Combination Of Stem Cell Factor Research Articles

Single-Cell RNA Sequencing Reveals Immunomodulatory Effects of Stem Cell Factor and Granulocyte Colony-Stimulating Factor Treatment in the Brains of Aged APP/PS1 Mice.

Alzheimer's disease (AD) leads to progressive neurodegeneration and dementia. AD primarily affects older adults with neuropathological changes including amyloid-beta (Aβ) deposition, neuroinflammation, and neurodegeneration. We have previously demonstrated that systemic treatment with combined stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) reduces the Aβ load, increases Aβ uptake by activated microglia and macrophages, reduces neuroinflammation, and restores dendrites and synapses in the brains of aged APPswe/PS1dE9 (APP/PS1) mice. However, the mechanisms underlying SCF+G-CSF-enhanced brain repair in aged APP/PS1 mice remain unclear. This study used a transcriptomic approach to identify the potential mechanisms by which SCF+G-CSF treatment modulates microglia and peripheral myeloid cells to mitigate AD pathology in the aged brain. After injections of SCF+G-CSF for 5 consecutive days, single-cell RNA sequencing was performed on CD11b+ cells isolated from the brains of 28-month-old APP/PS1 mice. The vast majority of cell clusters aligned with transcriptional profiles of microglia in various activation states. However, SCF+G-CSF treatment dramatically increased a cell population showing upregulation of marker genes related to peripheral myeloid cells. Flow cytometry data also revealed an SCF+G-CSF-induced increase of cerebral CD45high/CD11b+ active phagocytes. SCF+G-CSF treatment robustly increased the transcription of genes implicated in immune cell activation, including gene sets that regulate inflammatory processes and cell migration. The expression of S100a8 and S100a9 was robustly enhanced following SCF+G-CSF treatment in all CD11b+ cell clusters. Moreover, the topmost genes differentially expressed with SCF+G-CSF treatment were largely upregulated in S100a8/9-positive cells, suggesting a well-conserved transcriptional profile related to SCF+G-CSF treatment in resident and peripherally derived CD11b+ immune cells. This S100a8/9-associated transcriptional profile contained notable genes related to pro-inflammatory and anti-inflammatory responses, neuroprotection, and Aβ plaque inhibition or clearance. Altogether, this study reveals the immunomodulatory effects of SCF+G-CSF treatment in the aged brain with AD pathology, which will guide future studies to further uncover the therapeutic mechanisms.

Single cell RNA sequencing reveals immunomodulatory effects of stem cell factor and granulocyte colony-stimulating factor treatment in the brains of aged APP/PS1 mice.

Alzheimers disease leads to progressive neurodegeneration and dementia. Alzheimers disease primarily affects older adults with neuropathological changes including amyloid beta deposition, neuroinflammation, and neurodegeneration. We have previously demonstrated that systemic treatment with combined stem cell factor, SCF, and granulocyte colony stimulating factor, GCSF, reduces amyloid beta load, increases amyloid beta uptake by activated microglia and macrophages, reduces neuroinflammation, and restores dendrites and synapses in the brains of aged APP-PS1 mice. However, the mechanisms underlying SCF-GCSF-enhanced brain repair in aged APP-PS1 mice remain unclear. This study used a transcriptomic approach to identify potential mechanisms by which SCF-GCSF treatment modulates microglia and peripheral myeloid cells to mitigate Alzheimers disease pathology in the aged brain. After injections of SCF-GCSF for 5 consecutive days, single cell RNA sequencing was performed on CD11b positive cells isolated from the brains of 28-month-old APP-PS1 mice. The vast majority of cell clusters aligned with transcriptional profiles of microglia in various activation states. However, SCF-GCSF treatment dramatically increased a cell population showing upregulation of marker genes related to peripheral myeloid cells. Flow cytometry data also revealed an SCF-GCSF-induced increase of cerebral CD45high-CD11b positive active phagocytes. SCF-GCSF treatment robustly increased the transcription of genes implicated in immune cell activation, including gene sets that regulate inflammatory processes and cell migration. Expression of S100a8 and S100a9 were robustly enhanced following SCF-GCSF treatment in all CD11b positive cell clusters. Moreover, the topmost genes differentially expressed with SCF-GCSF treatment were largely upregulated in S100a8-S100a9 positive cells, suggesting a well-conserved transcriptional profile related to SCF-GCSF treatment in resident and peripherally derived CD11b positive immune cells. This S100a8-S100a9-associated transcriptional profile contained notable genes related to proinflammatory and antiinflammatory responses, neuroprotection, and amyloid beta plaque inhibition or clearance. Altogether, this study reveals immunomodulatory effects of SCF-GCSF treatment in the aged brain with Alzheimers disease pathology, which will guide future studies to further uncover the therapeutic mechanisms.

Effects of stem cell factor in follicular fluid and granulosa cells on oocyte maturity and clinical pregnancy.

Stem cell factor (SCF) is implicated in cell growth, proliferation, differentiation, migration, and apoptosis. SCF in follicular fluid (FF) and granulosa cells (GCs) plays a key role in oocyte maturation and clinical pregnancy; however, the exact mechanism is unclear. We aimed to investigate SCF potential in predicting oocyte maturity and clinical pregnancy. We collected 60 FF and 60 GCs samples from different patients with infertility. Real-time polymerase chain reaction and cellular immunofluorescence analyses were used to quantitatively and qualitatively determine SCF concentration in GCs; enzyme-linked immunosorbent assay was used to determine SCF concentration in FF. GC and FF SCF concentrations were positively correlated with metaphase (M)II oocyte proportion and clinical pregnancy (R = 0.280, 0.735 vs R = 0.257, 0.354). SCF concentrations in GCs were significantly higher in the clinical pregnancy group than in the nonclinical pregnancy group. Immunofluorescence analysis showed that SCF expression was higher in the clinical pregnancy and high-MII -oocyte proportion groups. Receiver operating characteristic curve analysis showed that combined SCF and serum anti-Müllerian hormone levels could predict oocyte maturity and clinical pregnancy better than either of these factors alone. SCF concentration in GCs and FF can serve as a predictor of oocyte maturity and clinical pregnancy.

Stem Cell Factor and Granulocyte Colony-Stimulating Factor Promote Remyelination in the Chronic Phase of Severe Traumatic Brain Injury.

Severe traumatic brain injury (TBI) causes long-term disability and death in young adults. White matter is vulnerable to TBI damage. Demyelination is a major pathological change of white matter injury after TBI. Demyelination, which is characterized by myelin sheath disruption and oligodendrocyte cell death, leads to long-term neurological function deficits. Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) treatments have shown neuroprotective and neurorestorative effects in the subacute and chronic phases of experimental TBI. Our previous study has revealed that combined SCF and G-CSF treatment (SCF + G-CSF) enhances myelin repair in the chronic phase of TBI. However, the long-term effect and mechanism of SCF + G-CSF-enhanced myelin repair remain unclear. In this study, we uncovered persistent and progressive myelin loss in the chronic phase of severe TBI. SCF + G-CSF treatment in the chronic phase of severe TBI enhanced remyelination in the ipsilateral external capsule and striatum. The SCF + G-CSF-enhanced myelin repair is positively correlated with the proliferation of oligodendrocyte progenitor cells in the subventricular zone. These findings reveal the therapeutic potential of SCF + G-CSF in myelin repair in the chronic phase of severe TBI and shed light on the mechanism underlying SCF + G-CSF-enhanced remyelination in chronic TBI.

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.

Stem Cell Factor in Combination With Granulocyte Colony-Stimulating Factor Protects the Brain From Capillary Thrombosis-Induced Ischemic Neuron Loss in a Mouse Model of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy (CADASIL) is a Notch3 mutation-induced cerebral small vessel disease, leading to recurrent ischemic stroke and vascular dementia. There is currently no treatment that can stop or delay CADASIL progression. We have demonstrated the efficacy of treatment with combined stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) in reducing cerebral small vessel thrombosis in a TgNotch3R90C mouse model of CADASIL. However, it remains unknown whether SCF+G-CSF treatment protects neurons from microvascular thrombosis-induced ischemic damage. Using bone marrow transplantation to track thrombosis, we observed that capillary thrombosis was widely distributed in the cortex, striatum and hippocampus of 22-month-old TgNotch3R90C mice. However, the capillary thrombosis mainly occurred in the cortex. Neuron loss was seen in the area next to the thrombotic capillaries, and severe neuron loss was found in the areas adjacent to the thrombotic capillaries with bifurcations. SCF+G-CSF repeated treatment significantly attenuated neuron loss in the areas next to the thrombotic capillaries in the cortex of the 22-month-old TgNotch3R90C mice. Neuron loss caused by capillary thrombosis in the cerebral cortex may play a crucial role in the pathogenesis of CADASIL. SCF+G-CSF treatment ameliorates the capillary thrombosis-induced ischemic neuron loss in TgNotch3R90C mice. This study provides new insight into the understanding of CADASIL progression and therapeutic potential of SCF+G-CSF in neuroprotection under microvascular ischemia in CADASIL.

Interaction of Follicle-Stimulating Hormone and Stem Cell Factor to Promote Primordial Follicle Assembly in the Chicken.

Follicle-stimulating hormone (FSH) and KIT signaling are required for ovarian development. In this study the interactive effect of FSH and stem cell factor (SCF) on folliculogenesis was investigated in the chicken. Correlated changes between the FSH receptor and the expression of KIT signaling genes were seen to be involved in the formation of the chicken primordial follicles. Follicle-stimulating hormone and SCF displayed a reciprocal stimulating effect in the promotion of folliculogenesis involving elevated phosphorylation of mitogen-activated protein kinases (MAPK) and protein kinase B (AKT) proteins. Knockdown of c-KIT or SCF reduced the stimulatory effect of FSH on KIT signaling as well as upon MAPK and AKT phosphorylation. Treatment of FSH and SCF in combination enhanced ovarian cell proliferation and N-cadherin expression, but inhibited cell apoptosis and E-cadherin expression. Overall, the reciprocal stimulating effect of FSH and SCF in promoting chicken follicle assembly involving accelerated ovarian cell proliferation, N-cadherin expression, inhibited cell apoptosis, and E-cadherin expression is demonstrated.

Stem Cell Factor in Combination with Granulocyte Colony-Stimulating Factor reduces Cerebral Capillary Thrombosis in a Mouse Model of CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is a cerebral small vascular disease caused by NOTCH3 mutation-induced vascular smooth muscle cell (VSMC) degeneration, leading to ischemic stroke and vascular dementia. Our previous study has demonstrated that repeated treatment with a combination of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) reduces VSMC degeneration and cerebral endothelial cell (EC) damage and improves cognitive function in a mouse model of CADASIL (TgNotch3R90C). This study aimed to determine whether cerebral thrombosis occurs in TgNotch3R90C mice and whether repeated SCF+G-CSF treatment reduces cerebral thrombosis in TgNotch3R90C mice. Using the approaches of bone marrow transplantation to track bone marrow-derived cells and confocal imaging, we observed bone marrow-derived blood cell occlusion in cerebral small vessels and capillaries (thrombosis). Most thrombosis occurred in the cerebral capillaries (93% of total occluded vessels), and the thrombosis showed an increased frequency in the regions of capillary bifurcation. Degenerated capillary ECs were seen inside and surrounding the thrombosis, and the bone marrow-derived ECs were also found next to the thrombosis. IgG extravasation was seen in and next to the areas of thrombosis. SCF+G-CSF treatment significantly reduced cerebral capillary thrombosis and IgG extravasation. These data suggest that the EC damage is associated with thrombosis and blood–brain barrier leakage in the cerebral capillaries under the CADASIL-like condition, whereas SCF+G-CSF treatment diminishes these pathological alterations. This study provides new insight into the involvement of cerebral capillary thrombosis in the development of CADASIL and potential approaches to reduce the thrombosis, which may restrict the pathological progression of CADASIL.

THE THERAPEUTIC EFFECTS OF HEMATOPOIETIC GROWTH FACTORS IN A MOUSE MODEL OF CEREBRAL AMYLOIDOSIS

Emerging evidence has revealed that dysfunction of the innate immune system for Aβ clearance plays a key role in cerebral Aβ deposition and in Alzheimer's disease progression. Our earlier study has demonstrated that the combination of stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) (SCF+G-CSF), the two essential hematopoietic growth factors, increases bone marrow-derived monocyte/macrophage (BMDM) recruitment, enhances the association of BMDMs with amyloid plagues, and reduces Aβ deposits in APP/PS1 mice 9 months after treatment. The aim of this study is to further identify the therapeutic efficacy of SCF, G-CSF, or SCF+G-CSF in APP/PS1 mice. Male APP/PS1 mice (10 month old) were randomized to receive 12- day injections (s.c.) of either vehicle solution, SCF (200μg/kg), G-CSF (50μg/kg), or SCF+G-CSF. Age-matched wild-type (WT) mice served as normal controls. The spatial learning and memory was evaluated by water maze testing at 3 and 7 months post-treatment. The Aβ load in the brain was determined by ELISA, immunohistochemistry (IHC) and Thioflavin-S staining 8 months after treatment. APP/PS1 control mice spent significantly longer time to find the hidden platform than WT mice. SCF+G-CSF-treated APP/PS1mice found the platform significantly faster than vehicle controls, SCF alone- or G-CSF alone-treated APP/PS1 mice 3 months after treatment. At this time point, no difference was found between SCF+G-CSF-treated APP/PS1mice and WT mice, but significant difference was observed between SCF- or G-CSF-treated APP/PS1mice and WT mice. At 7 months post-treatment, the latency to find the platform was significantly reduced in SCF+G-CSF-treated or SCF-treated APP/PS1 mice. Eight months after treatment, only SCF+G-CSF treatment showed a significant reduction of Aβ1–40, Aβ 1–42. The deposition of Aβ (4G8+, Thioflavin-S+) was significantly decreased by SCF+G-CSF in both the cortex and hippocampus, while G-CSF alone only showed reduction in the hippocampus of APP/PS1 mice. SCF+G-CSF has synergistic effects in long-term improvement of cognitive function, and long-lasting reduction of brain Aβ load in APP/PS1mice. This study suggests a robust therapeutic potential of SCF and G-CSF combination over SCF or G-CSF alone in treatment of Alzheimer's disease. This study was supported by the NIA/NIH in the United States (R01AG051674).

The Ex-Vivo Expansion of Megakaryocytic Progenitors from Hematopoietic Stem Cells for Thrombocytopenia in NOD/SCID Mice

Objective: Thrombocytopenia is a common clinical problem in patients with cancer or bone marrow transplantation. Currently it is mainly managed by platelet transfusion. Repeated platelet transfusions are associated with the risks of transfusion reactions/alloimmunisation and may lead to platelet refractoriness. Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is other strategy for the treatment of thrombocytopenia. This study aimed to establish efficient conditions for the expansion of the MK progenitors from enriched CD34(+) cells of umbilical cord blood.Methods: This study investigated the effect of flt-3 ligang (FL), stem cell factor (SCF) and platelet-derived growth factor (PDGF) in combination with other megakaryocyte-promoting cytokines such as thrombopoietin (TPO) on the differentiation and proliferation of megakaryocytic progenitors. As an early acting growth factor, FL may promote the ex vivo expansion of hematopoietic stem and progenitor cells. We compared the effects of FL and SCF in combination with other megakaryocyte-promoting cytokines in megakaryocytic progenitors.Results: In liquid cultures of enriched CD34+ cells from human umbilical cord blood for 14 days, FL plus TPO, interleukin-3 (IL-3), and IL-6 promoted the expansion of nucleated cells, CD34+ cells, CD34+ CD38- cells, and megakaryocyte colony-forming units (CFU-MK) by 300 +/- 115-, 23.8 +/- 11.3-, 33.9 +/- 28.6-, and 584 +/- 220-fold, respectively. Replacing FL with SCF significantly decreased the yield of all cell types. While one human acute lymphoblastic leukemia sample expressed high levels of flt-3 receptor, the four megakaryocytic cell lines (Meg-01, CHRF-288-11, M-07e, and Dami) did not show any positive expression. Our data suggest that the effect of FL in augmenting the expansion of MK progenitors might be due to the early action of FL at the pluripotent stem cell stage. Our results also demonstrated that TPO alone produced a high proportion of CD61(+)CD41(+) cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of in IL-1 beta, FL and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400-fold with the highest yield observed in the presence of TPO, IL-1 beta, IL-3, IL-6 and FL. CD34(+) cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61(+)CD41(+) cells, CFU-MK and CD34(+) cells increased by 101%, 134% and 70%, respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in NOD/SCID mice.Conclusions: This study showed that the present cytokine combination and expansion conditions provide an effective and potentially useful system for the clinical expansion of cord blood for bone marrow transplantation (BMT). PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications. DisclosuresYang:National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).

Effect of bone marrow stem cell mobilisation on the expression levels of cellular growth factors in a rat model of acute tubular necrosis.

The aim of the present study was to observe the mobilisation effects of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) on bone marrow stem cells (BMSCs) in rats with renal ischaemia-reperfusion injury. In addition, the effects of the BMSCs on the expression levels of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were investigated, with the aim to further the understanding of the protective mechanisms of SCF and G-CSF in renal ischaemia-reperfusion injury. The model and treatment groups were established using a model of unilateral renal ischaemia-reperfusion injury, in which the treatment group and the treatment control group were subcutaneously injected once a day with 200 µg/kg SCF and 50 µg/kg G-CSF, 24 h after the modelling, for five consecutive days. The CD34+ cell count was measured in the peripheral blood using flow cytometry. The mRNA expression levels of HGF and EGF were determined using polymerase chain reaction analysis, while the protein expression levels of HGF and EGF were detected using immunohistochemistry. The CD34+ cell count in the peripheral blood of the treatment and treatment control groups was significantly higher compared with that in the model group (P<0.05). However, CD34 expression levels in the cells from the renal tissues of the model and treatment groups were significantly higher compared with that of the control and treatment control groups (P<0.05), with the greatest increase observed in the treatment group. The mRNA and protein expression levels of HGF and EGF in the treatment group were significantly higher compared with the model group (P<0.05). Therefore, the results indicated that a combination of SCF and G-CSF can promote the repair of acute tubular necrosis. This combination, which can mobilise sufficient numbers of BMSCs to migrate back to the injured site, is a key factor in promoting the repair of renal tubular injury. Upregulation of HGF and EGF was also shown to promote the repair of renal tubular injury.

Effects of associated SCF and G-CSF on liver injury two weeks after liver damage: A model induced by thioacetamide administration.

The present study aimed at investigating the beneficial effects of co-administering granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) in a model of chronic liver injury induced by thioacetamide (TAA). Biochemical and histopathology- cal examinations were performed on serum and liver specimens. At the end of the treatment period, the rats were anesthetized with ether, serum was collected and sections of randomly selected fixed liver specimens from each group were embedded in paraffin and processed for light microscopy by staining individual sections with hematoxylin- eosin (HE) stain. Administration of a combination of G-CSF+SCF was carried out two weeks after the TAA treatment. Livers of rats treated with TAA alone exhibited damage, which was significantly less in the group treated with the combination of SCF and G-CSF. Albumin level was 2.35 (g/dl) in the G-CSF+SCF and 1.03 in the TAA- alone group. These differences were statistically significant (P<0.05). Also, in the G- CSF+SCF and TAA group the total protein means (7.16 versus 3.57 mg/dl, respective- ly) were higher than those of the TAA-alone group, and the differences were statistically significant (P<0.05). In the G-CSF+SCF and TAA group the total bilirubin content mean (0.15 versus 0.14 mg/dl, respectively) this difference was not statistically significant (P>0.05).

The Effects of Hematopoietic Growth Factors on Neurite Outgrowth

Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) are initially discovered as the essential hematopoietic growth factors regulating bone marrow stem cell proliferation and differentiation, and SCF in combination with G-CSF (SCF+G-CSF) has synergistic effects on bone marrow stem cell mobilization. In this study we have determined the effect of SCF and G-CSF on neurite outgrowth in rat cortical neurons. Using molecular and cellular biology and live cell imaging approaches, we have revealed that receptors for SCF and G-CSF are expressed on the growth core of cortical neurons, and that SCF+G-CSF synergistically enhances neurite extension through PI3K/AKT and NFκB signaling pathways. Moreover, SCF+G-CSF induces much greater NFκB activation, NFκB transcriptional binding and brain-derived neurotrophic factor (BDNF) production than SCF or G-CSF alone. In addition, we have also observed that BDNF, the target gene of NFκB, is required for SCF+G-CSF-induced neurite outgrowth. These data suggest that SCF+G-CSF has synergistic effects to promote neurite growth. This study provides new insights into the contribution of hematopoietic growth factors in neuronal plasticity.

Effect of mobilization bone marrow stem cells on hypoxia inducible factor-1α and epidermal growth factor in rats with renal injury induced by ischemic reperfusion

Objective To investigate the mobilization of granulocyte-colony stimulating factor(G-CSF) with stem cell factor(SCF) in bone marrow stem cells(BMSC), and to observe the therapeutic effects of BMSC mobilization on repairing ischemia/reperfusion(I/R) renal injury induced by hypoxia inducible factor-1α(HIF-1α) and epidermal growth factor(EGF), and to investigate the mechanism for BMSC mobilization for repairing I/R renal injury. Methods One hundred and sixty male Sprague-Dawley rats(age: 8-10 weeks) were randomly allocated into 4 groups(40 rats in each group): control group(group A), I/R group(group B), SCF+ G-CSF treatment group(group C) and SCF+ G-CSF control group(group D). The expressions of CD34+ and EGF were measured by immunohistochemistry technique, and the expression of EGF mRNA was measured by reverse transcriptase-polymerase chain reaction. Results 1.After I/R injury, the renal tubular epithelial cells were in a state of degeneration, necrosis, even exfoliation.As the time prolonged, the necrotic renal tubules were repaired gradually, but the repair in group C was not good compared with that in group B. The level of group C was obviously lower than that of group B on the 5th, 10th, 17th and 24th day(P 0.05). On 5 d postoperative, the level of CD34+ cells of group B and group C was conspicuous higher than group A and D(P<0.05), and that of group C was conspicuous higher than that of group B(P<0.05). They descended gradually from the 5th day with the time prolonged.3.The expressions of HIF-1α of group B and group C had a highly positive reaction on the 5th day postoperatively and the level of HIF-1α of group B and group C decended to the normal level with the time prolonged, and at each time, the expression of HIF-1α of group C was significantly higher than that of other groups(P<0.05).4.On the 5th day postoperative, the expression of EGF of group B and group C showed a higher positive reaction on the 5th day postoperatively and the level of EGF in group B and group C descended to the normal level with the time prolonged, and at each time the expression of EGF of group C was significantly higher than that of the other groups(P<0.05).5.The expressions of EGF mRNA of group B and C showed higher positive reaction on the 5th day postoperatively(P<0.05). The level of EGF mRNA of group B was higher significantly than that of group A on the 17th day posto-peratively(P<0.05). The level of EGF mRNA of group C was a peak and higher than that of other groups on the 10th day postoperatively(P<0.05). The level of EGF of group B and group C descended to a normal level with the time prolonged.6.There was a positive correlation between the expressions of HIF-1α and EGF(r=0.815, P<0.01). Conclusions Combination of SCF with the mobilization of G-CSF sufficiently homed BMSC to injured renal tissue was an important factor for the recovery of injured renal tubules.Combination of SCF with G-CSF may promote the expression of HIF-1α and EGF in the kidney may be one of the mechanisms of why combination of SCF with G-CSF can promote recovery from renal ischemic reperfusion injury. Key words: Bone marrow stem cell; Ischemic reperfusion, renal; Hypoxia inducible factor-1α; Epidermal growth factor; Rat

Reestablishing Neuronal Networks in the Aged Brain by Stem Cell Factor and Granulocyte-Colony Stimulating Factor in a Mouse Model of Chronic Stroke

Stroke has a high incidence in the elderly. Stroke enters the chronic phase 3 months after initial stroke onset. Currently, there is no pharmaceutical treatment available for chronic stroke. We have demonstrated the therapeutic effects of the combination of stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) (SCF+G-CSF) on chronic stroke. However, it remains unclear how SCF+G-CSF repairs the brain in chronic stroke. In this study, we determined the effects of SCF+G-CSF on neuronal network remodeling in the aged brain of chronic stroke. Cortical brain ischemia was produced in 16–18 month-old transgenic mice expressing yellow fluorescent protein in layer V pyramidal neurons. SCF+G-CSF was subcutaneously injected for 7 days beginning at 3.5 months post-ischemia. Using both live brain imaging and immunohistochemistry, we observed that SCF+G-CSF increased the mushroom-type spines on the apical dendrites of layer V pyramidal neurons adjacent to the infarct cavities 2 and 6 weeks after treatment. SCF+G-CSF also augmented dendritic branches and post-synaptic density protein 95 puncta in the peri-infarct cortex 6 weeks after treatment. These data suggest that SCF+G-CSF treatment in chronic stroke remodels neural circuits in the aged brain. This study provides evidence to support the development of a new therapeutic strategy for chronic stroke.

Biochemical measurements on single erythroid progenitor cells shed light on the combinatorial regulation of red blood cell production

Adult bone marrow (BM) erythrocyte colony-forming units (CFU-Es) are important cellular targets for the treatment of anemia and also for the manufacture of red blood cells (RBCs) ex vivo. We obtained quantitative biochemical measurements from single and small numbers of CFU-Es by isolating and analyzing c-Kit(+)CD71(high)Ter119(-) cells from adult mouse BM and this allowed us to identify two mechanisms that can be manipulated to increase RBC production. As expected, maximum RBC output was obtained when CFU-Es were stimulated with a combination of Stem Cell Factor (SCF) and Erythropoietin (EPO) mainly because SCF supports a transient CFU-E expansion and EPO promotes the survival and terminal differentiation of erythroid progenitors. However, we found that one of the main factors limiting the output in RBCs was that EPO induces a downregulation of c-Kit expression which limits the transient expansion of CFU-Es. In the presence of SCF, the EPO-mediated downregulation of c-Kit on CFU-Es is delayed but still significant. Moreover, treatment of CFU-Es with 1-Naphthyl PP1 could partially inhibit the downregulation of c-Kit induced by EPO, suggesting that this process is dependent on a Src family kinase, v-Src and/or c-Fyn. We also found that CFU-E survival and proliferation was dependent on the level of time-integrated extracellular-regulated kinase (ERK) activation in these cells, all of which could be significantly increased when SCF and EPO were combined with mouse fetal liver-derived factors. Taken together, these results suggest two novel molecular strategies to increase RBC production and regeneration.

The combination of stem cell factor and granulocyte-colony stimulating factor for chronic stroke treatment in aged animals

BackgroundStroke occurs more frequently in the elderly population and presents the number one leading cause of persistent disability worldwide. Lack of effective treatment to enhance brain repair and improve functional restoration in chronic stroke, the recovery phase of stroke, is a challenging medical problem to be solved in stroke research. Our early study has revealed the therapeutic effects of stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) (SCF+G-CSF) on chronic stroke in young animals. However, whether this treatment is effective and safe to the aged population remains to be determined.MethodsCortical brain ischemia was produced in aged C57BL mice or aged spontaneously hypertensive rats. SCF+G-CSF or equal volume of vehicle solution was subcutaneously injected for 7 days beginning at 3–4 months after induction of cortical brain ischemia. Using the approaches of biochemistry assays, flow cytometry, pathology, and evaluation of functional outcome, several doses of SCF+G-CSF have been examined for their safety and efficiency on chronic stroke in aged animals.ResultsAll tested doses did not show acute or chronic toxicity in the aged animals. Additionally, SCF+G-CSF treatment in chronic stroke of aged animals mobilized bone marrow stem cells and improved functional outcome in a dose-dependent manner.ConclusionsSCF+G-CSF treatment is a safe and effective approach to chronic stroke in the aged condition. This study provides important information needed for developing a new therapeutic strategy to improve the health of older adults with chronic stroke.

C-Kit Signaling Blocks B Cell Differentiation of Common Lymphoid Progenitor Cells by Negatively Regulating Stat5-Dependent Activation of Ebf Expressionc-Kit Signaling Blocks B Cell Differentiation of Common Lymphoid Progenitor Cells by Negatively Regulating Stat5-Dependent Activation of Ebf1 Expression

Abstract 1209B-lymphocyte commitment from common lymphoid progenitor cells (CLP) is orchestrated by a number of transcription factors and external cues such as IL-7, stem cell factor (SCF) and Flt3 ligand. IL-7 signaling plays a critical role in promoting B-cell differentiation by inducing the expression of transcription factor Ebf1 via the Jak-Stat5 signaling pathway (Kikuchi et al., 2007; Tsapogas et al., 2011).Previously, it has been shown that Flt3 ligand acts synergistically with IL-7 during the earliest stages of B-cell commitment (CLP and pre-pro-B fraction)(Åhsberg et al., 2010), but is downregulated by the pre-B stage to ensure normal development of B lymphocytes (Holmes et al., 2006). The role of SCF signaling in this early cell fate decision, however, remains unclear.Here, we demonstrate that SCF dominantly inhibits IL-7-induced B-cell differentiation from CLP by negatively regulating the Jak-Stat5 signaling pathway.Co-culture of bone marrow Lin-Kit+Sca-1+(LSK) cells with OP9 feeder cells in the presence of IL-7 (10 ng/ml) and SCF (10 ng/ml) resulted in 1.5–2-fold reduction in the frequency and absolute numbers of B220+CD19+ cells compared to cultures stimulated with IL-7 alone. To test whether c-Kit signaling inhibits B cell commitment in vivo, BM progenitor cells overexpressing a constitutively active form of KIT (MSCV-Kit-V814D-IRES-VEX) were transplanted into lethally irradiated mice. VEX+ cells in reconstituted mice had a very limited potential for B-lymphoid lineage differentiation, as judged by severe reduction in the frequency of B220+ cells in BM at 7 weeks post-transplant [B220+CD19-IgM-: 1.5%±0.3 (control) vs 0.6%±0.2 (V814D), p<0.05, n=6); B220+CD19+: 19.4%± 2.7 vs 2.1%±0.6, p<0.001; CD19+IgM+: 6.7%±1.3 vs 0.9%±0.3, p<0.01)].Analysis of CLP cells isolated from IL-7−/− mice revealed that co-culture with IL-7 and SCF inhibited IL-7-induced upregulation of Ebf1 as well as other Stat5 target genes (Socs3, Cish, Osm). Furthermore, short-term treatment of BM cells isolated from IL-7−/− mice with a combination of SCF and IL-7 resulted in a significant reduction of phospho-Stat5 in CLP cells compared to cells treated with IL-7 alone.Class III receptor tyrosine kinases such as Flt3 and c-Kit are able to activate multiple signaling pathways via a juxtamembrane SH2-docking site. Mutation of the SH2 domain in the context of the Kit-V814D mutation (Kit-V814D-Y567/569F) did not rescue B-cell development when this mutant protein was expressed in primary murine bone marrow cells in vivo. In contrast, expression of a mutant form of Kit-V814D that inhibited binding of the SH2B adapter protein 2 (APS) (Kit-V814D-I570A/L937A) rescued B-lymphopoiesis, indicating that a signaling pathway coupled to APS dominantly suppresses B cell commitment when CLP are co-stimulated with SCF and IL-7.Based on the observations that APS is a known adapter for the E2-ubiquitin ligase, c-Cbl, which has been shown to negatively regulate Stat5 in other systems, we speculate that c-Kit signaling inhibits IL-7-induced Stat5 activation via an APS-Cbl pathway during early B-cell development. Further characterization of this inhibitory developmental pathway will be discussed. Disclosures:No relevant conflicts of interest to declare.

Stem cell factor and granulocyte colony-stimulating factor promote neuronal lineage commitment of neural stem cells

Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) were originally discovered as growth factors for hematopoietic stem cells (HSCs). It has been well defined that SCF and G-CSF contribute to regulation of lineage commitment for HSCs. However, little is known about whether SCF and G-CSF play roles in the determination and differentiation of neural stem cells (NSCs). Here we demonstrate the novel function of SCF and G-CSF in controlling cell cycle and cell fate determination of NSCs. We also observe that SCF and G-CSF promote neuronal differentiation and inhibit astroglial differentiation at the early stage of differentiation. In addition, our research data reveal that SCF in combination with G-CSF has a dual function in promoting cell cycle exit and directing neuronal fate commitment at the stage of NSC dividing. This coordination effect of SCF+G-CSF on cell cycle arrest and neuronal differentiation is through enhancing neurogenin 1 (Ngn1) activity. These findings extend current knowledge regarding the role of SCF and G-CSF in the regulation of neurogenesis and provide insights into the contribution of hematopoietic growth factors to brain development and remodeling.

Stem cell factor and granulocyte colony-stimulating factor reduce β-amyloid deposits in the brains of APP/PS1 transgenic mice.

IntroductionAlzheimer's disease (AD) is widely recognized as a serious public health problem and heavy financial burden. Currently, there is no treatment that can delay or stop the progressive brain damage in AD. Recently, we demonstrated that stem cell factor (SCF) in combination with granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) has therapeutic effects on chronic stroke. The purpose of the present study is to determine whether SCF+G-CSF can reduce the burden of β-amyloid deposits in a mouse model of AD.MethodsAPP/PS1 transgenic mice were used as the model of AD. To track bone marrow-derived cells in the brain, the bone marrow of the APP/PS1 mice was replaced with the bone marrow from mice expressing green fluorescent protein (GFP). Six weeks after bone marrow transplantation, mice were randomly divided into a saline control group and a SCF+G-CSF-treated group. SCF in combination with G-CSF was administered subcutaneously for 12 days. Circulating bone marrow stem cells (CD117+ cells) were quantified 1 day after the final injection. Nine months after treatment, at the age of 18 months, mice were sacrificed. Brain sections were processed for immunohistochemistry to identify β-amyloid deposits and GFP expressing bone marrow-derived microglia in the brain.ResultsSystemic administration of SCF+G-CSF to APP/PS1 transgenic mice leads to long-term reduction of β-amyloid deposition in the brain. In addition, we have also observed that the SCF+G-CSF treatment increases circulating bone marrow stem cells and augments bone marrow-derived microglial cells in the brains of APP/PS1 mice. Moreover, SCF+G-CSF treatment results in enhancement of the co-localization of bone marrow-derived microglia and β-amyloid deposits in the brain.ConclusionsThese data suggest that bone marrow-derived microglia play a role in SCF+G-CSF-induced long-term effects to reduce β-amyloid deposits. This study provides insights into the contribution of the hematopoeitic growth factors, SCF and G-CSF, to limit β-amyloid accumulation in AD and may offer a new therapeutic approach for AD.