative to individual use of two hematopoietic factors in clinics. Future studies should determine the purification strategies, folding status and mechanism of action of the recombinant proteins. Communicated by Ramaswamy H. Sarma

Computational Design for the Production of Secretory Recombinant Codon-Optimized Human Stem Cell Factor (SCF) in Chinese Hamster Ovary (CHO) Cells With an Appropriate Signal Peptide: An Intensive In Silico Study

Background: Production of the recombinant proteins in mammalian cells is an important issue with a bio-therapeutic purpose. Numerous efforts have been focused on the improvement of the yields of recombinant proteins, which include optimization of conventional biological processes, selection of appropriate signal peptides, codon optimization, and re-engineering of cells to produce more proteins. Stem cell factor (SCF) is a blood cytokine which activates the c-Kit receptor. This factor is crucial not only for the differentiation of hematopoietic progenitor cells but also for the survival, proliferation, and differentiation of mast cells. Recently, its therapeutic role in several diseases such as Alzheimer’s and myocardial infarction has been investigated. Therefore, the aim of this study was to design a secretory recombinant human SCF with the maximal yield in an appropriate mammalian host cell as Chinese hamster ovary (CHO) cells using the computational studies. Methods: As the first step, computational simulation studies were carried out to design the appropriate signal peptide for the human SCF protein. Codon optimized coding sequence of hSCF was transferred into a eukaryotic expression vector (pBudCE4.1). Recombinant vector (pBudCE4.1/SCF) was transfected into CHO cells and the stably transformed cells were screened and isolated. Subsequently, the expression of SCF was defined by reverse transcription polymerase chain reaction (RT-qPCR) in stably transformed cells. Results: Our bioinformatics studies indicated that Azurocidin signal peptide could be a suitable signal peptide for the production of SCF proteins in the CHO cells. Accordingly, computational studies revealed that the presence of 6×His-tag did not have a significant impact on the three-dimensional structure of the protein. Furthermore, the expression of hSCF was significant in the stable CHO cells. Conclusion: The use of this approach may, therefore, lead to the production of highly efficient recombinant hSCF, which would be feasible for the mass production of this factor for therapeutic purposes.

High activation and skewed T cell differentiation are associated with low IL-17A levels in a hu-PBL-NSG-SGM3 mouse model of HIV infection.

The humanized NOD/SCID/IL-2 receptor γ-chainnull (NSG) mouse model has been widely used for the study of HIV pathogenesis. Here, NSG mice with transgenic expression of human stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 (NSG-SGM3) were injected with peripheral blood leukocytes (PBL mice) from two HIV-infected (HIV+ ) patients who were under anti-retroviral therapy (ART; referred as HIV+ mice) or one HIV-seronegative healthy volunteer (HIV- ). Such mice are either hu-PBL-NSG-SGM3 HIV+ or HIV- mice, depending on the source of PBL. The kinetics of HIV replication and T cell responses following engraftment were evaluated in peripheral blood and secondary lymphoid tissues. High HIV replication and low CD4:CD8 ratios were observed in HIV+ mice in the absence of anti-retroviral therapy (ART). Consistent with high activation and skewed differentiation of T cells from the HIV-infected donor, HIV+ mice exhibited a higher T cell co-expression of human leukocyte antigen D-related (HLA-DR) and CD38 than HIV- mice, as well as a shifted differentiation to a CCR7- CD45RA+ terminal effector profile, even in the presence of ART. In addition, HIV replication and the activation/differentiation disturbances of T cells were associated with decreased plasma levels of IL-17A. Thus, this hu-PBL-NSG-SGM3 mouse model recapitulates some immune disturbances occurring in HIV-infected patients, underlying its potential use for studying pathogenic events during this infection.

Mesenchymal stem cells restore the sperm motility from testicular torsion-detorsion injury by regulation of glucose metabolism in sperm

BackgroundTesticular torsion is an urological emergency that may lead to infertility due to ischemic injury. Promptly surgical correction by orchiopexy is the only way to avoid infertility and no effective treatment for restoration of spermatogenesis. We previously reported that mesenchymal stem cells (MSCs), through local injection upon testicular torsion-detorsion, restored the spermatogenesis without differentiation into sperm. In this study, molecular mechanisms of MSCs in regulating germ cell activity induced by testicular torsion-detorsion were investigated.MethodsSixteen male Sprague-Dawley rats 6–8 weeks old received left testis 720° torsion for 3 h followed by detorsion with or without MSCs. Right inguinal skin incision without testicular torsion served as control. MSCs with 3 × 104 cells were locally injected into left testis 30 min before detorsion. Three days after the surgery, orchiectomy was executed and the testis, epididymis, and sperm were separated to each other. Functional assessments on sperm included counting sperm amount and sperm motility, staining F-actin, and quantifying adenosine triphosphate (ATP) content. The hallmarks of glycogenesis and glycolysis in each tissue segment were measured by Western blot.ResultsTesticular torsion-detorsion significantly decreased the amount of sperm, inhibited the motility, declined the F-actin expression, and reduced the content of ATP in sperm. Local injection of MSCs improved sperm function, particularly in sperm motility. With MSCs, ATP content and F-actin were preserved after testicular torsion-detorsion. MSCs significantly reversed the imbalance of glycolysis in sperm and testis induced by testicular torsion-detorsion, as evidenced by increasing the expression of phosphoglycerate kinase 2 and glyceraldehyde-3-phosphate dehydrogenase-spermatogenic, activating Akt, and increasing glycogen synthase kinase 3 (GSK3), which led to the increase in glycolysis cascades and ATP production. Human stem cell factor contributed the activation of Akt/GSK3 axis when sperm suffered from testicular torsion-detorsion-induced germ cell injury.ConclusionsLocal injection of MSCs into a testis damaged by testicular torsion-detorsion restores sperm function mainly through the improvement of sperm motility and energy. MSCs reversed the imbalance of glycogenesis and glycolysis in sperm by regulating Akt/GSK3 axis. Thus, MSCs may potentially rescue torsion-detorsion-induced infertility via local injection.

BRD4 Degradation Is a Potent Approach to Block MYC Expression and to Overcome Multiple Forms of Stem Cell Resistance in Ph+ CML

Chronic myeloid leukemia (CML) is a hematopoietic stem cell neoplasm in which BCR-ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients, leukemic cells can be kept under control by BCR-ABL1 tyrosine kinase inhibitors (TKI). Nevertheless, resistance against one or more TKI may occur. Therefore, research is focusing on novel potential drug targets in CML. We have recently identified the epigenetic reader bromodomain-containing protein 4 (BRD4) as a new therapeutic target in leukemic stem cells (LSC) in acute myeloid leukemia. In the present study, we examine the expression of BRD4 and its downstream effector MYC in CML cells and asked whether BRD4 serves as a drug target in CML cells and whether BRD4-targeting drugs, including JQ1 and newly developed BRD4 degraders (dBET1 and dBET6) are able to overcome LSC resistance in CML. Primary CML cells were obtained from 22 patients with chronic phase (CP) CML and 3 with blast phase (BP) CML. As determined by qPCR and/or immunocytochemistry, the CML cell lines KU812 and K562 as well as primary CML cells expressed BRD4 and MYC. All three BRD4-targeting drugs (JQ1, dBET1 and dBET6) were found to decrease MYC expression in KU812 and K562 cells as assessed by Western blotting. In 3H-thymidine uptake experiments, JQ1 and dBET6 were found to inhibit the proliferation of KU812 in a dose-dependent manner (IC50, JQ1: 100-500 nM; dBET6: 50-100 nM) whereas dBET1 showed only little if any effects on growth of KU812 cells (IC50: 1-5 µM), and in K562 cells, only dBET6 was found to inhibit growth with a reasonable IC50 value (250-500 nM). Corresponding results were obtained when examining drug effects on survival of CML cell lines by Annexin-V/PI staining. All three BRD4-targeting drugs were found to inhibit proliferation of primary CP CML cells with varying IC50 values. As expected, growth-inhibitory effects of dBET6 were more pronounced (IC50: <100 nM) compared to effects seen with JQ1 and dBET1. dBET1 and dBET6 were also found to inhibit growth of primary CML cells obtained from patients with BP CML, whereas JQ1 was not effective. JQ1 also failed to suppress survival on CML CD34+/CD38− LSC. By contrast, dBET1 induced apoptosis in CML LSC at 1 µM and dBET6 induced apoptosis in CML LSC at 0.1 µM. dBET6 induced apoptosis in CML LSC obtained from patients with imatinib-sensitive CML as well as patients with imatinib-resistant CML harboring BCR-ABL1 T315I or BCR-ABL1 F317L. Finally, pre-incubation of CD34+ CP CML cells with dBET6 resulted in reduced leukemic engraftment in NSG mice exhibiting human membrane-bound stem cell factor, SCF [NSG-Tg(hu-mSCF)] 6 months after transplantation (engraftment with CD45+/CD33+/CD19−cells in control mice receiving DMSO-treated cells: 8.1±6.6% vs mice receiving dBET6-treated cells: 1.1±0.6%). To further explore the ability of dBET6 to interfere with LSC resistance in CML, we established a co-culture system mimicking LSC-niche interactions in the osteoblastic niche. In this model, co-culturing K562 cells, KU812 cells or primary CML LSC with the osteoblast-like osteosarcoma cell line CAL-72 resulted in resistance against nilotinib and ponatinib. In this culture system, JQ1 was found to partially restore TKI effects in K562 cells and completely restored TKI effects in KU812 cells. Interestingly, JQ1 was not able to restore TKI effects in primary CML LSC in these co-cultures. However, dBET6 was found to overcome niche cell-induced TKI-resistance of primary CML LSC. Finally, we were able to demonstrate that JQ1, dBET1 and dBET6 inhibit interferon-gamma-induced upregulation of PD-L1 expression in CML LSC. Together we show that BRD4 and MYC are potential new therapeutic drug targets in CML and that the BET-degrader dBET6 overcomes multiple forms of LSC resistance, including i) intrinsic resistance, ii) mutation-induced resistance, iii) niche induced resistance and iv) checkpoint-mediated resistance. Whether BRD4 degradation is also able to overcome TKI-resistance of BCR-ABL1+ LSC in vivo in patients with CML remains to be determined in clinical trials. DisclosuresHoermann:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria. Wolf:BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding. Mayer:Amgen: Research Funding; Novartis: Research Funding. Zuber:Mirimus Inc.: Consultancy, Other: Shareholder; Boehringer Ingelheim GmbH & Co KG: Research Funding. Sperr:Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Valent:Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria.

Reprogrammed Adult Human Endothelium into Hematopoietic Stem Cells Yields Functional T Cells In Vivo

During development, the hematopoietic stem cells that go on to populate the bone marrow and give rise to all blood cell lineages emerge from a specialized endothelial subpopulation. We have previously harnessed this vestigial identity to achieve the direct conversion of adult endothelial cells (ECs) into long-term engraftable hematopoietic stem and progenitor cells (rEC-HSPCs); however, to date, we had only detected and characterized functional T cells that result from the transplantation, engraftment, and differentiation of mouse rEC-HSPCs (Lis, R. et al., Conversion of adult endothelium to immunocompetent haematopoietic stem cells. Nature, 545:439-445. 2017).We first reprogrammed adult human endothelium using lentiviral vectors encoding FOSB, GFI1, SPI1, and RUNX1 (FGRS) under constitutive promoters and an inductive vascular niche (Sandler V. et al., Reprogramming of human endothelium into hematopoietic cells requires vascular niche induction. Nature, 511:312-8. 2014). The resulting rEC-HSPCs maintained exogenous expression of the four transcription factors for over 20 weeks post-transplantation into immuno-compromised NSG mice. Constitutive expression of Spi1, however, has been shown to hinder lymphoid differentiation in vivo by blocking T lymphopoiesis (Anderson, M. et al. Constitutive expression of PU.1 in fetal hematopoietic progenitors blocks T cell development at the pro-T cell stage. Immunity 16:285-296. 2002). In addition, mice of the NSG strain cannot educate native B or T cells to maturity. Our system of constitutive exogenous FGRS expression was therefore unable to confer transplanted immuno-compromised mice the ability to generate an adaptive immune response.Here, we obtained human rEC-HSPCs making use of (i) doxycycline-inducible vectors to temporarily overexpress FGRS and (ii) transgenic substrains of NSG mice (one carrying human stem cell factor, granulocyte/macrophage colony-stimulating factor, and interleukin 3; the other, human major histocompatibility complex class I as well as beta-2 microglobulin) for transplantation assays. We show that human rEC-HSPCs engraft primary- and secondary-transplanted mice for over a year at levels of up to 20% in the spleen or bone marrow. Engrafted cells differentiate into all blood lineages including phenotypically and functionally mature T cells in the absence of exogenous FGRS expression in vivo. Notably, the resulting T cells undergo TCR rearrangement and are able to clear viral particles one week post-LCMV infection. Functional and phenotypic analyses are presented in juxtaposition with experiments using cord blood-transplanted mice. These results demonstrate that our present direct conversion strategy generates bona fide human hematopoietic stem cells from adult endothelial cells. DisclosuresRafii:Angiocrine Bioscience: Equity Ownership.

Therapeutic Effect of Recombinant Human Stem Cell Factor on Rhesus Monkeys with Severe Acute Radiation Sickness

To study the therapeutic effect of rhSCF early administration on rhesus monkeys with severe acute radiation sickness(ARS). Twelve adult monkeys totally exposed to 7.0 Gy 60Co were divided into radiation control and SCF groups, and monkeys in SCF group were subcutaneously injected recombinant human SCF(rhSCF) 200 µg/kg at half an hour and 24 hour after irradiation, while the radiation control monkeys were injected physiological saline. Survival was monitored and hematopoiesis was evaluated at 40 days following early treatment. 6 animals treated with rhSCF all survived, while 2 in irradiated controls survived on 40 day after radiation. rhSCF treatment promoted hematopoiesis recovery significantly, increased the nadir of white blood cells, neutrophils and platelets, and simplified supportive care in ARS rhesus monkeys. RhSCF injection soon after TBI taken shows an significant therapeutic efficiency on rhesus monkeys with severe acute radiation sickness.