Expression Of SCF Research Articles

Human embryonic stem cell-derived immunity-and-matrix-regulatory cells on collagen scaffold effectively treat rat corneal alkali burn

Corneal alkali burns (CAB) are a severe form of ocular injury that often leads to significant vision loss, with limited effective treatment options available beyond corneal transplantation. Immunity and matrix-regulatory cells (IMRCs) have emerged as a promising alternative due to their ability to modulate immune responses and support tissue repair. This study investigates the efficacy of IMRCs on collagen scaffolds (IMRCs-col) for treating CAB in a rat model. We developed a novel treatment combining IMRCs with a collagen scaffold to align with the ocular surface structure. In vitro analyses showed that IMRCs-col significantly upregulated the expression of immune regulatory molecules, including IL-1RA and SCF. Additionally, IMRCs-col effectively inhibited the production of pro-inflammatory cytokines (IL-8 and Gro-a/CXCL1) while promoting pro-regenerative cytokines (bFGF, HGF, and PDGF). In an animal model of CAB, IMRCs-col transplantation demonstrated substantial efficacy in restoring corneal opacity and reducing neovascularization. Histological examination revealed reduced inflammation and improved corneal tissue regeneration compared to untreated CAB. Enhanced activation of pathways associated with anti-inflammatory responses and tissue repair was observed at days 3, 7, and 21 post-treatment.

Induction of Thymus Atrophy and Disruption of Thymocyte Development by Fipronil through Dysregulation of IL-7-Associated Genes.

The susceptibility of the immune system to immunotoxic chemicals is evident, particularly in the thymus, a vital primary immune organ prone to atrophy due to exposure to toxicants. Fipronil (FPN), a widely used insecticide, is of concern due to its potential neurotoxicity, hepatotoxicity, and immunotoxicity. Our previous study showed that FPN disturbed the antigen-specific T-cell functionality in vivo. As T-cell lineage commitment and thymopoiesis are closely interconnected with the normal function of the T-cell-mediated immune responses, this study aims to further examine the toxic effects of FPN on thymocyte development. In this study, 4-week-old BALB/c mice received seven doses of FPN (1, 5, 10 mg/kg) by gavage. Thymus size, medulla/cortex ratio, total thymocyte counts, double-positive thymocyte population, and IL-7-positive cells decreased dose-dependently. IL-7 aids the differentiation of early T-cell precursors into mature T cells, and several essential genes contribute to the maturation of T cells in the thymus. Foxn1 ensures that the thymic microenvironment is suitable for the maturation of T-cell precursors. Lyl1 is involved in specifying lymphoid cells and maintaining T-cell development in the thymus. The c-Kit/SCF collaboration fosters a supportive thymic milieu to promote the formation of functional T cells. The expression of IL-7, IL-7R, c-Kit, SCF, Foxn1, and Lyl1 genes in the thymus was significantly diminished in FPN-treated groups with the concordance with the reduction of IL-7 signaling proteins (IL-7, IL-7R, c-KIT, SCF, LYL1, FOXO3A, and GABPA), suggesting that the dysregulation of T-cell lineage-related genes may contribute to the thymic atrophy induced by FPN. In addition, FPN disturbed the functionality of thymocytes with an increase of IL-4 and IFN-γ production and a decrease of IL-2 secretion after T-cell mitogen stimulation ex vivo. Collectively, FPN significantly deregulated genes related to T-cell progenitor differentiation, survival, and expansion, potentially leading to impaired thymopoiesis.

Hypoxic Preconditioning Promotes Survival of Human Adipose Derived Mesenchymal Stem Cell

Background: Contributing factors for improved survival of human adipocytes mesenchymal stem cells (h-AMSCs) cultured through hypoxia preconditioning, in example apoptosis inhibition involving BCL2 and HSP27 expression, trigger signal expression (VEGF), SCF expression, OCT-4 expression, and CD44+ expression. The objective if this study was to explain the mechanism and role of hypoxic preconditioning and the optimal duration of hypoxic preconditioning exposure to improve survival of h-AMSCs. Methods: An experimental laboratory explorative study (in vitro) with hypoxic preconditioning in h-AMSCs cultures. This research was conducted through four stages. First, isolation of h-AMSCs culture from adipose tissue of patients. Second, the characterization of h-AMSCs from adipose tissue by phenotype (flowcytometry) through CD44+, CD90+ and CD45-expression before being pre-conditioned for hypoxic treatment. Third, the hypoxic preconditioning in h-AMSCs culture (in vitro) was performed with an oxygen concentration of 1% for 24, 48 and 72 hours. Fourth, observation of survival from h-AMSCs culture was tested on the role of CD44+, VEGF, SCF, OCT-4, BCL2, HSP27 with Flowcytometry and apoptotic inhibition by Tunnel Assay method. Results: The result of regression test showed that time difference had an effect on VEGF expression (p<0.001;β=-0.482) and hypoxia condition also influenced VEGF expression (p<0.001;β=0.774). The result of path analysis showed that SCF had effect on OCT-4 expression (p<0.001; β=0.985). The regression test results showed that time effects on HSP27 expression (p<0.001; β=0.398) and hypoxia precondition also affects HSP27 expression (p<0.001; β=0.847). Pathway analysis showed that BCL2 expression inhibited apoptosis (p=0.030; β=-0.442) and HSP27 expression also inhibited apoptosis (p<0,001;β=-0.487). Conclusion: Hypoxic preconditioning of h-AMSC culture has proven to increase the expression of VEGF, SCF, OCT-4, and BCL2 and HSP27. This study demonstrated and explained the existence of a new mechanism of increased h-AMSC survival in cultures with hypoxic preconditioning (O2 1%) via VEGF, SCF, OCT-4, BCL2, and HSP 27.

Prunus persica (L.) Batsch blossom soluble dietary fiber synergia polyphenol improving loperamide-induced constipation in mice via regulating stem cell factor/C-kit, NF-κB signaling pathway and gut microbiota

This study aimed to investigate the ameliorating effects of peach blossom soluble dietary fiber (PBSDF) and polyphenol (PBP) combinations on loperamide (Lop)-induced constipation in mice, together with the possible mechanism of action. The results demonstrated that the combined use of PBSDF and PBP could synergistically accelerate the gastrointestinal transit rate and gastric emptying rate, shorten first red fecal defecation time, accelerate the frequency of defecation, regulate the abnormal secretion of gastrointestinal neurotransmitters and pro-inflammatory cytokines, and down-regulate the expressions of AQP3 and AQP8. Western blotting and RT-qPCR analysis confirmed that PBSDF + PBP up-regulated the protein and mRNA expressions of SCF and C-kit in SCF/C-kit signaling pathway, and down-regulated pro-inflammatory mediator expressions in NF-κB signaling pathway. 16S rRNA sequencing showed that the diversity of gut microbiota and the relative abundance of specific strains, including Akkermansia, Bacteroides, Ruminococcus, Lachnospiraceae_NK4A136_group, and Turicibacter, rehabilitated after PBSDF + PBP intervention. These findings suggested that the combination of a certain dose of PBSDF and PBP had a synergistic effect on attenuating Lop-induced constipation, and the synergistic mechanism in improving constipation might associated with the regulating NF-κB and SCF/C-kit signaling pathway, and modulating the specific gut strains on constipation-related systemic types. The present study provided a novel strategy via dietary fiber and polyphenol interactions for the treatment of constipation.

Network pharmacology combined with experimental validation reveals the mechanism of action of erpixing granules on functional dyspepsia

Ethnopharmacological relevanceFunctional dyspepsia (FD) is a prevalent gastrointestinal disorder characterised by high incidence and recurrence rates, posing significant health risks. Erpixing Granules (EPX), approved by the National Food and Drug Administration in 2002, are known for their spleen and stomach invigorating properties, effectively treating FD. However, its mechanism of action remains unclear. Aim of the studyThis study aims to elucidate EPX's mechanism of treating FD through network pharmacology, and experimental validation using FD animal models. MethodsIn this study, the chemical composition of EPX in positive and negative ion modes was analyzed by UHPLC-Q-TOF MS. The mass spectral data were processed and analyzed using MS-DIAL software to automatically match compound fragment information and identify the known components with the compound database to obtain the active components of EPX. SwissTargetPrediction was used to obtain EPX targets, while FD-related targets were sourced from GeneCards, OMIM and DisGeNET databases. A protein-protein interaction (PPI) network was constructed using the STRING platform, and potential signalling pathways of EPX were determined through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Finally, an FD model was established in rates by administering a 0.1% iodoacetamide sucrose solution, followed by tail clamp stimulation to experimentally validate the network pharmacology findings. ResultsOur results revealed 139 effective ingredients in EPX, targeting 60 core FD-related genes. PPI network analysis identified EGFR, CTNNB1 and NFκB1 as core target genes. The KEGG pathway analysis indicated that EPX can modulate FD progression through the PI3K/AKT signalling pathway. Animal experiments demonstrated EPX's capacity to increase body mass, food intake and food utilisation efficiency in FD rats, alongside increased gastric juice secretion, pepsin activity, trypsin activity, cholesterol, bile acid and bilirubin activity. HE examination revealed that EPX improved the inflammatory infiltration of gastric mucosal cells in rats. Furthermore, EPX also promoted gastric emptying and intestinal propulsion in mice. These results suggest that EPX improves spleen and stomach function, enhances the protective effect on the spleen and stomach and promotes food digestion and absorption. Immunofluorescence studies revealed upregulated expression of PI3K, AKT and ANO1 proteins in gastric tissue following EPX administration, while Western blotting indicated increased expression of SCF and C-kit proteins. ConclusionSuggesting EPX's anti-FD effect may involve the regulation of the SCF/C-kit signalling pathway and activation of downstream PI3K/AKT signalling pathway, thereby promoting gastrointestinal motility and improving FD symptoms.

Loss of c-Kit in Endothelial Cells Protects against Hindlimb Ischemia.

Critical limb ischemia (CLI) is the end stage of peripheral artery disease (PAD), and around 30% of CLI patients are ineligible for current treatments. The angiogenic benefits of c-Kit have been reported in the ischemia scenario; however, the present study demonstrates the effects of specific endothelial c-Kit signaling in arteriogenesis during hindlimb ischemia. We created conditional knockout mouse models that decrease c-Kit (c-Kit VE-Cadherin CreERT2-c-Kit) or its ligand (SCF VE-Cadherin CreERT2-SCF) specifically in endothelial cells (ECs) after tamoxifen treatment. These mice and a control group (wild-type VE-Cadherin CreERT2-WT) were subjected to hindlimb ischemia or aortic crush to evaluate perfusion/arteriogenesis and endothelial barrier permeability, respectively. Our data confirmed the lower gene expression of c-Kit and SCF in the ECs of c-Kit and SCF mice, respectively. In addition, we confirmed the lower percentage of ECs positive for c-Kit in c-Kit mice. Further, we found that c-Kit and SCF mice had better limb perfusion and arteriogenesis compared to WT mice. We also demonstrated that c-Kit and SCF mice had a preserved endothelial barrier after aortic crush compared to WT. Our data demonstrate the deleterious effects of endothelial SCF/c-Kit signaling on arteriogenesis and endothelial barrier integrity.

Hypoxic Preconditioning Promotes Survival of Human Adipose Derived Mesenchymal Stem Cell.

Background: Contributing factors for improved survival of human adipocytes mesenchymal stem cells (h-AMSCs) cultured through hypoxia preconditioning, in example apoptosis inhibition involving BCL2 and HSP27 expression, trigger signal expression (VEGF), SCF expression, OCT-4 expression, and CD44+ expression. The objective if this study was to explain the mechanism and role of hypoxic preconditioning and the optimal duration of hypoxic preconditioning exposure to improve survival of h-AMSCs. Methods: An experimental laboratory explorative study ( in vitro) with hypoxic preconditioning in h-AMSCs cultures. This research was conducted through four stages. First, isolation of h-AMSCs culture from adipose tissue of patients. Second, the characterization of h-AMSCs from adipose tissue by phenotype (flowcytometry) through CD44+, CD90+ and CD45-expression before being pre-conditioned for hypoxic treatment. Third, the hypoxic preconditioning in h-AMSCs culture ( in vitro) was performed with an oxygen concentration of 1% for 24, 48 and 72 hours. Fourth, observation of survival from h-AMSCs culture was tested on the role of CD44+, VEGF, SCF, OCT-4, BCL2, HSP27 with Flowcytometry and apoptotic inhibition by Tunnel Assay method. Results: The result of regression test showed that time difference had an effect on VEGF expression ( p<0.001; β=-0.482) and hypoxia condition also influenced VEGF expression ( p<0.001; β=0.774). The result of path analysis showed that SCF had effect on OCT-4 expression ( p<0.001; β=0.985). The regression test results showed that time effects on HSP27 expression ( p<0.001; β=0.398) and hypoxia precondition also affects HSP27 expression ( p<0.001; β=0.847). Pathway analysis showed that BCL2 expression inhibited apoptosis ( p=0.030; β=-0.442) and HSP27 expression also inhibited apoptosis ( p<0,001; β=-0.487). Conclusion: Hypoxic preconditioning of h-AMSC culture has proven to increase the expression of VEGF, SCF, OCT-4, and BCL2 and HSP27. This study demonstrated and explained the existence of a new mechanism of increased h-AMSC survival in cultures with hypoxic preconditioning (O2 1%) via VEGF, SCF, OCT-4, BCL2, and HSP 27.

Vascular Cell Adhesion Molecule-1 Regulates Bone Marrow Mesenchymal Stem Cell Maintenance and Niche Function

Bone marrow (BM) mesenchymal stem cells (MSCs) are rare non-hematopoietic perivascular stromal cells characterized by their unique ability to self-renew and differentiate into bone, cartilage, and fat, ensuring proper skeletal development and maintenance. Different subsets of BM MSCs associate with arteriolar and sinusoidal vessels to form specialized niches that regulate hematopoietic stem cell (HSC) function by secreting high levels of niche factors such as CXC-chemokine ligand 12 (CXCL12), stem cell factor (SCF), and Vascular Cell Adhesion Molecule-1 (VCAM1). VCAM1 is classically expressed on endothelial and stromal cells where it acts as an adhesion molecule that preferentially binds to α4β1 integrin on HSCs and progenitor cells (HSPCs). Deletion of Vcam1 in endothelial and hematopoietic cells induces HSPC mobilization into the peripheral blood without affecting endothelial cells homeostasis. Recent work from our lab further demonstrated that VCAM1 expression on HSCs and leukemic stem cells confers innate immune tolerance ( Nat Cell Biol 2022). While the contribution of HSC and endothelial-derived VCAM1 to homeostasis has been well studied, the role of MSC-derived VCAM1 on HSC and MSC maintenance and multilineage potency remains unknown. Our supporting data suggest that MSCs are the BM's main source of Vcam1 (p&amp;lt;0.0001) and that VCAM1 is critical for the maintenance, survival, and function of MSCs. Using a mouse model in which Vcam1 can be conditionally deleted in BM MSCs by a tamoxifen-inducible N-Cadherin-Cre ER line (referred to as Vcam1 N-CadCreER), we found that deletion of Vcam1 significantly reduces the number (4-fold, p=0.0030), frequency (4-fold, p=0.0042), and viability (2-fold, p=0.0039) of BM MSCs. Previous findings in breast cancer revealed that VCAM1 promotes PI3K/Akt survival signaling via Akt phosphorylation. Accordingly, Vcam1 deletion in N-Cadherin-Cre ER MSCs, demonstrated a significant reduction in phospho-Akt (Ser473) (p&amp;lt;0.05), supporting the role of VCAM1 in mediating PI3K/Akt survival signaling in MSCs. Intriguingly, the remaining viable VCAM1-null MSCs isolated from Vcam1 N-CadCreER mice exhibited reduced clonogenic potential (determined by CFU-F assays; 3-fold, p=0.0001). Furthermore, micro-CT analysis of 6-month-old Vcam1 N-CadCreER mice presented an altered bone phenotype compared to the control as evidenced by their significantly increased bone thickness and volume (p&amp;lt;0.05), yet significantly decreased tissue mineral density and medullary cavity area (p&amp;lt;0.05). Bulk RNA sequencing (RNAseq) of sorted viable MSCs revealed distinct grouping between Control and Vcam1 N-CadCreER MSCs. Gene ontology analysis demonstrated that deletion of Vcam1 caused an upregulation of pathways associated with inflammation and cytokine release while pathways associated with ossification, extracellular matrix organization, and skeletal development were all downregulated. Surprisingly, both bulk RNAseq and qPCR analysis confirmed a significant reduction in the expression of HSC niche factors ( Vcam1 Cxcl12 and Scf) following Vcam1 deletion, indicative of reduced niche activity. As expected, Vcam1 deletion in N-Cadherin-Cre ER MSCs, caused significant mobilization of HSCs into the blood (p&amp;lt;0.0001) and BM homing defects (p&amp;lt;0.05), however the number and repopulation capacity of BM HSCs remained unaltered, as seen by BM transplantation experiments. Surprisingly, imaging analysis of whole-mount bone marrow sternum of phenotypic HSCs revealed no differences in HSC niche localization between Vcam1-null and Control mice. Accordingly, ELISA analysis of BM extracellular fluid revealed no differences in overall CXCL12 and SCF levels suggesting that there may be some local or systemic niche compensation at play. Single-cell (sc)RNAseq of pooled BM and compact bone sorted non-hematopoietic stromal cells (DAPI -Lineage -CD45 -Ter119 -CD19 -CD71 -)from Vcam1 N-CadCreERand Control mice further confirmed the downregulation of HSC niche factors in MSCs, and overall remodeling of the BM stromal cell composition following Vcam1 deletion. Interestingly, our scRNAseq analysis revealed increased expression of Cxcl12 and Scf in specific vascular and stromal cell populations, supporting the notion of HSC niche compensation. Overall, these findings demonstrate that MSC-derived Vcam1 is critical for BM MSC survival and their HSC niche function.

Targeting Normal Myelopoiesis Negatively Affects CAR T Cell Activity

Chimeric antigen receptor (CAR) T cells have not been successful for the treatment of acute myeloid leukemia (AML) to date, and the reason for this is not clear. While CAR T cells have been traditionally studied as independent agents targeting tumor, in reality they work in the context of the endogenous host immune system, which may influence outcomes of therapy. We hypothesized that targeting normal myelopoiesis is detrimental to CAR T cell activity, and this creates a unique obstacle to effective CAR T cell therapy for AML. We generated prototypical CAR T cells targeting B cell (CD19) and myeloid (CD33) antigens, using a standard 2 nd generation CAR design with 4-1BB and CD3ζ costimulatory domains. In parallel we created tumor cell lines expressing both targets by adding CD33 expression to a CD19+ B cell line (Ramos +33), and by adding CD19 expression to a CD33+ myeloid cell line (THP1 +19). For ease of tracking tumor cells were labeled with GFP, and CAR T cells were labeled with RFP. CD19-targeting CAR T cells (CART19) and CD33-targeting CAR T cells (CART33) exhibited similar anti-tumor activity in standard in vitro cytotoxicity assays and in vivo NSG mouse models. We then proceeded to add PBMCs to CAR T cell and tumor co-culture and measured tumor cell clearance and CAR T cell expansion using flow cytometry and the Incucyte Live-Cell analysis system. Addition of PBMCs enhanced the tumor clearance and CAR T cell expansion of both CART19 and CART33 in short-term co-culture assays (7-10 days). Fractionation of PBMCs revealed that CD33+ classical monocytes were able to replicate the effect of PBMCs, while the presence of CD19+ B cells did not have any effect. Monocytes are viable in vitro for less than 7 days. To assess the long-term effects of monocytes on CAR T cells, we performed serial replating assays, in which fresh tumor cells and monocytes were added to CAR T cells every 7 days. Ongoing monocyte support further improved CART19 expansion and tumor clearance. However, the continuous presence of monocytes was detrimental to CART33 activity upon serial replating, as evidenced by suboptimal T cell expansion and loss of tumor control. To study the effects of normal myelopoiesis on CAR T cell activity in vivo, NSGS mice (NOD/SCID/IL2Rg null mice with human SCF, IL-3 and GM-CSF expression) were injected with human cord blood CD34+ cells to engraft human hematopoiesis. Mice were then injected with THP1 +19 tumor cells, followed by administration of untransduced (UTD) control T cells, CART19 or CART33. To prevent the confounding effect of allogeneic killing, CAR T cells were generated from the same cord blood product as the CD34+ cells. Consistent with our in vitro data, CART19 and CART33 showed better anti-tumor activity in the presence of human hematopoiesis. Notably, while the anti-tumor activity of CART19 and CART33 was equivalent in non-humanized mice, CART19 had better long-term activity in humanized mice, as mice treated with CART33 had late tumor relapses. To directly compare the effects of myeloid cells as bystanders versus targets, we lentivirally transduced cord blood CD34+ cells with human CD19 so that they would be targets for CART19. We then differentiated these cells into myeloid progenitors (MP) by culturing in IMDM with 20% FBS and the following cytokines: SCF (100ng/ml), FLT3L (50ng/ml), IL-3 (10ng/ml), and GM-CSF (10ng/ml). After 10 days of culture &amp;gt;95% of MP expressed CD33, and were selected for &amp;gt;95% CD19 expression (Fig A). Addition of non-transduced MP to CAR T cell and Ramos +33 co-culture enhanced CART19-mediated tumor clearance without affecting CART19 expansion. However, the presence of MP CD19+ had a detrimental effect on both tumor control and expansion of CART19, confirming that CAR T cells are less effective when targeting myeloid cells. In contrast, both MP and MP CD19+ had similar effects on CART33 that mirrored the negative effects of MP CD19+ on CART19 (Fig B). To summarize, we show that normal myeloid cells have beneficial effects on CAR T cell function, and targeting these same myeloid cells is detrimental to CAR T cell activity. These findings provide insight into the reason for suboptimal CAR T cell activity in AML, and show that independent of target antigen and CAR construct, the nature of the cells being targeted can change the effectiveness of this therapy.

Fenchone Ameliorates Constipation-Predominant Irritable Bowel Syndrome via Modulation of SCF/c-Kit Pathway and Gut Microbiota.

In this study we sought to elucidate the therapeutic effects of fenchone on constipation-predominant irritable bowel syndrome (IBS-C) and the underlying mechanisms. An IBS-C model was established in rats by administration of ice water by gavage for 14 days. Fenchone increased the reduced body weight, number of fecal pellets, fecal moisture, and intestinal transit rate, and decreased the enhanced visceral hypersensitivity in the rat model of IBS-C. In addition, fenchone increased the serum content of excitatory neurotransmitters and decreased the serum content of inhibitory neurotransmitters in the IBS-C rat model. Meanwhile, western blot and immunofluorescence experiments indicated that fenchone increased the expressions of SCF and c-Kit. Furthermore, compared with the IBS-C model group, fenchone increased the relative abundance of Lactobacillus, Blautia, Allobaculum, Subdoligranulum, and Ruminococcaceae_UCG-008, and reduced the relative abundance of Bacteroides, Enterococcus, Alistipes, and Escherichia-Shigella on the genus level. Overall, fenchone ameliorates IBS-C via modulation of the SCF/c-Kit pathway and gut microbiota, and could therefore serve as a novel drug candidate against IBS-C.

Salidroside affects the Th17/Treg cell balance in aplastic anemia via the STAT3/HIF-1α/RORγt pathway

ABSTRACTBackgroundAcquired aplastic anemia (AA) is a life-threatening disease associated with an imbalance in Th17/Treg cells. Regulating this balance may be an effective treatment approach for AA. Rhodiola rosea has shown efficacy in AA treatment, but its mechanisms remain unclear.PurposeWe investigated salidroside's effect (a component of Rhodiola rosea) on Th17/Treg balance in adult AA patients and a mouse model.MethodsHIF-1α mRNA and protein levels were measured in AA patients' peripheral blood. Flow cytometry, qRT-PCR, and WB analyzed salidroside's impact on T cell differentiation, Th17 cells, Treg cells, STAT3, HIF-1α, and RORγt expression. ELISA measured hematopoietic growth factors in mouse serum.ResultsAA patients exhibited elevated HIF-1α levels. Salidroside improved hematopoietic function, increasing blood cell count and enhancing bone marrow. Salidroside induced SCF, TPO, and IL-3 expression while inhibiting IL-2 in mice. Salidroside reduced STAT3, HIF-1α, RORγt, and IL-17a, while increasing FoxP3 expression, correcting the Th17/Treg imbalance in vitro and in vivo.ConclusionSalidroside has potential as a novel AA treatment by correcting the Th17/Treg imbalance through the STAT3/HIF-1α/RORγt pathway.

Expression analysis of genes involved in the expansion of hematopoietic stem cells (SCF, Flt3-L, TPO, IL-3, and IL-6) in unrestricted somatic stem cells cultured on fibrin

Umbilical cord blood (UCB) transplantation is a promising therapeutic approach for patients lacking HLA-matched donors. A main limitation to the use of UCB-derived HSCs (UCB–HSCs) is the low number of transplantable cells. Novel culture strategies are being developed to increase the number of HSCs. Unrestricted somatic stem cells (USSCs) have been identified as promising stromal cells for supporting HSC expansion. The current study aimed to explore the effect of fibrin on the expression of hematopoiesis-related genes (SCF, Flt3-L, TPO, IL-3, and IL-6) in USSCs. USSCs were isolated from UCB and characterized by flow cytometry and in vitro multilineage differentiation ability. DAPI staining and the MTT assay were used to assess the effect of fibrin on USSC viability. The cell attachment was evaluated using SEM. qRT-PCR was performed to evaluate the expression of SCF, Flt3-L, TPO, IL-3, and IL-6 in USSCs cultured on 3D fibrin scaffolds. USSCs were positive for CD73, CD105, and CD166 and negative for CD45. Alizarin red and Oil red O stains confirmed calcium deposition and lipid vacuoles in USSCs. Results obtained from DAPI and MTT assays revealed a positive effect of fibrin on USSC viability. Cells cultured on fibrin express significantly higher levels of SCF and TPO compared to those grown in a 2D environment. The positive effect of fibrin on IL-6 levels was reversed. Fibrin did not affect Flt3-L expression and IL-3 mRNA expression was not detected in either group. The results of this study provide the basis for developing further research on the ex vivo expansion of HSCs with USSCs.

Longitudinal transcriptional analysis of peripheral blood leukocytes in COVID-19 convalescent donors

BackgroundSARS-CoV2 can induce a strong host immune response. Many studies have evaluated antibody response following SARS-CoV2 infections. This study investigated the immune response and T cell receptor diversity in people who had recovered from SARS-CoV2 infection (COVID-19).MethodsUsing the nCounter platform, we compared transcriptomic profiles of 162 COVID-19 convalescent donors (CCD) and 40 healthy donors (HD). 69 of the 162 CCDs had two or more time points sampled.ResultsAfter eliminating the effects of demographic factors, we found extensive differential gene expression up to 241 days into the convalescent period. The differentially expressed genes were involved in several pathways, including virus-host interaction, interleukin and JAK-STAT signaling, T-cell co-stimulation, and immune exhaustion. A subset of 21 CCD samples was found to be highly “perturbed,” characterized by overexpression of PLAU, IL1B, NFKB1, PLEK, LCP2, IRF3, MTOR, IL18BP, RACK1, TGFB1, and others. In addition, one of the clusters, P1 (n = 8) CCD samples, showed enhanced TCR diversity in 7 VJ pairs (TRAV9.1_TCRVA_014.1, TRBV6.8_TCRVB_016.1, TRAV7_TCRVA_008.1, TRGV9_ENST00000444775.1, TRAV18_TCRVA_026.1, TRGV4_ENST00000390345.1, TRAV11_TCRVA_017.1). Multiplexed cytokine analysis revealed anomalies in SCF, SCGF-b, and MCP-1 expression in this subset.ConclusionsPersistent alterations in inflammatory pathways and T-cell activation/exhaustion markers for months after active infection may help shed light on the pathophysiology of a prolonged post-viral syndrome observed following recovery from COVID-19 infection. Future studies may inform the ability to identify druggable targets involving these pathways to mitigate the long-term effects of COVID-19 infection.Trial Registration: https://clinicaltrials.gov/ct2/show/NCT04360278 Registered April 24, 2020.

OR25-3 Sperm-carried IGF2 Downregulates Mitogens Released by Sertoli Cells: A Paracrine Mechanism of Spermatogenetic Regulation?

Abstract The presence of insulin-like growth factor 2 (IGF2) mRNA has been demonstrated in human and mouse spermatozoa. However, the expression of the IGF2 protein in spermatozoa and its function is not known. To accomplish this, we searched for IGF2 protein in spermatozoa of four healthy Caucasian men by Western blot (WB) and immunofluorescence (IM). Semen samples were collected by masturbation into a sterile container after 3-4 days of sexual abstinence. The spermatozoa were separated from leucocytes and epithelial cells by swim-up. The WB demonstrated the presence of the IGF2 protein in each sample examined. IM showed that IGF2 is a cytoplasmic protein with a variable degree of expression in each spermatozoon and localized in the equatorial and post-acrosomal segments. To evaluate the effects of IGF2, porcine Sertoli cells (SCs) were isolated from neonatal Large White pigs (7-15 days) using established methods. SCs were incubated with increasing concentrations (0.33, 3.33, and 10 ng/mL) of recombinant human IGF2 (rhIGF2) for 48 hours. At the end of incubation, the following endpoints were evaluated: a) expression of the GDNF, FGF2, and SCF genes, known to be mitogens that promote gonocyte proliferation and differentiation towards their spermatogonial fate, by real-time PCR (RT-PCR); b) gene (RT-PCR) and protein (WB and IM) expression of the follicle-stimulating hormone receptor (FSHR); and c) proliferation of SCs by flow cytometry. Subsequently, the effects of IGF2 were re-evaluated by pre-treating SCs with NVP-AEW541, a not competitive inhibitor of the insulin-like growth factor 1 receptor (IGF1R), known to be activated by IGF2. For this purpose, the NVP-AEW541 was added to the culture medium at a concentration of 1 µg/mL one hour before the IGF2 and was left for the entire duration of the incubation. The results of this study showed that IGF2 significantly downregulates GDNF gene expression in a concentration-dependent manner (-40.3% at 0.33 ng/mL, -48.2% at 3.33 ng/mL, -55.5%, respectively, p&amp;lt;0.01). Gene expression of FGF2 and SCF was significantly downregulated only by IGF2 at the highest concentration used (-23.6% and -39.1%, respectively, p&amp;lt;0.01). Similarly, IGF2 incubation downregulated FSHR mRNA (-27.1% at 0.33 ng/mL, -45.4.2% at 3.33 ng/mL, -21.1%, respectively, p&amp;lt;0.01) and protein (for both WB and IM) expression. Finally, SC proliferation significantly decreased after incubation with IGF2 (2.82±0.1% in untreated controls, -1.87±0.1% at 0.33 ng/mL, -2.19±0.2% at 3.33 ng/mL, and -2.36±0.1% at 10 ng/mL; p&amp;lt;0.01). In the presence of NVP-AEW541, all these effects were counteracted, thus suggesting that they are mediated by the IGF1R. In conclusion, these results suggest the presence of a paracrine regulatory mechanism between germ cells and SCs by which the former can influence the proliferation of SCs, their sensitivity to FSH, and their function by reducing the secretion of germ cell mitogens. Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 a.m.

Hypoxic preconditioning promotes survival of human adipose derived mesenchymal stem cell via expression of prosurvival and proangiogenic biomarkers

Background: Contributing factors for improved survival of human adipose derived mesenchymal stem cells (h-AMSCs) cultured through hypoxia preconditioning, in example apoptosis inhibition involving BCL2 and HSP27 expression, trigger signal expression (VEGF), SCF expression, OCT-4 expression, and CD44+ expression. The objective if this study was to explain the mechanism and role of hypoxic preconditioning and the optimal duration of hypoxic preconditioning exposure to improve survival of h-AMSCs. Methods: An experimental laboratory explorative study (in vitro) with hypoxic preconditioning in h-AMSCs cultures. This research was conducted through four stages. First, isolation of h-AMSCs culture from adipose tissue of patients. Second, the characterization of h-AMSCs from adipose tissue by phenotype (flowcytometry) through CD44+, CD90+ and CD45-expression before being pre-conditioned for hypoxic treatment. Third, the hypoxic preconditioning in h-AMSCs culture (in vitro) was performed with an oxygen concentration of 1% for 24, 48 and 72 hours. Fourth, observation of survival from h-AMSCs culture was tested on the role of CD44+, VEGF, SCF, OCT-4, BCL2, HSP27 with Flowcytometry and apoptotic inhibition by Tunnel Assay method. Results: The result of regression test showed that time difference had an effect on VEGF expression (p&lt;0.001;β=-0.482) and hypoxia condition also influenced VEGF expression (p&lt;0.001;β=0.774). The result of path analysis showed that SCF had effect on OCT-4 expression (p&lt;0.001; β=0.985). The regression test results showed that time effects on HSP27 expression (p&lt;0.001; β=0.398) and hypoxia precondition also affects HSP27 expression (p&lt;0.001; β=0.847). Pathway analysis showed that BCL2 expression inhibited apoptosis (p=0.030; β=-0.442) and HSP27 expression also inhibited apoptosis (p&lt;0,001;β=-0.487). Conclusion: Hypoxic preconditioning of h-AMSC culture has proven to increase the expression of VEGF, SCF, OCT-4, and BCL2 and HSP27. This study demonstrated and explained the existence of a new mechanism of increased h-AMSC survival in cultures with hypoxic preconditioning (O2 1%) via VEGF, SCF, OCT-4, BCL2, and HSP 27.

Aberrant BMP15/HIF-1α/SCF signaling pathway in human granulosa cells is involved in the PCOS related abnormal follicular development

Aims To investigate the regulatory mechanism of SCF expression in human GCs of PCOS related follicles. Materials and Methods SCF, BMP15 and HIF-1α were evaluated in human serums, follicular fluids (FFs) and GCs, which were collected from 69 PCOS patients and 74 normal ovulatory patients. KGN cell line was used in this study. Results Our results showed that the rate of MII oocyte and 2PN fertilization was lower in PCOS group, though PCOS patients retrieved much more oocytes. The level of BMP15 in FF and the level of SCF in serum and FF were also lower in PCOS patients. We found a weakened expression of HIF-1α and SCF in GCs from PCOS patients when compared with the non-PCOS patients. The expression of HIF-1α and SCF was significantly increased in KGN cells after treating cells with rhBMP15, however, this promotion effects of BMP15 on HIF-1α and SCF expression were obviously abolished by co-treatment with BMP-I receptor inhibitor (DM). Moreover, knock down of HIF-1α expression in KGN cells significantly reduced the expression of SCF in human GCs, in spite of activating BMP15 signaling pathway. Conclusions The present study suggest that BMP15 could induce SCF expression by up-regulating HIF-1α expression in human GCs, the aberrance of this signaling pathway might be involved in the PCOS related abnormal follicular development.

Effect of miR-129-3p on autophagy of interstitial cells of Cajal in slow transit constipation through SCF C-kit signaling pathway.

To explore the mechanism by which miR-129-3p affected the autophagy of interstitial cells of Cajal (ICCs) in slow transit constipation tissues through the SCF C-kit signaling pathway. Colon samples from 20 Slow transit constipation (STC) patients who underwent total colectomy plus ileorectal anastomosis or subtotal colon resection plus anti-peristaltic rectal anastomosis were collected in our hospital. The colon of 20 non-STC patients was used as control. The control of this study was 20 patients undergoing radical surgery for colon cancer (left colon cancer) in our hospital. Fifty healthy SPF Kunming mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The mRNA expression of miR-129-3p in the STC group was lower than that in the control group (CTLR) group (P<0.05). The mRNA expression of miR-129-3p in STC group was lower than that in the NC group (P<0.05), and mRNA expression in STC+miR-129-3p group was higher than that in STC+miR-NC group (P<0.05). In the first week, the weight of dry and wet feces of the STC group was lower than that of the NC mice (P<0.05), and the weight of dry feces and wet feces of the STC group was lower than that of the NC group at the 2, 3, and 4 weeks, STC+miR-129 -3p was higher than that in the STC group (P<0.05). The increased expression of C-kit and SCF regulated by miR-129-3p contributed to the protection of interstitial cells. Knockdown of miR-129-3p expression could inhibit the activation of AKT/mTOR signaling pathway, reduce cell proliferation activity.

Modulating the Siah2-PHD3-HIF1α axis and/or autophagy potentially retard colon cancer proliferation possibly, due to the damping of colon cancer stem cells

BackgroundHypoxic microenvironment of colon cancer is associated with HIF-1α upregulation. HIF-1α response elements are responsible for autophagy induction that promotes tumor proliferation. Moreover, HIF-1α induces tumor cell proliferation via maintaining cancer stem cells (CSCs) survival. Siah2 is E3 ubiquitin ligase that indirectly stabilizes HIF-1α. We hypothesized that dual inhibition of Siah2 as well as autophagy could be a promising approach that may inhibit CSCs growth. Aim of the workThis study investigated the possible effect of vitamin K3 as a Siah2 inhibitor and hydroxychloroquine as an autophagy inhibitor in colon cancer management. The effect (if any) of these agents on CSCs growth will be also manipulated. MethodsColon cancer was induced by dimethylhydrazine. MDA and GSH were selected as oxidative stress markers, Expression of HIF-1α, Caspase-3, VEGF, MMP-9, EpCAM, SCF, and CA19.9 were assayed using immunoassay. The Western blot technique was used to assess LC3Ⅰ, CD44, and CD133 whereas RT-PCR was used to investigate PHD3 and CD44 in colon tissues. Additionally, Ki-67 and Siah2 were detected immunohistochemically. Resultsvitamin K3 and hydroxychloroquine either alone or in combination downregulated the expression of Siah2 and HIF-1α through upregulating PHD3 in colon tissues. This combination significantly downregulated MDA, Ki-67, VEGF, and MMP-9 expression and upregulated the expression of GSH and caspase-3. LC3Ⅰ was also upregulated. Interestingly, these therapeutic options were correlated with down-regulation of the cancer stem cell marker such as CD44 and EpCAM. ConclusionOur results suggested that suppression of both Siah2-PHD3-HIF-1α axis and autophagy retard colon cancer proliferation and dampened CSCs

Poster 106: Commonly Used Orthopaedic Clinical Therapeutics Affect the Cytokine Activity of Culture-Expanded Mesenchymal Stem Cells

Objectives:Hyaluronic acid (HA), platelet-rich plasma (PRP), and methylprednisolone are widely used for the non-operative management of osteoarthritis (OA). More recently, human bone marrow derived mesenchymal stem cells (BM-MSCs) have garnered significant interest as a non-operative means for symptom modification of OA. Anti-inflammatory cytokine secretion is a major component of proposed BM-MSC action. We sought to describe the effect of HA, PRP, and methylprednisolone on the cytokine profile of BM-MSCs at multiple stages of culture expansion.Methods:Following Institutional Review Board and United States Food and Drug Administration approval, nine BM-MSC cell lines from 4 human donors underwent three stages of culture expansion: passage 2 (P2), passage 3 (P3), and passage 4 (P4). Levels of OA-related cytokines (IL-1β, IL-6, IL-8, IL-10, Stem cell Factor [SCF], Stem Cell Derived Factor-alpha [SDF-α]) were measured using Luminex multiplexing technology. Each of the BM-MSC preparations were evaluated at each passage, 24 hours following exposure to HA, PRP, or methylprednisolone. Paired T-tests and Wilcoxon signed-rank testing were performed as appropriate following assessment of normality. Significance was set at p<0.05.Results:Culture expansion significantly altered cytokine production, however treatment with current OA therapeutics further altered these responses implicating the potential of combined treatment of culture expanded hMSCs and OA therapeutics. Figure 1 demonstrates the behavior of selected cytokines after exposure to HA, PRP or methylprednisolone preparations. Further, we tested different concentrations of each of the therapeutics to determine optimal dosing. Exposure to increasing doses of HA led to reductions in BM-MSC expression of SCF, SDF-α, VEGF, CCL20, and adiponectin (p<0.05 for all). PRP increased IL-1 levels in P2 and P3 (p<0.05 for both), IL-6 in P2 (100 pg/mL vs 5500 pg/mL, p<0.001), and IL-8 in P3 (900 pg/mL vs 3900 pg/mL, p<0.001). PRP did not affect IL-10 expression (p>0.05). Aside from an increase in IL-6 production for P2 (200 pg/mL vs 1500 pg/mL, p<0.05), methylprednisolone did not have significant effects on cytokine expression.Conclusions:Orthopaedic therapeutic adjuvants influence the inflammatory cytokine profile of BM-MSCs at multiple stages of culture expansion. Although further study is needed, these therapeutics have potential to be used in tandem with BM-MSCs to create an optimal cytokine environment for treatment of degenerative joint disease.Figure 1.hMSCs and Osteoarthritis Adjuvants. Human bone marrow derived hMSCs were culture expanded over 4 passages and treated with hyaluronan (HA), platelet rich plasma (PRP) and methylprednisolone (DEPO). Culture expansion significantly altered cytokine production, however treatment with current OA therapeutics further altered these responses implicating the potential of combined treatment of culture expanded hMSCs with OA therapeutics.

S245: ALTERATION OF BONE MARROW NICHE BY ALLOGENEIC IMMUNE REACTION AFTER HSCT

Background: Allogeneic stem cell transplantation (allo-SCT) has been considered as a curable therapy for hematologic diseases. Post-transplant myelosuppression is one of the life-threatening complications. Hematopoietic stem cell (HSC) functions are tightly regulated by a specialized microenvironment called “niche” in the bone marrow (BM) and we have identified differential contributions of perivascular stromal cells as HSC niche (Asada et al. Nat Cell Biol 2017). Recent studies indicate that BM niche can be targeted by allo-immune reaction but it remains unclear how the perivascular niche cells are involved in the mechanism of HSC dysfunction after allo-SCT. Aims: In this study, we aim to investigate the mechanisms of the impairment of the niche cells by allo-immune reactions, and to explore a targetable pathway for myelosuppression after allo-SCT. Methods: To evaluate the alterations in niche cells after allo-SCT, we utilized allo-SCT mice models (C3H/HeJ into C57BL/6, C57BL/6 into B6D2F1). Hematopoietic cells, including HSCs and mature cells, and perivascular stromal cells in BM were analyzed by flow cytometry (FACS) after transplantation. In imaging studies, we visualized perivascular stromal cells using Nestin-GFP (Nes-GFP), Myh11-CreER/tdTomato, LeptinR-Cre/tdTomato transgenic mice and observed them with confocal laser scanning microscopy. The gene expression analysis of niche factors was performed by real-time PCR. To assess the landscape of transcriptional remodeling in BM after allo-SCT, we performed single-cell RNA sequencing (scRNAseq) of BM cells. Results: FACS analyses showed that Nes-GFP+ stromal cells were dramatically reduced, and HSC recovery was severely impaired in allo-SCT mice compared to control mice at day 21 after SCT. Imaging studies also revealed that perivascular stromal cells are morphologically damaged in allo-SCT mice. Nes-GFP+ stromal cells in allo-SCT mice presented a decreased expression of niche factors, including Cxcl12 and Scf, which are essential for HSC maintenance, indicating the dysfunction of niche cells. We confirmed the impaired niche function in allo-SCT mice by tandem transplantation analysis in vivo, in which the recovery of syngeneic HSCs was also impaired in damaged microenvironment in allo-SCT mice. Prevention of allo-reactive T cell expansion by post-transplant cyclophosphamide ameliorated the niche cell reduction along with improved recovery of HSCs. These results indicate that niche impairment caused by allo-immune reaction leads to HSC dysfunction. The scRNAseq of BM highlighted the elevated expression levels of inflammation response genes, including Cxcl9 and Cxcl10, and MHC classⅡ, together with the deceased expression of niche factors in niche cells in allo-SCT mice. These alterations seemed to be induced by proinflammatory cytokines such as interferon-gamma (INF-γ) derived from allo-T cells, as the increased number of T cells and high levels of INF-γ in BM were observed in allo-SCT mice. Additionally, evaluation of T cell localization by using imaging techniques of BM revealed that allo-T cells reside significant closer to LepR+ niche cells in allo-SCT mice. Since activation of CXCR3, which is a receptor for chemokines Cxcl9 and Cxcl10, has been shown to induce chemotaxis of activated T cells, we treated allo-SCT mice with AMG487, an antagonist of CXCR3, resulted in an improved recovery of both perivascular niche cells and hematopoiesis in allo-SCT mice. Summary/Conclusion: Collectively, allo-immune reaction severely damages perivascular HSC niche cells, leading to impaired recovery of hematopoiesis after allo-SCT.